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S.No GSE ID Profile Summary
affy_fsh_human - affy_fsh_human - - G protein-coupled receptors (GPCR) are centrally involved in most physiological processes and are a major drug targets. They transduce extracellular signals inside the cells through at least two different mechanisms: i) the classical coupling to heterotrimeric G proteins and ii) a newly discovered beta-arrestin-dependent pathway. The fundamental issue of the respective impacts that these two transduction mechanisms exert on gene regulation has not been clearly addressed to date. To tackle this question we have developed two mutants of the follicle stimulating hormone (FSH) receptors which do not couple to G proteins upon FSH activation but continue to recruit beta-arrestins and signal through them.-In the present study, we compare the wild-type FSH receptor to either the R466A or the T469F mutants. These two mutations are localized in the second intra cellular loop of the FSH receptor and prevent G protein coupling to the active FSH receptor. Each receptor was permanently expressed in HEK-293 cells at comparable levels. Cells were treated or not for 6 hours with 3 nM FSH. Keywords: treated vs untreated comparison,wt vs mutant comparison
In vertebrates non-lens bg-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit) and trefoil factor (b-subunit), named bg-CAT, was identified from frog Bombina maxima skin secretions. Its a-subunit and b-subunit (containing three trefoil factor domains), with a non-covalently linked form of ab2, show significant sequence homology to ep37 proteins, a group of non-lens bg-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. The bg-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of bg-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. The bg-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. However, what exactly target of bg-CAT act on HUVECs nuclear is still unknown. Primary cultured HUVECs treated with bg-CAT (25 nM, 2 h) were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genome wide level of significant differential gene expression induced by bg-CAT on HUVECs in order to get clues about bg-CAT action mechanisms. These findings illustrate novel cellular functions of non-lens bg-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.
Microarray analysis revealed differential gene expression patterns of rhabdomyosarcoma (A-204) leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cells treated with TRAIL and/or taurolidine. To explore new therapeutic options in the treatment of sarcomas, we tested the antibiotic taurolidine (TRD) on A-204, SK-LMS-1 and VA-ES-BJ carcinoma cell lines alone and in combination with rhTRAIL (TNF related apoptosis-inducing ligand). Gene expression was analysed by RNA microarray.
Normal-appearing epithelium of cancer patients can harbor occult genetic abnormalities. Data comprehensively comparing gene expression between histologically normal breast epithelium of breast cancer patients and cancer-free controls are limited. The present study compares global gene expression between these groups. We performed microarrays using RNA from microdissected histologically normal terminal ductal-lobular units (TDLU) from 2 groups: (i) cancer normal (CN) (TDLUs adjacent to untreated ER1 breast cancers (n = 14)) and (ii) reduction mammoplasty (RM) (TDLUs of age-matched women without breast disease (n = 15)). Cyber-T identi?ed differentially expressed genes. Quantitative RT-PCR (qRT-PCR) immunohistochemistry (IHC), and comparison to independent microarray data including 6 carcinomas in situ (CIS), validated the results. Gene ontology (GO), UniProt and published literature evaluated gene function. About 127 probesets, corresponding to 105 genes, were differentially expressed between CN and RM (p < 0.0009, corresponding to FDR <0.10). 104/127 (82%) probesets were also differentially expressed between CIS and RM, nearly always (102/104 (98%)) in the same direction as in CN vs. RM. Two-thirds of the 105 genes were implicated previously in carcinogenesis. Overrepresented functional groups included transcription, G-protein coupled and chemokine receptor activity, the MAPK cascade and immediate early genes. Most genes in these categories were under-expressed in CN vs. RM. We conclude that global gene expression abnormalities exist in normal epithelium of breast cancer patients and are also present in early cancers. Thus, cancer-related pathways may be perturbed in normal epithelium. These abnormalities could be markers of disease risk, occult disease, or the tissue’s response to an existing tumor. Keywords: disease state analysis
Pathways that govern normal stem cell (SC) function are often subverted in cancer. Here we report the isolation to near purity of human normal mammary SC (hNMSCs), from cultured mammospheres, based on their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. We demonstrated that PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we could prospectively isolate SCs from the normal gland and from breast tumors. Poorly-differentiated aggressive (G3) cancers displayed higher content of prospectively isolated cancer SCs, than well-differentiated less aggressive (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in cancer SCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their SC content.
Experiments using xenografts show that some solid tumours and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSC). Despite promise the relevance of the CSC model to human disease remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model based on sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSC) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSC and HSC, revealing the molecular machinery underlying stemness properties. Both stem cell programs were highly significant independent predictors of patient survival and also found in existing prognostic signatures. Thus, determinants of stemness influence clinical outcome of AML establishing that LSC are clinically relevant and not mere artifacts of xenotransplantation.
In this study we have investigated the gene expression profiles of three different types of subclone all generated by single cell cloning of the same parental EBV positive Burkitt lymphoma cell line Awia-BL. These included EBV negative clones which have lost the virus episome EBV positive clones with a conventional Latency I form of infection and EBV positive clones with an atypical Wp-restricted form of infection.
Samples were prospectively collected during colonoscopic examination from 46 rectal cancer patients before starting preoperative chemoradiotherapy. The expression profiles were determined using Affymetrix Human Genome U133 Plus 2.0 arrays. Comparison between the sample groups allow to identify a set of discriminating genes that can be used for prediction of the response to radiotherapy in rectal cancer.
Background: Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures. Methods: We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of different potential stem cell markers: CD133 CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts mRNA expression of the investigated markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated in vivo. Results: All surface markers showed distinct expression patterns in the examined tumours. Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated markers. CD44 and CXCR4 mRNA expression correlated within the cell line panel and CD44 and CDCP1 within the xenograft panel, respectively. Small subpopulations of double and triple positive cells could be described. SW620 showed significantly higher take rates and shorter doubling times in vivo when sorted for CD133 positivity. Conclusion: Our data support the hypothesis of a small subset of cells with stem cell-like properties characterized by a distinct surface marker profile. In vivo growth kinetics give strong relevance for an important role of CD133 within the mentioned surface marker profile. Key words: colon cancer, tumour stem cell, CD133
Adipose tissue-derived mesenchymal stromal cells (ATSC) hold great promises in regenerative medicine due to their easy retrieval, their high proliferative capacity, and overall, their multi-lineages differentiation potential. In the last decade, several studies have reported the plasticity of ATSC toward a hepatic fate. Nonetheless, the molecular mechanisms allowing the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. In this study, we investigated the full genome expression profiles of ATSC cultured for 4 weeks under pro-hepatogenic condition in comparison to control ATSC. Sets of differentially expressed genes were then functionally categorized to understand which pathways trigger the hepatic conversion. We showed that ATSC-derived hepatocyte-like cells overexpress sets of genes associated with hepatic functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted the downregulation of several transcripts involved in stemness maintenance along with genes associated with the epithelial-mesenchymal-transition. Taken together, these data suggest that in vitro hepatogenic differentiation converts ATSC into immature hepatic cells, functionally related to liver progenitor cells.
The objective of this study is to identify the genes that are up-regulated amid proteasome dysfunction to facilitate the discovery of proteolytic pathways that are activated as a compensatory response to proteasome inhibition. Proteasome is a large multi-component proteolytic complex in the cell. It is responsible for the constitutive turn-over of many cellular proteins as well as the degradation of oxidized and/or unfolded proteins. With such a fundamental role in the cell disruption of proteasome understandably can lead to disastrous outcome. Oxidative stress has been postulated as the driving mechanism for aging. Oxidatively modified proteins, which usually have lost their activity, require immediate removal by proteasome to maintain normal cellular function. Dysfunction of proteasome has also been linked to neuro-degenerative diseases such as Alzheimer’s and Parkinson’s diseases, those that are most commonly seen in aged population. There is more than one proteolytic pathway in the cell, and it has been reported that obstruction of any one of these pathways may enhance the activity of the others. Proteasomal function has been found to have decreased during aging, prompting researchers to hypothesize that failure to remove oxidized proteins may play an important role in aging. It would be interesting to determine the other proteolytic pathways that are activated after proteasome inhibition by a relatively specific inhibitor epoxomicin to help understand their roles in aging processes. Keywords: time course, proteasome, inhibitor, oxidative stress, epoxomicin
Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. More-over, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. We used microarrays to detail the global programme of gene expression after transduction of AKT and RAS
Our previous studies have shown that inhibition of the IGF-1R pathway by the IGF-1RTK inhibitor picropodophyllin (PPP) can be achieved and also constitutes a favorable therapeutic window in multiple myeloma (MM). As no complete remission using in vivo models of MM could be obtained a combinatorial drug screen (HTS) was performed to select the most performant combination with PPP. The HDAC inhibitor LBH589 was shown to act in synergy with PPP on survival of MM cells. The contribution from both drugs and the combination were further monitored for apoptosis, cell cycle distribution, and the impact on downstream gene and protein expression in human and mouse MM models in vitro. In the RPMI 8226 human MM cell line, simultaneous treatment with both compounds for 48h caused a 5-fold increase of apoptotic and late apoptotic/necrotic cells as compared to controls, while treatment with either compound alone only induced a 3-fold increase. After 24h cleavage of apoptotic proteins caspase -9, -8 and -3 could be found in RPMI 8226 cells treated with both drugs individually, but in the combination we observed an additive effect on the cleavage of the active forms of caspase 8 as compared to single drug treatments. The combination of LBH589 and PPP could be monitored as an accumulation of cells in the G2/M phase, and subsequent down-regulation of cell cycle regulated proteins. The effect of both compounds on the expression of cyclin B1, -E and -D2 was additive, as demonstrated by western blot. These data were also confirmed in the mouse 5T33MM cells in vitro. Gene expression analysis and validations of the RPMI 8226 cells reveal that the drug combination has better effects than the single drug alone. Combined treatment in vivo resulted in a significant prolonged survival of 5T33MM inoculated mice when compared to the control group and to treatment with the drugs alone. In conclusion, the results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589. We used whole genome Microarray to decipher the changes occuring after treatment of the single and combination of the drugs. And see if the combination has better effects.
We used microarrays to investigate the transcription profile of FOXC2 expression in a human mammary epithelial cell line.
cDNA and cRNA hybridization technologies have different probe-specific sensitivities. We used samples from an etanercept trial (GSE11903) to explore in a real-life setting the uniqueness of each platform.
Background: pregnancy is associated with reduced activity of multiple sclerosis (MS). However the biological mechanisms underlying this pregnancy-related decrease in disease activity are poorly understood. This data series contains the subset of data used to generate a healthy donors signature comparing female healthy specimens before pregnancy with respect to female healthy specimens at ninth month pregnancy.
Genome-wide mRNA expression profiles of 25 unique gastric cancer cell lines (GCCLs). Gastric cancer (GC) is the second leading cause of global cancer mortality with individual gastric tumors displaying significant heterogeneity in their deregulation of various oncogenic pathways. We aim to identify major oncogenic pathways in GC that robustly impact patient survival and treatment response. We used an in silico strategy based on gene expression signatures and connectivity analytics to map patterns of oncogenic pathway activation in 25 unique GCCLs, and in 301 primary gastric cancers from three independent patient cohorts. Of 11 oncogenic pathways previously implicated in GC, we identified three predominant pathways (proliferation/stem cell, NF-kB, and Wnt/b-catenin) deregulated in the majority (>70%) of gastric tumors. Using a variety of proliferative, Wnt, and NF-kB-related assays, we experimentally validated the pathway predictions in multiple GC cell lines showing similar pathway activation patterns in vitro. Patients stratified at the level of individual pathways did not exhibit consistent differences in clinical outcome. However, patients grouped by oncogenic pathway combinations demonstrated robust and significant survival differences (e.g., high proliferation/high NF-kB vs. low proliferation/low NF-kB), suggesting that tumor behavior in GC is likely influenced by the combined effects of multiple oncogenic pathways. Our results demonstrate that GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Keywords: gastric cancer, cell culture
Stable knockdown of NET1 a RhoGEF, was achieved in AGS Gastric Cancer cells. This gene is known to be overexpressed in the disease. Knockdown was achieved using lentiviral shRNA particles. Gene expression was compared between knockdown and scrambled shRNA treated control cells. Cells were treated with and without LPA, a known activator of RhoA.
The patients who underwent surgery for primary lesions were examined. All patients had metastatic or recurrent CRC and received bevacizumab therapy as first line or second line treatment. Responders and nonresponders were determined based on RECIST and confirmed by CT or MRI. Gene-expression profiles of primary CRC were determined using Human Genome GeneChip arrays U133.
Analysis of mobilized peripheral blood CD34+ cells from a healthy volunteer under erythroid differentiation conditions with and without stimulation to the BMP or Wnt signaling pathways. For erythroid differentiation expanded CD34+ cells were placed in Stemspan SFEM medium supplemented with 2% pen/strep, 20ng/ml SCF, 1U/ml Epo, 5ng/ml IL3, 2uM dexamethasone, and 1uM beta-estradiol. Arrays were performed 2 hours after addition of cytokines. For signaling pathway stimulation, cells were exposed to 0.5uM BIO (a GSK3 inhibitor) for Wnt pathway activation, 25ng/ml rhBMP4 for BMP pathway activation, or vehicle control for 2 hours. Three biological replicates were performed per treatment group. We used microarrays to detail the global program of gene expression changes after Wnt or BMP pathway stimulation in human CD34+ hematopoietic progenitors under erythroid differentiation conditions.
As part of a large genetic evolution study we also acquired 3'UTR expression arrays at two time points for the same 18 patients with CLL. We have analysed the data to evaluate whether genetic evolution (somatic mutations and Somatic copy number alterations) also manifested at the transcriptome level either globally, or at the level of pre-defined curated geneset that correspond to specfic evolving genetic lesions.
human foreskin fibroblasts were infected with HCMV We monitor cellular gene expression network altered by HCMV entry using Affymetrix Human Genome U133 Plus 2.0 Array
Background: Glioblastoma multiforme (GBM) is the most aggressive and most lethal primary malignant brain tumor correlated with survival rates of less than one year from the time of diagnosis. Current surgical procedure attempts to remove the bulk of the tumor mass, whereas GBM frequently recurs within 1-3cm from the primary tumor resection site. Molecular mechanisms involved in the recurrence of the tumor are still poorly understood. The aim of the study was to define the molecular signature of GBM surrounding white matter (WM) in order to better understand the molecular mechanisms involved with tumor relapse. Material & Methods: Human GBM tumor bulk and surrounding tissue (1-3cm from the border of the tumor) were obtained from five patients who underwent total tumour resection, while normal white matter was harvested from patients who underwent surgical procedure for nonmalignant pathologies. Samples were processed for hybridization on the Affymetrix Human U133A arrays and data were examined with the GeneSpring analysis software. Results: Gene expression analysis of the samples was done in 2 independent steps. First, molecular profiling comparison of GBM surrounding WM and normal WM resulted in 59 genes differentially expressed between both tissues. Among these, numerous genes expressed by mature neural cells were down-regulated in GBM surrounding WM, while gene products supporting invasion were overexpressed. Moreover, KLRC1, a specific natural killer receptor naturally involved in the activation of antitumoral cells was drastically repressed in GBM surrounding WM, suggesting that the antitumoral immune surveillance is compromised in this tissue. Second, we focused our study on genes specifically regulated in GBM periphery respectively to GBM core. The highest up-regulated gene in GBM surrounding tissue encodes for DTX4, a regulator of NOTCH signalling pathway described for its key role in maintaining neural progenitors in an uncommitted state. Conclusion: This study revealed unique molecular characteristics of GBM surrounding tissue, showing the dysregulation of genes involved in immune surveillance along with genes associated to stemness maintenance. All together, these data may help to understand the molecular mechanisms associated with GBM recurrence
Cyclin T1-dependent genes in PMA-activated MM6 cells. HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4+ T cells and macrophages the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4+ T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that are Cyclin T1-dependent for their induction in activated CD4+ T cells and during macrophage differentiation and activation. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value < 0.00021). SiRNA depletions of two CTDGs identified here, CDK11 and Casein kinase1gamma1, suggest that these genes are also involved in HIV-1 replication. It is therefore likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors. Keywords: shrna knockdown
We performed a time-course microarray experiment to define the transcriptional response to carboplatin in vitro and to correlate this with clinical outcome in epithelial ovarian cancer (EOC). RNA was isolated from carboplatin and control-treated 36M2 ovarian cancer cells at several time points, followed by oligonucleotide microarray hybridization. Carboplatin induced changes in gene expression were assessed at the single gene as well as at the pathway level. Clinical validation was performed in publicly available microarray datasets using disease free and overall survival endpoints. Time-course and pathway analyses identified 317 genes and 40 pathways (designated time-course and pathway signatures) deregulated following carboplatin exposure. Both types of signatures were validated in two separate platinum-treated ovarian and NSCLC cell lines using published microarray data. Expression of time-course and pathway signature genes distinguished between patients with unfavorable and favorable survival in two independent ovarian cancer datasets. Among the pathways most highly induced by carboplatin in vitro, the NRF2, NF-kB, and cytokine and inflammatory response pathways were also found to be upregulated prior to chemotherapy exposure in poor prognosis tumors.
In osteosarcoma patients the development of metastases, often to the lungs, is the most frequent cause of death. To improve this situation, a deeper understanding of the molecular mechanisms governing osteosarcoma development and dissemination and the identification of novel drug targets for an improved treatment are needed. Towards this aim, we characterized osteosarcoma tissue samples compared to primary osteoblast cells using Affymetrix HG U133A microarrays.
We used microarrays to examine gene expression levels in individuals from different populations (cephgp chb, jpt, yri). The chb and jpt populations are combined in the analysis.
Hypoxia is a low oxygen condition that occurs in the developing tumor mass and that is associated with poor prognosis and resistance to chemo- and radio-therapy. The definition of the hypoxia gene signature is fundamental for the understanding of tumor biology as in the case of neuroblastoma, the most common pediatric solid tumor. The issue of identifying a significant group of variables in microarray gene expression experiments is particularly difficult due to the typical high dimensional nature of the data and great effort has been spent in the development of feature selection techniques. Our main goal is to define a robust hypoxia gene signature in neuroblastoma cell lines. A set of 11 neuroblastoma cell lines were cultured under normoxic and hypoxic conditions for 18 hours, and their gene expression profiles were measured with Affymetrix GeneChip HG-U133 Plus 2.0. We used the l1-l2 regularization framework in order to select the significant probesets defining hypoxic versus normoxic cell lines.
3D cultivation of cells lead to changes in morphology of the cells. This is likely to explain the higher radioresistance of cells growing in 3D compared to cells growing in 2D cell culture. Whole genome gene expression is performed to determine genes involved in changes of cell moroholgy and radioresistance. Keywords: comparison of 2D vs. 3D cell culture
Evaluation of specific coordinated pattern of transcriptional events consistent with anti-myeloma activity of FK866 (chemical Nampt inhibitor)
Multipotent progenitor cells (MPs) have been observed in human kidneys and particularly in Bowman's capsule and proximal tubules. The kidney owns the ability to repair local damage and renal MPs may play a role in the regenerative processes. Microarray technology was applied to identify differentially expressed genes among resident MPs isolated from glomeruli and tubules of normal renal tissue renal proximal tubular epithelial cells (RPTECs) and mesenchymal stem cells (MSCs). The results of our analysis represent a starting point for further functional studies. Keywords: cell type comparison
The purpose of this study was to use global gene expression analysis to determine major functional pathways and genes dysregulated in the skin of active rashes of dermatomyositis patients. These data will be used to help assign a diagnosis to skin biopsies from patients with rashes that are not clearly dermatomyositis. In addition we will correlate gene expression changes with specific histopathologic changes in skin biopsies that are taken adjacent to those analyzed for gene expression analysis. Finally, these data will be used to search for genes and pathways that are associated with clinical outcomes and autoantibody status in this cohort of patients.
Spontaneous cell fusion of MDA-MB-231 bone-metastatic subline Bm (i.e. SCP2) and lung metastatic subline Lm (i.e., LM2) gave rise to hybrid lines BLm-FACS or BLm-DRUG, as well as its single clones (#8, #12, #18). The hybrids acquired the metastasis tropisms from both parental cells. Expression profiles of the parental cells, the hybrids and several previously characterized MDA-MB-231 metastatic derivatives were compared. Hierarchical clustering showed the hybrids assimilated the organ-specific metastasis gene signatures from both parental cells. Keywords: Cell type comparison
Study designed to explore the effects of endothelial cell/MSC co-culture on individual gene expression profile of each cell type
Iron-deficiency affects 500 million people yet the molecular role of iron in gene expression remains poorly characterized. Moreover, the alterations in global gene expression after iron chelation remains unclear and are important to assess for understanding the molecular pathology of iron-deficiency and the biological effects of iron chelators. We assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both chelators, while a further 50 genes were regulated by either ligand. Most of the genes identified in this study have not been previously described to be iron-regulated and are important for understanding the molecular and cellular effects of iron-deficiency.
We used gene expression profiling of human DCIS and IBC to discover uniquely expressed genes that may also regulate progression.
In order to ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC) we analyzed the anti-tumour effects of Trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via MTT assay. TSA and vorinostat both displayed strong anti-tumor activities in a proportion of NSCLC cell lines, and suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified NCI program. In addition, pathway analysis was performed with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine (SVM) algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the SVM model was validated by an additional seven cell lines, resulting in a prediction value of 100% in respect to determining response to TSA. Our results suggested that [1] HDAC inhibitors may be promising anticancer drugs to NSCLC, and [2] the nine gene classifer is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients. training sample set: GSM94303 PC9 GSM94304 PC7 GSM94305 PC14 GSM94306 A549 GSM94308 LK2 GSM94313 RERF LC-KJ GSM94314 RERF LC-MS GSM94315 RERF-LC-AI GSM94316 PC-1 GSM94317 PC-3 GSM94319 PC-10 GSM94323 ABC-1 GSM94324 EBC-1 GSM94325 LC2/ad GSM94328 SQ-5 GSM94329 QG-56 test sample set: GSM94307 LU65 GSM94326 LC1/sq GSM94327 LC-1F GSM254967 LCOK GSM254968 LCD GSM254969 H1650 GSM254970 H1975 Keywords: HDAC inhibitor, NSCLC, cDNA array, drug sensitivity test, pathway analysis
Overall study: Identification of PDGF-dependent patterns of gene expression in U87 glioblastoma cells. RNA was obtained from triplicate dishes of 5 different groups of U87 cells each (total 15) analyzed with one U95 microarray chip. Three different comparisons were made: 1) Clone 3.1 (34580-34582) vs. clone 3.3 (34583-34585) vs. parent U87 (34592-34594). Purpose: demonstrate that the gene expression profiles between these 3 cell lines are not different, so they could be pooled as a single untreated group. 2) Pooled control group (34580-34585, 34592-34594) vs. clone 8.1 (34586-34588). Purpose: identify genes specifically controlled by autocrine PDGF activity. 3) Clone 8.1 (34586-34588) vs. clone 8.1 treated with PDGF (34589-34591) Purpose: Identify genes specifically induced by exogenous PDGF. Keywords = platelet-derived growth factor Keywords = glioblastoma Keywords = brain cancer Keywords = sterol regulatory element binding protein Keywords = SREBP Keywords: ordered
Adult T-cell leukemia (ATL) is a fatal neoplasia derived from HTLV-1 infected T lymphocytes exhibiting constitutive activation of NF-kB. To elucidate the complex molecular mechanism of anti-tumor effect of the proteasome inhibitor bortezomib in ATL cells, we attempted to perform gene expression profiling. Keywords: dose response
Layer II stellate neurons (entorhinal cortex) and layer III cortical neurons (hippocampus CA1 middle temporal gyrus, posterior cingulate, superior frontal gyrus, primary visual cortex) were gene expression profiled. Brain regions are from non-demented individuals with intermediate Alzheimer's disease neuropathologies Keywords: neuronal gene expression profiling
The human nm23-H1 was discovered as a tumor metastasis suppressor based on its reduced expression in melanoma cell lines with low versus high metastatic potential. It encodes for one of two subunits of the nucleoside-diphosphate kinase. Besides its role in the maintenance of the cells NTP pool nm23 plays a key role in different cellular processes. The role of nm23-H1 in these processes still has to be elucidated. Our goal was to identify Nm23-H1 downstream targets by subjecting Nm23-H1 overexpressing CAL 27 cells oral squamous cell carcinoma (OSSC) to microarray analysis. The genes with changed expression patterns could be clustered into several groups: transforming growth factor (TGF) signaling pathway, cell adhesion, invasion and motility, proteasome machinery, cell-cycle, epithelial structural and related molecules and others. Based on the expression patterns observed we presume that nm23-H1 might have a role in OSSCs, which should be confirmed by future experiments. Keywords: disease state analysis
We used gene expression profiling of human DCIS and IBC to discover uniquely expressed genes that may also regulate progression.
Using a transcriptomics approach we explored the mechanism(s) of synergy observed between CDKI-73 and fludarabine in primary CLL cells. The cytotoxic effects of CDKI-73 were associated with transcriptional inhibition of cdk9 target genes including MCL1 and XIAP. In contrast fludarabine induced the transcription of these genes, an effect that was reversed by the combination of CDKI-73 and fludarabine. We used microarrays to explore the cytoxic synergy observed in primary CLL cells when we combined a novel CDK9 inhibitor with the purine nucleoside analogue fludarabine
GIST is considered to invariably arise through gain-of-function KIT or PDGFRA mutation of the interstitial cells of Cajal (ICC). However the genetic basis of the malignant progression of GIST is poorly understood. We analysed the expression levels of 54,613 probe sets in 32 surgical samples of untreated GIST of the stomach and small intestine with GeneChip Human Genome U133 Plus 2.0 arrays. Keywords: gene expression array-based, count
Metastasis of tumor consists of a complex serias of cascades . Molecular mechanisms needs to be elucidated. We used microarrays to explore the global change of gene expression betwwen two pairs of osteosarcoma sublines screened by different method.
CD8+ T cells in chronic viral infections like HIV develop functional defects such as loss of IL-2 secretion and decreased proliferative potential that are collectively termed exhaustion1. Exhausted T cells express increased levels of multiple inhibitory receptors such as Programmed Death 1 (PD-1). PD-1 inhibition contributes to impaired virus-specific T cell function in chronic infection because antibody-mediated blockade of its ligand, Programmed Death Ligand 1 (PD-L1) is sufficient to improve T cell function and reduce viral replication in animal models. Reversing PD-1 inhibition is therefore an attractive therapeutic target, but the cellular mechanisms by which PD-1 ligation results in T cell inhibition are not fully understood. PD-1 is thought to limit T cell activation by attenuating T cell receptor (TCR) signaling. It is not known whether PD-1 ligation also acts by upregulating genes in exhausted T cells that impair their function. Here, we analyzed gene-expression profiles from HIV-specific CD8+ T cells in patients with HIV and show that PD-1 coordinately upregulates a program of genes in exhausted CD8+ T cells from humans and mice. This program includes upregulation of basic leucine transcription factor, ATF-like (BATF), a transcription factor in the AP-1 family. Enforced expression of BATF was sufficient to impair T cell proliferation and cytokine secretion, while BATF knockdown reduced PD-1 inhibition. Silencing BATF in CD4+ and CD8+ T cells from chronic viremic patients rescued HIV-specific T cell function. Thus inhibitory receptors can cause T cell exhaustion by upregulating genes – such as BATF – that inhibit T cell function.
Gene expression data from AML cell lines MOLM-14, U937, THP-1 and HL-60, that were infected with a scrambled control hairpin (shControl), two shRNAs directed against GSK-3B (shGSK3B_1 and shGSK3B_2), or two shRNAs directed against GSK-3A (shGSK3A_5 and shGSK3A_6). Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetic-based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3A (GSK-3A) in AML by performing two independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3A induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3A–specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3B has been well studied in cancer development, these studies support a role for GSK-3A in AML.
The purpose of this study was to characterize the transcriptional effects induced by intramuscular IFN-beta-1a treatment (Avonex 30 µg once weekly) in patients with relapsing-remitting form of multiple sclerosis (MS). By using Affymetrix DNA microarrays, we obtained genome-wide expression profiles of peripheral blood mononuclear cells from 24 MS patients within the first four weeks of IFN-beta administration. Keywords: Multiple sclerosis, Interferon, Pharmacogenomics, Affymetrix
Primary colon CSC cultures were transduced with a Wnt responsive construct (TOP-GFP) and were single cell cloned. 10% highest and lowest TOP-GFP cell fractions were FACS sorted and arrayed.
Down syndrome (DS) is the result of trisomy chromosome 21 but the mechanisms by which the genotype leads to the characteristic disease phenotype are unclear. We performed a microarray study using human adult brain tissue (dorsolateral prefrontal cortex) from DS subjects and healthy controls to characterise for the first time the human adult Down syndrome brain Keywords: disease state analysis
Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC (leucine-rich PPR-motif containing), a poorly studied gene that is mutated in Leigh Syndrome, French Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. Using expression profiling, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, without affecting nuclear genes encoding mitochondrial proteins.
The RPMI-8226 human multiple myeloma cell line was stably infected with either a validated shRNA against BMI1 or a control shRNA. RNA was prepared from these lines +/- doxycycline induction and at various time points post-induction. Samples were hybridized on the Affymetrix U133plus2 human genome expression microarray.
To identify molecular singnal alterations between androgen dependent prostate cancer and castration resistant prostate cancer we performed interspecies comparative microarray analyses using RNAs prepared from uncastrasion and castration tumor from LNCAP Orhotopic xenograft models of prostate cancer. microarray data from uncastrasion and castration tumor revealed that the gene expression profile is most significantly altered in between androgen dependent prostate cancer and castration resistant prostate cancer. Comparative analyses of LNCAP Orhotopic xenograft models of prostate cancer showed that genes involved in androgen dependent and androgen independent tumor were significantly altered.
We used microarrays to detail the global program of gene expression underlying Parkinson's disease Keywords: normal versus diseased
Activation of the immune system is a way for host tissue to defend itself against tumor growth. Hence treatment strategies that are based on immunomodulation are on the rise. Conventional cytostatic drugs such as the anthracycline doxorubicin can also activate immune cell functions of macrophages and natural killer cells. In addition, cytotoxicity of doxorubicin can be enhanced by combining this drug with the cytokine IFN-alpha. Although doxorubicin is one of the most applied cytostatics, the molecular mechanisms of its immunomodulation ability are not investigated thoroughly. In microarray analyses of HeLa cells, a set of 19 genes related to interferon signaling was significantly overrepresented among genes regulated by doxorubicin exposure including STAT-1, -2, IRF9, NMI, and caspase 1. Regulation of these genes by doxorubicin was verified with Real-Time PCR and immunoblotting. An enhanced secretion of IFN-alpha was observed when HeLa cells were exposed to doxorubicin as compared to untreated cells. IFN-alpha neutralizing antibodies and inhibitors of JAK-STAT signaling (ATA and AG490) significantly abolished doxorubicin-stimulated expression of interferon signaling-related genes. Furthermore, inhibition of JAK-STAT signaling significantly reduced doxorubicin induced caspase 3 activation and desensitized HeLa cells to doxorubicin cytotoxicity. In conclusion, we demonstrate that doxorubicin induces interferon-responsive genes via IFN-alpha-JAK-STAT1 signaling and that this pathway is relevant for doxorubicin’s cytotoxicity in HeLa cells. As immunomodulation is a promising strategy in anticancer treatment, this novel mode of action of doxorubicin may help to further improve the use of this drug among different types of anticancer treatment strategies.
We used microarrays to determine which genes are upregulated by IFNbeta stimulation in 293T cells.
This study investigates the molecular signatures that drive Renal Cell Carcinoma (RCC) metastatic conversion using the metastatic (LM2) and non-metastatic (SN12C) RCC cell lines. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process.
We performed microarray analysis to evaluate differences in the transcriptome of type 2 diabetic human islets compared to non-diabetic islet samples.
Chemotherapy may cause DNA damage within the oral mucosa of cancer patients leading to mucositis a dose-limiting side effect for effective cancer treatment. We used whole genome gene expression analysis to identify cellular damage to the mucosal tissue occuring two days post induction chemotherapy and identified gene expression patterns that may or may not be predictive of oral mucositis. Keywords: Treatment effect
Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognisable by its distinctive red skin lesions. The lesions are KSHV-infected spindle cells most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The effects of KSHV infection of both LEC and BEC were assayed using Affymetrix hgu133plus2 chips at 72 hours post infection.
Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease with complex etiopathogenesis. Here we use Affymetrix U133 plus 2.0 microarray gene expression data from human parotid tissue. Parotid gland tissues were harvested from 17 pSS and 14 14 non-pSS sicca patients and 18 controls. The data were used in the following article: Nazmul-Hossain ANM Pollard RPE, Kroese FGM, Vissink A, Kallenberg CGM, Spijkervet FKL, Bootsma H, Michie SA, Gorr SU, Peck AB, Cai C, Zhou H, Horvath S, Wong DTW (2012) Systems Analysis of Primary Sjögren’s Syndrome Pathogenesis in Salivary Glands: Comparative Pathways and Molecular Events in Humans and a Mouse Model.
We performed microarray to compare gene expression patterns of PBMC treated with rATG or hATG. Fold changes were compared using 2-way ANOVA tests for untreated rATG- and hATG-treated PBMC. In PBMC treated with 10 ug/mL rATG, compared with untreated PBMC, 478 genes showed up-regulation, and 341 genes showed down-regulation at 24 hours using 10% FDR and 2-fold change cutoff. Immediately striking was that 10 ug/mL hATG had affected many fewer genes than did rATG: only 3 genes were up-regulated and 6 genes were down-regulated at 24 hours in hATG-treated PBMC. When we compared rATG with hATG, rATG induced up-regulation of 268 genes and down-regulation of 95 genes. These genes belong to the categories of immune response (64 genes), cytokine-cytokine receptor interaction (36 genes), regulation of cell proliferation (24 genes), cell cycle (23 genes), cell growth (8 genes), apoptosis (7 genes), and others. Keywords: different treatment
Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here we report the identification and characterization of a novel heterochromatinization factor in vertebrates, Bromo Adjacent Homology Domain-containing protein 1 (BAHD1). BAHD1 interacts with HP1, MBD1, HDAC5 and with several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes formation of large heterochromatic domains, which lack acetyl histone H3 and are enriched in H3 trimethylated at lysine 27. Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic X inactive chromosome. As highlighted by whole genome microarray analysis of BAHD1 knock down cells, BAHD1 represses several proliferation and survival genes and in particular, the insulin-like growth factor II gene (IGF2). BAHD1 specifically binds the CpG-rich P3 promoter of IGF2. This region contains DNA binding sequences for the transcription factor SP1, with which BAHD1 co-immunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting proteins that coordinate heterochromatin assembly at specific sites in the genome. We used microarrays to identify BAHD1 gene targets. We compared the transcriptome profile of BAHD1 depleted cells with siRNA to that of cells treated with control siRNA.
As an oncogene use of HER2 vaccines in humans requires the development of HER2 immunotherapies with maximal immunologic potential, but minimal oncologic potential. To address these issues, we developed a recombinant adenoviral vector expressing a mutated HER2 inactivated for kinase function (Ad-HER2-ki). Ad-HER2-ki was highly expressed, but non-phosphorylated and elicited minimal transcription dysregulation in primary cells. In contrast, Ad-HER2-wt elicited a strong oncogenic signature associated with tumorigenesis.
Upregulation of Expression of the Ubiquitin Carboxyl Terminal Hydrolase L1 Gene in Human Airway Epithelium of Cigarette Smokers The microarray data deposited here is from 39 HG-U133 Plus 2.0 GeneChips from 12 normal non-smokers, 12 phenotypic normal smokers, 9 Early COPD and 6 COPD individuals, all small airways, all small airway. A subset of these samples have been already submitted under GEO Accession Number GSE 4498. These are: 12 non-smokers samples (GSM101095-GSM101106) and 10 smoker samples (GSM101107-GSM101116). These 22 samples that are also in GSE4498 were described in Harvey, B-G; Heguy, A.; Leopold, P.L.; Carolan, B.; Ferris, B. and Crystal R.G. Modification of Gene Expression of the Small Airway Epithelium in Response to Cigarette Smoking. J. Mol. Med (in press). These data are part of a study aimed at understanding how cigarette smoking modifies neuroendocrine cells, in which microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals (normal nonsmokers, normal smokers, smokers with early COPD and smokers with established COPD). Of 11 genes considered to be neuroendocrine cell-specific, only ubiquitin C-terminal hydrolase L1(UCHL1), a member of the ubiquitin proteasome pathway, was consistently upregulated in smokers compared to nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemistry of bronchial biopsies of smokers compared to nonsmokers. Interestingly, however, while UCHL1 expression was present only in neuroendocrine cells of the airway epithelium in nonsmokers, UCHL1 expression was also expressed in ciliated epithelial cells in smokers, an intriguing observation in light of recent observations that ciliated cells can are capable of transdifferentiating to other airway epithelium. In the context that UCHL1 is involved in the degradation of unwanted, misfolded or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy. Keywords: non-smokers vs phenotypic normal smokers, smokers with early COPD, and smokers with COPD
Pirin (PIR) is a putative transcriptional regulator abundantly expressed in melanocytes and in a subset of primary and metastatic melanomas. Ablation of PIR in the melanoma cell lines results in induction of a senescence-like phenotype. Keywords: Transcriptional regulation knock-down using siRNA
Expression analysis of wild-type SAOS cells and SAOS cells transiently transfected with RB SMYD2, or RB and SMYD2.
Identification of genes involved in lumen formation: gene chip analysis was performed on mRNA isolated from both wild type and T457AS459A mutated CEACAM1-4S transfected MCF7 cells grown in Matrigel
The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding selection of therapy, and monitoring interventions. Previously, we determined that plasma of recent-onset (RO) Type 1 diabetes (T1D) patients induce a proinflammatory transcriptional signature in fresh peripheral blood mononuclear cells (PBMC) relative to that of unrelated healthy controls (HC). Here, using an optimized cryopreserved PBMC-based protocol, we compared the signature found between unrelated healthy controls and non-diabetic cystic fibrosis patients possessing Pseudomonas aeruginosa pulmonary tract infection.
we performed genome-wide screening using SW480 cells with Gankyrin knockdown on an Affymetrix gene expression array to identify the transcriptional targets of Gankyrin
We used microarrays to determine the gene expression profile of different types of glioma Keywords: Disease state analysis
In the marrow and lymphatic tissues chronic lymphocytic leukemia (CLL) cells interact with accessory cells that constitute the leukemia microenvironment. In lymphatic tissues, CLL cells are interspersed with CD68+ nurselike cells (NLC) and T cells. However, the mechanism regulating co-localization of CLL cells and these accessory cells are largely unknown. To dissect the molecular cross-talk between CLL and NLC, we profiled the gene expression of CD19-purified CLL cells before and after co-culture with NLC. NLC co-culture induced high-level expression of B cell maturation antigen (BCMA) and two chemoattractants (CCL3, CCL4) by CLL cells. Supernatants from CLL-NLC co-cultures revealed high CCL3/CCL4 protein levels. B cell receptor triggering also induced a robust induction of CCL3 and CCL4 expression by CLL cells, which was almost completely abrogated by a specific Syc inhibitor, R406. High CCL3 and CCL4 plasma levels in CLL patients suggest that activation of this pathway plays a role in vivo. These studies reveal a novel mechanism of cross-talk between CLL cells and their microenvironment, namely the secretion of two T cell chemokines by CLL-NLC interaction and in response to BCR stimulation. Through these chemokines, CLL cells can recruit accessory cells, and thereby actively create a microenvironment that favors their growth and survival.
Specific vulnerability of neurons in the human entorhinal cortex has been associated with the onset of disease. Gene expression is analyzed to define the molecular characteristic of those neurons.
Response to allergen was studied in epithelial cells derived from allergic pantients and from healthy controls. Cells were cultured after isolation from a nasal biopsy. Cells were exposed to Housed dust mite or vessel (saline) Microarray data was analysed using bioinformatics and biostaistics. We conclude that a marked difference in basal expression and in response to hous dust mite exists Keywords: cellular response to allergen
To establish a robust cellular model system for screening genes associated with cell invasion we over-expressed the oncogenic translocated promoter region (Tpr)-MET proteins in SCC23 cells (SCC23/MET). Using a functional siRNA screen, we identified that the histone demethylase KDM4A played a critical role in the invasive growth and metastasis of SCC mediated by the Oncogenic MET. To investigate the molecular mechanism through which KDM4A inhibit the tumor cell invasion, we knock-down KDM4A in SCC23/MET cell and performed a gene microarray to examine which genes may be regulaged by kDM4A.
Mixed-lineage leukemias represent about 3-5% of acute leukemias occurring in patients of all ages and comprise several different subtypes (biphenotypic bilineal, and lineage switch). The optimal therapeutic approach to these cases, especially in pediatric patients, has not been defined. We used microarrays to detail the gene expression of pediatric patients with biophenotypic leukemia. Keywords: Patient sample accumulation
Little is known about the early transcriptional events in innate immune signaling in immature and tolerogenic monocyte-derived dendritic cells (DCs) the professional antigen-presenting cells of our immune system. TLR ligands usually induce a proinflammatory transcriptional response, whereas IL10 and/or dexamethasone induce a more tolerogenic phenotype. We used Affymetrix microarrays to obtain detailed information underlying pro- and anti-inflammatory transcriptional responsesand transcriptional networks in DCs
Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1 pT2, pT3) and in 3 normal testes. Keywords: ordered
The clinical features of psoriasis characterized by sharply demarcated scaly erythematous plaques, are typically so distinctive that a diagnosis can easily be made on these grounds alone. However, there is great variability in treatment response between individual patients, and this may reflect heterogeneity of inflammatory networks driving the disease. In this study, whole-genome transcriptional profiling was used to characterize inflammatory and cytokine networks in 62 lesional skin samples obtained from patients with stable chronic plaque psoriasis. We were able to stratify lesions according to their inflammatory gene expression signatures, identifying those associated with strong (37% of patients), moderate (39%) and weak inflammatory infiltrates (24%). Additionally, we identified differences in cytokine signatures with heightened cytokine-response patterns in one sub-group of lesions (IL-13-strong; 50%) and attenuation of these patterns in a second sub-group (IL-13-weak; 50%). These sub-groups correlated with the composition of the inflammatory infiltrate, but were only weakly associated with increased risk allele frequency at some psoriasis susceptibility loci (e.g., REL, TRAF3IP2 and NOS2). Our findings highlight variable points in the inflammatory and cytokine networks known to drive chronic plaque psoriasis. Such heterogeneous aspects may shape clinical course and treatment responses, and can provide avenues for development of personalized treatments. We used Affymetrix microarrays to evaluate genome-wide expression in primary human keratinocytes exposed to cytokines. Cytokine activity signatures were used to interpret the shifts in gene expression that occur in psoriasis plaques relative to normal uninvolved skin.
Introduction: microRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and may play a causal role in invasive breast cancer. Since many genetic aberrations of invasive disease are detectable in earlier stages we hypothesized that miRNA expression dysregulation and the predicted changes in gene expression would also be found in early breast neoplasias. Methods: Expression profiling of 365 miRNAs by RT-qPCR was combined with laser-capture microdissection to obtain an epithelial specific miRNA expression signature of normal breast epithelium (n=9) and of paired samples of histologically normal epithelium (HN) and ductal carcinoma in situ (DCIS) (n=16). To determine how miRNAs may control the expression of co-dysregulated mRNAs we also performed gene expression microarray analysis in the same paired HN and DCIS samples and integrated this with miRNA-target prediction. We further validated several target pairs by modulating the expression levels of miRNAs in MCF7 cells and measured the expression of target mRNAs and proteins. Results: Thirty-five miRNAs were aberrantly expressed between RM, HN and DCIS. Twenty-nine miRNAs and 420 mRNAs were aberrantly expressed between HN and DCIS. Combining these two datasets with miRNA-target prediction we identified two established target pairs (miR-195:CCND1 and miR-21:NFIB) and tested several novel miRNA:mRNA target pairs. Over-expression of the putative tumor-suppressor miR-125b, under-expressed in DCIS, repressed the expression of MEMO1, which is required for ErbB2-driven cell motility (also a target of miR-125b); and NRIP1/RIP140, which modulates the transcriptional activity of the estrogen receptor. Knockdown of the putative oncogenic miRNAs miR-182 and miR-183, both highly over-expressed in DCIS, increased the expression of CBX7 (which regulates E-cadherin expression), DOK4, NMT2, and EGR1. Augmentation of CBX7 by knockdown of miR-182 expression, in turn, positively regulated the expression of E-cadherin, a key protein involved in maintaining normal epithelial cell morphology which is commonly lost during neoplastic progression. Conclusions: These data provide the first miRNA expression profile of normal breast epithelium and of pre-invasive breast carcinoma. Further, we demonstrate that altered miRNA expression can modulate gene expression changes that characterize these early cancers. We conclude that miRNA dysregulation likely plays a substantial role in early breast cancer development.
We studied transcriptional changes by Affymetrix human microarrays in DLBCL cell lines as a result of treatment with GSK126 a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 In eukaryotes, epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.
Scoliosis is a common disorder affecting one out of every twenty females. Some forms of scoliosis are congenital, resulting from disruptions in early spinal development. The spinal column is patterned during a cycling process called somitogenesis, and is the first musculoskeletal structure formed during development. Genes in the notch signaling pathway regulate somitogenesis, and display oscillatory expression in synchrony with somite formation. During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. The periodicity and components of the human clock have recently been described by our group (William et al., Dev Biol, 2007). In that publication, we showed that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression, including known cycling genes such as HES1 that displayed a period of 5 hours. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 hours, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Studies in mouse cell lines, including fibroblasts and PC12 neuronal cells, by R. Kageyema (Kyoto University, Hirata et al., Science, 2002) have shown that a number of mouse cell types can be synchronized to display oscillatory expression. In this study, we extend our analysis of human cells to human primary fibroblasts. Developing an in vitro model of somitogenesis using human mesenchymal stem cells and fibroblasts a. Induce cycling gene expression in human mesenchymal stem cells (MSC) and fibroblasts and identify oscillatory genes (using Affymetrix U133 GeneChips®). Compare lists of oscillatory genes identified in these two human cell types. b. Characterize and validate mouse homologues of oscillatory genes for expression during somitogenesis. Using notch and wnt pathway mutants and cultured half embryos, determine if these oscillatory genes also display cycling expression during somitogenesis. We hypothesized that human fibroblasts could be synchronized to display oscillatory expression of notch and wnt pathway genes, including the marker gene HES1. Preliminary quantitative analysis by Taqman-based Real-Time PCR for these samples confirms that HES1 expression displays oscillatory expression. Homo sapiens fibroblasts were maintained in culture medium (DMEM high glucose with 10% fetal bovine serum, FBS), which was replaced twice weekly. The cells were then seeded at a density of 6,000-10,000 cells/cm2 and expanded in DMEM high glucose with 10% FBS. After 5-7 days of incubation at 37C in a humidified atmosphere containing 5% carbon dioxide, cells were detached with 0.05% trypsin for 2 minutes at confluency. Cells were synchronized using low serum treatment, which has been described previously for Hes1 (Hirata et al., 2002). Cell culture synchronization is required to assay oscillatory expression levels, otherwise the oscillations of individual cells would be out of phase and cancel one another. Synchronized cells will gradually desynchronize, leading to diminished oscillatory amplitude. Briefly, synchronization was carried out as follows: T-25 cm flasks were set up in parallel. These cells were grown to 90% confluence in DMEM supplemented with 10% FBS (UCB-MSC), then incubated in DMEM with only 0.2% FBS for 24 hours, and returned to DMEM supplemented with FBS. Samples were collected at 1 hr. intervals from 0 to 12 hours. In addition, an unsynchronized cell culture was sampled. Total RNA was isolated from cells in culture using the RNeasy Mini Kit (Qiagen) and quantitated using a Nanodrop 2000. Keywords: time-course
Although not an affected cell type skin fibroblasts from individuals with CC-ALD, an early onset X-linked neurological disorder, show defects in very long chain fatty acid (VLCFA) metabolism that provide the basis for clinical diagnostic tests. Skin fibroblasts from CC-ALD patients can be reprogrammed into iPS cells with all the hallmark properties of pluripotency. The iPS cell phenotypes may reflect the tissue-specificity of the lipid metabolic defects found in CC-ALD patients. We report the gene expression profiles of fibroblasts and fibroblast-reprogrammed iPSCs from childhood cerebral adrenoleukodystrophy patients and healthy controls
Early immature T-cell acute lymphoblastic leukemias (T-ALLs) account for about 5-10% of pediatric T-ALLs and are associated with poor prognosis. However the genetic defects that drive the biology of these tumors remain largely unknown. Analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Consistently, adult immature T- ALLs showed characteristic mutations in myeloid specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3 and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. All together, our results demonstrate that early immature adult T- ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics and highlight the role of ETV6 mutations in these tumors.
Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells including sequence-specific RNA interference, sequence-independent interferon response and editing by adenosine deaminases. To assess the potential of expressed dsRNA to induce interferon stimulated genes in somatic cells, we performed microarray analysis of HEK293 and HeLa cells transfected with a MosIR plasmid expressing an mRNA with a long inverted repeat structure in its 3’UTR (MosIR) or with a parental MosIR plasmid (without inverted repeat) as a control. Clustering analysis based on differentially expressed genes suggested that there was no common transcriptome signature in cells expressing dsRNA. Overall, the number of genes with altered expression upon transfection of the MosIR plasmid was rather small and only 19 probe sets, corresponding to 17 genes, were changed more than two-fold in both cell lines.
Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hematopoiesis the conclusions of such studies are quite controversial since they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion while, in others, implicate this transcription factor in the induction of monocyte - macrophage differentiation. To clarify this issue we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hematopoietic progenitors by retroviral transduction of a full length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of MafB gene, recently identified as master regulator of such maturation pathway. By using a combined approach based on computational analysis, EMSA experiments and luciferase assays, we were able to demonstrate the presence of a Hox-A10 binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Interestingly, stimulation of the same cells with the Vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving VDR, Hox-A10 and MafB transcription factors. Altogether these data allow to conclude that the Vitamin D3 / Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.
Dendritic cells (DCs) are the sentinels of the mammalian immune system and they undergo a complex maturation process mediated by activation upon pathogen detection. Recent studies described the analysis of activated DCs by transcriptional profiling but translation regulation was never taken in account. Therefore, the nature of the mRNAs being translated at various stages of DC activation was determined with the help of translational profiling, which is the sucrose gradient fractionation of polysomal-bound mRNAs combined to microarrays analysis. Total and polysomal-bound mRNA populations were compared in immature (0h) and LPS-stimulated (4h and 16h) human monocyte-derived DCs with the help of Affymetrix microarrays. Biostatistical analysis indicated that 296 mRNA molecules are translationally regulated during DC-activation. The most abundant biological process among the regulated mRNAs was protein biosynthesis, indicating the existence of a negative feedback loop regulating translation. Interestingly, a cluster of 17 ribosomal proteins were part of the regulated mRNAs, indicating that translation may be fine-tuned by particular components of the translational machinery. Our observations highlight the importance of translation regulation during the immune response, and may favour the identification of novel gene clusters or protein networks relevant for immunity. Our study also provides information on the possible absence of correlation between gene expression and real protein production in DCs.
RNA was isolated from testicular biopsies of cryptorchid boys. Gene expression profiles for these samples were investigated using Affymetrix arrays
Gene expression profiling was performed on primary human erythroid progenitor cells expressing a control shRNA (luciferase) two different HDAC1 shRNAs, and two different HDAC2 shRNAs. The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a novel bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.
The retinal pigment epithelium (RPE) is a polarized cell layer that is critical for photoreceptor function and survival. It’s unique relationship to the photoreceptors and its specific physiology makes the RPE a critical determinant of human vision. Therefore we performed global expression profiling of native and cultured human fetal and adult RPE and determined a unique set of highly-expressed genes (called the “signature” set) by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues.
Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential. From genomic expression profiles comparing clones derived from the NPC cell line CNE-2 serglycin (SRGN) was identified as one of the most up-regulated genes in the high-metastasis clone. Serglycin protein was secreted by the high-metastasis clone, but not by any of the low-metastasis clones. Suppression of serglycin by shRNA diminished serglycin secretion and subsequently inhibited the migration and invasion of high-metastasis clone, and also reduced its metastasis rate in vivo. Overexpression of serglycin in low-metastasis cells resulted in an increased metastasis rate in vivo. Moreover, secreted serglycin promoted cellular motility in the wild-type low-metastasis cells. Interestingly, suppression of serglycin reduced the protein level of vimentin but did not influence the level of E-cadherin in high-metastasis clone. Proliferation was not influenced by serglycin in both high-and low-metastasis clones. Clinically, serglycin expression was significantly elevated in liver metastases from NPC relative to its expression in primary tumors. The prognostic value of serglycin was evaluated by immunohistochemical staining of tissue microarrays of NPC tissues from 263 patients, followed by multivariate analyses. A high level of serglycin expression in primary NPC was found to be an independent unfavorable indicator for distant-metastasis-free survival and disease-free survival. In summary, serglycin regulates NPC metastasis via autocrine and paracrine means without influencing proliferation, and it serves as a prognostic indicator of metastasis-free survival and disease-free survival for NPC patients. Keywords: Gene Expression experiment
ACSL4 expression appears to be inversely associated with steroid hormone and growth factor receptor expression in breast cancer and positively correlated with an aggressive breast cancer phenotype. Neither MCF-7 nor SKBr3 cells normally express ACSL4 and when manipulated to do so, develop basal-like characteristics, including increased proliferation, migration and anchorage independent growth. We used an Affymetrix array platform to assess changes in individual gene expression as a function of conditional and stable expression of ACSL4 in MCF-7 and SKBr3 cells.
Invasion of cytotrophoblasts into uterine tissues is essential for placental development. To identify molecules regulating trophoblast invasion mRNA signatures of purified villous (CTB, poor invasiveness) and extravillous (EVT, high invasiveness) trophoblasts isolated from first trimester human placentae and villous explant cultures, respectively, were compared using GeneChip analyses yielding 991 invasion/migration related transcripts. Several genes involved in physiological and pathologic cell invasion, including ADAM-12,-19,-28 as well as Spondin-2, were upregulated in EVT. Pathway prediction analyses identified several functional modules associated with either the invasive or the non-invasive trophoblast phenotype. One of the genes which were downregulated in the invasive mRNA pool, heme oxygenase-1 (HO-1), was selected for functional analyses. Real-time PCR analyses, Western blottting, and immunofluorescene of first trimester placentae and differentiating villous explant cultures demonstrated downregulation of HO-1 in invasive EVT as compared to CTB. Modulation of HO-1 expression in loss-of as well as gain-of function cell models (BeWo and HTR8/SVneo, respectively) demonstrated an inverse relationship of HO-1 expression with trophoblast migration in transwell and wound healing assays. Importantly, HO-1 expression led to an increase in protein levels and activity of the nuclear hormone receptor PPARgamma. Pharmacological inhibition of PPARgamma abrogated the inhibitory effects of HO-1 on trophoblast migration. Collectively, our results demonstrate that gene expression profiling of EVT and CTB can be used to unravel novel regulators of cell invasion. Accordingly, we identify heme oxygenase-1 as a negative regulator of trophoblast motility acting via upregulation of PPARgamma.
CD11c+ Myeloid Dendritic Cells (mDCs) were isolated from the peripheral blood mononuclear cells (PBMCs) of HIV uninfected and HIV infected subjects. The expression of CD11c+ mDCs was accessed to determine how HIV infection may play a role on the expression profiles of these cells. mDCs are known to play a role in antigen presentation and thus are pivotal in immune sensing and priming of the adaptive immune response. We wanted to see if the change in immune system function during chronic HIV infection may be due to defects in this cell subtype. We used microarray analysis to detail the global program of gene expression underlying changes in mDC function during HIV infection.
Analysis of MDA-MB-453 breast cancer cells treated with the androgen 5a-dihydrotestosterone (DHT) for 6h 16h and 48h to define the genes that are differentially regulated in response to DHT.
Therapies targeting the androgen receptor are critical for treatment of hormone refractory prostate cancer. We have previously demonstrated that Ebp1 a protein isolated by its ability to bind ErbB3, is a downstream effector of heregulin activated pathways and an AR corepressor. As Ebp1 is decreased in preclinical models of hormone refractory prostate cancer, we studied the ability of Ebp1 to mitigate the hormone refractory phenotype. As we previously found that Ebp1 affected the expression of some androgen receptor target genes, we sought to determine a full spectrum of genes changed using an unbiased appraoch by microarry analysis.
Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer and is associated with a poor prognosis. We have developed a new model of IBC derivated from the pleural effusion of a 49-year-old woman with metastatic secondary IBC. FC-IBC02 tumor cells were isolated from the pleural effusion and cultured under non-adherent conditions resulting in the formation of spheroids or mammospheres. FC-IBC02 are triple negative (estrogen receptor negative, progesterone receptor negative and ErbB2 negative) and strongly positive for E-cadherin, beta-catenin and vimentin. FC-IBC02 cells developed breast tumors when they were injected into the mammary fat pad of SCID mice and characteristic tumor emboli were detected. Breast tumor xenografts were poorly differentiated triple negative carcinomas and all injected mice developed metastasis in the lungs and lymph nodes. These IBC tumor cells showed genomic alterations in all chromosomes, with the gains/amplifications more common than the deletions/losses. Duplicated regions were on 1q, 2p, 3q, 8q and 18p and chromosomes 7 and 9. The 8q chromosome arm where the MYC oncogene resides was amplified up to seven fold. Chromothripsis (local chromosome shattering) was observed on chromosome 11q and losses were found on 8p, 11q, 16q and 17p (location of TP53). FC-IBC-02 cells expressed the stem cell marker CD44, EpCAM and strongly expressed EGFR and ALK. In summary, this novel preclinical model demonstrated that IBC is a disease enriched for highly tumorigenic cells which harbor a stem cell phenotype. This IBC model is ideal for the study of the metastatic process and to evaluate targeting therapeutic modalities.
Mounting evidence points to a link between a cancer possessing stem-like properties and a worse prognosis. To understand the biology a common approach is to integrate network biology with signal processing mechanics. That said, even with the right tools, predicting the risk for a highly susceptible target using only a handful of gene signatures remains very difficult. By compiling the expression profiles of a panel of tumor stem-like cells (TSLCs) originating in different tissues, comparing these to their parental tumor cells (PTCs) and the human embryonic stem cells (hESCs), and integrating network analysis with signaling mechanics, we propose that network topologically-weighted signaling processing measurements under tissue-specific conditions can provide scalable and predicable target identification. All function codes related to this project could be accessed at the supplementary website.
Thermal injury incites inflammatory responses that often transcend the local environment and lead to structural deficiencies in skin that give way to scar formation. We hypothesized that extensive perturbations within burned skin following thermal insult and during subsequent events of wound repair induce vast alterations in gene expression that likely serve as a wound and systemic healing deterrent. A high-throughput microarray experiment was designed to analyze genetic expression patterns and identify potential genes to target for therapeutic augmentation or silencing. The study compares gene expression from burn wound margins at various times following thermal injury to expression observed in normal skin. Utilizing this design we report that the totality of gene expression alterations is indeed enormous. Further, we observed that the differential expression of many inflammatory and immune response genes appear to be continually up-regulated in burn wound margins seven days or more after initial thermal insult. As it is well established that the inflammatory process must abate for wound healing to proceed, the finding of ongoing local inflammation is cause for further investigation. To our knowledge, this is the first report of the gene expression alterations induced by thermal injury of human skin. As such, it provides a wealth of data to mine with the ultimate goal of better understanding the local pathophysiologic changes at the site of thermal injury that not only affect wound healing capacity, but may also contribute to systemic derangements within the burn patient. Keywords: time course, disease state analysis
Analysis of mRNA profiles after MEK1/2 inhibition in human pancreatic cancer cell lines reveals pathways involved in drug sensitivity. We used microarrays to find gene expression patterns associated with drug response and also identified genes regulated by the MAP kinase pathway
Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Keywords: Expression changes following pharmacological reversal of epigenetic silencing
Lymphatic endothelial cells were grown under normoxia hypoxia (1% 0xygen) and conditioned medio from NSLCN growth under normoxia or hypoxia. Gene expression was measured and comparition between samples performed Keywords: Comparative genomic hybridization
24 standard human Neuroblastoma Cell lines were profiled without applying any transfections in order to measure the expression profiles. Keywords: cell line neuroblastoma, mRNA profiling
Gene expression analysis identified a MLL translocation-specific signature of differentially expressed genes discriminating ALL and AML with and without MLL rearrangements. Gene expression signatures of acute lymphoblastic and myeloblastic leukemia samples with and without MLL rearrangements were analyzed using paired supervised analyses.
Neutrophil recruitment is pivotal to host defense against microbial infection but also contributes to the immunopathology of disease. We investigated the mechanism of neutrophil recruitment in human infectious disease by bioinformatic pathways analysis of the gene expression profiles in the skin lesions of leprosy. In erythema nodosum leprosum (ENL), which occurs in patients with lepromatous leprosy (L-lep), and is characterized by neutrophil infiltration in lesions, the most overrepresented biologic functional group was 'cell movement' including E-selectin, which was coordinately regulated with IL-1beta. In vitro activation of TLR2, upregulated in ENL lesions, triggered induction of IL-1beta, which together with IFN-gamma, induced E-selectin expression on, and neutrophil adhesion to endothelial cells. Thalidomide, an effective treatment for ENL, inhibited this neutrophil recruitment pathway. The gene expression profile of ENL lesions comprised an integrated pathway of TLR2/FcR activation, neutrophil migration and inflammation, providing insight into mechanisms of neutrophil recruitment in human infectious disease.
We performed expression profiling of 36 types of normal human tissues and identified 2503 tissue-specific genes. We then systematically studied the expression of these genes in cancers by re-analyzing a large collection of published DNA microarray datasets. Our study shows that integration of each gene's breadth of expression (BOE) in normal tissues is important for biological interpretation of the expression profiles of cancers in terms of tumor differentiation, cell lineage and metastasis. Twenty five total RNA specimens were purchased from Clontech (Palo Alto, CA), Ambion (Austin, TX) and Strategene (La Jolla, CA). We tried to cover as many tissue types as possible by using pooled RNA samples. In order to define breadth-of-expression (BOE) accurately at a reasonable cost, we tried to cover as many tissue types as possible by using pooled RNA samples. Each specimen represents a human organ. We used RNA samples pooled from 2 to 84 donors to avoid differences at the individual level. Keywords = Normal human tissues Keywords = Tissue-specificity Detailed sample information and Affymetrix .CEL files are available at http://www.genome.rcast.u-tokyo.ac.jp/normal/ Publication:Ge X et al., Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues. Genomics. 2005 Aug;86(2):127-141. PMID: 15950434 Keywords: other
Morphogenesis of epithelial tissues relies on the precise developmental control of cell polarity and architecture. In the early Drosophila embryo the primary epithelium forms during cellularisation, following a tightly controlled genetic programme where specific sets of genes are up-regulated. Some of them, for instance, control membrane invagination between the nuclei anchored at the apical surface of the syncytium.
Human Neural progenitor cells have been found to have karyotypic abnormalities. In this study we explored the gene changes between hNPCs that are karyotypically normal and hNPCs with a trisomy of chromosome 7. We used Affymetrix GeneChips to study gene expression changes between karyotypically normal hNPCs and trisomy 7 hNPCs.
Characterization of genes associated with adipose tissue is key to understanding the pathogenesis of obesity and developing treatments for this disorder. Differential gene expression in the adipose tissue has been described in adulthood but none studies have been developed on childhood. The purpose of this study was to compare gene expression in omental adipose tissue from obese prepubertal and normal weight children. We selected 5 obese (BMI adjusted for age and sex z score >2) and 6 normal weight children. RNA was extracted from omental adipose tissue biopsies and cRNA was hybridizated on the human genome U133 Plus 2.0 Arrays (Affymetrix®). Microarray experiments were performed for each sample and selected group of gene expression values were confirmed with real-time RT-PCR in 10 obese and 10 normal weigth prepubertal children. 1276 genes were found to be differentially expressed at P<0.05. Of those differential genes, 201 were upregulated (Fc>2) and 42 were downregulated (Fc<-2). Genes involved in metabolic and signalling pathways were altered in childhood obesity. Keywords: disease state analysis
Anaysis of mRNA changes in HeLa cells following knockdown of Drosha or DGCR8. Drosha is a nuclear RNase III that carries out microRNA (miRNA) processing by cleaving primary microRNA transcript (pri-miRNA). DGCR8 is an essential co-factor of Drosha. Keywords: gene expression array-based (RNA / in situ oligonucleotide)
The Human T-cell Leukemia Virus (HTLV)-type-I non-structural protein p30 plays an important role in virus transmission and gene regulation. p30 has been documented to inhibit the export of certain viral mRNA transcripts from the nucleus to the cytoplasm. This nuclear retainment of RNA molecules essentially results in gene silencing where protein products are not produced. Considering this unique function of p30, we used microarray analysis to assess the ability of p30 to inhibit not only the regulation of transcription of cellular genes, but also the ability of p30 to regulate the export of cellular transcripts to the cytoplasm.
The intracellular pathogen Trypanosoma cruzi secretes an activity that blocks TGF-β-dependent induction of connective tissue growth factor (CTGF/CCN2). Here we address the mechanistic basis for T. cruzi-mediated interference of CTGF/CCN2 expression by examining host cell signaling pathways and the global inhibitory effect on TGF-β-dependent gene expression. We show that the expression of a discrete subset of TGF-β-inducible genes involved in cell proliferation, wound repair, and immune regulation are blocked by the soluble T. cruzi activity, demonstrating that this parasite-derived activity has broad, but specific effects on fibroblast gene regulation.
Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in “active repression”. Interestingly approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. Keywords: Drug treatment with siRNA
We used microarrays to detail the global gene transcription underlying T cells activation during the first 24 hours after stimulation.
We analyzed gene expression in human peripheral blood mononuclear cells (PBMCs) from breast cancer patients patients with benign breast abnormalities, healthy cancer-free individuals as well as patients with other types of cancer (gastrointestinal and brain cancers).
T-cell prolymphocytic leukemina (T-PLL) is an agressive lymphoma derived from mature T-cells which is in most cases characterized by the presence of an inv(14)(q11q32) and a characteristic pattern of secondary chromosomal abberations. We used microarrays to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor derived peripheral blood cells with five highly purified inv(14)-positive T-PLL blood samples. Keywords: desease state analysis
Full title: Expression data from human primary subcutaneous preadipocytes treated with glucocorticoids prior to the initiation of differentiation. Preadipocytes are continuously exposed to glucocorticoids in situ due to both steroid present in the circulatory system as well as adipose tissue specific 11βHSD1 activity. While the effects of glucocorticoids during differentiation are well studied the effect of exposure of preadipocytes to glucocorticoids prior to differentiation is unknown. We therefore treated confluent human primary preadipocytes drived from subcutaneous adipose tissue with the synthetic glucocorticoid dexamethasone for 48 hours prior to the initiation of differentiation and assessed what effect this had on their subsequent potential to differentiate. We found that pretreatment with glucocorticoids had a priming effect and resulted in increased differentiation of these preadipocytes. Furthermore, this treatment was additive to the effects of glucocorticoids during the initial phase of adipogenesis. Microarray analysis performed subsequent to the pretreatment with glucocorticoids (at the time point at which preadipocytes would have been induced to differentiate) identified glucocorticoid-responsive, candidate genes whose altered expression could mediate these effects. keywords: glucocorticoids, glucocorticoid receptor, preadipocytes, adipogenesis, human primary preadipocytes, subcutaneous, adipose tissue
Acute myeloid leukemia (AML) with CEBPA mutations is determined as provisional entity in the current WHO. A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported whereupon dm cases were shown to be associated with longer overall survival (OS). The occurrence and prognostic impact of concomitant molecular mutations in addition to CEBPAdm has not been assessed until now. Here, we investigated a cohort of 95 AML CEBPAdm cases for concomitant mutations. TET2 was found to be the most frequent mutation (32/94, 34.0%), followed by GATA2 (20/95, 21.0%), WT1 (13/95, 13.7%), DNMT3A (9/94, 9.6%), ASXL1 (9/95, 9.5%), NRAS (8/95, 8.4%), KRAS (3/94, 3.2%), IDH1/2 (6/95, 6.3%), FLT3-ITD (6/95, 6.3%), FLT3-TKD (2/95, 2.1%), NPM1 (2/95, 2.1%), and RUNX1 (1/94). No mutation was detected in MLL-PTD and TP53. With respect to prognostic impact, we observed that those cases harboring additional mutations in TET2 showed significant worse survival than wild-type cases (P=0.035), whereas GATA2 mutated cases showed improved survival (P=0.032). Further, using gene expression microarray analysis we identified no clear different clustering within the CEBPAdm cases with the distinct concomitant mutated genes. In conclusion, we demonstrated that 76.8% of CEBPAdm cases harbored additional alterations in other molecular markers and that CEBPA is a suitable MRD marker to control therapy.
bulk breast tumor RNA from patient Abstract: Sporadic basal-like cancers (BLC) are a distinct class of human breast cancers that are phenotypically similar to BRCA1-associated cancers. Like BRCA1-deficient tumors most BLC lack markers of a normal inactive X chromosome (Xi). Duplication of the active X chromosome and loss of Xi characterized almost half of BLC cases tested. Others contained biparental but nonheterochromatinized X chromosomes or gains of X chromosomal DNA. These abnormalities did not lead to a global increase in X chromosome transcription but were associated with overexpression of a small subset of X chromosomal genes. Other, equally aneuploid, but non-BLC rarely displayed these X chromosome abnormalities. These results suggest that X chromosome abnormalities contribute to the pathogenesis of BLC, both inherited and sporadic. total 62 sample incudes 43 tumor, 7 normal breast and 12 normal organelle Keywords: disease state analysis
Tumors from pancreatic cancer specimens obtained at surgery were used for efficacy testing and biologic analysis Keywords: Pharmacogenetics
Polycystic ovary syndrome (PCOS) the most common cause of anovulatory infertility, is characterized by increased ovarian androgen production, arrested follicle development, and is frequently associated with insulin resistance. These PCOS phenotypes are associated with exaggerated ovarian responsiveness to FSH and increased pregnancy loss. To examine whether the perturbations in follicle growth and the intrafollicular environment affects development of the mature PCOS oocyte, genes that are differentially expressed in PCOS compared to normal oocytes were defined using microarray analysis. This analysis detected approximately 8000 transcripts. Hierarchical clustering and principal component analysis revealed differences in global gene expression profiles between normal and PCOS oocytes. 374 genes had a statistically-significant increase or decrease in mRNA abundance in PCOS oocytes. A subset of these genes was associated with chromosome alignment and segregation during mitosis and/or meiosis, suggesting that increased mRNAs for these proteins may negatively affect oocyte maturation and/or early embryonic development. Of the 374 differentially expressed genes, 68 contained putative androgen receptor, retinoic acid receptor, and/or peroxisome proliferating receptor gamma binding sites, including 9 of the genes involved in chromosome alignment and segregation. These analyses demonstrated that normal and PCOS oocytes that are morphologically indistinguishable and of high quality exhibit different gene expression profiles. Furthermore, altered mRNA levels in the PCOS oocyte may contribute to defects in meiosis and/or mitosis which might impair oocyte competence for early development and therefore contribute to poor pregnancy outcome in PCOS. Keywords: disease state analysis
The underlying mechanisms which are responsible and govern early haematopoietic differentiation during development are poorly understood. Gene expression comparison between pluripotent human embryonic stem cells and earliest haematopoietic progenitors may reveal novel transcripts and pathways and provide crucial insight into early haematopoietic lineage specification and development. Understanding of transcriptional cues that direct differentiation of human embryonic stem cells (hESC) to defined and functional cell types is essential for their future clinical applications. In this study we have undertaken a comparative transcriptional approach of haematopoietic progenitors derived from hESC at various stages of a feeder and serum free differentiation method and have shown that the largest transcriptional changes occur during the first four days of differentiation. Data mining based on molecular function pointed to RhoGTPase signalling as key regulator of this differentiation. Inhibition of this pathway using a chemical inhibitor (Y26732) resulted in a significant downregulation of haematopoietic progenitors throughout the differentiation window thus uncovering a previously unappreciated role for RhoGTPase signalling in differentiation of hESC to haematopoietic lineages.
Glioblastoma stem-like cells or their differentiated progeny were co-cultured for 48h with normal human astrocytes to detect if invasion associated genes were influenced
We used microarrays to detail transcriptional changes in cultured human smooth muscle cells in response to acute and chronic 2-methoxyestradiol treatment 2-ME an endogenous metabolite. of estradiol, not only exerts cytotoxic effects on cancer cells but it also protects against multiple proliferative disorders, including atherosclerosis and injury-induced intimal thickening Keywords: treatment vs. control
The TAP transporter is responsible for transferring cytosolic peptides into the ER where they can be loaded onto MHC molecules. Deletion of TAP results in a drastic reduction of MHC surface expression and alters the presented peptide pattern. Using the TAP deficient cell line LCL721.174 and its TAP expressing progenitor cell line LCL721.45 we have identified and quantified more than 160 HLA ligands, 50 out of which were presented TAP independently. Peptides which were predominantly presented on the TAP deficient LCL721.174 cell line had a decreased MHC binding affinity according to their SYFPEITHI and BIMAS score. About half of the identified TAP independently presented peptides were not derived from signal sequences and may partly be generated by the proteasome. Furthermore, we have excluded that different HLA presentation ratios were due to varying expression of the respective protein or due to changes in the antigen loading complex. Features of TAP-independently presented peptides as well as proteasomal contribution to their generation provides an insight into basic immunological mechanisms. Keywords: differential mass spectrometry, TAP independent, antigen presentation
The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding selection of therapy, and monitoring interventions. Previously we determined that plasma of recent-onset (RO) Type 1 diabetes (T1D) patients induce a proinflammatory transcriptional signature in fresh peripheral blood mononuclear cells (PBMC) relative to that of unrelated healthy controls (HC). Here, using an optimized cryopreserved PBMC-based protocol, we compared the signature found in pre H1N1 samples to the signature associated with active H1N1 flu.
From the results of gene expression analyses of HepG2 under the exposure of 23-Dimethoxy-1,4-naphthoquinone (DMNQ), N-nitrosodimethylamine (DMN), phenol and six heavy metals We showed that biological action of six heavy metals were clearly related to that of DMNQ and distinguishable from the other chemicals. These results suggest that oxidative stress is major apparent biological action of high dose heavy metals, supporting the previous reports. Keywords: other
Gene transfer into HSCs by gammaretroviral vectors (RV) is an effective treatment for inherited blood disorders although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T-lymphocytes from adenosine deaminase (ADA)-Severe combined immunodeficiency (SCID) patients 10 to 30 months after infusion of autologous, genetically-corrected CD34+ cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single cell level, primary T-cell clones were isolated from two patients. T-cell clones harboured either one or two vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism and TCR-driven functions. Analysis of retroviral integration sites (RIS) indicated a high diversity in T-cell origin, consistent with the polyclonal TCR-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200kb-window around RIS showed modest (2.8- to 5.2-fold) disregulation of 5.8% genes in 18.6% of the T-cell clones compared to controls. Nonetheless, affected clones maintained a stable phenotype and normal functions in vitro. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. Global gene expression profiling was performed on CD4+ and CD8+ T-cell subsets purified ex vivo from three ADA-SCID patients at different times after gene therapy. The microarray analysis showed a substantial overlap with the expression patterns of T-cells from controls, indicating the absence of gross abnormalities in the development and function of T-cells derived from genetically corrected hematopoietic stem/progenitor cells.
Pirin (PIR) is a putative transcriptional regulator whose expression is silenced in cells bearing the AML1/ETO and PML/RAR leukemogenic fusion proteins and is significantly repressed in a large proportion of acute myeloid leukemias. PIR expression increases during in vitro myeloid differentiation of primary hematopoietic precursor cells and ablation of PIR in the U937 myelomonocytic cell line or in murine primary hematopoietic precursor cells results in impairment of terminal myeloid differentiation. Keywords: Transcriptional regulation, knock-down using shRNA
The hypothesis was that 6 months of dietary n-3 FA supplementation to humans affected expression of genes that might be of significance for inflammatory processes and for Alzheimer´s disease.
Modification of Gene Expression of the Small Airway Epithelium in Response to Cigarette Smoking The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (HG-133 Plus 2.0 array) in phenotypically normal smokers (n=10, 33 ± 7 pack-yr) compared to matched non-smokers (n=12). Even though the smokers were phenotypically normal, analysis of the small airway epithelium of the smokers compared to the non-smokers demonstrated up- and -down-regulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD. Keywords: smokers vs non-smokers
Identification of the molecular changes that promote viability and metastatic behavior of prostate cancer cells is critical for the development of improved therapeutic interventions for prostate cancer. Stat5a/b and Stat3 are both constitutively active in locally-confined and advanced prostate cancer and both transcription factors have been reported to be critical for the viability and growth of prostate cancer cells. We used microarrays to compare gene expression profiles regulated by Stat5a/b vs. Stat3 in human prostate cancer cells.
Human induced pluripotent stem (iPS) cells derived from somatic cells of patients hold great promise for modelling human diseases. Dermal fibroblasts are frequently used for reprogramming but require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Here, we report the derivation of iPS cells from multiple human blood sources including peripheral blood mononuclear cells (PBMCs) harvested by routine venipuncture. Peripheral blood-derived human iPS lines are comparable to human embryonic stem (ES) cells with respect to morphology, expression of surface antigens, activation of endogenous pluripotency genes, DNA methylation and differentiation potential. Analysis of Immunoglobulin and T-cell receptor gene rearrangement revealed that some of the PBMC iPS cells were derived from T-cells, documenting derivation of iPS cells from terminally differentiated cell types. Importantly, peripheral blood cells can be isolated with minimal risk to the donor and can be obtained in sufficient numbers to enable reprogramming without the need for prolonged expansion in culture. Reprogramming from blood cells thus represents a fast, safe and efficient way of generating patient-specific iPS cells.
Peripheral blood leukocytes of three human donors (Donors 2 4, and 5) were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns. The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 millions cells/mL at room temperature overnight. T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 mg/mL Con A + 4 mg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s (vg). T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment. After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and beta-mercaptoethanol was added to lyse cells and stabilize RNA. Keywords: ordered
Background: While BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH) only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Further, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself. Methods: To resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses. Results: By gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. Analysis of overrepresented gene ontology groups suggests that it is pathway-specific, not gene-specific changes that carry increased risk of disease. Conclusions: B-cell lines are a valuable and accessible tool for assaying alterations in gene expression free from drug and disease effects. Predisposition to disease within BMPR2 mutation carriers was linked to several pathways, including proliferation, GTP signaling, and stress response. Keywords: Search for modifier genes
Abstract. Deregulated c-MYC is found in a variety of cancers where it promotes proliferation as well as apoptosis. In many hematological malignancies enhanced NF-kB exerts prosurvival functions. Here we investigated the role of NF-kB in mouse and human c-MYC-transformed lymphomas. The NF-kB-pathway is extinguished in murine lymphoma cells and extrinsic stimuli typically inducing NF-kB activity fail to activate this pathway. Genetic activation of the NF-kB pathway induces apoptosis in these cells while inhibition of NF-kB by an IkBa superrepressor provides a selective advantage in vivo. Furthermore, in human Burkitt´s lymphoma cells we find that NF-kB activation induces apoptosis. NF-kB upregulates Fas and predisposes to Fas-induced cell death, which is caspase 8 mediated and can be prevented by CFLAR overexpression. We conclude that c-MYC overexpression sensitizes cells to NF-kB-induced apoptosis and persistent inacvtivity of NF-kB signaling is a prerequisite for MYC-mediated tumorigenesis. We could also show that low immunogenicity and Fas insensitivity of MYC driven lymphoma cells is reversed by activation of NF-kB. Our observations provide a molecular explanation for the described absence of the NF-kB signaling in Burkitt´s lymphoma and question the applicability of NF-kB inhibitors as candidates for treatment of this cancer.
The goal is the characterization of the off-target activity of BKM120 observed in A2058 human melanoma cell line at IC90 concentration (3.606 µM) but not at lower concentrations. Controls are BEZ235 GDC0941, showing no off-target activity. A secondary objective is the characterizations of drug effects on transcript expression with respect to a control treatment (DMSO) of those PI3K inhibitors.
This study aimed to understand the transcriptional networks regulating endoderm specification from HESC and therefore explored the phenotype of CA1 and CA2 HESC constitutively over-expressing SOX7 or SOX17. Cell lines were created using an inducible construct whereby clonal populations containing transgene integration are selected by Neomycin resistance without expressing of the gene of interest (NoCre controls). Transgene expression is induced via Cre-mediated recombination and selected for puromycin resistance (SOX O/E). The phenotype of the resulting cells suggests that SOX7 expressing HESC represent stable extraembryonic endoderm progenitors while SOX17 expressing HESC represent early definitive endoderm progenitors. Both in vitro and in vivo SOX7 expressing HESC are restricted to the extraembryonic endoderm lineage, while SOX17 expressing HESC demonstrate mesendodermal specificity. In vitro, SOX17 expressing HESC efficiently produce mature definitive endoderm derivatives. The molecular phenotype of the resulting SOX7 and SOX17 expressing HESC was characterized by microarray analysis Keywords: cell line comparison
Transcription mediated by hypoxia inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in the activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compared the genome-wide effects of three molecules that each interfere with the HIF-1-DNA interaction: a polyamide targeted to the hypoxia response element (HRE) siRNA targeted to HIF-1α, and echinomycin, a DNA binding natural product with a similar but less specific sequence preference to the polyamide. The polyamide affects a subset of hypoxia-induced genes that are consistent with the binding site preferences of the polyamide. For comparison, siRNA targeted to HIF-1α and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data shows how polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression. Keywords: Gene expression changes in cultured U251 cells after DFO-stimulation and various treatment conditions
Glioblastoma (GBM) is an incurable brain tumor carrying a dismal prognosis which displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical positions of histone H3.3 (K27, G34) in one-third of pediatric GBM. Here we show that each of these H3F3A mutations defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and are mutually exclusive with IDH1 mutation (characterizing a CpG-Island Methylator Phenotype (CIMP) subgroup). Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM (EGFR amplification, CDKN2A/B deletion) and/or known transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of OLIG1/2 and FOXG1, possibly reflecting different cellular origins. To further dissect the biological differences between epigenetic glioblastoma subgroups, we looked at the transcriptomic profiles of glioblastoma samples.
Genome-wide transcriptome analysis of expression changes in laser-dissected SNpc neurons from Parkinson's disease brain tissue versus control brain tissue.
Cholangiocarcinomac ells have bee treated with felodipine and gene expression analyzed. In order to identify genes with differential gene expression between the groups cb-f and cb-g we study 2 hybridizations on the HG-U133_Plus_2_IVT array using analysis of variance. The analysis identified 7068 genes with significant gene expression differences between the groups (p<0.01).
RNA expression analysis was performed to compare patterns to DNA copy number changes and sensitivity to BCL2 inhibitors. Keywords: cell line comparison
To identify the genes and pathways regulated by FOXF2 we investigated potential FOXF2 gene targets by microarray analyses of primary prostate stromal cells (PrSC) in which FOXF2 was knocked down by siRNA. 190 differentially expressed genes were selected, of which 104 genes were more highly expressed in PrSC cells treated with FOXF2 siRNA and 86 were more highly expressed in PRSC cells treated with negative control siRNA.
We FACS sorted Ras-transformed human mammary epithelial cells (HMLER cells) into GD2+ and GD2- as well as CD44high/CD24low and CD44low/Cd24highcells and comapred the four different population by array.
Although high mammographic density (MD) is considered one of the strongest risk factors for invasive breast cancer the genes involved in modulating this clinical feature are unknown. Histologically, areas of high MD are associated with low adipocyte content and high matrix content, both stromal phenotypes. We hypothesized that fibroblasts purified from low and high MD tissues would show gene expression differences responsible for these histologic differences.
Imatinib as the first-line agent of chronic myeloid leukemia (CML), is ineffective in eradicating CML stem/progenitor cells, thus unable to prevent late relapse. Here we present data indicating that fenretinide preferentially targets CD34+ CML cells and enhances the efficacy of imatinib in CML. As tested by colony forming cell assays, both number and size of total colonies derived from CD34+ CML cells were significantly reduced by fenretinide, and by combining fenretinide with imatinib. In particular, colonies derived from erythroid progenitors and those derived from more primitive pluripotent progenitor cells were highly sensitive to fenretinide/fenretinide plus immtinib. Further data showed that fenretinide was able to induce apoptosis in CD34+ CML cells which were refractory to imatinib. Through transcriptome analysis and followed by molecular validation, we further showed that apoptosis induced by fenretinide in CD34+ CML cells was mediated by complex mechanisms of stress responses, probably triggered by elevated levels of intracellular reactive oxygen species. Thus, fenretinide combines with imatinib may represent a new strategy for the treatment of CML, in which fenretinide targets primitive CD34+ CML cells whereas imatinib targets leukemic blasts. This strategy may eventually reduce the risk of relapse and probably resistant as well in CML patients.
The epithelial-mesenchymal transition (EMT) is an embryonic transdiffrentiation program which consists of the conversion of polarized epithelial cells into a motile mesenchymal phenotype. EMT is aberrantly reactivated during tumor progression promoting metastatic dissemination. Herein, we demonstrate that EMT permissive conditions also favor tumor initiation by minimizing the number of events required for neoplastic transformation. We further demonstrated that even partial commitment of human mammary epithelial cells into an EMT program is sufficient to confer malignant properties, suggesting that the reactivation of embryonic EMT inducers participate to the primary tumor growth long before the initiation of the invasion-metastasis cascade.
Our results suggest that HCMV infection disrupts the self-renewal capacity of NPCs and influences their differentiation. Whole genome expression analysis revealed many changes in cellular gene expression including downregulation of genes pertinent to the neuronal lineage.
This SuperSeries is composed of the SubSeries listed below.
To better understand the role of tumor microenvironment in breast cancer progression we combined laser capture microdissection and microarray analysis to provide a comprehensive catalog of gene expression changes in both tumor and tumor-associated stroma. Keywords: cancer vs. normal
Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognizable by its distinctive red skin lesions. The lesions are KSHV infected spindle cells most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. Here we examine KSHVs modulation of Notch signaling using wild-type LEC cells co-cultured with DLL4 and JAG1 expressing LEC cells.
In these microarray experiments we characterize the gene expression of mammary epithelial cells (MCF10A cells) grown in either a traditional monolayer cell culture setting (2D) or on Matrigel, which induces single MCF10A cells to form organized acinar structures (3D). Morphogenesis of mammary epithelial cells into organized acinar structures in vitro is accompanied by widespread changes in gene expression patterns, including a substantial decrease in expression of Myc. The purpose of this study was to analyze the impact of morphogenesis and organization on gene expression with respect to changes in overall gene expression and Myc target gene expression.
Gene Expression of muscle tissue during microsurgical free tissue transfer was basicly studied in animal models. There was found activation of inflammatory and apoptotic cascades and of genes regulating intracellular metabolism. We used microarrays to detail the programme of gene expression in human muscle tissue which is activated during this process.
MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.
The underlying change of gene network expression of Guillain-Barre syndrome (GBS) remains elusive. We sought to identify GBS-associated gene networks and signalling pathways by analyzing the transcriptional profile of leukocytes in the patients with GBS.
African-American (AA) men experience increased risk of developing prostate cancers as well as increased mortality following treatment as compared to European-American (EA) men. The aim of our study was to identify biological factors with potential to predispose AA men to prostate tumor progression and metastasis. High-throughput microarrays were used to investigate differences in global gene expression comparing the two groups.
The association between chronic inflammation and the development and progression of malignancy is exemplified in the biliary tract where persistent inflammation strongly predisposes to cholangiocarcinoma. The inflammatory cytokine interleukin-6 (IL-6) enhances tumor growth in cholangiocarcinoma by altered gene expression via autocrine mechanisms. We therefore investigated the effect of chronic exposure to IL-6 on gene expression using malignant cholangiocytes stably transfected to overexpress IL-6. Comparison of gene expression identified several genes that were altered by enforced IL-6 expression.
Examine gene expression for meningioma cases by hormone receptor status and indicate a stronger association with progesterone than with estrogen receptors Keywords: comparative gene expression
This experiment was designed to study if there are differences in gene expression in the adipose tissue of women affected by polycystic ovary syndrome (PCOS) compared to non-hyperandrogenic women. PCOS is the most common endocrinopathy in women of reproductive age and is characterized by hyperandrogenism and chronic anovulation. This disease is frequently associated with obesity, insulin resistance, and defects in insulin secretion, predisposing these women to type 2 diabetes, atherosclerosis, and cardiovascular disease. We have applied high-density oligonucleotide arrays to omental adipose tissue samples obtained from eight morbidly obese PCOS patients and seven morbidly obese non-PCOS women at the time of bariatric surgery. Keywords: Disease state analysis
Tissue inhibitor of metalloproteinase 1 (TIMP-1) controls matrix metalloproteinase (MMP) activity through 1:1 stochiometric binding. Human TIMP-1 fused to a glycosylphosphatidylinositol (GPI) anchor (TIMP-1-GPI) shifts the activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1-GPI treated renal cell carcinoma cells (RCC) show increased apoptosis and reduced proliferation. Transcriptomic profiling and regulatory pathway mapping were used to identify potential mechanisms driving these effects. Significant changes in inhibitor of DNA binding (IDs) TGF-β1/SMAD and BMP pathways resulted from TIMP-1-GPI treatment. These events were linked to reduced TGF-β1 signaling mediated by inhibition of proteolytic processing of latent TGF-β1 by TIMP-1-GPI. Activity of TIMP-1 from the extracellular matrix to the cell surface. TIMP-1-GPI treated renal cell carcinoma cells (RCC) show increased apoptosis and reduced proliferation. Transcriptomic profiling and regulatory pathway mapping were used to identify potential mechanisms driving these effects. Significant changes in inhibitor of DNA binding (IDs), TGF-β1/SMAD and BMP pathways resulted from TIMP-1-GPI treatment. These events were linked to reduced TGF-β1 signaling mediated by inhibition of proteolytic processing of latent TGF-β1 by TIMP-1-GPI.
The rapid development in septic patients of features of marked immunosuppression associated with increased risk of nosocomial infections and mortality represents the rational for the initiation of immune targeted treatments in sepsis. However as there is no clinical sign of immune dysfunctions, the current challenge is to develop biomarkers that will help clinicians identify the patients that would benefit from immunotherapy and monitor its efficacy. Using an in vitro model of endotoxin tolerance (ET), a pivotal feature of sepsis-induced immunosuppression in monocytes, we identified using gene expression profiling by microarray a panel of transcripts associated with the development of ET which expression was restored after immunostimulation with interferon-gamma (IFN-γ). These results were confirmed by qRT-PCR. Importantly, this short-list of markers was further evaluated in patients. Of these transcripts, six (TNFAIP6, FCN1, CXCL10, GBP1, CXCL5 and PID1) were differentially expressed in septic shock patients’ blood compared to healthy blood upon ex vivo LPS stimulation and were restored by IFN-γ. In this study, by combining a microarray approach in an in vitro model and a validation in clinical samples, we identified a panel of six transcripts that could be used for the identification of septic patients eligible for IFNg therapy. The potential value of these markers should now be evaluated in a larger cohort of patients. Upon favorable results, they could serve as stratification tools prior to immunostimulatory treatment and to monitor drug efficacy.
RNA microarray profiling of 45 tissue samples was carried out using the Affymetrix (U133) gene expression platform. Laser capture microdissection (LCM) was employed to isolate cancer cells from the tumors of 18 serous ovarian cancer patients (Cepi). For 7 of these patients a matched set of surrounding cancer stroma (CS) was also collected. For controls, surface ovarian epithelial cells (OSE) were isolated from the normal (non-cancerous) ovaries of 12 individuals including matched sets of samples of OSE and normal stroma (NS) from 8 of these patients. Unsupervised hierarchical clustering of the microarray data resulted in the expected separation between the OSE and Cepi samples. Consistent with models of stromal activation, we also observed significant separation between the NS and CS samples. Unexpectedly, the CS samples sub-divided into two distinct groups. Analysis of expression patterns of genes encoding signaling molecules and compatible receptors in the CS and Cepi samples are consistent with the hypothesis that the two CS sub-groups differ significantly in their relative propensities to support tumor growth.The results indicate the existence of distinct categories of ovarian cancer stroma and suggest that functionally significant variability exists among ovarian cancer patients in the ability of the microenvironment to modulate cancer development.
Alefacept treatment is highly effective in a select group patients with moderate-to-severe psoriasis and is an ideal candidate to develop systems to predict who will respond to therapy. A clinical trial of 22 patients with moderate to severe psoriasis treated with alefacept (7.5mg weekly i.v. x12 weeks) was conducted in 2002-2003, as a mechanism of action study. Patients were classified as responders or non-responders to alefacept based on histological criteria. Microarray data from PBMCs of 16 of these patients was analyzed to generate a treatment response classifier. We used a discriminant analysis method that performs sample classification from gene expression data, via nearest shrunken centroid method''. A disease response classifier using 23 genes was created to accurately predict response to alefacept (12.3% error rate in favour of responders). This preliminary study may provide a useful tool to predict response of psoriatic patients with alefacept. Keywords: class prediction
Cellular senescence is an irreversible proliferative arrest and can be triggered in many cell types in response to diverse forms of cellular damage or stress. We used microarrays to compare gene expression profile between growing and senescent human activated hepatic stellate cells. Keywords: cell type comparison
A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance we constructed Biological Association Networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX.
It is widely believed that the molecular and cellular features of a tumor reflect its cell-of-origin and can thus provide clues about treatment targets. The retinoblastoma cell-of-origin has been debated for over a century. Here we report that human and mouse retinoblastomas have molecular cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type–specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Importantly, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro. Our finding that retinoblastoma tumor cells express multiple neuronal differentiation programs that are normally incompatible in development suggests that the pathways that control retinal development and establish distinct cell types are perturbed during tumorigenesis. Therefore, the cell-of-origin for retinoblastoma cannot be inferred from the features of the tumor cells themselves. However, we now have a detailed understanding of the neuronal pathways that are deregulated in retinoblastoma and targeting the catecholamine and indolamine receptors or downstream components could provide useful therapeutic approaches in future studies. This example highlights the importance of comprehensive molecular, cellular and physiological characterization of human cancers with single cell resolution as we incorporate molecular targeted therapy into treatment regimens.
We used microarrays to analyze the global expression patterns for 22 commercially available pancreatic cancer cell lines
some genes associated with cell growth cell differentiation, meiosis, migration, and apoptosis are regulated in ECC-1 after DEHP exposure. We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated genes during this process.
FaDu treated with citric buffer vs. rCTGF
Tissue response following implantation determines the success of the healing process. This response is not only dependent on the chemical properties of the implant surface but also by the surface topography or its roughness. Although in vitro and in vivo studies show improved results with rough- and fluoride-modified implants the mechanisms behind these findings are still unknown. Here, we have used a two step procedure to identify novel genes related to the early cell response of primary human osteoblasts to roughness and fluoride-modified titanium implants. 217 genes were identified by microarray analysis as response genes to roughness and 198 genes as response genes to fluoride. 11 of these identified genes have been related to bone and mineralization and were further investigated by real-time RT-PCR. After one day of culture, TLR3, ANKH, DCN, OC and RUNX2 were classified as responsive genes to roughness; DLX2 and TUFT1 as responsive genes to fluoride treatment. COLL-I, PTHLH, HES1, FST, ENPP1 and THRA as responsive genes to both, roughness and fluoride treatment. In conclusion, our strategy was useful for identifying novel genes that might be involved in the early response of osteoblasts to roughness and fluoride treatment of titanium implants. Tissue response following implantation determines the success of the healing process. This response is not only dependent on the chemical properties of the implant surface but also by the surface topography or its roughness. Although in vitro and in vivo studies show improved results with rough- and fluoride-modified implants, the mechanisms behind these findings are still unknown. Here, we have used a two step procedure to identify novel genes related to the early cell response of primary human osteoblasts to roughness and fluoride-modified titanium implants. 217 genes were identified by microarray analysis as response genes to roughness and 198 genes as response genes to fluoride. 11 of these identified genes have been related to bone and mineralization and were further investigated by real-time RT-PCR. After one day of culture, TLR3, ANKH, DCN, OC and RUNX2 were classified as responsive genes to roughness; DLX2 and TUFT1 as responsive genes to fluoride treatment. COLL-I, PTHLH, HES1, FST, ENPP1 and THRA as responsive genes to both, roughness and fluoride treatment. In conclusion, our strategy was useful for identifying novel genes that might be involved in the early response of osteoblasts to roughness and fluoride treatment of titanium implants.
We compared PBMC genomic response to exercise in both early (EG) and late-pubertal girls (LG) Keywords: stress response
Recently global gene expression profiling of patients samples lead to a molecular definition of Burkitt Lymphoma (BL) with lymphocyte enhancer-binding factor 1 (LEF1) as a signature gene. Here we report the discovery of nucleic LEF1 in a very high proportion of BL cases (15/18) and LEF1 target genes. Germinal center B cells were devoid of detectable nuclear LEF1 expression as mantle cell lymphoma (0/5) marginal zone lymphoma (0/6), follicular lymphoma (0/12) or diffuse large B cell lymphoma (DLBCL) (1/31). Using whole genome gene expression profiling after transient knockdown of LEF1 in BL cell lines, new LEF1 target genes were identified. The joint expression of these genes in primary BL samples shows that LEF1 is not only expressed aberrantly in BL but also transcriptionally active. Our study identified aberrantly expressed LEF1 and its target genes suggesting an important functional role in BLs.
Most of the breast cancer samples used in clinical research contain multiple cell types other than epithelial cells alone. The non-epithelial cell types have have a substantial effect on the gene expression-profile which is used to define molecular subtypes of the tumours. The purpose of this data set is to retrieve gene-expression profile within tumour epithelial cells. We collected 9 breast cancer epithelial cell lines and 5 tumour sampes from which epithelial cells were sorted and enriched using BerEp4 antibody coated beads. We profiled the mRNA expression level of these samples and classified probe sets into epithelial genes which were those genes with present calls in at least 50% of the samples. Then we derived an 23-gene signature based on only the epithelial genes to stratify breast cancer.
Integrated microarray and multiplex cytokine analyses of Kaposi's Sarcoma Asssociated Herpesvirus viral FLICE Inhibitory Protein K13 affected genes and cytokines in human blood vascular endothelial cells. The KSHV-encoded K13 protein is one of the few proteins to be expressed in latently-infected spindle cells and the ectopic expression of K13 in human vascular endothelial cells is sufficient to transform them into spindle cells.
Overexpression of the AP-2γ transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy even in ER positive, ErbB2 negative tumours; markers of a more favourable prognosis. To understand further the role of AP-2γ in breast carcinoma, we have used an RNA interference and gene expression profiling strategy using the MCF-7 cell line as a model for ER positive, ErbB2 negative tumours with AP-2γ overexpression. Gene expression changes between control and silenced cells implicate AP-2γ in the control of cell cycle progression and developmental signalling. Keywords: RNA interference
Diabetic neuropathy (DN) is a common complication of diabetes. While multiple pathways are implicated in the pathophysiology of DN there are no specific treatments for DN and currently it is not possible to predict DN onset or progression. To examine gene expression signatures related to DN, microarray experiments were performed on a subset of human sural nerves collected during a 52-week clinical trial of acetyl-L-carnitine. A series of bioinformatics analyses analyzed differential gene expression and identified gene networks and pathways potentially responsible for the progression of DN. We identified 532 differentially expressed genes (DEGs) between patient samples with progressing or non-progressing DN, which were functionally enriched in pathways involving defense and inflammatory responses and lipid metabolism. A literature-derived co-citation network of the DEGs revealed gene sub-networks centered on apolipoprotein E (APOE), jun oncogene (JUN), leptin (LEP), serpin peptidase inhibitor E Type 1 (SERPINE1) and peroxisome proliferator-activated receptor gamma (PPARG). DEGs were used to predict DN progression in a test set of patients. Ridge-regression classification models with 14 DEGs achieved an overall accuracy of 92%, correctly classifying the progression status of 11 out of 12 patients. To our knowledge, this is the first study to identify transcriptional changes associated with DN progression in human sural nerves biopsies and describe their potential utility for molecular prediction of DN. Our results identifying the unique gene signature of patients with progressive DN will facilitate the development of new mechanism-based diagnostics and therapies.
MCF-7aro cells were used to generate a cell culture model system that is resistant to 3 aromatase inhibitors (AIs) letrozole, anastrozole and exemestane. For comparison, the MCF-7aro cells were also used to generate the tamoxifen-resistant cells as well as long-term estrogen deprived, LTEDaro. Affymetrix microarray analysis was performed to determine changes in gene expression that are unique to AI-resistance. Keywords: cell lines, aromatase inhibitor resistance, tamoxifen resistance
The whole-genome oligonucleotide microarray analysis of NS398-treated HT29 colon adenocarcinoma cells samples can give an insight into global molecular background of selective COX2 inhibitor administration in order to find other target molecules and pathways influenced by NS398 selective COX2 inhibitor treatment in the epithelial cells.
Active HUMSC with distinct binding rate to MDA MB-231 breast cancer cells distinct ability in suppressing tumorigenesis,distinct cell in cell features and distinct features under TEM then inactive HUMSC We used microarrays to detail the difference gene expression between active HUMSC and inactive HUMSC
Organophosphorus compounds induce cardiotoxicity through currently unknown mechanisms which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. We used microarrays to study gene expression changes in human cardiomyocytes after exposure to VX, and identified pathways underlying these changes.
In this study we sought to determine how IL-17 and TNF influence normal human melanocytes, either alone, or with both cytokines together. We reveal a dichotomous effect of IL-17 and TNF, which not only elicit essential mitogenic cytokines but also suppress melanogenesis by down-regulating genes of melanogenesis pathway
In this study we have investigated the effect of LMP1 on gene expression in normal human GC B cells using a non-viral vector based system Keywords: transfection of viral oncogene in normal human B cells
Aberrant methylation has been postulated to play an important role in tumorigenesis. We report the use of methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to investigate methylation changes in testicular germ cell tumor (TGCT) cells. Coupled to expression profiling changes we found that only 22-26% of differentially methylated genes were also expressed differentially. This phenomenon was independent of the presence of CpG islands in the promoter. Differential methylation and expression of some of these genes were confirmed in testicular tumor tissue. A substantial number of differentially methylated regions in the human genome were not linked to annotated gene loci. Subsequent analysis indicated several microRNAs and small nucleolar RNAs were regulated by these differentially methylated regions. Our results demonstrate the power of the combination of MeDIP-chip analysis and expression profiling for discovery in cancer cells of epigenetically regulated genes and non-coding RNAs in cancer cells.
The proto-oncogenes ETV1 ETV4, and ETV5 encode members of the E26 transformation-specific (ETS) transcription factor family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COnstitutive Photomorphogenic-1 (COP1, also called RFWD2) as a tumor suppressor that negatively regulates ETV1, ETV4, and ETV5. ETV1, which is the member mutated more frequently in prostate cancer, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs (degrons) and was 50-fold more stable than wild-type ETV1. Almost all patient translocations eliminate these ETV1 degrons, implying that translocations rendering ETV1 insensitive to COP1 confer a significant selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 levels and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. The combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, relatively rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein expression, and abnormally elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a bona fide tumor suppressor whose down-regulation promotes prostatic epithelial cell proliferation and tumorigenesis.
Huh7/5-2 cells (Binder et al. Hepatology 2007) were mock infected (DMEM) (time points 4 and 48 h) or infected with the chimeric HCV virus Jc1 (Pietschmann et al., PNAS 2006) (all time points). Multiplicity of infection was 15 (TCID50). Cells were lysed after 4, 12, 24, 48 and 72 hours post infection and total cellular RNA was prepared.
Placental trophoblasts are key determinants of in utero development. Mouse trophoblast stem cells (mTSCs) which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and trophoblast fate determinants in the early-gestation placenta, amnion and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing FGF and an inhibitor of activin/nodal signaling, which is required for human embryonic SC self-renewal. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human trophoblast lineages—multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic trophoblast progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2 and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.
Non-union skeletal fractures are characterized by their inability to heal six months after injury. Left untreated the non-union fracture may cause advanced arthritis or loss of function in the affected limb. Arrays were used to identify the mechanisms that lead to lack of skeletal repair in non-unions. These profiles are the non-union callous samples pooled from five individuals Keywords: other
Cancer cells were MACS sorted from tumor tissue specimem 05-179. Self replicates of CD26+ cancer cells were generated and the expression profiles were determined using Affymetrix U133 Plus 2.0 arrays. These data represent cancer cell type specific transcriptome. Keywords: disease state analysis
In this study we focussed on malignant post-transplant lymphomas. Post-transplant lymphoma is strongly associated with Epstein-Barr Virus (EBV) infection in contrast to lymphoma arising in an immunocompetent population. Nevertheless about 1 in 3 PTLD cases are negative for EBV. We used a microarray to define the gene expression profile of different PTLDs to elucidate the pathogenesis of EBV(+) and EBV(-) PTLD and to define whether EBV(-) PTLD is biologically different from EBV(-) lymphoma arising in an immunocompetent host.
We developed a general approach to small molecule library screening called GE-HTS (Gene Expression-Based High Throughput Screening) in which a gene expression signature is used as a surrogate for cellular states and applied it to the identification of compounds inducing the differentiation of acute myeloid leukemia cells. In screening 1739 compounds, we identified 8 that reliably induced the differentiation signature, and furthermore yielded functional evidence of bona fide differentiation. We tested several of these in duplicate replicates in blasts from a patient with APL. Also included in this data set are a collection of 6 primary patient AML cells, 3 normal neutrophils samples, and 3 normal monocyte samples. This data was used to evaluate whole genome effects of the compounds on APL cells in relation to AML versus normal neutrophils and monocytes. Keywords = Leukemia Keywords = APL Keywords = AML Keywords = chemical genomics Keywords: repeat sample
We defined the C/EBPa signature characterized by a set of genes which are upregulated upon C/EBPa activation. In order to identify the C/EBPa signature we performed microarray gene expression analysis of K562 cells stably expressing p42-C/EBPa-ER after activating the C/EBPa construct to translocate to the nucleus for 6 hours with beta-estradiol.
Analysis of gene expression changes due to nonviral gene delivery of DNA lipoplexes versus control in human HEK293T cells.
The FAT1 gene was knocked down using 2 independent siRNAs in immortalized human astrocytes and U87 and U251 glioma cell lines. A non-targeted scramble siRNA was used as a control.
Cancer cells interact with surrounding stromal fibroblasts during tumorigenesis but the complex molecular rules that govern these interactions remain poorly understood, thus hindering the development of therapeutic strategies to target cancer stroma. We have taken a mathematical approach to begin defining these rules by performing large-scale quantitative analysis of fibroblast effects on cancer cell proliferation across more than four hundred heterotypic cell line pairings. Systems-level modeling of this complex dataset using singular value decomposition revealed that normal tissue fibroblasts variably express at least two functionally distinct activities, one which reflects transcriptional programs associated with activated mesenchyme, that act either coordinately or at cross-purposes to modulate cancer cell proliferation. To gain insight into the molecular identity of these fibroblast activities, we isolated RNA from 36 human skin and lung fibroblast cell line monocultures from Coriell Repositories or ATCC and performed microarray-based gene expression profiling using Affymetrix gene chips.
Alkaline phosphatase (ALP) is known to be a marker for several somatic stem cells and cancer cells. We found that human squamous cell carcinoma HeLa cells are comprised by ALP-positive and negative cells. Single cell-derived colony assay revealed that the former cells are labile with respect to ALP activity but the latter are stable. We cloned ALP-negative cells from the HeLa cells, and named H-1 clone. DNA microarray analysis revealed that gene expression pattern of H-1 cells is almost the same with that of their parental HeLa cells, but several genes for glycoprotein hormone alpha chain, ras-related and estrogen-regulated growth inhibitor, ALP, and Frizzled-10 was respectively 18.2, 9.6, 9.2 and 10.5–fold are upregulated in the HeLa cells. Although there is no evidence that the ALP-positive cells are cancer stem cells (CSCs) at present, HeLa cells comprised by ALP-positive and -negative cells may be a good model for CSC study in future.
GATA2 is well recognized as a key transcription factor and regulator of cell type specificity and differentiation. Here we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2 mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2 associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity.
We retrospectively analyzed AML patients enrolled in the AIEOP since 2000 42 patients with 11q23 rearrangement were analyzed by gene expression profile Gene expression analyses were performed to compare AML MLL partner genes (AF9, AF10, AF6, ENL, ELL, Septin 6, and AF1q) Keywords: Expression data
Class IIa histone deacetylases (HDACs) are signal-responsive regulators of gene expression involved in vascular homeostasis. To investigate the differential role of class IIa HDACs for the regulation of angiogenesis we used siRNA to specifically suppress the individual HDAC isoenzymes. Among the HDAC isoforms tested, silencing of HDAC5 exhibited a unique pro-angiogenic effect evidenced by increased endothelial cell migration, sprouting and tube formation. Consistently, overexpression of HDAC5 decreased sprout formation, indicating that HDAC5 is a negative regulator of angiogenesis. The anti-angiogenic activity of HDAC5 was independent of MEF2 binding and its deacetylase activity, but required a nuclear localization indicating that HDAC5 might affect the transcriptional regulation of gene expression. To identify putative HDAC5 targets, we performed microarray expression analysis. Silencing of HDAC5 increased the expression of fibroblast growth factor 2 (FGF2) and angiogenic guidance factors including Slit2. Antagonization of FGF2 or Slit2 reduced sprout induction in response to HDAC5 siRNA. ChIP assays demonstrate that HDAC5 binds to the promoter of FGF2 and Slit2. In summary, HDAC5 represses angiogenic genes, like FGF2 and Slit2, which causally contribute to capillary-like sprouting of endothelial cells. The de-repression of angiogenic genes by HDAC5 inactivation may provide a useful therapeutic target for induction of angiogenesis.
Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality. Previous studies have suggested that T cell responses may contribute to RSV immunopathology which could be driven by dendritic cells (DCs). DCs are productively infected by RSV, and during RSV infections, there is an increase of DCs in the lungs with a decrease in the blood. Pediatric populations are particularly susceptible to severe RSV infections, however DC responses to RSV from pediatric populations have not been examined. In this study, primary isolated DCs from cord blood and adult peripheral blood were compared after RSV-infection. Transcriptional profiling and biological network analysis identified transforming growth factor (TGF)-b and associated signaling molecules as differentially regulated in the two age groups. TGF-b1 was decreased in RSV-infected adult blood DCs, but increased in RSV-infected cord blood DCs. Co-culture of adult RSV-infected DCs with autologous T-cells induced secretion of interferon gamma (IFNg), IL-12p70, IL-2, and tumor necrosis factor alpha (TNFa). Conversely, co-culture of cord RSV-infected DCs and autologous T-cells induced secretion of IL-4, IL-6, IL-1b, and IL-17. Addition of purified TGF-b1 to adult DC-T cell co-cultures reduced secretion of IFNg, IL-12p70, IL-2, and TNFa, which addition of a TGF-b chemical inhibitor to cord DC-T cell co-cultures increased secretion of IL-12p70. These data suggest that TGF-b acts as a major regulator of RSV DC-T cell responses, which could contribute to immunopathology during infancy.
The Tesi system allows analysis of HTLV-1 Tax's impact on the transcriptome of a human CD4+ T-cell which is not derived from leukemia but directly from normal human lymphocytes. By comparing cells with and without Tax one can specifically filter for celluar genes that are either activated or repressed in the presence of Tax. Keywords: cell type comparison, Tax
Background :To evaluate the impact of the duration of chronic inflammation on gene expression in skeletal muscle biopsies (MBx) from untreated children with juvenile dermatomyositis (JDM) and identify genes and biological processes associated with the disease progression expression profiling data from 16 girls with active symptoms of JDM greater or equal to 2 months were compared with 3 girls with active symptoms less than 2 months. Results: Seventy-nine genes were differentially expressed between the groups with long or short duration of untreated disease. Genes involved in immune responses and vasculature remodeling were expressed at a higher level in muscle biopsies from children with greater or equal to 2 months of symptoms, while genes involved in stress responses and protein turnover were expressed at a lower level. Among the 79 genes, expression of 9 genes showed a significant linear regression relationship with the duration of untreated disease. Five differentially expressed genes--HLA-DQA1, smooth muscle myosin heavy chain, clustering, plexin D1 and tenomodulin--were verified by quantitative RT-PCR. The chronic inflammation of longer disease duration was also associated with increased DC-LAMP+ and BDCA2+ mature dendritic cells, identified by immunohistochemistry. Conclusions: We conclude that chronic inflammation alters the gene expression patterns in muscle of untreated children with JDM. Symptoms lasting greater or equal to 2 months were associated with dendritic cell maturation and anti-angiogenic vascular remodelling, directly contributing to disease pathophysiology. Keywords: Disease progression; time course
The transcription factor OTX2 has been implicated as an oncogene in medulloblastoma which is the most common malignant brain tumor in children. It is highly expressed in most medulloblastomas and amplified in a subset of them. The role of OTX2 in medulloblastoma and its downstream targets are unclear. Therefore, we generated D425 medulloblastoma cells in which we can silence endogenous OTX2 by inducible shRNA. Silencing of OTX2 strongly inhibited cell proliferation and resulted in a neuronal-like differentiation. Expression profiling of time courses after silencing showed a progressive change in gene expression for many cellular processes. Down regulated genes were highly enriched for cell cycle and visual perception genes, while up regulated genes were enriched for genes involved in development and differentiation. This shift in expression profiles is reminiscent to changes described to occur during normal cerebellum development. OTX2 is expressed in proliferating granular progenitor cells, but the expression diminishes when these cells exit the cell cycle and start differentiating. ChIP-on-chip analyses of OTX2 in D425 cells showed that cell cycle and perception genes were direct OTX2 targets, while regulation of most differentiation genes appears to be indirect. These analyses provide the first insight in the molecular network of OTX2, demonstrating that OTX2 is essential in medulloblastoma and directly drives proliferation by regulating the expression of cell cycle genes. Since many of these genes also correlate in expression with OTX2 in primary tumors, they might be potential targets for therapy in medulloblastoma patients. Keywords: OTX2, medulloblastoma, mRNA profiling
The effects of diverse stresses on promoter selectivity and transcription regulation by the tumor suppressor p53 are poorly understood. We have taken a comprehensive approach to characterizing the human p53 network that includes p53 levels binding, expression and chromatin changes under diverse stresses. Human osteosarcoma U2OS cells treated with anti-cancer drugs Doxorubicin or Nutlin-3 led to strikingly different p53 gene binding patterns based on ChIP-seq experiments. While two contiguous RRRCWWGYYY decamers is the consensus binding motif, p53 can bind a single decamer and function in vivo. Although the number of sites bound by p53 was 6-times greater for Nutlin-3 than Doxorubicin, expression changes induced by Nutlin-3 were much less dramatic compared to Doxorubicin. Unexpectedly, the solvent DMSO alone induced p53 binding to many sites common to Doxorubicin; however, this binding had no effect on target gene expression. Together, these data imply a two-stage mechanism for p53 transactivation where p53 binding only constitutes the first stage. Furthermore, both p53 binding and transactivation were associated with increased active histone modification H3K4me3. We discovered 149 putative new p53 target genes including several that are relevant to tumor suppression, revealing potential new targets for cancer therapy and expanding our understanding of the p53 regulatory network.
The multifunctional protein lipopolysaccharide-induced TNFalpha factor (LITAF) induces the secretion of inflammatory cytokines in monocytes and regulates protein degradation in neural cells. In B-cell lymphomas LITAF is frequently inactivated by epigenetic mechanisms, but beyond these data little is known about its regulation and function. Immunohistochemical and gene expression profiling analyses of normal and malignant B-cells revealed that LITAF and BCL6 exhibited opposite expression patterns. Accordingly, chromatin immunoprecipitation and luciferase experiments showed that LITAF is transcriptionally repressed by BCL6 in germinal center (GC) lymphocytes and in B-cell lymphoma cells. Gain- and-loss-of-function assays demonstrated that LITAF does not exert any of its previous roles. Conversely, LITAF co-localized with autophagosomes in B-cells whereby activated autophagic responses, which were abrogated upon LITAF silencing. Therefore, BCL6-mediated transcriptional repression of LITAF may contribute to an appropriate GC reaction by suppressing autophagy in GC lymphocytes, whereas constitutive repression of autophagic responses may promote B-cell lymphoma development.
Although the prognosis for childhood Acute Lymphoblastic Leukemia (ALL) in general has improved tremendously over the last decades the survival chances for infants (<1 year of age) with ALL remains poor. A major obstacle hampering successful treatment results in infant ALL is cellular resistance to several drugs currently used in the treatment of ALL, especially to prednisolone (or prednisone). Therefore we set out to search for genes differentially expressed between from infant (children <1 year of age) and non-infant (children >1 year of age) ALL samples either resistant or sensitive to prednisolone.
Toxin A and B from Clostridium difficile are the primary virulence factors in Clostridium difficile disease. The changes in gene transcription of human colon epithelial cells were investigated in vitro in order to better understand the many effects of both toxins.
Gene expression profiling was carried out on peripheral blood leukocytes from 14 healthy older adults. The primary research question is whether gene expression differs in individuals experiencing chronically high levels of social isolation (by UCLA Loneliness Scale) vs chronically low levels of social isolation. Keywords: Risk prediction
Identification of genes up or down regulated in LPS stimulated samples in comparison to control samples.
The effects of ethanol may vary from induction of apoptosis to the inhibition of proliferation differentiation, migration or other function. The complex and diverse response of fetal cells to ethanol has prompted us to use a bioinformatics approach to study the effect of ethanol on fetal stem cells derived from the amniotic fluid-derived (AFSC). To characterize the global response of human AFSC to ethanol, gene expression profiles of AFSC treated with or without 100mM ethanol for 48 hours were analyzed. Keywords: stem cells, amniotic fluid-derived stem cells, ethanol, alcohol
Multiple DNA methylation changes have been associated with the acquisition of drug resistance; however it remains uncertain how many of these changes may represent critical DNA methylation drivers of chemoresistance. Using gene expression profiling method on HGU133plus2 array we identified a total of 1370 genes showing significant gene expression changes with 687 genes going up and 683 genes going down in the resistant (cp70) versus sensitive cell lines (A2780) by Rank Product (FDR<5%). Combining expression profiling with methylation profiling data we found out of 245 hypermethylated and down-regulated genes in the resistant cell line, 41 genes were up-regulated following Decitabine treatment alone, 45 genes up-regulated following combined treatment of Decitabine and PXD101, and only 10 genes up-regulated following PXD101 treatment alone. Altogether we found a small set of genes as being potential key drivers of chemoresistance and should be further evaluated as predictive biomarkers, both to existing chemotherapies, but also to epigenetic therapies used to modulate drug resistance.
RNA was isolated from ectopically sFRP1-expressing MDA-MB-231 cells and control MDA-MB-231 cells and as well from tumor lysates arising from these cells as nude mouse xenograft. Gene expression profiles for these samples were investigated using Affymetrix arrays.
ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events) the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapib’s effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.
The second leading cause of cancer death for women in the U.S. is breast cancer. Moreover a significant number of patients with breast tumors acquire resistance to drugs during therapy. To develop targeted therapeutic strategies to combat drug resistance it is essential to understand the basic molecular mechanisms through which cancer cells control sensitivity to chemotherapeutics. To identify new candidate genes and facilitate the discovery of novel drug resistance pathways, we have generated a resistance profile or ‘resistome’ of etoposide resistant MCF7 breast cancer cells. Differential expression of over 5000 genes (fold change > 2, P value < 0.05) indicate that several drug resistance mechanisms may be operating in these cells, including up-regulation of ABC transporter genes, down-regulation of the drug target and down-regulation of apoptotic genes. Several transcription factors such as RUNX2, SOX9, ETS1 and SMAD3 were up-regulated in the drug resistant cells. Targeted RUNX2 knockdown in the resistant cells using siRNA increased sensitivity to etoposide and also upregulated expression of pro-apoptotic genes indicating that RUNX2 could be a molecular target against etoposide resistance. Differential miRNA (microRNA) expression was observed among the drug resistant and sensitive cells suggesting that miRNA may also play a role in regulation of drug resistance. Hsa-miR-218, which targets ABCC6, was down-regulated in the drug resistant cell line. Transfection of a miR-218 mimic could down-regulate the expression of the efflux pump ABCC6 by 65% in drug resistant cells suggesting that regulation of miRNA may play an important role in etoposide resistance.
Comparison of mRNA expression profiles in W12 Series 1 cervical ectokeratinocytes at passage number 22 versus 19 (during which time the cells gain an invasive phenotype) As these cells demonstrate gain of chromosome 5p during this time mRNA expression profiling data interrogated for over-expressed genes on 5p that may be important in cervical neoplastic progression.
Background: Dengue virus (DENV) infection often leads to acute illness lasting 2-7 days with severity ranging from dengue fever (DF) to hemorrhagic fever (DHF) and fatal dengue shock syndrome (DSS). The dynamic changes of host responses on the gene transcription level that accompany DENV infection and differences between DF and DHF cases have been poorly understood particularly for South American population. Methodology/Principal Findings: Supported by a longitudinal active surveillance program for dengue transmission in Maracay, Venezuela, we conducted a prospective study to investigate host responses in dengue patients. Blood specimens and clinical information were collected on a daily basis from febrile cases confirmed with DENV infection from their first day of enrollment to early defervescence together with one convalescent sample. A total of 49 and 13 study participants were defined as DF and DHF cases respectively based on their clinical and hematological information. Using convalescent specimens as baseline, day-to-day gene expression was evaluated ex vivo in peripheral blood mononuclear cells of the study participants. Two waves of gene expression were detected: the first wave peaked at day 1 from the onset of fever (day 0) then declined at days 3-4; the second wave emerged from day 4 and peaked around day 5-6. Genes associated with innate immune process, including type I interferon signaling, cytokine-mediated signaling, chemotaxis, and antiviral responses, dominated the first wave; whereas genes involved in cell cycle processes, including cell division, mitosis, DNA replication, chromosome, and spindle organization dominated the second wave. Measureable genomic markers predicted early acute, late acute and convalescent phases with 91% accuracy. Gene signatures expressed in early acute phase predicted disease severity (DF vs DHF) with 96% accuracy. Conclusions/Significance: Our study established a dynamic pattern of detailed host immune responses to DENV infection and revealed genomic signatures valuable for diagnostics purposes.
[original title] Gene expression response to the implantation of drug (paclitaxel)-eluting or bare metal stents in denuded human LIMA arteries. Different clinical outcomes have been observed for paclitaxel-eluting and bare metal cardiovascular stents. The aim of this project was to identify genes that might be associated with the observed clinical outcomes.
Nasopharyngeal carcinoma is an Epstein-Barr virus-associated epithelial cancer with high prevalence in Southeast Asia. mRNA expression levels were measured for essentially all human genes and all latent Epstein-Barr virus (EBV) genes in nasopharyngeal carcinoma tissue samples and normal nasopharyngeal tissues. Data were analyzed for differential gene expression between tumor and normal tissue and for correlations with levels of viral gene expression. Primary publications: Sengupta et al 2006, Cancer Research 66(16): 7999-8006. Dodd et al, 2006, Cancer Epidemiology, Biomarkers & Prevention 15(11): 2216-2225. In subsequent studies using the same set of tissue samples, microRNA levels were measured in tumors and normal tissues and analyzed for correlations with differential target gene expression (Sengupta et al, 2008, Proc. Nat. Acad. Sci. USA 105: 5874-5878.) Keywords: mRNA expression profiling
Decoy receptor 3 (DcR3) a member of the tumor necrosis factor receptor (TNFR) superfamily, competitively binds and inhibits members of the TNF family, including Fas ligand (FasL), LIGHT, and TL1A. DcR3 was recently reported not only to act as a decoy receptor for these TNFRs but also to play a role as a ligand for the pathogenesis of RA. We hypothesized that DcR3 regulates the gene expression in RA-FLS. We used to search for genes in which expression in RA-FLS is regulated by the ligation of DcR3.
Carbonic anhydrase IX (CA 9) is a transmembrane isoform of carbonic anhydrase (CA) that contributes to an acidification of tumor microenvironment. The expression of CA 9 in cervical tumors was shown to be strongly involved in high incidence of metastasis and poor prognosis. To search for the key regulators of metastasis related to ectopic expression of CA 9 we investigated differentially expressed gene profiles in CA 9- transfected cervix carcinoma cell line C33-A (CF) compared with mock-transfected (CP) cell line, using Affymetrix Human Genome U133 Plus 2.0 Array.
Mammalian cells were grown as multicellular aggregates (spheroids) in an effort to determine the signaling events required for two cellular transformations states; primary foreskin fibroblasts (HFF-2) and glioblastoma cancer (T98G) cells to survive at room temperature under oxygen and nutrient-deprived conditions for extended periods of time (2 weeks) and subsequently grown out from the arrested state as adherent monolayers. HFF-2 cells were cultured in DMEM supplemented with 15% fetal bovine serum and 5% carbon dioxide humidified air at 37 degrees C. T98G cells were cultured in EMEM with 10% FBS, 5% non-essential amino acids and 5% carbon dioxide humidified air at 37 degreesC. Monolayers were grown in T-185 flasks to 60% confluency then split into T-185 flasks coated with a 1% agarose mix in a 2:1 media/water ratio. Cells were suspended in 30 ml of supplemented media and grown for 4 days in order to form multicellular spheroids as described previously by our group (J. Cell. Physiol., 206 [2006] 526-536; see GSE1364 and GSE1455 for similar experiments with HEK293 cells). The suspension was removed from the flasks and centrifuged (1500 x g, 2 min) and the media removed. The pellet was returned to the flasks and then placed in vacuum bags (Dri-shield 2000 moisture barrier bag from Surmount Inc., USA; Cat. number 70068), which were sealed immediately under vacuum (Deni Magic Vac, Champion model; Keystone Manufacturing, USA). Vacuum-sealed flasks were stored for 2 weeks (in the dark) at room temperature. Recovery was initiated by removing the flask from the bag and resuspending the spheroids in supplemented media and placing the flasks in a 5% CO2/humidified air incubator maintained at 37 degreesC. Timepoints for transcriptional analysis were monolayer (control), 4 day growth spheroids, 2 week stored spheroids and 7 day growth back to monolayers. There were 67 HFF-2 and 50 T98G genes that exhibited at least a 10-fold change in expression during the course of arrest and recovery. Eleven of these genes were common to both cell lines. While the trends were the same between the two cell lines for genes that were in common, the timing of the responses were clearly different. The primary fibroblasts showed significant gene expression changes during spheroid formation and returned to their initial monolayer state after the two week arrest, whereas the glioblastoma cells exhibited major changes in gene expression during the recovery process. Furthermore, the T98G cells never returned to the monolayer expression levels during the time course of the recovery phase (7 days growth under monolayer conditions). Major differences in the transcriptional data were also found between the cell lines with regards to the putative cellular location of the encoded proteins. Of the genes with significant fold changes (+/- 10-fold) for which location information is available, the primary fibroblasts were significantly enriched in genes encoding extracellular (32%) and membrane-associated (26%) proteins. The glioblastoma cells, on the other hand, had only 17% extracellular and 13% membrane-associated. The expression level trends for all genes above the 10-fold cutoff were as observed for the "in common" genes; HFF-2 genes had returned to monolayer expression levels after 7 days of recovery whereas the T98G line was still exhibiting expression values much different from that of the monolayer control. Keywords = human foreskin fibroblasts, desiccation, rehydration, monolayer, spheroid, stabilization, ambient temperature Keywords: other
In our lab we detected focal genomic amplification of PDE1C in 90% of short term GBM cultures. Knocking down of PDE1C was associated with compromised capacity opf these cultures to proliferate migrate and invade. Therefore we carried out affymetric whole genome expression analysis to identify the down stream gene effectors of this function effects.
We investigated the ability of HDAC inhibitors (HDACi) to target CML stem cells. Treatment with HDACi combined with IM effectively induced apoptosis in quiescent CML progenitors resistant to elimination by IM alone and eliminated CML stem cells capable of engrafting immunodeficient mice. In vivo administration of HDACi with IM markedly diminished LSC in a transgenic mouse model of CML. The interaction of IM and HDACi inhibited genes regulating hematopoietic stem cell maintenance and survival. HDACi treatment represents a novel and effective strategy to target LSC in CML patients receiving tyrosine kinase inhibitors.
There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in DFCI-1 medium retain a fraction with progenitor cell properties. These cells co-express basal, luminal and stem/progenitor cell markers. Clonal derivatives of progenitors co-expressing these markers fall into two distinct types: K5+/K19- (Type I) and K5+/K19+ (Type II). We show that both types of progenitor cells have self-renewal and differentiation ability. Through microarray analysis, we want to identify genes and pathways linked to human mammary epithelial stem/progenitor cell self-renewal and differentiation.
Microarray based mRNA profiling was used to identify the mechanism of action for the small molecule b-AP15.
Nematode derived substances are known to down regulate host immune responses in order to survive in the human host. Brugia malayi is a parasitic nematode responsible for long lasting and disabling infection known as lymphatic filariasis in humans. The therapeutic benefit of a controlled parasitic nematode infection on the course of inflammatory bowel disease (IBD) has been demonstrated in both animal and human models. However the inability of individual purified nematode proteins to recreate this beneficial effect has limited the application of component immunotherapy to human disease. This experiment addresses the hypothesis that the genes regulated by IL8 and recombinant Brugia malayi AsnRS (rBmAsnRS) are different even though it is known that both molecules interact with IL-8 receptors. Furthermore we theorize that the signal transduction pathways activated by IL-8 and rBmAsnRS are different because it is known that the extracellular G protein loops utilized by IL-8 and rBmAsnRS to activate IL8 receptors, are different. These results obtained with a single recombinant nematode protein, rBmAsnRS, share immunological features with those observed in a whole nematode infection and include desirable features for treatment of idiopathic inflammatory diseases, such as IBD.
Peripheral T-cell lymphoma unspecified (PTCL/U) the most common form of PTCL, displays heterogeneous morphology and phenotype, poor response to treatment, and dismal prognosis. We demonstrate that PTCL/U shows a gene expression profile clearly distinct from that of normal T-cells. Comparison with the profiles of purified T-cell subpopulations [CD4+, CD8+, resting (HLA-DR-), and activated (HLA-DR+)] reveals that PTCLs/U are most closely related to activated peripheral T-lymphocytes, either CD4+ or CD8+. Interestingly, the global gene expression profile cannot be surrogated by routine CD4/CD8 immunohistochemistry. When compared with normal T-cells, PTCLs/U display deregulation of functional programs often involved in tumorigenesis (e.g. apoptosis, proliferation, cell adhesion, and matrix remodeling). Products of deregulated genes can be detected in PTCLs/U by immunohistochemistry with an ectopic, paraphysiologic or stromal location. Among others, PTCLs/U aberrantly express PDGFRA, a tyrosine-kinase receptor, whose deregulation is often related to a malignant phenotype. Notably, both phosphorylation of PDGFRA and sensitivity of cultured PTCL cells to imatinib (as well as to an inhibitor of histone-deacetylase) are found. These results, which might be extended to other rarer PTCL categories, are provided with implications for tumor pathogenesis and clinical management. Keywords: Molecular pathogenesis, molecular classification
We used the microarray data to analyze host cells response on A549 cells infected with A/Duck/Malaysia/01 (H9N2)
The origin and function of human double negative (DN) TCR-alpha/beta T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. Here we provide evidence that human TCR-alpha/beta CD4- CD8- DN T cells derive exclusively from activated CD8+ T cells. Freshly isolated TCR-alpha/beta DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of pro-inflammatory mediators that includes IL-1, IL-17, IFN-gama, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity.
The goal of this study is to define a gene expression signature unique to DS-AMKL (acute megakaryoblastic leukemia or FAB M7 leukemia). A similar study was done previously but using unfractionated patient leukemic samples. In this study, we sorted megakaryocytic leukemia blasts from patients and then compared their gene expression signatures to those from similarly sorted blasts from patients with non-DS AMKL. This allowed us to identify a gene expression signature more unique to DS-AMKL samples.
Tazarotene-induced gene 1 (TIG1) also named as retinoic acid receptor responder 1 (RARRES1), is a retinoid inducible type II tumor suppressor gene; the TIG1B isoform inhibits growth and invasion of cancer cells. Expression of TIG1B is frequently downregulated in various cancer tissues; however, the expression and activities of the TIG1A isoform has yet to be analyzed. This study investigated the effects of TIG1A and TIG1B isoforms on gene expression profiles of colon cancer cells. TIG1A, TIG1B and control stable clones derived from HCT116 colon cells were established using the GeneSwitch system. TIG1 isoform expression was induced upon 5 micro Molar of mifepristone (MFP) treatment for 24 hr. Biological triplicate samples were prepared and gene expression profiles were determined by microarray using human genome HGU133 plus 2 array (Affymatrix). Upon induction of TIG1A and TIG1B expression for 24 hr, a total of 129 and 55 genes were significantly altered, respectively. Of the genes analyzed, 23 and 6 genes were up- and downregulated, respectively in both TIG1A and TIG1B expressing cells.
EVI1 is one of the famous poor prognostic markers for a chemotherapy-resistant acute myeloid leukemia (AML). To identify molecular targets on the surface of leukemia cells with EVI1high expression we compared the gene expression profiles of several AML cell lines by DNA microarray
Relatively brief bouts of exercise alter gene expression in peripheral blood mononuclear cells (PBMCs) but whether or not exercise changes gene expression in circulating neutrophils (whose numbers, like PBMCs, increase) is not known. We hypothesized that exercise would activate neutrophil genes involved in apoptosis, inflammation, and cell growth and repair, since these functions in leukocytes are known to be influenced by exercise. Blood was sampled before and immediately after 30-min of constant, heavy (about 80% peak O2 uptake) cycle-ergometer exercise in 12 healthy men (19-29 yr old) of average fitness. Neutrophils were isolated using density gradients; RNA was hybridized to Affymetrix U133+2 Genechip arrays. Using FDR<0.05 with 95% confidence a total of 526 genes were differentially expressed between before and after exercise. 316 genes had higher expression after exercise. The Jak/STAT pathway, known to inhibit apoptosis, was significantly activated (EASE score, p<0.005), but 14 genes were altered in a way likely to accelerate apoptosis as well. Similarly, both proinflammatory (e.g., IL32, TNFSF8 and CCR5) and anti-inflammatory (e.g., ANXA1) were affected. Growth and repair genes like AREG and FGF2 receptor genes (involved in angiogenesis) were also activated. Finally, a number of neutrophil genes known to be involved in pathological conditions like asthma and arthritis were altered by exercise, suggesting novel links between physical activity and disease or its prevention. In summary, brief heavy exercise leads to a previously unknown substantial and significant alteration in neutrophil gene expression. Keywords: stress response
Exposure to polychlorobiphenyls (PCBs) is known to cause serious health effects in human but the gene expression profiles leading to development of different diseases and disorders are not fully understood. The knowledge of global gene expression will help us to develop early disease or disorder biomarkers for PCB-induced health effects. We used microarrays to detail the global gene expression profile underlying the effects of high PCB exposure to Slovak 45-month-old children leading to identification of distinct classes of up-regulated and down-regulated genes and cellular processes.
DZIP (DAZ-Interacting Protein) containing a C2H2 zinc-finger domain is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; DZIP colocalizes with DAZ and/or DAZL proteins in these tissues. DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both embryonic stem cells and germ cells. To identify mRNAs associated with human DZIP1 protein in HeLa cells we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip).DZIP1 ribonucleoprotein (RNP) complexes were performed with 2 µg of anti-DZIP1 antibody (rabbit polyclonal, Santa Cruz Biotechnology, CA, USA) bound to protein A-agarose beads (Sigma, Deisenhofen, Germany). HeLa cells were lysed in polysome lysis buffer (Tris-HCl pH 7,4 15mM, MgCl2 15 mM, NaCl 0,3 M, 1% Triton X-100, 1 mM DTT, 100 U / ml RNase Out, PMSF 1mM and E64 10uM) for one hour at 4°C. Beads were washed, then buffer and cell lysate were added, and the reaction mixtures were tumbled for 2 hours at 4°C. After this incubation, the beads were thoroughly washed again with polysome lysis buffer and then either RNA extracted for microarray and RT-PCR experiments using the RNeasy mini kit (Qiagen). To control for non-specifically enriched RNAs, identical IPs were performed with beads precoated with rabbit IgG as a negative control. RNA was processed for hybridization with GeneChip 3? IVT Express (Affymetrix - Santa Clara, USA), according to the manufacturers instruction. Briefly, cDNA was synthesized from immunoprecipitated RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. After purification and fragmentation, cRNA was hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were preformed on an Affymetrix GeneChip® Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip® Scanner 3000. Scanned arrays were normalized using GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Differentially enriched RNAs were found after performing One-way ANOVA analysis comparing immunoprecipitated samples against control samples. Final lists of genes were obtained by filtering the data from the statistical results according to fold enrichment more than 2 and false discovery rate (FDR 5%).
Background: The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the MELAS syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. Objective: To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Methods: Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Results and Interpretation: Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM) but also detected the hypoxia-inducible factor 1α (HIF-1α)/HIF-1β complex, nuclear factor Y (NF-Y) and CREB-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.
The present study has focused on the identification of the differences between expression patterns of kinin-dependent genes in endometrial cancer
Although practiced clinically for more than 40 years the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy. LGC006, a less potent SR1 analog, was also examined. KEYWORDS: two compounds, multiple doses, one time point
Hyperthermia is widely used to treat patients with various cancers. The 42.5˚C is well known as inflection point of hyperthermia and generally up to 42˚C of hyperthermia is used in clinical case to combine with other therapy. Here the effects of heat stress at 42 or 44˚C for 90 min on the gene expression in HSC-3 human oral squamous carcinoma cells were investigated using an Affymetrix GeneChip system. The cells were treated with heat stress (42 or 44°C for 90 min) and followed by incubation for 0, 6, or 12 h at 37°C. The percentage of cell death was 5.0 ± 1.5 (mean ± SD) at 42°C for 12 h and 17.4 ± 0.6 at 44°C for 12 h. Of approximately 47,000 probe sets analyzed, many genes that were differentially expressed by a factor 2.0 or greater were identified in the cells treated with heat stress at 42 and 44°C.
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst stage embryos but have very different biological properties and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by exogenous induction of Oct4, Klf4 and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (ERK) pathway. Forskolin, a protein kinase A pathway agonist that induces Klf4 and Klf2 expression, can transiently substitute for the requirement for ectopib transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X chromosome activation state (XaXa), a gene expression profile, and signaling pathway dependence that are highly similar to that of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of “naïve” human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans, and may open up new opportunities for patient-specific, disease-relevant research.
Differential expression in human peripheral blood monocytes between F. novicida-infected and uninfected and between Francisella tularensis tularensis isolate Schu S4 and uninfected. The goal was to examine genomewide transcriptional reponses to these two strains, and identify differentially-regulated genes that may help explain the virulence of Schu S4. Keywords: Immune Response, Human Monocytes, Bacteria, Francisella
To compare the transcriptome profiles of the two principal histological variants of malignant germ cell tumor that occur in childhood Keywords: Disease state analysis
SIRT6 has been implicated in a range of biological processes including inflammation aging and the control of metabolism. Hence inhibitors or activators of SIRT6 have the potential to be therapeutics for a number of indications. Genome wide expression studies were used to investigate the effect of overexpression of SIRT6 and mutant SIRT6 on a wide range of NFκB dependent gene expression
Expression data from DHT stimulation vs. control in LNCaP cells
Comparison of human iPSC lines ESC and fibroblasts to determine their expression patterns. All early passage female lines profiled expressed XIST RNA which is an indicator of an inactive X chromosome. Genes on the X-chromosome were also analyzed for overall levels of gene expression compared to human fibroblasts.
To identify genes associated with lung cancer progression we examined gene expression profiles of tumor cells from 20 patients with primary, untreated non-small cell lung cancer (10 adenocarcinomas (AC) and 10 squamous cell carcinomas (SCC)) in comparison to lung tissue of 23 patients with stage IIIB or stage IV non-small cell lung cancer (15 AC and 8 SCC). Bronchoscopical biopsies from patient with recurrent lung tumor were taken after initial treatment. Cancer cells were isolated using laser capture microdissection in order to obtain pure samples of tumor cells. For expression analysis, microarrays covering 8793 defined genes (Human HG Focus Array, Affymetrix) were used. Array data were normalized and analysed for significant differences using variance stabilizing transformation (VSN) and significance analysis of microarrays (SAM), respectively. Genes were considered to be up- or down-regulated when the ratio between primary and recurrent tumor samples were at least 1.5-fold differentially expressed with an estimated false discovery rate: < 5%. Based on differentially expressed genes, primary cancer samples could be separated from recurrent tumor samples. We identified 115 and 124 significantly regulates genes in AC and SCC, respectively. For example, in recurrent AC we found increased expression of genes related to the wingless (FZD6, RYK, MYC) and calcium (CALM1, ATB2B1, S100A2) signalling pathways which might play a role in metastasis of tumor cells. Other differentially expressed genes were related to cell cycle (CCND1, CDK2), transcription factors (TTF1, TAF2, YY1), nuclear mRNA splicing and mRNA processing (SFRS1, HNRPL), protein-nucleus import (NUTF2, KPNB1, NUP50) and chromatin modification (HIST1H4C, SMARCC1). In SCC, we found an increased expression of CTNNB1, an important mediator in wingless signalling pathway. Among the down-regulated genes in SCC, the utmost fraction belonged to genes coding for ubiquitin mediated proteolysis (UCHL1, PSMA3, COPS6) and ribosomal proteins (RPS26, RPL7A, RPS15). Other down regulated genes were related to transcription factors (TCEA2, TAF10), nuclear mRNA splicing and mRNA processing (SNRPD2, HNRPM). In conclusion, a distinct pattern of gene expression is found during the progression from primary carcinoma to recurrent NSCLC. Our microarray-based expression profiling revealed interesting novel candidate genes and pathways that may contribute to lung cancer progression. Keywords: Lung cancer, NSCLC, gene expression, progression, Wnt signalling pathway
For the microarray experiments MV4-11 and MOLM-14 cells were treated with DMSO control, ABT-869 3 nM, SAHA 6 uM and combination therapy for 24 hours. Cells were then washed in PBS and high-quality total RNA was extracted RNeasy Midi Kit, according to the manufacturer’s instruction (Qiagen, Valencia, USA). RNA quantity, quality, and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA, USA). Gene expression profiling was performed using Affymetric U133plus2.0 gene chip (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol.
Human lymphoblastoid cell lines (EBV-immortalised B cells LcL) obtained from subjects of different age (young 28-40 years, centenarians >95 years) were analysed for gene expression at basal culture conditions and after 48 hours of serum starvation. Lymphoid B cells from centenarians were more resistant to apoptosis induction and displayed a more developed lysosomal compartment, the most critical component of phagic machinery. In addition, cells from centenarians were capable of engulfing and digesting other cells, i.e. their siblings (even entire cells). This behavior was improved by nutrient deprivation, but strikingly, it was unaffected by the autophagy-modulating drugs rapamycin, an autophagy inducer, and 3-methyladenine, an autophagy inhibitor.
Organophosphorus compounds may induce neurotoxicity through mechanisms other than the cholinergic pathway which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. We used microarrays to study gene expression changes in human neural cells after exposure to VX, and identified pathways underlying these changes.
Pharmacogenomic identification of targets for adjuvant therapy with the topoisomerase poison camptothecin. The response of tumor cells to the unusual form of DNA damage caused by topoisomerase poisons such as camptothecin (CPT) is poorly understood and knowledge regarding which drugs can be effectively combined with CPT is lacking. To better understand the response of tumor cells to CPT and to identify potential targets for adjuvant therapy, we examined global changes in mRNA abundance in HeLa cells after CPT treatment using Affymetrix U133A GeneChips, which include all annotated human genes (22,283 probe sets). Statistical analysis of the data using a Bayesian/Cyber t test and a modified Benjamini and Hochberg correction for multiple hypotheses testing identified 188 probe sets that are induced and 495 that are repressed 8 h after CPT treatment at a False Discovery Rate of <0.05 and a minimum 3-fold change. This pharmacogenomic approach led us to identify two pathways that are CPT induced: (a) the epidermal growth factor receptor; and (b) nuclear factor-kappaB-regulated antiapoptotic factors. Experiments using HeLa cells in our lab and prior animal model studies performed elsewhere confirm that inhibitors of these respective pathways super-additively enhance CPT's cytotoxicity, suggesting their potential as targets for adjuvant therapy with CPT. Cancer Res. 2004 Mar 15;64(6):2096-104 Keywords = HeLa Keywords = Camptothecin Keywords: ordered
Although EWS/FLI-1 fusion protein is responsible for most Ewing’s sarcoma family tumors (ESFT) the function of native EWS remains largely unknown. Here, we first showed that EWS repressed protein expression in a tethering assay. mRNAs bound to EWS were determined by RNA-immunoprecipitation Chip assay, and one of them, proline-rich Akt substrate of 40 kDa (PRAS40) mRNA, directly interacted with EWS. The inhibitor of AKT, API-2, repressed ESFT cell proliferation. We demonstrate that EWS negatively regulated PRAS40 protein expression through binding to PRAS40 3’UTR. Furthermore, PRAS40 knockdown inhibited the proliferation and metastatic potential of ESFT cells.
Characterization of the underlying genetic defects in patients with a rare and peculiar phenotype is challenging. Here we have utilized whole genome expression profiling and identified a homozygous germline mutation in the DDB2 gene in a patient with several facial tumors. The feasibility of using blood derived RNA, diminishing costs of the technology, and the limited number of samples needed provide this approach a powerful new tool that may substantially aid in such gene identification efforts. Keywords: disease vs control
Gene transfer vectors based on gamma-retroviruses target actively transcribing genes indicating that the cellular gene expression profile can be predictive of their integration pattern. Therefore different culture conditions leading to different transcriptional activity may translate into differences in the profile of targeted genes in cells transduced with these vectors. Recent data from two gene therapy trials for SCID-X1 conducted in France and the UK suggested that small differences between in vitro stimulation conditions could explain the disparity in the frequency of common integrations sites observed in the two studies. We set out to compare the transcriptional activity of human CD34+ bone marrow-derived cells cultured under the French (S1) or British (S2) culture conditions.
The prognosis of pancreatic cancer is still very poor how to detect pancreatic cancer from high-risk group in an early stage is essential for improving its long-time survival. Therefore, the purpose of this study was to explore specific biomarkers that can differentiate pancreatic cancer-associated diabetes from type-2 diabetes for the early detection of pancreatic cancer. In the current study, we used global gene transcription analysis with affymetrix gene chip to identify genes specifically expressed in pancreatic cancer-associated diabetes mellitus from peripheral blood samples in stead of from tissue samples.
Molecular profiles of dystophin-deficient patients and normal human skeletal muscles on Affymetrix HG-U95A arrays Keywords = DMD Keywords = Duchenne muscular dystrophy Keywords = dystrophin Keywords = Affymetrix U95A array Keywords = skeletal muscle Keywords = gene expression profiles Keywords: other
Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous illness with a variable clinical course. Loss of chromosomal material on chromosome 13 at cytoband 13q14 is the most frequent genetic abnormality in CLL but the molecular aberrations underlying del13q14 in CLL remain incompletely characterized. We analyzed 171 CLL cases for LOH and sub-chromosomal copy loss on chromosome 13 in DNA from FACS-sorted CD19+ cells and paired buccal cells using the Affymetrix XbaI 50K SNP-array platform. The resulting high-resolution genomic maps, together with array-based measurements of expression levels of RNA in CLL cases with and without del13q14 and Q-PCR-based expression analysis of selected genes support the following conclusions: i) del13q14 is heterogeneous and composed of multiple subtypes with deletion of Rb or the miR15a/16 loci serving as anatomic landmarks, respectively ii) del13q14 type Ia deletions are relatively uniform in length and extend from breakpoints close to the miR15a/16 cluster to a newly identified telomeric breakpoint cluster at ~50.2-50.5 Mb physical position iii) LATS2 RNA levels are ~2.6-2.8-fold lower in cases with del13q14 type I that do not delete Rb as opposed to all other CLL cases and iv) ~15% of CLL cases display marked reductions in miR15a/16 expression often but not invariably associated with bi-allelic miR15a/16 loss. This data should aid future investigations into biological differences imparted on CLL by different del13q14 subtypes including investigations into LATS2 as one of the genes found deregulated as part of del13q14. Keywords: Chronic lymphocytic leukemia, CLL, gene expression comparison, del13q14, del13q
To explore the psoriasis phenotype and pathways involved in psoriasis we characterized gene expression in lesional and non-lesional skin from psoriasis patients.
We used microarrays to examine the impact of AF1q/MLLT11 on the gene expression profile of CD34+CD45RA-Lin- and CD34+CD45RA+Lin- HPCs isolated from umbilical cord blood CD34+CD45RA-Lin- and CD34+CD45RA+Lin- HPCs correspond respectively to early multipotent and lympho-granulo-macrophagic precursors. A2M is a nuclear mutant-derivative of AF1q/MLLT11
Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where pluripotent stem cells gradually limit their differentiation potential generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization. The genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions. Keywords: cell differentiation
We here report transcriptome profiles of human embryos at six successive developmental stages (i.e. Carnegie Stages 9 to 14), representing the first comprehensive gene expression database of early human organogenesis. Through a series of data mining and comparisons with the transcriptome during mouse embryogenesis and the multi-layered genomic data in human embryonic stem cells, we revealed that development potential during early human organogenesis is orchestrated by two dominant categories of genes. Specifically, most gradually induced genes are largely differentiation-related and indicative of diverse organ formation, whereas those gradually repressed are involved in both stemness- and differentiation-relevant aspects of the developmental potential, which may be important for the initiation of organogenesis. Further through integrative mining we uncovered a molecular network (including a stemness-relevant module and a differentiation-relevant module) that may provide a framework for the regulation of early human organogenesis. Preliminary analysis of published data showed that the network could serve to evaluate various in vitro differentiation models. Our results make a significant step towards understanding of human embryogenesis at a molecular level and suggest that developmental potentials of early embryonic cells are under control of shared regulatory events.
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program we employed a genome-wide RNAi screen for Myc-synthetic-lethal (MySL) genes and uncovered a role for the SUMO-activating-enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc-switchers (SMS genes) governs mitotic spindle function and is required to support the Myc oncogenic program.
Comparisons among breast cancer metastases at different organs revealed distinct microenvironments as characterized by cytokine content. Such microenvironment distinction might be important to dictate how the cancer cells adapt to survival before they successfully colonize. Keywords: Disease state analyses
While identification of genes mutated in high penetrance tumor predisposition syndromes has been a success story much less progress has been made in characterizing the genetic basis of low penetrance tumor susceptibility. Combining recently introduced chip-based technologies with traditional genealogy work we have identified inactivating germline mutations in patients with pituitary adenoma predisposition (PAP). The experiment consisted of a collection of blood samples from identified families where PAP was observed and analysis of gene expression data used together with SNP genoptyping and linkage analysis. The findings were further studied using direct screening and other supporting methods. Keywords: affected and obligatory carriers vs controls
Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory lung disease. The abnormal inflammatory response of the lung mainly to cigarette smoke, causes multiple cellular and structural changes affecting all of its compartments, which leads to disease progression. The molecular mechanisms underlying these pathological changes are still not fully understood The aim of this study was to identify genes and molecular pathways potentially involved in the pathogenesis of COPD
We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4 T cells in peripheral blood was observed while gut mucosal CD4 T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients.
Inflammatory mechanisms and immune cells are involved in acute coronary syndromes (ACS) and may lead to acute plaque rupture. However the local expression of the different genes potentially involved is largely unknown. We therefore performed an Affymetrix analysis of genes expressed in white blood cells obtained from an occluding coronary thrombus or peripheral blood of patients with ST-elevation myocardial infarction.
Micro RNAs (miRNAs) miR-130a miR-203 and miR-205 are jointly downregulated in prostate cancer and act as repressors of AR-signaling. MiRNAs are small non-coding RNAs that regulate the expression of specific mRNA targets mainly by translational repression, mRNA deadenylation or cleavage. Reconstitution of these lost miRNAs in the LNCaP PCa cell line cause morphology changes, growth arrest, and apoptosis, increasing when the miRNAs were co-expressed. This series identifies direct targets of miR-130a, miR-203, and miR-205 by AGO2-RNA co-immunoprecipitation as described by (Beitzinger et al. 2007) upon miRNA reconstitution in LNCaP cells and analyzing AGO2-bound mRNAs using Affymetrix Genechips. Relative levels of AGO2 bound versus total RNA expression were compared between miRNA reconstituted and miR-scr transfected samples.
Expression profiles of aggressive versus non-aggressive ovarian breast, melanoma, and prostate cancer cell lines
Recently the p53-miR-34a network was identified to play an important role in tumorigenesis. As in acute myeloid leukemia with complex karyotype (CK-AML) TP53 alterations are the most common known molecular lesion, we further analyzed the p53-miR-34a axis in CK-AML with known TP53 status. Clinically, low miR-34a expression and TP53 alterations predicted for chemotherapy resistance and inferior outcome. Notably, in TP53unaltered CK-AML high miR-34a expression predicted for inferior overall survival (OS), whereas in TP53biallelic altered CK-AML high miR-34a expression pointed to better OS. To further investigate miR-34a-associated gene expression patterns, we analyzed distinct subgroups defined by TP53 alteration and miR-34a expression status. Exemplary samples from TP53unaltered (n=6) and TP53biallelic altered (n=6) CK-AML characterized by either high (CK+/miR-34ahigh expression, above median miR-34a expression of the entire cohort), or low (CK+/miR-34alow expression, below median miR-34a expression of the entire cohort) miR-34a expression (n=3 in each group), were analyzed. This molecular profiling linked impaired p53 to decreased miR-34a expression but also identified p53-independent miR-34a induction mechanisms, as shown in TP53biallelic altered cell lines treated with 15-deoxy-∆12,14-prostaglandin (PGJ2). An improved understanding of this mechanism might provide novel therapeutic options to restore miR-34a function and thereby induce cell cycle arrest and apoptosis in TP53altered CK-AML.
The effects of the CDK inhibitor Flavopiridol on the A2780 human adenocarcinoma ovary cell line were analysed by gene expression profiling.
This SuperSeries is composed of the SubSeries listed below.
Skeletal muscle mitochondrial dysfunction is secondary to T2DM and can be improved by long-term regular exercise training Mitochondrial dysfunction has long been implicated to play a causative role in development of type 2 diabetes (T2DM). However a growing number of recent studies provide data that mitochondrial dysfunction is a consequence of T2DM development. The aim of our study is to clarify in further detail the causal role of mitochondrial dysfunction in T2DM by a comprehensive ex vivo analysis of mitochondrial function combined with global gene expression analysis in muscle of pre-diabetic newly diagnosed untreated T2DM subjects and long-standing insulin treated T2DM subjects compared with age- and BMI-matched controls. In addition, we assessed the impact of long-term interval exercise training on physical activity performance, mitochondrial function and glycemic control in long-standing insulin-treated T2DM subjects. Ex vivo mitochondrial density, quality and functioning was comparable between pre-diabetic subjects and matched controls, however, gene expression analysis showed a switch from carbohydrate toward lipids as energy source in pre-diabetes subjects. In contrast, long-term insulin treated T2DM subjects had slightly decreased mitochondrial density and ex vivo function. Expression of Krebs cycle and OXPHOS related genes were decreased, indicating a decreased capacity to use lipids as an energy source. The insulin-treated T2DM subjects had a lower physical activity level than pre-diabetic and normoglycemic subjects. A 52 weeks exercise training of these subjects increased submaximal oxidative efficiency, increased in vivo PCr recovery rate, as well as mildly increased in vitro mitochondrial function. Gene expression of β-oxidation, Krebs cycle and OXPHOS-related genes was increased. Our data demonstrate that mitochondrial dysfunction is rather a consequence than a causative factor in T2DM development as it was only detected in overt diabetes and not in early diabetes. Regular exercise training stabilized exogenous insulin requirement and improved mitochondrial functioning, fatty acid oxidation and general physical work load capacity in long-standing insulin-treated T2DM subjects. As such, the present study shows for the first time that long-term exercise interventions are beneficial in this group of complex diabetes patient and may prevent further metabolic deterioration.
Alterations in the presence of sperm RNAs have been identified using microarrays in teratozoospermic (abnormal morphology) or other types of infertile patients. However so far no studies had been reported on the sperm RNA content using microarrays in asthenozoospermic patients (low motility). We started the present project to with the goal to characterize the RNA expression in asthenozoospermic infertile patients as compared to normozoospermic fertile controls.
In this study we have investigated the effect of LMP2A on gene expression in normal human GC B cells using a non-viral vector based system Keywords: transfection of viral oncogene in normal human B cells
The aim of this study was to identify genes regulated by IL-12 IL-18 and IFN-alpha during early differentiation of human Th1 cells
The activation of endothelium by tumor cells is one of the main steps by tumor metastasis. The role of the blood components (platelets and leukocytes) in this process remain unclear. We used microarrays to detail the repsonse of endothelial cells to tumor cells in the presence or absence of blood components.
Gene expression analyis of primary MCL including IGHV mutated and unmutated cases Gene set analysis was perfomed in MCL samples comparing IGHV mutated cases vs. IGHV unmutated cases
DUSP1 is involved in different cellular pathways including cancer cell proliferation angiogenesis, invasion and resistance to chemotherapy. To gain insight into the cellular signaling pathways involving DUSP1 actions and the response to Cisplatin (CDDP) in non small cell lung cancer (NSCLC), we have used a double strategy that combines microarray and SiRNA technology. This strategy provided a differential expression profile of genes involved in CDDP response in NSCLC cell line regulated by DUSP1 using H460 and H460cri and a time course to CDDP. KEYWORDS: Expression profiling by array in cells with genetic modification in response to CCDP treatment
The generation of induced pluripotent stem (iPS) cells 1-4 has spawned unprecedented opportunities for investigating the molecular logic that underlies cellular pluripotency and reprogramming as well as for obtaining patient-specific cells for future clinical applications. However, both prospects are hampered by the low efficiency of the reprogramming process. Here, we show that juvenile human primary keratinocytes can be efficiently reprogrammed to pluripotency by retroviral transduction with Oct4, Sox2, Klf4 and c-Myc. Keratinocyte-derived iPS (KiPS) cells appear indistinguishable from human embryonic stem (hES) cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, as well as in vitro and in vivo differentiation potential. Notably, keratinocyte reprogramming to pluripotency is, at least, 100-fold more efficient and 2-fold faster than that of fibroblasts. This increase in reprogramming efficiency allowed us to expand the practicability of the technology and to generate KiPS cells from single plucked hairs from adult individuals.
This SuperSeries is composed of the SubSeries listed below.
We used cDNA microarray to study the gene expression profile between the tumor and non-tumor tissue samples from patients with esophageal cancer
Senescent is an irreversible form of cell cycle arrest initiated by damaged cell constituents and subsequent pro-oncogenic signaling. Replicative senescence in vitro can be considered a model for human aging. When fibroblasts are cultured under atmospheric oxygen conditions of 20% typical of normal tissue culture procedure, fibroblasts generally reach their replicative capacity at 50-60 population doublings (PDs). When fibroblasts are cultured under normal physiological oxygen conditions of 3%, PDs increase about 30% relative to atmospheric levels. Hence while oxygen is a requirement for normal aerobic respiration, it can contribute to the total amount of oxidative stress to which cells are exposed to, leading to a long-term adverse effect in vitro. Inasmuch, cultures maintained under hyperoxic and hypoxic conditions provide a convenient model system for assessing the relationship between oxygen/oxidative stress and senescence. We used microarrays to profile the changes in global gene expression during aging and senescence of Imr90 cells under growth oxygen conditions of 3% and 20%.
Microarrays were used to determine relative global gene expression changes upon introduction of EMT-inducing or control vectors.
Th17 cells are believed to be a critical cell population for driving autoimmune diseases. However environmental factors that are directly related to the development of Th17 cells are largely unknown. High-salt (NaCl) concentrations enhance Th17 differentiation of human naive CD4+ T cells in vitro. The aim of the study was to analyse the changes in gene expression induced by high-salt conditions during Th17 differentiation.
Gene expressions relate to the pathogenesis of periodontitis and have a crucial role in local tissue destruction and susceptibility to the disease. The aims of the present study were to explore comprehensive gene expressions/transcriptomes in periodontitis-affected gingival tissues and to identify specific biological processes. The purpose of the present study was 1) to compare comprehensive gene expression/transcriptomes of periodontitis-affected gingival tissues with those of healthy tissues by using microarray and data mining technologies, and 2) to analyze significantly differentially expressed genes which belong to pathological pathways in periodontitis by qRT-PCR.
Different human adipose tissue depots may have functional differences. Subcutaneous human adipose tissue has been extensively studied but less is known about other depots. Perithyroid (PT) adipose tissue contains not only white adipocytes but also brown adipocytes. The aim of this study was to compare the expression of brown adipocyte containing perithyroid adipose tissue with s.c. adipose tissue.role in the development of obesity. Expression profiling of adipose tissue may give insights into mechanisms contributing to obesity and obesity-related disorders.
The objective of the study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy.
Major depressive disorder (MDD) is a clinically defined entity with little understanding as to the underlying pathological substrate. Biologically MDD is characterized by disruption of neurotransmitters, especially serotonin and noradrenaline, which are the main targets of antidepressants. We previously demonstrated significant reduction of glial cell number in the cingulate and dorsolateral prefrontal cortical regions. Unfortunately, individuals living with HIV still have very high rates of MDD, despite the fact that mortality rates have fallen sharply with effective antiretroviral treatment. It is possible that in this treatment era, living with chronic HIV infection may result in long-term neuropathological changes that predispose to MDD. For example, it is known that HIV is associated with a range of inflammatory pathologies, neuronal loss, and dendrite-synaptic damage. In HIV, these neurodegenerative changes have been linked to neurocognitive impairments, however it is also possible that these changes potentiate MDD. In the current study, we sought to determine whether there are changes in gene expression in the MDD brain in the frontal cortex in HIV-context. We identify a large number of genes dysregulated at p<0.05 significance.
NDRG1 functions as a metastasis suppressor in pancreatic and other cancers. To determine the molecular function of NDRG1 in MIAPaCa-2 pancreatic cancer cells we performed a whole genome gene array analysis on these cells stably transfected with NDRG1 when compared to empty vector-transfected controls. The differentially expressed genes indetified in this microarray may represent potential molecular targets of NDRG1 in pancreatic cancer.
Gene expression profiling of normal hematopoietic cell subpopulations RNA extracted from flow sorted normal hematopietic cells were hybridized onto Affymetrix U133 Plus 2.0 arrays.
This SuperSeries is composed of the SubSeries listed below.
tumor microenviroment facilitates metastatic spread by eliciting reversible changes in the phenotypes of cancer cells Keywords: expression analysis of breast tumor samples
Aberrant co-expression of LMO2 and TAL1 is regularly found in T cell acute lymphoblastic leukemia (T-ALL). Here we describe a xenotransplant model for primary T-ALL cells derived from a patient who developed LMO2/TAL1 associated T-ALL after gene therapy for X-SCID (gamma c deficiency) due to insertional activation and a SIL-TAL1 fusion as an additional independent event. We identified a protein complex containing LMO2 TAL1 and E47 in the patient’s T-ALL cells. This complex functions as a transcription regulation complex in undifferentiated hematopoietic cells. However, its role in T-ALL is not fully understood. By comparative gene expression profiling we identified retinaldehyde dehydrogenase (RALDH2) as one of the genes which was highly up regulated in the primary T-ALL cells; the gene product was N-terminally truncated (RALDH2-T), but enzymatically active. To investigate the interference of LMO2 in RALDH2-T expression as well as the role of RALDH2-T in T-ALL development, LMO1 (analogue of LMO2) was down regulated by RNA interference in the T-ALL cell line Jurkat which led to a decrease of RALDH2-T expression and cell viability, indicating that positive regulation of RALDH2-T through an LMO1 or 2 containing transcription regulation complex might be essential for maintaining the T-ALL phenotype. Keywords: Primary human T-ALL cells and CD8 cells of normal donors
Our compound (PRIMA) selectively kills tumor cells expressing mutant p53. Here in our study we decided to investigate the impact of our compound on the transcriptome of Saos2 cells lacking p53.
The goal of this study is to identify genes that are differentially expressed by E6 in oral epithelial cells that are stably expressing Bmi-1. Keywords: oncogenesis.
Aplidin (plitidepsin) is a novel marine-derived antitumor agent presently undergoing phase II clinical trials in hematological malignancies and solid tumors. Lack of bone marrow toxicity has encouraged further development of this drug for treatment of leukemia and lymphoma. Multiple signaling pathways have been shown to be involved in Aplidin-induced apoptosis and cell cycle arrest in G1 and G2 phase. However the exact mechanism(s) of Aplidin action remains to be elucidated. Here we demonstrate that mitochondria-associated or -localized processes are the potential cellular targets of Aplidin. Whole genome gene-expression profiling (GEP) revealed that fatty acid metabolism, sterol biosynthesis and energy metabolism, including the tricarboxylic acid cycle and ATP synthesis are affected by Aplidin treatment. Moreover, mutant MOLT-4, human leukemia cells lacking functional mitochondria, were found to be resistant to Aplidin. Cytosine arabinoside (araC), which also generates oxidative stress but does not affect the ATP pool, showed synergism with Aplidin in our leukemia and lymphoma models in vitro and in vivo. These studies provide new insights into the mechanism of action of Aplidin. The efficacy of the combination of Aplidin and araC is currently being evaluated in clinical phase I/II program for the treatment of patients with relapsed leukemia and high-grade lymphoma. Keywords: Aplidin, araC, drug combination, hematological malignancies
Reduction in the cellular levels of the cyclin kinase inhibitor p27kip1 are frequently found in many human cancers and correlate directly with patient prognosis. Specifically ubiquitin dependent proteasomal turnover has been shown to cause reduced p27 expression in many human cancers. We recently demonstated that expression of a stabilized version of p27kip1 (p27kip1T187A) in a genetically modified mouse significantly reduced the number of intestinal adenomatous polyps which progressed to invasive carcinomas. Based on this work we set out to identify compounds which lead to a re-expression of p27 in cancer tissues. In this work we identify Argyrin A a compound derived from myxobacterium archangium gephyra as a potent inducer of p27kip1 expression. Argyrin A induces apoptosis in human colon cancer xenografts and tumor vasculature in vivo leading to a profound reduction in tumor size at well tolerated levels. Argyrin A functions are strictly dependent on the expression of p27kip1 as neither tumor cells nor endothelial cells which do not express p27kip1 respond to this compound. Surprisingly the molecular mechanism by which Argyrin A exerts its p27 dependent biological function is through a potent inhibition of the 20S proteasome. Keywords: protasome inhibitor time course, MCF7 cells, genome wide gene expression response, siRNA
Splenic marginal zone lymphoma (SMZL) is an indolent B-cell lymphoproliferative disorder characterised by 7q32 deletion but the target genes of this deletion remain unknown. In order to elucidate the genetic target of this deletion, we performed an integrative analysis of the genetic, epigenetic, transcriptomic and miRNomic data. High resolution array comparative genomic hybridization of 56 cases of SMZL delineated a minimally deleted region (2.8Mb) at 7q32, but showed no evidence of any cryptic homozygous deletion or recurrent breakpoint in this region. Integrative transcriptomic analysis confirmed significant under-expression of a number of genes in this region in cases of SMZL with deletion, several of which showed hypermethylation. In addition, a cluster of 8 miRNA in this region showed under-expression in cases with the deletion, and three (miR-182/96/183) were also significantly under-expressed (P <0.05) in SMZL relative to other lymphomas. Genomic sequencing of these miRNA and IRF5, a strong candidate gene, did not show any evidence of somatic mutation in SMZL.
Angiogenesis the formation of new capillaries by sprouting from preexisting vessels, is mainly induced by VEGF-A. To identify genes which are induced by VEGF-A in endothelial cells, HUVEC were starved and induced by VEGF-A165 for 30, 60 and 150min. RNA of induced and uninduced cells was isolated and subjected to microarray analysis using Affymetrix microarray.
Gene expression was measured on the Affymetrix platform in iPrEC cell line transfected with wild-type USP2A mutant USP2A, or empty vector.
Samples collected from human subjects in clinical trials possess a level of complexity arising from multiple cell types, that can obfuscate the analysis of data derived from them. Blood, for example, contains many different cell types that are derived from a distinct lineage and carry out a different immunological purpose. Failure to identify, quantify, and incorporate sources of heterogeneity into an analysis can have widespread and detrimental effects on subsequent statistical studies. We used microarrays to detail a statistical approach to model expression from a mixed cell population as the weighted average of expression from different cell types. Consequently, we can accurately and efficiently estimate the abundance of various cell populations. Favoring computation over manual purification has its advantages, such as measuring responses of multiple cell types simultaneously, keeping samples intact, and identifying biologically relevant differentially expressed genes.
The role of acid and bile salts in the pathogenesis of esophageal carcinoma arising from Barrett's metaplasia has been well established. Cell proliferation of Barrett's epithelium in response to pulsatile acid exposure has since been confirmed in vivo using endoscopy specimens. Histone deactylases (HDACs) modulate nucleosomal packaging of DNA thereby influencing gene transcription and multiple cancer-associated processes. Thus, we conducted microarray analysis to assess the ability of HDAC-42 to modulate key acid-induced changes as well as to impact other genes altered as the normal esophageal epithelium progresses along the metaplasia-dysplasia-esophageal adenocarcinoma continuum. Keywords: acid-pulsed cells pretreated with HDAC inhibitor or vehicle
In this study we tested the hypothesis that pregnancy a physiologic condition of transient, organ system-based immune evasion, can integrate known and identify new mechanisms of immune modulation pertinent to metastatic melanoma.
Expression data from microdissected glomeruli to examine the role of hypoxia in glomerulosclerosis of human Nephrosclerosis (NSC).
Adenoid cystic carcinoma (ACC) is one of the most common malignancies that arise in the salivary glands with an incidence of 4.5 per 1,000,000. It can also arise in glandular tissue closely related to salivary glands in the lacrimal gland, nasal passages and tracheobronchial tree, as well as in glands of the breast and vulva. At all of these sites, it is characterized by a distinctive histology of basaloid epithelial cells arranged in cribriform or tubular patterns, usually demonstrating abundant hyaline extracellular matrix secretion and some degree of myoepithelial differentiation. ACC is generally a slow-growing tumor characterized by a protracted clinical course, usually well over 5 years in duration, marked by regional recurrence, distant metastasis and/or spread along peripheral nerves. A recurrent chromosomal translocation, t(6;9)(q23;p21), has been identified in ACC, and recently it has been discovered that in a majority of ACC the MYB gene on chromosome 6 is fused to the 3’ terminus of the NFIB gene on chromosome 9, creating a fusion gene product resulting in increased MYB-related transcriptional activation. Recently it has been determined that most cell lines with attribution of ACC derivation are either contaminants of other cell lines or do not have the characteristic MYB-NFIB translocation. Also, there are no animal models of this histologically and genetically defined tumor type. To address the paucity of experimental and pre-clinical models systems of ACC, we have for several years been establishing xenograft tumor lines from clinical samples of ACC. We describe our experience with these models and their characterization here.
Clinical application of induced pluripotent stem (iPS) cells is limited by low efficiency of iPS derivation and protocols that permanently modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-mutagenic strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem (RiPS) cells into terminally differentiated myogenic cells. Our method represents a safe, efficient strategy for somatic cell reprogramming and directing cell fates that has broad applicability for basic research, disease modeling and regenerative medicine.
Understanding the systemic regulation of normal prostate gene expression by cholesterol diet is critical for deciphering the mechanisms responsible for the transition from healthy to pathogenic prostate conditions. To understand mechanism under cholesterol effect on prostate we performed microarray analysis using LNCaP human prostate cells cultured in low cholesterol medium.
Despite advances in contemporary chemotherapeutic strategies long term survival still remains elusive for patients with metastatic colorectal cancer. A better understanding of the molecular markers of drug sensitivity to match therapy with patient is needed to improve clinical outcomes. In this study, we used in vitro drug sensitivity data from the NCI-60 cell lines together with their Affymetrix microarray data to develop a gene expression signature to predict sensitivity to oxaliplatin. In order to validate our oxaliplatin sensitivity signature, Patient-Derived Colorectal Cancer Explants (PDCCEs) were developed in NOD-SCID mice from resected human colorectal tumors. Analysis of gene expression profiles found similarities between the PDCCEs and their parental human tumors, suggesting their utility to study drug sensitivity in vivo. The oxaliplatin sensitivity signature was then validated in vivo with response data from 14 PDCCEs treated with oxaliplatin and was found to have an accuracy of 92.9% (Sensitivity=87.5%; Specificity=100%). Our findings suggest that PDCCEs can be a novel source to study drug sensitivity in colorectal cancer. Furthermore, genomic-based analysis has the potential to be incorporated into future strategies to optimize individual therapy for patients with metastatic colorectal cancer.
Kaposi’s sarcoma-associated hepesvirus (KSHV) encodes four genes with homology to human interferon regulatory factors (IRFs). One of these IRFs the viral interferon regulatory factor 3 (vIRF-3) is expressed in latently infected PEL cells and required for their continuous proliferation. Moreover, vIRF-3 is known to be involved in modulation of the type I interferon response. In order to identify cellular genes regulated by vIRF-3, cells from the PEL cell line BC-3 (KSHV positive) were transfected with either siRNAs targeted at vIRF-3 (si463 or si716s) or a negative control siRNA without homology to a known gene (siN, siNs).
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC affected human myocardium reveals fibrolipomatous replacement the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of 6 ARVC specimen derived from heart transplantation candidates and compared our findings to 6 non-failing donor hearts (NF) which could not be transplanted for technical reasons. In addition, we compared our findings to 7 hearts from patients with idiopathic dilated cardiomyopathy. From each heart left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the six sample groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, unsupervised cluster analyses revealed no distinct expression pattern in LV and RV samples from ARVC-hearts. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only about 15-16% of the genes are commonly regulated compared to NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts.
Fourteen human subjects (18-40 years of age) were studied. The control group (saline + LPS [n = 7]) received a 12 hour infusion of saline and the experimental group (cortisol + LPS [n = 7]) received a 12 hour infusion of cortisol (3 µg/kg/min). After 6 hours of saline or cortisol infusion intravenous lipopolysaccharide (2 ng/kg) was administered to all subjects. Blood samples were collected at -24 hours, 0 hours, 6 hours and 24 hours relative to LPS administration at 0 hours. Leukocytes and monocytes were isolated from these blood samples, total RNA extracted, and microarray analyses performed using Affymetrix Focus GeneChips®.
The immune mechanisms that control resistance vs. susceptibility to mycobacterial infection in humans were investigated by studying leprosy skin lesions the site where the battle between the host and the pathogen is joined. Using an integrative genomics approach, we found an inverse correlation between of IFN-beta and IFN-gamma gene expression programs at the site of disease. The Type II IFN, IFN-gamma and its downstream vitamin D-dependent antimicrobial genes were preferentially expressed in the lesions from patients with the self-healing tuberculoid form of the disease and mediated antimicrobial activity against the pathogen, Mycobacterium leprae in vitro. In contrast, the Type I IFN, IFN-beta and its downstream genes, including IL-27 and IL-10, were induced in monocytes by M. leprae in vitro, and were preferentially expressed in the lesions of disseminated and progressive lepromatous form. The IFN-gamma induced macrophage antimicrobial response was inhibited by IFN-beta/IL-10, by a mechanism involving blocking the generation of bioactive 1,25-dihyroxy vitamin D as well as inhibiting induction of antimicrobial peptides cathelicidin and DEFB4. The ability of IFN-B to inhibit the IFN-gamma induced vitamin D pathway including antimicrobial activity was reversed by neutralization of IL-10, suggesting a possible target for therapeutic intervention. Finally, a common IFN-beta and IL-10 gene signature was identified in both the skin lesions of leprosy patients and in the peripheral blood of active tuberculosis patients. Together these data suggest that the ability of IFN-beta to downregulate protective IFN-gamma responses provides one general mechanism by which some bacterial pathogens of humans evade protective host responses and contribute to pathogenesis.
To determine cardiac transcription profiles we collected myocardial samples immediately after institution of cardiopulmonary bypass from acyanotic or cyanotic Tetralogy of Fallot patients undergoing corrective surgery. The transcriptional profile of the mRNA in these samples was measured with gene array technology.
Cultured epidermal keratinocytes treated with OsM 1 4, 24 & 48hrs, and Skinethic epidermal substitutes treated 1, 4, 24, 48h & 7days, each with untreated control Keywords: Time course
Microarray deconvolution is a technique for quantifying the relative abundance of constituent cells in a mixture based on that mixture's microarray signature and the signatures of the purified constituents. Its ability to discriminate related human cells is unknown. Here we test the ability of this technique to determine the fractions of transformed cells of immune origin in mixed samples. Keywords: cell type comparison
Patterns of expressed genes examined in cryopreserved peripheral blood mononuclear cells (PBMCs) of seropositive persons electing to stop antiretroviral therapy in the AIDS Clinical Trials Group Study A 5170 were scrutinized to identify markers capable of predicting the likelihood of CD4+ T cell depletion after cessation of ART. Two cohorts were matched for clinical characteristics in a study of the effects of therapy interruption (TI) in ART-treated patients with immunological preservation. Disease progression was determined by CD4+ T cell count decline 24 weeks after TI.Gene Set Enrichment Analysis (GSEA) identified a set of genes in the Ras signaling pathway, associated with the downregulation of apoptosis, as significantly upregulated in the good outcome group at cessation of ART. Keywords: Disease state analysis
Estradiol Timecourse of MDA-MB-231ER+ cells containing a WT-ER and DBDmut-ER
Under conditions of hormonal adjuvant treatment the estrogen receptor apoprotein supports breast cancer cell cycling through the retinoic acid receptor α1 apoprotein. Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using siRNA inhibited basal proliferation by promoting cell cycle arrest. The basal expression of RARα1 the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition.
This SuperSeries is composed of the SubSeries listed below.
We recently mapped 605 chromosomal breakpoints in 61 ATL cases by spectral karyotyping and identified chromosome 14q11 as one of the most common chromosomal breakpoint regions. To map the precise location of chromosomal breakpoints at 14q11 we performed single-nucleotide polymorphism (SNP)-based comparative genomic hybridization on leukemia cells from acute-type ATL patients. The breakpoints accumulated frequently adjacent to the T cell receptor alpha/delta chain locus (TCRα/δ) with chromosomal deletions at 14q11 and a recurrent 0.9 Mb interstitial deletion was identified at a region including part of the TCRα/δ locus. Because leukemia-associated genes are frequently located near the breakpoint cluster regions, we then analyzed the gene expression profiles of ATL cells and identified N-myc downstream regulated gene 2 (NDRG2) as one of the genes that are down-regulated in ATLL cells among the 25 genes mapped to the region adjacent to the recurrently deleted regions at 14q11.
A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance we constructed Biological Association Networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX.
Background: microRNAs (miRNAs) are approximately 21 nucleotide non-coding transcripts capable of regulating gene expression. The most widely studied mechanism of regulation involves binding of the miRNA to a target mRNA usually in its 3’ untranslated region (UTR). As a result, translation of the target mRNA is inhibited and sometimes the mRNA itself can be de-stabilized. The inhibitory effects of miRNAs have been linked to many diverse cellular processes including malignant proliferation and apoptosis, development and differentiation, metabolic processes and neural plasticity. We asked whether endogenous fluctuations in a set of mRNA and miRNA profiles contain correlated changes that are statistically distinguishable from the many other fluctuations in the data set. Methodology/Principal Findings: Biopsies from 12 human primary brain tumors were used to extract RNA. These samples were used to determine genome-wide mRNA expression levels by microarray analysis and a miRNA profile by real-time reverse transcription PCR (RT-PCR). Correlation coefficients were determined for all possible mRNA-miRNA pairs and the distribution of these correlations compared to the random distribution. An excess of high positive and negative correlation pairs were observed at the tails of these distributions. Most of these highest correlation pairs do not contain sufficiently complementary sequences to predict a target relationship; nor do they lie in physical proximity to each other. However, by examining pairs in which the significance of the correlation coefficients is modestly relaxed, negative correlations do tend to predict targets and positive correlations do tend to predict physically proximate pairs. A subset of high correlation pairs were experimentally validated by over-expressing or suppressing a miRNA and measuring the correlated mRNAs. Conclusions/Significance: Sufficient information exists within a set of tumor samples to detect endogenous correlations between miRNA and mRNA levels. Based on the validations of the correlations the causal arrow for these correlations is likely to be directed from the miRNAs to the mRNAs. From these data sets, we inferred and validated a tumor suppression pathway linked to miR-181c. Keywords: cell type comparison
Background: Intrinsic glioma subtypes (IGS) are molecularly similar tumors that can be identified based on unsupervised gene-expression analysis. Here we have evaluated the clinical relevance of these subtypes within EORTC26951, a randomized phase III clinical trial investigating adjuvant procarbazine, CCNU (lomustine) and vincristine (PCV) chemotherapy in anaplastic oligodendroglial tumors. Our study is the first to include gene-expression profiles of formalin-fixed and paraffin-embedded (FFPE) clinical trial samples. Methods: Gene-expression profiling was performed in 140 samples: 47 fresh frozen and 93 FFPE, on HU133_Plus_2.0 and HuEx_1.0_st arrays (Affymetrix), respectively. Results: All previously identified six intrinsic glioma subtypes are present in EORTC26951. This confirms that different molecular subtypes are present within a well-defined histological subtype. Intrinsic subtypes are highly prognostic for overall- (OS) and progression-free survival (PFS). They are prognostic for PFS independent of clinical (age, performance, tumor location), molecular (1p19qLOH, IDH1 mutation, MGMT methylation) and histological parameters. Combining known molecular (1p19LOH, IDH1) prognostic parameters with intrinsic subtypes improves outcome prediction (Proportion of Explained Variation 30% v 23%). Specific genetic changes (IDH1, 1p19qLOH and EGFR amplification) segregate into different subtypes. We identified one subtype, IGS-9 (characterized by a high percentage of 1p19qLOH and IDH1 mutations), that especially benefits from PCV chemotherapy. Median OS in this subtype was 5.5 years after radiotherapy (RT) alone v 12.8 years after RT/PCV; P=0.0349; HR 2.18, 95% CI [1.06, 4.50]. Conclusion: Intrinsic subtypes are highly prognostic in EORTC26951 and improve outcome prediction when combined with other prognostic factors. Tumors assigned to IGS-9 benefit from adjuvant PCV
Natural Killer cells (NK) a major constituent of innate immune system, have the ability to kill the transformed and infected cells without prior sensitization; can be put to immunotherapeutic use against various malignancies. NK cells discriminate between normal cells and transformed cells via a balance of inhibitory and activating signals induced by interactions between NK cell receptors and target cell ligands. Present study investigates whether expansion of NK cells could augment their anti-myeloma (MM) activity. For NK cell expansion, peripheral blood mononuclear cells from healthy donors and myeloma patients were co-cultured with irradiated K562 cells transfected with 4-1BBL and membrane-bound IL15 (K562-mb15-41BBL). A genome-wide profiling approach was performed to identify gene expression signature in expanded NK (ENK) cells and non-expanded NK cells isolated from healthy donors and myeloma patients. A specific set of genes involved in proliferation, migration, adhesion, cytotoxicity, and activation were up regulated post expansion, also confirmed by flow cytometry. Exp-NK cells killed both allogeneic and autologous primary MM cells more avidly than non-exp-NK cells in vitro. Multiple receptors, particularly NKG2D, natural cytotoxicity receptors, and DNAM-1 contributed to target lysis, via a perforin mediated mechanism. In summary, vigorous expansion and high anti-MM activity both in vitro and in vivo provide the rationale for testing exp-NK cells in a clinical trial for high risk MM. Differential gene expression profile in expanded natural killer (ENK) cells and non-expanded natural killer (NK) cells from healthy donors and myeloma patients
The statsitcal model latent pathway identification analysis (LPIA), was implemented for the analysis of A549 lung carcinoma cells treated with geldanamycin. Control and treated samples were assayed with Affymetrix HG_U133_plus_2 arrays and analyzed using LPIA. LPIA looks for statistically signficant evidence of dysregulation in a network of pathways constructed in a manner that explicitly links pathways through their common function in the cell. Geldanamycin (geld) is known to inhibit the molecular chaperone protein, Hsp90, and plays a role in preventing the malignant transformation and proliferation of healthy cells during oncogenesis. LPIA successfully identified pathways specific to geldanamycin effects at the gene transcription level.
First experiment: Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys) Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys). Keywords: amino acid deprivation
We compared PBMC genomic response to exercise in both early (EB) and late-pubertal boys (LB) Keywords: stress response
Rationale: Immortalized cells may exhibit important differences relative to their primary cell counterparts. Microarrays were used compare primary human umbilical vein endothelial cells (HUVECs) and the immortalized HUVEC cell line EA.hy926 in their response to inhibition of the mevalonate pathway by a HMG-CoA reductase inhibitor (atorvastatin). The effects of atorvastatin were reversed by the addition of mevalonate, to subtract non-specific changes in gene expression. Methods: Confluent cell cultures of HUVECs and EA.hy926 cells (two independent experiments in each cell type) were incubated with 1) statin-free media; 2) 10 microM atorvastatin; 3) 500 microM mevalonate; or 4) a combination of 10 microM atorvastatin and 500 microM mevalonate. All cells were harvested at 24 h. Total RNA was isolated using Trizol reagent (Invitrogen). cDNA was prepared from 10 microgram total RNA using a double-stranded cDNA synthesis kit (Life Technologies) with a T7-dT24 primer for first-strand synthesis. cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Twenty microgram of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer, containing 40 mM Tris-acetate, pH 8.1, 125 mM KOAc, 30 mM MgOAc. Fifteen micrgram of fragmented cRNA, together with control cRNAs and grid alignment oligonucleotides, were hybridized overnight to GeneChip Human Genome U133A 2.0 arrays (Affymetrix) at 45°C under constant rotation. Arrays were washed and incubated with an anti-streptavidin antibody. Fluorescent signals were measured using an Agilent Gene Array Laser Scanner and analyzed with MicroArray Suite 5.0 (Affymetrix). Microarrays were scaled using default settings. Keywords: other
Every year more than 42000 women die of endometrial cancer, mainly due to recurrent or metastatic disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been correlated with improved prognosis. This study describes two mechanisms by which progesterone inhibits metastatic spread of endometrial cancer: by stimulating T-cell infiltration and by inhibiting epithelial-to-mesenchymal cell transition (EMT). Paraffin sections from patients with (n=9) or without (n=10) progressive endometrial cancer (recurrent or metastatic disease) were assessed for the presence of CD4+ (helper), CD8+ (cytotoxic) and Foxp3+ (regulatory) T-lymphocytes and PR expression. Progressive disease was observed to be associated with significant loss of TILs and loss of PR expression. Frozen tumor samples, used for genome-wide expression analysis, showed significant regulation of pathways involved in immunosurveillance, EMT and metastasis. For a number of genes, such as CXCL14, DKK1, DKK4 and WIF1, quantitive RT-PCR was performed to verify down regulation in progressive disease. To corroborate the role of progesterone in regulating invasion, Ishikawa (IK) endometrial cancer cell lines stably transfected with PRA (IKPRA), PRB (IKPRB) and PRA+PRB (IKPRAB) were cultured in the presence/absence of progesterone (MPA) and used for genome-wide expression analysis, Boyden- and wound healing migration assays, and IHC for known EMT makers. IKPRB and IKPRAB cell lines showed MPA induced inhibition of migration and loss of the mesenchymal marker vimentin at the invasive front of the wound healing assay. Furthermore, pathway analysis of significantly MPA-regulated genes showed significant down regulation of important pathways involved in EMT, immunesuppression and metastasis: such as IL6-, TGF-β and Wnt/β-catenin signalling. Intact progesterone signaling in non-progressive endometrial cancer seems to be an important factor stimulating immunosurveillance and inhibiting transition from an epithelial to a more mesenchymal, more invasive phenotype.
Human fetal and adult hematopoietic stem cells (HSC) were obtained from fetal liver fetal bone marrow (BM), and adult BM. These were injected into human fetal thymic implants in SCID-hu Thy/Liv mice (4-6 separate mice per HSC donor) and allowed to mature into single positive CD4+ (SP4) thymocytes over the course of 7-8 weeks. SP4 thymocytes from injected stem cells were subsequently sort-purified from thymic implants and gene expression was performed.
We performed the oligonucleotide microarray analysis in bipolar disorder major depression, schizophrenia, and control subjects using postmortem prefrontal cortices provided by the Stanley Foundation Brain Collection. By comparing the gene expression profiles of similar but distinctive mental disorders, we explored the uniqueness of bipolar disorder and its similarity to other mental disorders at the molecular level. Notably, most of the altered gene expressions in each disease were not shared by one another, suggesting the molecular distinctiveness of these mental disorders. We found a tendency of downregulation of the genes encoding receptor, channels or transporters, and upregulation of the genes encoding stress response proteins or molecular chaperons in bipolar disorder. Altered expressions in bipolar disorder shared by other mental disorders mainly consisted of upregulation of the genes encoding proteins for transcription or translation. The genes identified in this study would be useful for the understanding of the pathophysiology of bipolar disorder, as well as the common pathophysiological background in major mental disorders at the molecular level. Keywords: disease state analysis
Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and South East Asia where more than 50000 new cases are diagnosed each year. We used microarrays to identify down or upregulated genes in NPC compared with non-malignant controls. Keywords: Diseased versus control
EpCAM is frequently overexpressed in human invasive breast cancer. We reported EpCAM overexpression to be an independent prognostic marker for poor overall survival in node-positive breast cancer. We used microarrays in order to investigate changes of the transcriptome on EpCAM gene overexpression in human breast cancer cells
Follicular Lymphomas are blood tumors growing as spheres in patients. Before this study there was no experimental model mimicking the 3D organization of these in vivo tumors. We develop such a model, called MALC, and observed a progressive enrichment in quiescent cells in these with time of culture; these cells were sorted, as their cycling counterparts, and their transcriptomes were compared. We used microarrays to detail the differential global gene expression profile between quiescent and cycling cells isolated from MALC.
Background: Diesel exhaust (DE) is the primary source of urban fine particulate matter which has been associated with cardiovascular disease in epidemiological studies. These effects may be related to oxidative stress and systemic inflammation with resulting perturbation of vascular homeostasis. Peripheral leukocytes are involved in both inflammation and control of vascular homeostasis. Objectives: We conducted an exploratory study using microarray techniques to analyze whether global gene expression in peripheral blood mononuclear cells (PBMCs) can inform on potential mechanisms of effect of DE inhalation. Methods: In a double-blind, crossover, controlled exposure study, healthy adult volunteers were exposed in randomized order to filtered air (FA) and diluted DE in two-hour sessions. We isolated RNA (Trizol/Qiagen method) form PBMCs before, and two times after each exposure. RNA samples were arrayed using the Affymetrix® platform (GeneChip® Human Genome U133 Plus 2.0 Array). Results: Microarray analyses were conducted on five subjects with available RNA sample form exposures to FA and to the highest DE inhalation (200 µg/m³ of fine particulate matter). Following data normalization and statistical analysis, a total of 1290 out of 54,675 probe sets with significant evidence for differential expression (more than 1.5-fold up or down regulated with p < 0.05) were identified. These include genes involved in inflammatory response (e.g., IL8RA, TNFAIP6, FOS), oxidative stress (e.g., HMOX1, BAX, PRDX1,), and in biochemical pathways like mitogen-activated protein kinases (MAPK) and tight junction pathways. Conclusions: These data suggest that DE may exert time-dependent changes in gene expression in PBMCs in healthy individuals. Genes that may be affected by DE inhalation are involved in inflammatory and oxidative stress processes. Keywords: time course
Human peripheral blood monocytes (Mo) consist of subsets distinguished by expression of CD16 (FCGRIII) and chemokine receptors. Classical CD16- Mo express CCR2 and migrate in response to CCL2 while a minor CD16+ Mo subset expresses CX3CR1 and migrates into tissues expressing CX3CL1. CD16+ Mo produce pro-inflammatory cytokines and are expanded in certain inflammatory conditions including HIV infection. To gain insight into the developmental relationship and functions of CD16+ and CD16- Mo, we examined transcriptional profiles of these Mo subsets in peripheral blood from healthy individuals. Of 16,328 expressed genes, 2,759 genes were differentially expressed and 228 and 250 were >2-fold upregulated and downregulated, respectively, in CD16+ compared to CD16- Mo. CD16+ Mo were distinguished by upregulation of dendritic cell (DC) (SIGLEC10, CD43, RARA) and macrophage (MF) (CSF1R/CD115, MafB, CD97, C3aR) markers together with transcripts relevant for DC-T cell interaction (CXCL16, ICAM-2, LFA-1), cell activation (LTB, TNFRSF8, LST1, IFITM1-3, HMOX1, SOD-1, WARS, MGLL), and negative regulation of the cell cycle (CDKN1C, MTSS1), whereas CD16- Mo were distinguished by upregulation of myeloid (CD14, MNDA, TREM1, CD1d, C1qR/CD93) and granulocyte markers (FPR1, GCSFR/CD114, S100A8-9/12). Differential gene expression in CD16+ and CD16- Mo was confirmed by quantitative real time RT-PCR (i.e., CD16, C3AR1, C1QR1, ICAM-2, CSF1R, CSF3R, CDKN1C, TNFRSF1, and LTB) and flow cytometry (i.e., CSF1R, CSF3R, C1QR1, C3AR1, CD1d, CD43, CXCL16, and CX3CR1). Furthermore, increased expression of RARA and KLF2 transcripts in CD16+ Mo coincided with absence of cutaneous lymphocyte associated antigen (CLA) expression, indicating potential imprinting for non-skin homing. These results suggest that CD16+ and CD16- Mo originate from a common myeloid precursor, with CD16+ Mo having a more MF- and DC-like transcription program suggesting a more advanced stage of differentiation. Distinct transcriptional programs, together with their recruitment into tissues via different mechanisms, also suggest that CD16+ and CD16- Mo give rise to functionally distinct DC and MF in vivo.
By comparing to common tumor cells genes were differencially expressed in pseudopalisading cells in human glioblastoma Keywords: parallel sample
Analysis of HEK293T cells overexpressing ZAPS (zinc finger antiviral protein short form; NP_078901), which is a member of the PARP (poly (ADP-ribose) polymerase)-superfamily. Results of gene profiles provide insight into the role of ZAPS in innate immunity.
Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen.
Pompe disease is caused by autosomal recessive mutations in the GAA gene which encodes acid alpha-glucosidase. Although enzyme replacement therapy has recently improved patient survival greatly, the results in skeletal muscles and for advanced disease are still not satisfactory. Here, we report the derivation of Pompe disease induced pluripotent stem cells (PomD-iPSCs) and their potential for pathogenesis modeling, drug testing and disease marker identification. PomD-iPSCs maintained pluripotent features, and had low GAA activity and high glycogen content. Cardiomyocyte-like cells (CMLCs) differentiated from PomD-iPSCs recapitulated the hallmark Pompe disease pathophysiological phenotypes, including high levels of glycogen, abundant intracellular LAMP-1- or LC3-positive granules, and multiple ultrastructural aberrances. Drug rescue assessment showed that exposure of PomD-iPSC-derived CMLCs to rhGAA reversed the major pathologic phenotypes. Further, L-carnitine and 3- methyladenine treatment reduced defective cellular respiration and buildup of phagolysosomes, respectively, in the diseased cells. By comparative transcriptome analysis, we identified glycogen metabolism, lysosome and mitochondria related marker genes whose expression robustly correlated with the therapeutic effect of drug treatment in PomD-iPSC-derived CMLCs. Collectively, these results demonstrate that PomD-iPSCs are a promising in vitro disease model for development of novel therapeutic strategies for Pompe disease.
Analysis of genes that were differentially expressed in MSC-derived hES cells (VUB01 and SA01) as compared to VUB01 and SA01 undifferentiated hES cells Keywords: differentiation state analysis
Duplication of chromosomal arm 20q occurs in prostate cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a key causal role in tumorigenesis. According to an alternative view, chromosomal instabilities are mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that deregulation of some key pathways, such as MAPK, p53, cell cycle regulation and Polycomb group factors, in addition to activation of several genes like Myc, AML, B-Catenin and the ETS family transcription factors, are key steps in cancer development driven by 20q amplification. Finally we identified 13 cancer initiating genes, located on 20q13, which were significantly overexpressed in many tumors, with expression levels correlated with tumor grade and outcome; these probably play key roles in inducing malignancy via20q amplification.
Gene expression profiling was performed on primary human erythroid progenitor cells left untreated or treated with 0.5uM SAHA. The worldwide burden of sickle cell disease is enormous with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a novel bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.
Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. We recently reported the identification of Importin 8 (Imp8) as a novel component of miRNA-guided regulatory pathways. Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P-bodies) structures involved in RNA metabolism. For this micro-array dataset, we used immunoprecipitations of Ago2-associated mRNAs followed by micro-array analysis. The results demonstrate that Imp8 is required for recruiting Ago protein complexes to a large set of Ago2-associated target mRNAs allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing.
Evaluation of pretreatment gene expression profiling features in elderly CLL patients; correlation with clinical outcome
Glomerular diseases account for the majority of cases with chronic renal failure. Several genes have been identified with key relevance for glomerular function. Quite a few of these genes show a specific or preferential mRNA expression in the renal glomerulus. To identify additional candidate genes involved in glomerular function in humans we generated a human renal glomerulus-specific transcript dataset (GTD) by comparing gene expression profiles from human glomeruli and tubulointerstitium obtained from six transplant living donors using Affymetrix HG-U133A arrays. This analysis resulted in 677 genes with prominent overrepresentation in the glomerulus. Genes with ‘a prior’i established known prominent glomerular expression served for validation and were all found in the novel expression library (e.g. CDKN1 DAG1, DDN, EHD3, MYH9, NES, NPHS1, NPHS2, PDPN, PLA2R1, PLCE1, PODXL, PTPRO, SYNPO, TCF21, TJP1, WT1). The mRNA expression for several novel glomerulus-enriched genes identified in REGGEL was validated by qRT-PCR. Gene ontology and pathway analysis identified biological processes previously not reported to be of relevance in glomeruli including among others axon guidance. This finding was further validated by assessing the expression of the axon guidance molecules neuritin (NRN1) and roundabout receptor ROBO1 and -2. Glomerular disease associated differential mRNA regulation of ROBO2 was found in diabetic nephropathy. In summary, using a comparative strategy on microdissected nephrons novel transcripts with predominant expression in the human glomerulus could be identified. A systematic analysis of this glomerulus-specifc gene expression library allows the detection of target molecules and biological processes involved in glomerular biology and renal disease.
Notch1-IC Notch2-IC or EBNA2 have been induced in a conditionally immortalized human B cell line (EREB2-5) in order to identify similar and unique target genes in B cells. CAT was used as a control. Keywords: time course
To investigate the role of TAZ downstream of the abberrant Wnt signaling in CRC cells we compared the expression profiles of parental SW480 cells (empty vector) transfected with siControl, siTAZ, sibeta-catenin or reconstituted with wild type APC and transfected with siControl Keywords: expression profiling by array
The derivation of molecular signatures indicative of disease status and predictive of subsequent behavior could facilitate the optimal choice of treatment for prostate cancer patients. In this study we conducted a computational analysis of gene expression profile data obtained from 79 cases, 39 of which were classified as having disease recurrence, to investigate whether advanced computational algorithms can derive more accurate prognostic signatures for prostate cancer. At the 90% sensitivity level, a newly derived prognostic genetic signature achieved 85% specificity. This is the first reported genetic signature to outperform a clinically used postoperative nomogram. Furthermore, a hybrid prognostic signature derived by combination of the nomogram and gene expression data significantly outperformed both genetic and clinical signatures, and achieved a specificity of 95%. Our study demonstrates the feasibility of utilizing gene expression information for highly accurate prostate cancer prognosis beyond the current clinical systems, and shows that more advanced computational modeling of tissue-derived microarray data is warranted before clinical application of molecular signatures is considered.
Reliable clinical tests for predicting cancer chemotherapy response are not available and individual markers failed to correctly predict resistance against anticancer agents. We hypothesized that gene expression patterns attributable to chemotherapy-resistant cells can be used as a classification tool for chemoresistance and provide novel candidate genes involved in anthracycline resistance mechanisms. We contrasted the expression profiles of 4 different human tumor cell lines of gastric pancreatic, colon and breast origin and of their counterparts resistant to the topoisomerase inhibitors daunorubicin or doxorubicin. We also profiled the sensitive parental cells treated with doxorubicin for 24h. We interrogated Affymetrix HGU133A and U95A arrays independently. We applied two independent methods for data normalization and used Prediction Analysis of Microarrays (PAM) for feature selection. In addition, we established data sets related to drug resistance by using a “virtual array” composed of features represented on both types of oligonucleotide arrays. We identified 71 candidate genes associated with doxorubicine/daunorubicine resistance. To validate the microarray data, we also analyzed the expression of 12 selected genes by quantitative RT-PCR or immunocytochemistry, respectively. While the comparison of drug-sensitive versus drug-resistant cells yields candidates associated with drug resistance, the 24h treatment of sensitive parental cells produced a distinct transcriptional profile related to short-term drug effects. Keywords: cell type and treatment comparison
To identify gene expression that distinguishes hematopoietic cells that express PRAME from those that do not normal CD34+ cells with forced PRAME expression were compared to cells without PRAME expression in culture over time (days 4, 7, 14) using Affymetrix HU-133A microarrays
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system and is generally considered to be autoimmune in nature. We previously demonstrated that the transcription factor Sp3 is significantly down-regulated in immune cells from MS patients. The potential role of Sp3 down-regulation in MS pathogenesis is not well understood. The function of endogenous Sp3 was assessed in vitro after siRNA-mediated knockdown of its transcript in Jurkat cells. Sp3 protein levels were reduced an average of 70%. ELISA studies demonstrated decreased endogenous production of IL-10 and TGFβ1 and increased endogenous production of TNFα (p<0.05 in all assays). Subsequent microarray analysis demonstrated significantly altered expression of 36 genes (p<0.001 for each gene) compared with control samples. Analysis showed differential expression (p<0.005) of 8 gene pathways. Many of the genes and pathways that were regulated by Sp3 are involved in immune function specifically with regard to apoptosis, cell-to-cell adhesion, integrin signaling, T-cell differentiation, and cytokine production. This study identifies mechanisms by which Sp3 may regulate immune function and suggests a basis for its potential contribution to MS disease pathogenesis. Keywords: siRNA knockdown; Jurkat T-cells; Multiple Sclerosis
Background: Causative genes for autosomal dominantly inherited familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) have been well characterized. There is however, another 10-15 % early onset colorectal cancer (CRC) whose genetic components are currently unknown. In this study, we used DNA chip technology to systematically search for genes differentially expressed in early onset CRC. Keywords: disease state analysis
Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA. We used microarrays to identify genes differentially expressed in U937 cells expressing AML1/ETO compared to vector transfected U937 cells. Keywords: Transcriptional regulation
Using Affymetrix microarray technology we analyzed the gene expression profiles of the most important pathological categories of bladder cancer in order to detect potential marker genes. Applying an unsupervised cluster algorithm we observed clear differences between tumor and control samples as well as between superficial and muscle invasive tumors. According to cluster results, the T1 high grade tumor type presented a global genetic profile which could not be distinguished from invasive cases. We described a new measure to classify differentially expressed genes and we compared it against the B-rank statistic as a standard method. According to this new classification method, the biological functions overrepresented in top differentially expressed genes when comparing tumor versus control samples were associated with growth, differentiation, immune system response, communication, cellular matrix and enzyme regulation. Comparing superficial versus invasive samples, the most important overrepresented biological category was growth and, specifically, DNA synthesis and mitotic cytoskeleton. On the other hand, some under expressed genes have been clearly related to muscular tissue contamination in control samples. Finally, we demonstrated that a pool strategy could be a good option to detect the best differentially expressed genes between two compared conditions. Keywords: disease state analysis
Several studies described a role for the E2F/Rb pathway in ovarian serous carcinomas (SCAs). Since E2F/Rb pathway deregulation is a general hallmark of human cancer it remains unclear whether this deregulation is of particular importance in SCAs or whether it reflects a common oncologic feature. Here, we attempted to clarify this issue by the examination of microarray expression profiles of SCAs (10 SCA1s, 15 SCA3s) and particularly by the comparison with another, less malignant, ovarian cancer type, serous borderline tumours (13 SBTs). Results were further validated by quantitative RT-PCR, both on the microarray samples and an independent panel. In addition, TP53 mutation analysis was performed. This integrated analysis revealed a significant increase in the expression of the transcription factors E2F1 and E2F3 in SCAs, when compared to SBTs. This was associated with a vast overexpression of E2F target genes in SCAs compared to SBTs. Overall, at least 45% of those genes with a significantly higher expression in SCAs were E2F targets. When taking into account the different SCA tumour grades, particularly SCA3s exhibited a major deregulated E2F target expression pattern, compared to SBTs. To a lesser extent, this was also the case for SCA1s, although the E2F target expression pattern of several SCA1s appeared to be more similar to SBTs. Generally, overexpression of E2F targets in SCAs appeared to be well-structured since those targets considered as negative regulators of the cell cycle or promoters of apoptosis were usually not overexpressed in SCAs. Similar to E2F target deregulation, TP53 mutations were identified in SCA3s, to a lesser extent in SCA1s, and not in SBTs. These results suggest that a structured, generally upregulated, E2F transcription factor activity is associated with a global cell cycle disturbance in high grade SCAs, and exceeds typical E2F/Rb pathway disruption in tumours, at least compared with SBTs
Affymetrix Hu133 GeneCHIP Microarray data for Control and c-MYC knock-down (KD) human cancer cell lines. Data related to article 'Novel c-MYC target genes mediate differential effects on cell proliferation and migration' from D. CAPPELLEN et al., EMBO Reports Note: Samples GSM136093 and GSM136094 (Hela cell line, expt 1) were hybridized and normalized separately from the other 18 Samples. Keywords: siRNA analysis
Knock-down of LSD1 using siRNA approach induced regulation of several proliferation-associated genes in ER-negative breast cancer cells MDA-MB-231.
Microarrays were used to evaluate the effects of azithromycin and an inflammatory stimulus (SMM) on human airway epithelium. Effects of azithromycin treatment were evaluated at 6 24 and 48 hours. Effects of SMM were evaluated at 6 and 24 hours. In addition, pretreatment with azithromycin was used to evaluate the modulatory effects on SMM-induced inflammation. SMM=supernatant from microcorpulent material from human cystic fibrosis airways. Keywords: timecourse, treatment comparisons.
Endothelial cells were transduced with different genes modulating signaling pathways and compared to GFP transduced control group to identify changes in the expression of the angiocrine factors.
Results from 29 samples tested on the Affymetrix HG-focused target array identified transcriptional changes characteristic of ocular disease and ocular Chlamydia trachomatis infection phenotypes. Large numbers of differentially regulated genes were demonstrated. These were characteristic of the host defence response and typical of innate responses at epithelial surfaces and infiltrating leukocytes. These results provide an insight into the complexity of the acute response in trachoma.
Study objectives: The objectives of this study was perform the global gene expression profiling aimed at identifying differentially expressed genes in the circulating lympho-monocytes of NRLCP patients affected by Narcolepsy with Cataplexy (NRLCP). Based on the tight association to the HLA-DQB1*0602 haplotype in caucasians it could be hypotesized an immunological dysregulation underlying the pathogenesis of the disease. Design: 10 NRLCP patients with 10 healthy controls were compared. Total RNA isolated from blood specimens was analyzed using microarray technology followed by statistical data analysis to detect genome-wide differential gene expression between patients and controls. Functional analysis of the genelist was performed in order to interpret the biological significance of the data. Results: 173 genes showed significant (p<0.01) differential expression between the two tested conditions. The biological interpretation allowed to categorize differentially expressed genes into main functional groups including includes genes involved in brain development, which could be possibly regarded as peripheral markers of the disease, along with molecular markers of the NRLCP-related dysmetabolic syndrome and immuregulatory molecules. Moreover a striking correlation within selected HLA haplotypes and HLA gene expression was detected, indicating an allele-specific trend of gene expression for the DQB1, DQA1 and DRB4 genes across all the tested subject, regardless of the disease status. Conclusions: the molecular profile associated to NRLCP suggested that molecular markers of neural, metabolic and immunological dysregulation can be detected in blood of NRLCP patients. Moreover, the allele-specific HLA gene expression could suggest a possible direct role of MHCII as a co-factor in the disease etio-pathogenesis
The goal of our study was to molecularly dissect mesothelioma tumor pathways by mean of microarray technologies in order to identify new tumor biomarkers that could be used as early diagnostic markers and possibly as specific molecular therapeutic targets. We performed Affymetrix U133A plus 2.0 microarray analysis comparing 9 human pleural mesotheliomas with 4 normal pleural specimen. Stringent statistical feature selection detected a set of differentially expressed genes that were further evaluated to identify potential biomarkers to be used in early diagnostics. Selected genes were confirmed by RT-PCR. As reported by other mesothelioma profiling studies, most of genes are involved in G2/M transition. Our list contains several genes previously described as prognostic classifier. Furthermore, we found novel genes never associated before to mesothelioma and could be involved in tumor progression. Notable, the identification of MMP-14, a member of matrix metalloproteinase family. This molecule has been described as a new disease marker and could be used as biomarker also for mesothelioma early diagnosis and prognosis and that can be viewed as new and effective therapeutic target to test. Accepted for pubblication in PLoS ONE
We measured the baseline expression levels of genes in members of the CEPH pedigrees using Affymetrix Human Genome Focus Arrays. Arrays were analyzed using MAS 5.0 software (Affymetrix). Keywords = natural variation Keywords = human Keywords: other
It has been demonstrated that Ring finger protein 43 (RNF43) is overexpressed in colorectal cancer and mediates cancer cell proliferation. We found that RNF43 was frequently overexpressed in HCC and knockdown of RNF43 could induce apoptosis and inhibit proliferation, invasion, colony formation and xenograft growth of HCC cells. Suggesting that RNF43 is involved in tumorigenesis and progression of HCC. We used microarrays to profile gene expression patterns before and after RNF43 knockdown, and identified differentially expressed genes during this process.
Background Homeobox gene associated regulatory networks are among the key determinants of early hematopoietic development. Previously the ‘hematopoietic PBX interacting protein’ (HPIP) has been identified as a novel interacting partner of the TALE homeodomain protein PBX1, forming a microtubule signalling complex. Expression of HPIP has been associated with increased tumorigenicity of the MCF7 breast cancer cell line. We now demonstrate that HPIP is a novel regulatory protein in human hematopoiesis: constitutive expression of HPIP in human umbilical cord blood derived CD34+ cells increased the absolute number of clonogenic progenitors in liquid expansion culture as well as in methylcellulose assays with a significantly enhanced formation of erythroid colonies compared to the control (p≤0.01, n=6). Limiting dilution LTC-IC assays confirmed the hematopoietic activity of the protein on primitive human progenitor cells with an over 5fold increase in the absolute number of LTC-ICs compared to non-transduced cells (n=8; p<0.05). In vivo HPIP expression induced a significant shift towards myeloid engraftment (n=8;p<0.05) and doubled the proportion of hCD34+CD38+ human cells in transplanted mice (p≤0.05, n=8). Structure – function analyses identified the C - terminal nuclear receptor/PBX interacting domain (NRPID; LXXLL domain) as a critical domain for the hematopoietic activity of HPIP. Gene expression data by microarray and Q-RT-PCR analysis demonstrated that HPIP induced particularly differential expression of genes involved in the MAPK pathway and cytokine-cytokine interaction. Taken together, these data demonstrate that proteins involved in the organization of microtubular signalling complexes such as HPIP can act as regulators of early human hematopoiesis.
Klinefelter’s Syndrome (KS) is one of the common chromosome aneuploidy diseases in males with unexplained physiological mechanism. iPSCs are similar to ESCs in terms of indefinitive self-renewal and pluripotency, provided an alternative choice for modeling disease to facilitate the disease research in vitro. We used microarray to detect the global reprogramming of KS and normal fibroblast cells to iPSCs. Also we used microarray to explore the possible molecular varieties between KS patient and normal person in the early development.
Stem cells from various lineages have well known for its migration tendency toward glioma and become attractive vehicles to deliver therapeutic genes to brain tumors. However which factors and mechanisms work in these function is not yet known. To identify those factors and mechanisms, we analyzed the brain tumor-specific gene expression profile using microarray analysis
Human umbilical vein endothelial cells (HUVECs) were transduced with either MIY-N1IC (Notch1 intracellular domain) or MIY vector control. The cells were sorted for YFP and RNA was extracted using Trizol (Invitrogen) and analyzed by the Affymetrix Human Genome U133 Plus 2.0 Array. Results were analyzed using the GCRMA algorithm to identify genes with a minimum of 2-fold induction or reduction. This global gene expression study was used to identify Notch targets in the endothelium.
In the chorionic villi of placenta trophoblasts and endothelial cells are present, and moreover mesenchymal cells (stromal cells) can be obtained. We generated cells with the mesenchymal phenotype from the chorionic mesoderm, and showed that: a) physiologically functioning cardiomyocytes were transdifferentiated from human placenta-derived chorionic villi cells, but these cells did not induce to osteoblasts and adipocytes ; b) the cardiomyogenic induction rate obtained using our system was relatively high compared to that obtained using the previously described method ; c) co-cultivation with fetal murine cardiomyocytes alone without transdifferentiation factors such as 5-azaC or oxytocin is sufficient for cardiomyogenesis in our system; d) Chorionic villi cells have the electrophysiological properties of 'working' cardiomyocytes. The chorionic mesoderm contained a large number of cells with a cardiomyogenic potential. Keywords: Cardiomyogenic induction
We used a whole genome approach to identify major functional gene categories (including xenobiotic transporters and metabolizing enzymes) whose expression depends on gestational age. STUDY DESIGN: We compared gene expression profiles of 1st (45-59 days) and 2nd trimester (109-115 days) and C-section term placentae. RESULTS: In 1st trimester placentae, genes related to cell cycle, DNA, aminoacids and carbohydrate metabolism were significantly overrepresented, while genes related to signal transduction were downregulated. In the organism defense category, we identified genes involved in chemical response, metabolism, and transport. Analysis of signal transduction pathways suggested, and subsequently confirmed independently, that the Wnt pathway was regulated by gestational age. CONCLUSIONS: Our study will serve as a reference database to gain insight into the regulation of gene expression in the developing placentae and, thus, allow comparisons with placentae from complicated pregnancies such as those in women experiencing gestational diabetes, pre-eclampsia and teratogenic sequelae. Keywords: time series
To determine cardiac transcription profile in acyanotic Tetralogy of Fallot patients we collected myocardial samples immediately after institution of cardiopulmonary bypass from acyanotic Tetralogy of Fallot patients undergoing corrective surgery. The transcriptional profile of the mRNA in these samples was measured with gene array technology.
We hypothesized that tissue genome-wide gene expression analysis coupled with gene network analyses of differentially expressed genes, would provide novel insights into the pathogenesis of pulmonary sarcoidosis. Keywords: Disease state analysis
Many human oncogenes are challenging therapeutic targets. An alternative to direct targeting of oncogenes is to perform “synthetic lethality” screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of the broad spectrum of mutant KRAS-driven cancers, and demonstrate the potential of RNAi screens for discovering critical functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers associated with “undruggable” genetic alterations.
Gene expression in HeLa cells was profiled using Affymetrix gene expression Human HG-U133_Plus_2 array. Transcript signal was mapped against the chromosome coordinates (probe-by-probe basis) using the HG-U133A_2 Annotations CSV file for hg18 build of the human genome provided by Affymetrix. Karnani N Taylor C, Malhotra A, Dutta A (2007) Pan-S replication patterns and chromosomal domains defined by genome-tiling arrays of ENCODE genomic areas. Genome Res 17: 865-876.
CD44+/CD24- subpopulation of normal and cancerous breast epithelial cells are suggested to have stem cell properties. The goal of this study was to identify gene expression differences between CD44+/CD24- and CD44-/CD24+ subpopulation of cells from a same cell lines. We selected MCF-10A cells which are immortalized derived from a fibrocystic breast disease. These cells are immortalized but not transformed and express basal cell markers. Cells were from a single sort but plated into four 100 mm plates. RNA was prepared from each plate separately for the analysis.
The molecular role of iron in gene expression remains poorly characterized. Moreover the alterations in global gene expression after iron chelation remains unclear and are important to assess for understanding the molecular pathology of iron-depletion and the biological effects of iron chelators. We assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and Dp44mT). These studies are important for understanding the molecular and cellular effects of iron-depletion.
This SuperSeries is composed of the SubSeries listed below.
Membrane-bound transcription factor CREB3L1 undergoes Regulated Intramembrane Proteolysis (RIP) in response to Hepatitis C infection. RIP activates CREB3L1 so that it can prevent the growth of HCV infected cells through the action of downstream genes. We over-expressed a truncated form of CREB3L1 that does not require RIP to enter the nucleus. Cells over-expressing this truncated form were isolated by Fluorescence Activated Cell Sorting (FACS). We used microarray to determine the downstream genes of CREB3L1 in comparison to a flow sorted empty vector control.
Brain tumor neurospheres (BTCSs) are cancer cells with neural stem cell-like properties found in the fatal brain tumor glioblastoma multiforme (GBM). These cells account for less than 1% of total tumor cells are poorly differentiated and are believed to be involved in tumor induction, progression, treatment resistance and relapse. Specific miRNAs play important roles in modulating the proliferation and differentiation of neural stem cells, therefore, we aimed to identify miRNAs controlling differentiation in GBM-BTSCs through high throughput screening miRNA array profiling. We compared the miRNA expression profiles at the neurosphere state and upon 4 and 14days of differentiation by using LIMMA, finding 21 differentially expressed miRNAs : hsa-miR-103, hsa-miR-106a, hsa-miR-106b, hsa-miR-15b, hsa-miR-17, hsa-miR-19a, hsa-miR-20a, hsa-miR-25, hsa-miR-301a and hsa-miR-93 were found up-regulated upon differentiation, while hsa-miR-100, hsa-miR-1259, hsa-miR-21, hsa-miR-22, hsa-miR-221, hsa-miR-222, hsa-miR-23b, hsa-miR-27a, hsa-miR-27b, hsa-miR-29a and hsa-miR-29b were down-regulated. Expression of 11 of the 21 miRNAs was examined by qPCR and 7 of them were validated: hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222 increased their expression upon differentiation, while hsa-miR-93 and hsa-miR-106a were inhibited. Functional studies demonstrated that miR-21 over-expression induced the expression of glial and/or neuronal cell markers in the neurospheres, possibly due to SPRY1 targeting by miR-21 in these cells, while miR-221 and miR-222 inhibition at the differentiated state reduced the expression of those differentiation markers. On the other hand, miR-29a and miR-29b targeted MCL1 in the GBM neurospheres and increased apoptotic cell death.
Prostate cancer is characterized by heterogeneity in the clinical course that often does not to correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets. Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors. The metastatic samples are highly heterogeneous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodeling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1). We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer. Keywords: disease state analysis
Analysis of transcription response of undifferentiated human BE(2)-C neuronal cells to stimulation with purified antimycin A1a or unfractionated commercially available antimycin A (Sigma A8674).
We used microarrays to examine the impact of AF1q/MLLT11 on the gene expression profile of Notch-activated CD34+CD45RA-Lin- HPCs isolated from umbilical cord blood CD34+CD45RA-Lin- HPCs correspond to early multipotent progenitors. A2M is a nuclear mutant derivative of AF1q/MLLT11
Transgelin was the top-ranked marker of metastatic potential identified in the comparison of node-positive colorectal cancer (CRC) versus node-negative CRC in our previous study. Transgelin is localized in the nucleus of cultured CRC cells and microRNA-mediated knockdown of TAGLN (the gene encoding transgelin) expression modulates the expression of genes involved in the epithelial-to-mesenchymal transition. We performed gene expression profiling on control and transgelin-overexpressing RKO cells using Affymetrix microarray technology.
From the result of the gene expression analyses of human hepatoma cell line HepG2, a number of genes associated with cell proliferation and DNA repair were distinctively up-regulated by Ag-nanoparticle exposure, suggesting that Ag-nanoparticles might stimulate cell proliferation and DNA damage, which are considered to be mechanisms playing an important role for carcinogenesis and tumor progression. The inductions of these genes involved in cell proliferation were also observed in PS-nanoparticles and Ag2CO3-exposed cells. In addition, the inductions of DNA repair-associated genes were also observed in Ag2CO3-exposure. These results suggest that both “nanoshape” and “silver” can cause the inductions of these gene expression patterns. Furthermore, cysteine, a strong ionic silver ligand partially abolished these gene expressions induced by silver nanoparticles. Ionic silver sourced from Ag-nanoparticles could not fully explain these gene expressions.
Muscle biopsies taken from vastus lateralis muscle of 30 normal subjects and 19 FSHD subjects (see PubMed ID 17151338) Affymetrix U133A and U133B arrays were scanned both before (S1) and after (S2) antibody enhancement. Effects of age and sex in normal subjects reported previously under GSE362 GSE674, and GSE9676. Keywords: facioscapulohumeral dystrophy, skeletal muscle
Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy Keywords = Diabetes Keywords = kidney Keywords = glomeruli Keywords: other
In order to clarify the human response of re-epithelialization we biopsied split-thickness skin graft donor site wounds immediately before and after harvesting, as well as during the healing process 3 and 7 days thereafter. Altogether 25 biopsies from 8 patients qualified for the study. All samples were analysed by genome-wide microarrays. Here we identified the genes associated with normal skin re-epithelialization on time-scale, and organized them by similarities according to their induction or suppression patterns during wound healing.
mRNA expression differences between the liver and kidney of an adult male (homo sapien) were investigated using three technical replicates. The purpose of the experiment was to compare array data generated using Affymetrix with measures of expression obtained using RNAseq (a sequencing approach for measuring expression that utilizes Solexa technology). Keywords: kidney liver
We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples when compared to normal B cells.
Background Epigenetic changes are involved in the extinction of the B-cell gene expression program of classical Hodgkin lymphoma. However little is known regarding epigenetic similarities between classical Hodgkin lymphoma and plasma cell myeloma cells, both of which share an extinction of the gene expression program of mature B-cells. Design and methods Global histone H3 acetylation patterns were determined in cell lines derived from classical Hodgkin lymphoma, plasma cell myeloma and B-cell lymphoma by chromatin immunoprecipitation and subsequent hybridization onto promoter tiling arrays. H3K27 trimethylation was analyzed by chromatin immunoprecipitation and real-time DNA-PCR for selected genes. Epigenetic modifications were compared to gene expression data. Results B-cell characteristic genes were hypoacetylated in classical Hodgkin lymphoma and plasma cell myeloma cell lines, as demonstrated by comparison of their histone H3 acetylation patterns to those of B-cell lines. However, the number of genes jointly hyperacetylated and expressed in classical Hodgkin lymphoma and plasma cell myeloma cell lines, such as IFR4/MUM1 and RYBP, is limited. Moreover, H3K27 trimethylation for selected B-cell characteristic genes revealed that this additional epigenetic silencing is much more prevalent in classical Hodgkin lymphoma as compared to plasma cell myeloma. Conclusion Our epigenetic data support the view that classical Hodgkin lymphoma is characterized by an abortive plasma cell differentiation with a down-regulation of B-cell characteristic genes but without activation of most plasma cell typical genes.
Expression data from ERBB2 over-expression and EGF stimulation in MCF10A cells
Abstract Background: Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers how specific methylation profiles are initially established, or what determines tumor-type specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype identify pathways important as therapeutic targets. Methods and Findings: In an effort to identify new cancer-specific methylation markers, we employed a high throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5’ CpG islands, are induced from undetectable levels by 5-aza-2’-deoxycytidine (5-aza) in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (N=20) and adjacent non-malignant tissue showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or lymphocytes. We studied the eight most frequently and specifically methylated genes from our lung cancer data set in breast cancer (N=37), colon cancer (N=24), and prostate cancer (N=24) along with counterpart non-malignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. Conclusions: By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent potential targets for early detection screening or therapeutic intervention. Keywords: Cell line comparison
primary ALL cells (B-lineage) resistant to prednisolone by the MTT in vitro sensitivity assay Keywords = ALL Keywords: other
Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease modifying therapies. For Huntington's disease (HD) a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared to controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV et al. (2005) Proc Natl Acad Sci U S A 102: 11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies. Keywords: RNA biomarker screen
UCHL1 is related to apoptosis and proliferation in cancers and we sought to identify genes regulated by UCHL1
To accelerate the development of disease-modifying therapeutics for Huntington’s disease (HD) a dynamic biomarker of disease activity and treatment response is critically needed.
Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible. Keywords = oral carcinoma lymphatic metastasis predict Keywords: repeat sample
lincRNA-ST8SIA3 was depleted using siRNAs and associated gene expression changes were profiled on Affymentrix arrays
This study investigates the genes that promote clear cell renal cell carcimoma (ccRCC) metastasis using 4 primary metastatic and 5 non-metastatic tumor samples. U133 plus 2.0 array was used to identify the diffrently expressed genes between the primary metastatic and non metastatic ccRCC samples
Human intervertebral disc tissue was obtained from patients (average age 51 yrs) undergoing surgery for lumbar interbody fusion (n=3) or lumbar disc herniation (n=1). Cells were isolated by sequential pronase-collagenase digestion [3]. Cells were passaged twice in monolayer and suspended at a density of 2 x 106 cells/ml in 1.2% alginate (low viscosity Sigma Chemical, St Louis, MO) dissolved in 150 mM NaCl. Alginate beads were formed by dropwise addition of the alginate from a 22 gauge needle into 102 mM CaCl2, followed by 10 minutes of curing, as described previously [13, 27]. Cell-gel beads were incubated in cell culture media consisting of Ham’s F-12 medium (Gibco BRL, Grand Island, NY), supplemented with 10% FBS (HyClone, Logan, UT), 25 μg/ml ascorbic acid (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml Fungizone at 5% CO2 and 37° C. After 24 h, the cell culture medium was removed via pipette and exchanged for one of three osmotically active solutions, representing iso-osmotic, hyper-osmotic and hypo-osmotic media [12, 23, 34]. The iso-osmotic solution consisted of a defined cell culture medium (Ham’s F-12 with supplements as described above; 293 mOsm/kg H2O). The hypo-osmotic solution consisted of the same cell culture media diluted with de-ionized water to a final osmolarity of 250 mOsm/kg H2O. The hyper-osmotic solution consisted of the same cell culture media supplemented with sucrose to a final osmolarity of 450 mOsm/kg H20. The osmolarity of all solution formulations was determined using a freezing-point osmometer (Advanced Laboratory Wide Range 3W2, Advanced Instrument, Needham Heights, MA) as described previously [12]. Cell-alginate beads were cultured for a 4 hour period under one of these conditions, after which the cells were released from alginate in a dissolving buffer (55 mM Na-citrate and 150 mM NaCl), lysed and stored at –80°C. Intervertebral disc (IVD) cells experience a broad range of physical stimuli under physiologic conditions, including alterations in their osmotic environment. In this study, the gene expression profile of human IVD cells was quantified with gene array technology following exposure to varying osmolarity in order to capture the biological responses for a broad set of targets. A total of 42 genes were identified in IVD cells as significantly changed following culture under hyper-osmotic conditions, while a total of 18 genes were identified as significantly changed under hypo-osmotic conditions. Gene expression patterns were verified using RT-PCR. Genes identified in this study include those related to cytoskeleton remodeling and stabilization (ephrin-B2, sarcoglycan beta, IQGAP1), as well as membrane transport (ion transporter SLC21A12, osmolyte tranporter SLC5A3, monocarboxylic acid SLC16A6, and amino acid transporter SLC7A8). An unexpected finding was the differential regulation of the gene for the neurotrophin brain-derived neurotrophic factor by hyper-osmotic stimuli that may be indicative of a physiological response of IVD cells to physical stimuli important in regulating discogenic pain. Keywords = intervertebral disc Keywords = osmotic stimuli Keywords: other
In comparing gene expression of normal and CML CD34+ quiescent (G0) and proliferating (G1/S/G2/M) cells 292 genes were down-regulated and 192 genes were up-regulated in the CML G0 cells. The differentially expressed genes were grouped according to their reported functions and correlations were sought with biological differences previously observed between the same groups. The most apparent correlations include: i) Normal and CML G0 cells are more primitive than G1/S/G2/M cells; ii) CML G0 cells are in a more advanced stage of development and more poised to begin proliferating than normal G0 cells; iii) When CML G0 cells are stimulated to proliferate, they undergo further differentiation and maturation more rapidly than normal G0 cells, but both granulopoiesis and erythropoiesis are less efficient than normal; iv) Whereas normal G0 cells form only granulocyte/monocyte (GM) colonies when stimulated by cytokines, CML G0 cells consistently form a combination of GM and erythroid clusters and colonies; and v) Prominin-1 (CD133) is the gene most down-regulated in CML G0 cells and its down-regulation appears to be associated with the spontaneous formation of erythroid colonies by CML progenitors without EPO. The gene most over-expressed in CML G0 cells is LepR, but its role in contributing to the myeloid expansion and other abnormalities is unknown. It was hoped that LepR might serve as a therapeutic target, but leptin had no stimulatory or inhibitory effect on either normal or CML G0 cells, our attempts to make a specific LepR antibody were unsuccessful, and no other potentially targetable over-expressed surface antigens were identified.
Primary colon CSC cultures were transduced with a Wnt responsive construct (TOP-GFP). 10% highest and lowest TOP-GFP cell fractions were FACS sorted and arrayed.
VAF347 is a low molecular weight compound which inhibits allergic lung inflammation in vivo. This effect is likely due to a block of dendritic cell (DC) function to generate pro-inflammatory T-helper (Th) cells since VAF347 inhibits IL-6 CD86 and HLA-DR expression by human monocyte derived DC, three relevant molecules for Th-cell generation. Here we demonstrate that VAF347 interacts with the aryl hydrocarbon receptor (AhR) protein resulting in activation of the AhR signaling pathway. Functional AhR is responsible for the biological activity of VAF347 since, i) other AhR agonists display an identical activity profile in vitro, ii) gene silencing of wild type AhR expression or forced over-expression of a trans-dominant negative AhR ablates VAF347 activity to inhibit cytokine induced IL-6 expression in a human monocytic cell line and iii) AhR deficient mice are resistant to the compound’s ability to block allergic lung inflammation in vivo. These data identify the AhR protein as key molecular target of VAF347 and its essential role for mediating the anti-inflammatory effects of the compound in vitro and in vivo. Keywords: effect of compound
It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether MSCs transfer mitochondria to the cells under several different conditions of mitochondrial dysfunction including human pathogenic mitochondrial DNA (mtDNA) mutations. Using biochemical selection methods, we found that exponentially growing cells in restrictive media (uridine- and bromodeoxyuridine [BrdU]+) after coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mtDNA identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B rho0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. A chemotaxis inhibitory agent blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential “cell therapy-based mitochondrial restoration or mitochondrial gene therapy” for human diseases caused by mitochondrial dysfunction.
Study to identify genes associated with NSCLC recurrence in patients not receiving adjuvant chemotherapy. Purpose: Recent clinical trials suggest improvement in survival with adjuvant chemotherapy in non-small cell lung cancer (NSCLC). This study's aim is to identify genes associated with NSCLC recurrence in patients not receiving adjuvant chemotherapy. Experimental design: Banked NSCLC tumors of patients who underwent resection of stage Ia-IIIb disease were identified. Patients were stratified into two groups: recurrent (R n=11) or non-recurrent (NR, n=16) 2 years after surgery. Microarray profiling and Cox multivariate analysis were performed. Conclusion: Increased CYP3A5 gene expression correlates with NSCLC recurrence and promotes proliferation through mechanisms that may involve, in part, CYP3A5 epoxygenase activity. Keywords: comparison of gene expression profiles for recurrent and non-recurrent cancer
The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a rational target for therapeutic intervention. In order to investigate the primary phenotype(s) mediated by mutant PIK3CA in a clean and highly patient-relevant context we utilized a non-tumorigenic MCF10A parental and isogenic knock-in cell line that harbors a common activating PIK3CA kinase domain mutation (H1047R). We found that introduction of an endogenously mutated PIK3CA primarily results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective and selective on reversing this phenotype compared to cell-proliferation, highlighting a potential new paradigm for studying PI3K-pathway targeted agents. Keywords: Expression Array
We report a highly-penetrant form of obesity initially observed in 31 heterozygous carriers of a 593kb or larger deletion at 16p11.2 from amongst subjects ascertained for cognitive deficits. Nineteen similar deletions were identified from GWAS data in 16053 individuals from 8 European cohorts; such deletions was absent from healthy non-obese controls and accounted for 0.7% of our morbid obesity cases (p = 6.4x10-8, OR = 43). These findings highlight a promising strategy for identifying missing heritability in obesity and other complex traits, in which insights from rare extreme cases can be used to elucidate the basis for more common phenotypes.
MDA-MB-231 bone-metastatic subline 1833 and lung metastatic subline 4175 underwent spontaneous ploidy doubling in culture i.e. the genome approximately duplicated itself gradually. The modal- and hyper-ploid subpopulations during the ploidy transition were sorted into two separate sublines, 1833-Modal and 1833-Hyper for 1833, 4175-Modal and 4175-Hyper for 4175. Their expresssion patterns were compared to each other as well as to other MDA-MB-231 sublines isolated previously by Kang et al. 2003 and Minn et al. 2005. Keywords: Cell type comparison
We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM) identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation. Keywords: static expression
Ovarian clear cell carcinoma (OCCC) shows unique clinical features including an association with endometriosis and poor prognosis. We previously reported that the contents of endometriotic cysts especially high concentrations of free iron, are a possible cause of OCCC carcinogenesis through iron-induced persistent oxidative stress. In this study, we conducted gene expression microarray analysis using 38 ovarian cancer cell lines and identified genes commonly expressed in both OCCC cell lines and clinical samples, which comprise an OCCC gene signature. The OCCC signature reproducibly predicts OCCC specimens in other microarray data sets, suggesting that this gene profile reflects the inherent biological characteristics of OCCC. The OCCC signature contains known markers of OCCC, such as hepatocyte nuclear factor-1b (HNF-1b) and versican (VCAN), and other genes that reflect oxidative stress. Expression of OCCC signature genes was induced by treatment of immortalized ovarian surface epithelial cells with the contents of endometriotic cysts, indicating that the OCCC signature is largely dependent on the tumor microenvironment. Induction of OCCC signature genes is at least in part epigenetically regulated, as we found hypomethylation of HNF-1b and VCAN in OCCC cell lines. This genomewide study indicates that the tumor microenvironment induces specific gene expression profiles that contribute to the development of distinct cancer subtypes.
This SuperSeries is composed of the SubSeries listed below.
We used microarrays to detail the global program of gene expression underlying the effect of p17 on human plasmacytoid dendritic cells and was compared to CpG profile. Keywords: gene expression study
Schwann cells expanded in number by exposure to growth factors in vitro, could be useful in nervous system repair. Our previous results suggest that long term exposure to heregulin and forskolin changes the functional properties of the human Schwann cells, including the ability to myelinate axons after transplantation. Here, we propose to determine the molecular changes in the Schwann cells that occur as a result of extended growth with mitogenic factors. We believe that the information obtained in these studies will provide clues about mechanisms underlying the already observed changes in function. This information will aid in the prediction of the safety and efficacy of neural repair approaches that use cultured, expanded Schwann cells. Finally this data may provide clues into the mechanisms underlying normal human Schwann cell function. To use gene array analysis to compare gene expression profiles in early and late passage human Schwann cells exposed to the growth factors heregulin and forskolin. Observed changes in the function of human Schwann cells, including their capacity for growth and differentiation, after prolonged exposure to heregulin and forskolin, are caused by changes in the gene expression profiles in these cells. Nerves from four different donors were obtained within 12-15 h postmortem, with full consent for research from the families of the donors (faxed). IRB approval not required because subjects were officially dead at nerve harvest (IRB confirmation faxed). The nerve tissue obtained consisted of the lower 15 cm of nerve extending from mid calf to ankle. Endoneurial fascicles were dissected from short (1-2 cm) nerve fragments and cultured with heregulin and forskolin for 1 week. The nerve fascicles were then treated overnight with collagenase and dispase and dissociated by gentle trituration. Cells were plated at low density on laminin coated dishes and maintained in culture with mitogens for 3 passages. Schwann cell purities during this period were > 95%. RNA was extracted from the cells at passage 1 ( i.e. after 2 population doublings) and passage 3 (i.e. after 8 population doublings) from the following donors: SN HSC 330, age 51; SN HSC 317, age 29; SN HSC 329, age 20 and SN HSC 351, age 42. Thus eight samples (2 groups, 4 independent samples per group) are to be analyzed. Because both groups in this comparison have been exposed to mitogens, differences in gene expression profiles will be interpreted as indicative of changes caused by prolonged versus short term exposure to mitogens. More RNA was prepared from each group of cells than was needed for gene array analysis to allow confirmation of differentially-expressed gene transcripts by real-time RT-PCR. Since the main purpose of this project is to detect any major changes in the molecular properties of the Schwann cells, we propose that these samples be analyzed using Affymetrix U133 + 2 arrays containing the complete human genome and request Option 1: Start to Finish Profiling of all samples. We have contacted Dr Stanley Nelson at UCLA and discussed the project with him. We request that the samples be analyzed in the UCLA facility. Keywords: time-course
To investigate genes that might influence resistance to infection through IGF1R we screened human breast cancer-derived OCUB-M cells transfected with expression vector encoding IGF1R using microarray analysis. Keywords: overexpression comparison
Previous studies suggest that there may be age and gender related differences in salivary gland function. However the limited and often conflicting information available from healthy populations makes it difficult to confirm these differences. The purpose of the present study was to evaluate and compare changes in gene expression associated with age and gender in the human parotid gland. Differential expression, defined as statistically significant differences with at least 1.5 fold changes, was detected using the Affymetrix® GeneChip® HGU133plus2.0 microarray in 787 gene probe sets; 320 showed higher expression in males, while 467 showed higher expression in females. Several genes associated with saliva secretion were differentially expressed in male and female parotid gland including vesicle-associated membrane protein 3 VAMP3, synaptosomal-associated protein SNAP23, RAS oncogene family member RAB1A, syntaxin binding protein STXBP1. When the gene expression results from the youngest (19-38 years old) and the oldest (65-69 years old) female subjects were further evaluated, it was found that the expression of 228 genes were altered during aging; 155 genes were down-regulated, whereas 73 genes were up-regulated in the female parotid gland. Of the genes that were altered during aging, 24 of the 28 genes (86%) classified as being associated with immune responses were down-regulated in the aged parotid gland. A panel of differentially expressed, age- and gender-related genes was selected for further study by quantitative, real-time RT-PCR. Comparable differences in gene expression were detected by both Affymetrix array and quantitative, real-time RT-PCR methods. Taken together, our data suggest that salivary gland function may be adversely affected in the aged population due, at least in part, to the down regulation of several categories of genes. Moreover, the gender specific gene expressions identified in the present study correlates with the previously observed sexual dimorphism in salivary gland function. Keywords: Human Parotid Gland
The presence of the PUF (Pumilio/FBF) domain defines a conserved family of RNA-binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans Pumilio2 (PUM2) is expressed in embryonic stem cells and adult germ cells. To identify mRNAs associated with human PUM2 protein in adipose tissue stem cells (ADSC), we used a modified Ribonucleoprotein-ImmunoPrecipitation Microarray (RIP-Chip). PUM2 ribonucleoprotein (RNP) complexes were performed with 2 µg of anti-Pum2 antibody (goat polyclonal, Santa Cruz Biotechnology, CA, USA) bound to protein G-agarose beads (Sigma, Deisenhofen, Germany). ADSCs were lysed in polysome lysis buffer (Tris-HCl pH 7.4 15mM, MgCl2 15 mM, NaCl 0,3 M, 1% Triton X-100, 1 mM DTT, 100 U/ml RNase Out, PMSF 1mM and E64 10uM) for one hour at 4°C. Beads were washed, then buffer and cell lysate were added, and the reaction mixtures were tumbled for 2 hours at 4°C. After this incubation, the beads were thoroughly washed again with polysome lysis buffer and then either RNA extracted for microarray and RT-PCR experiments using the RNeasy mini kit (Qiagen). To control for non-specifically enriched RNAs, identical IPs were performed with beads precoated with preimmune goat serum as a negative control. RNA was processed for hybridization with GeneChip 3’ IVT Express (Affymetrix - Santa Clara, USA), according to the manufacturers instruction. Briefly, cDNA was synthesized from immunoprecipitated RNA using reverse transcriptase followed by second strand synthesis to generate double-stranded cDNA. An in vitro transcription reaction was used to generate biotinylated cRNA. After purification and fragmentation, cRNA was hybridized onto GeneChip Affymetrix Human Genome U133 Plus 2.0 arrays. Post hybridization washes were preformed on an Affymetrix GeneChip Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip Scanner 3000. Scanned arrays were normalized using GCRMA in Partek software (Partek Incorporated. St. Louis, MO). Differentially enriched RNAs were found after performing One-way ANOVA analysis comparing immunoprecipitated samples against control samples. Final lists of genes were obtained by filtering the data from the statistical results according to fold enrichment more than 2 and a p value associated of less than 0.05 for features with signal well above the background.
We compared differences in fetal and adult T cells by performing whole genome profiling on sort-purified T cells (naïve CD4+ and Treg cells) from human fetal specimens (18-22 gestational weeks) and adult specimens (age 25-40 years old). Fetal and Adult Naïve CD4+ T cells phenotype: CD3+CD4+CD45RA+CCR7+CD27+ Fetal and Adult CD4+CD25+ Treg phenotype: CD3+CD4+CD25bright
Gene expression analysis identified 27 of these 744 p300 and pol II associated genes as significantly increased (p≤ 0.05) within the first hour following mitogen stimulation Keywords: time course
Periodontal infections have been associated with systemic inflammation and risk for atherosclerosis and vascular disease. We investigated the effects of comprehensive periodontal therapy on gene expression of peripheral blood monocytes. Approximately 1/3 of the patients showed substantial changes in expression in genes relevant to innate immunity apoptosis, and cell signaling. We concluded that periodontal therapy may alter monocytic gene expression in a manner consistent with a systemic anti-inflammatory effect. Keywords: time course,disease state analysis
To understand the biological pathways involved in twin-twin transfusion syndrome (TTTS) by performing global gene expression analysis of amniotic fluid (AF) cell-free RNA Analysis of paired data from TTTS cases and controls revealed differential expression of 801 genes which were significantly enriched for neurological disease and cardiovascular system pathways. We also identified cardiovascular genes and pathways associated with the presence of critically abnormal Doppler measurements in Stage III TTTS recipients.
Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation,we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressingmarkers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non- HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.
Although it is well-recognized that apoptosis senescence, and increased production of inflammatory cytokines and catabolic products are important factors in the degeneration of the human intervertebral disc, there is poor understanding of the underlying cause. The objective of the present study was to analyze gene expression patterns in the human annulus disc tissue.
Heat-Shock Factor 1 (HSF1) master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their non-transformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, even these genes are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.
T cell dysfunction is an important feature of many chronic viral infections. In particular it was shown that PD-1 regulates T cell dysfunction during chronic LCMV infection in mice and PD-1 high cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, HCV and HBV. However, it is not known if PD-1 high cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function and gene expression profiles of PD-1 high versus PD-1 low CD8 T cells in the peripheral blood of healthy human adults as following: 1) The percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans and PD-1 was expressed by the memory CD8 T cells. 2) PD-1 high CD8 T cells in healthy humans did not significantly correlated with the PD-1 high exhausted gene signature of HIV specific human CD8 T cells or chronic LCMV specific CD8 T cells from mice. 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults. 4) PD-1 was expressed by the effector memory (TEM) compared to ‘terminally differentiated effector’ (TEMRA) CD8 T cells. 5) Finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed.
This study characterizes the effects of chronic Hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naive HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136 gene signature including 66 genes elevated in infected individuals. Most of the up-regulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15 and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. Validation of these genes was carried out using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the up-regulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of interferon-stimulated genes.
Microarray profiles of native HeLa cells and RNF185/BSK65 gene silenced HeLa cells using siRNAs LMG2 and LMG4 were monitored using Affymetrix U133 Plus 2.0 microarrays.
We investigated whether we could identify gene expression profiles in initial core biopsies of breast cancer samples that would permit to a) predict a clinically meaningful response to Epi/Doc in terms of tumor size reduction b) predict a profound reduction in intratumoral Ki67 protein expression, and c) predict an in vitro response to Epi/Doc in the ATP-TCA.
Responses of the Human Airway Epithelium Transcriptome to In Vivo Injury To identify genes participating in repair of the human airway epithelium following injury we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed the recovered epithelium for relative levels of gene expression using Affymetrix high-density oligonucleotide microarrays with TaqMan PCR confirmation. Histologic assessment showed that the epithelium was denuded immediately following injury, at 7 days the epithelium was completely covered but partially de-differentiated, and by 14 days there was close to normal proportions of differentiated cells. Gene expression analysis was carried out with both the Affymetrix Microarray Suite 5.0 and Robust Multi-array Average algorithms, applying a multiple test correction to identify bona fide changes in gene expression. At day 7, there were substantial differences in the gene expression pattern compared to the resting epithelium, with a distinctive airway epithelial “repair transcriptome” of actively proliferating cells in the process of re-differentiation. The repair transcriptome at 7 days was dominated by genes encoding proteins involved in cell cycle regulation, transcription, signal transduction, metabolism and transport. Interestingly, the majority of cell cycle genes differentially expressed at day 7 belonged to the G2 and M late phases of the cell cycle, suggesting that the proliferating cells are relatively synchronized 1 wk following injury. At 14 days post-injury, the majority of the gene expression changes observed at day 7 were no longer observed, with the expression profile similar to that of resting airway epithelium. Using a class prediction algorithm, a group of 50 genes dominated by cell cycle genes, that represent a human airway epithelial “repair signature” was identified. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in human airway epithelial diseases. Keywords: response to airway injury
Early onset sepsis due to Group B streptococcus (GBS) leads to neonatal morbidity increased mortality and long term neurological deficencies. Interaction between septicemic GBS and confluent monlayers of human coronary artery endothelial cells (HCAEC) was analyzed by a genome wide expression profiling. Regulation of selected genes and proteins identified in the gene array analysis was confirmed by Real Time RT-PCR assay (Granulocyte chemotactic protein 2 (CXCL6)), ELISA (Urokinase, Cyclooxygenase 2 (COX2), Granulocyte chemotactic protein 1 (IL8)) and Western Blotting (Heme oxygenase1, BCL2 interacting protein (BIM)) at various time points between 4 and 24 hours. In total, 124 genes were differentially regulated (89 upregulated, 35 downregulated) based on a more than 3-fold difference to unstimulated HCAEC. Regulated genes are involved in apoptosis, hemostasis, oxidative stress response, infection and inflammation. We confirmed upregulation of urokinase (UPA), COX2, HMOX1 and BCL2 interacting protein and downregulation of CXCL6 and IL8. These results indicate that GBS infection might lead to impaired function of the innate immune system and might contribute to hemorrhagic and inflammatory complications during GBS sepsis. Keywords: stimulated HCAEC (with S. agalactiae) vs unstimulated control cells
Analysis of differentiated Caco-2 intestinal epithelial cell line cocultured with probiotics L. acidophilus NCFM™ B. lactis 420, L. salivarius Ls-33 bacterial cells or treated with cell-free supernatant, and with E. coli O157:H7 cell-free supernatant. Lactobacillus and Bifidobacterium are important genera suggested to be beneficial for human health and E. coli O157:H7 is a pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. Results provide insight into the mechanisms underlying the beneficial effects of probiotics on intestinal epithelial cells and a comparison to pathogenic E. coli.
Hyperthermia is widely used to treat patients with various cancers. 42.5˚C is well known as the inflection point of hyperthermia and generally up to 42˚C of hyperthermia is used in clinical cases combined with other therapies. Here the effects of heat stress at 42 or 44˚C for 15 min on the gene expression in human lymphoma U937 cells were investigated using an Affymetrix GeneChip system. The cells were treated with heat stress (42 or 44°C for 15 min), followed by incubation for 0, 1, 3 or 6 h at 37°C. The percentage of DNA fragmentation was 8.4 ± 2.2 (mean ± SD) at 42°C for 6 h and 21.0 ± 2.0 at 44°C for 6 h. Of approximately 47,000 probe sets analyzed, many genes that were differentially expressed by a factor 2.0 or greater were identified in the cells treated with heat stress at 42 and 44°C.
This SuperSeries is composed of the SubSeries listed below.
To identify candidate genes involved in enhanced tumorigenicity of CD133+ liver tumor-initiating cells Affymetrix Human Genome U133 Plus GeneChip 2.0
Nine cigarette smoke condensates (CSCs) were produced under a standard ISO smoking machine regimen and one was produced by a more intense smoking machine regimen. These CSCs were used to treat primary normal human bronchial epithelial cells for 18 hours.
Identify Wnt3A responsive signature in lung adenocarcinoma cells
Embryos secrete preimplantation factor (PIF) a peptide present in maternal circulation during viable pregnancy. We compared downstream synthetic PIF effect on gene expression in non-pregnant Human Endometrial Stromal Cell (HESC) and First Trimester Decidual cell (FTDC) culture to mimic the maternal intrauterine environment during embryo implantation and trophoblast invasion.
Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independent of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin-responsiveness in primary human muscle cells from patients with type 2 diabetes. Results: No single gene was differently expressed after correction for multiple testing and no biological pathway was differently expressed using both approaches for global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis, PPARGC1A and NRF-1, or in mitochondrial mass between diabetic and control myotubes. Keywords: Cell culture, genetic, oxidative phosphorylation, microarray, skeletal muscle, type 2 diabetes
We have characterized gene expression changes in HeLa cells following long term depletion of Cyclin T2 or Cyclin T1 using shRNA
• The origin of biological samples (In vitro infection of HCT-8 cells with Cryptosporidium parvum) Monolayers of the HCT-8 cells (ATCC CCL-244 American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and additional nutritional supplements. Cells in log phase were plated at 2×106 cells per 150 mm tissue culture dish and infected with sterilised Cryptosporidium parvum oocysts (Iowa strain). Purified oocysts were suspended in phosphate buffered saline, sterilised in 33% bleach for 7 min on ice, washed in Hank's buffered saline solution, and added to HCT-8 cultures at a ratio of one oocyst per cell. Following a 2 h excystation period at 37 °C, cells were washed with warm Hank's buffered saline solution, and cells were incubated in fresh supplemented media. Mock-infected cultures were treated identically, with the exception that oocysts were not added to the cultures. Three independent mock- and C. parvum-infected cultures were prepared for analysis. • The origin of mRNA samples At 6, 12, 24, 48, and 72h post-inoculation, cell culture media were removed and cultures were immediately lysed by the addition of 3.5 ml of Trizol® reagent (GIBCO BRL Life Technologies) directly to the culture plate. Total RNA was prepared as described by the manufacturer, and poly(A) mRNA was isolated using oligo-dT cellulose columns (Amersham Pharmacia Biotech). The qualities of total RNA and poly(A) mRNA preparations were assessed by Northern blot. • Protocols for conducting microarray hybridization Poly(A) mRNA was converted into ‘target’ suitable for hybridisation to Affymetrix microarray chips according to protocols provided by Affymetrix Inc. (Santa Clara, CA). Briefly, 2.0 g of poly(A) mRNA was reverse-transcribed to prepare double-stranded cDNA using T7-(dT)24 primer (GENSET Corp., La Jolla, CA) and Superscript II reverse transcriptase (GIBCO). Approximately 1 g of cDNA was used for in vitro transcription in the presence of biotinylated UTP and CTP using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Inc). This labelled cRNA was fragmented in 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94 °C for 35 min. The integrity of cRNA and the efficiency of fragmentation were monitored by Northern blot analysis (not shown). Fragmented biotinylated cRNA targets were hybridised to HG-U95Av2 chips (Affymetrix) that contain probe sets for 12,600 human gene/transcripts. Fifteen micrograms of fragmented cRNA and appropriate controls were hybridised to the chips at 45 °C for 16 h with constant rotation at 60 rev./min. The chips were subsequently washed and stained with streptavidin–phycoerythrin conjugate using the GeneChip Fluidics station protocol EukGE_WS2 (Affymetrix). Following washing and staining, microarray chips were scanned twice at 3 m resolution using a Hewlett-Packard confocal scanner and hybridisation intensities for each of the genes/transcripts were collected from scanned images. • Analysis of microarray data Gene expression data were initially analysed with the GeneChip® expression analysis software (Affymetrix Microarray Suite, version 5.0). The fluorescence intensity of the genes/transcripts was measured for each probe array and, to minimise discrepancies due to non-biological variations, normalised by global scaling to 1000. Further data analyses were performed using GeneSpring software (version 6.0; Silicon Genetics, Redwood City, CA). Per gene normalisation was applied in which the expression signal of each gene in C. parvum-infected cells (raw data) was normalised to the median of its measurements in the mock-infected samples (control); and the ratio of expression levels between mock and infected samples was calculated as the mean value of normalised signal vs. control signal among three replicates. To identify genes with significantly altered expression, a series of statistical analyses (filtering) were performed: cut-off values for ratio of expression levels 1.80 and 0.55 were used to filter genes with expression level fold changes greater than ±1.8 in all three independent samples. Genes with fold change variations >1.5 across the three samples were excluded. Furthermore, a ‘statistical group comparison’ using Student's t-test/ANOVA was conducted to compare mean expression levels between mock-infected and infected samples, and the genes with significant differential expression (P<0.05) were selected. Identified differentially expressed genes were then annotated using GeneSpring's ‘Build Simplified Ontology’ constructor which hierarchically groups genes into meaningful biological categories (gene lists) based on the Gene Ontology Consortium Classifications. Various lists of regulated genes were created by cross-referencing annotated gene lists and applying assorted statistical and visualisation methods. Keywords: time-course
We studied transcriptional changes by Affymetrix human microarrays in 2 DLBCL cell lines as a result of shRNA mediated knockdown of EZH2. In eukaryotes epigenetic post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is responsible for repressing target gene expression through methylation of histone H3 on lysine 27 (H3K27). Over-expression of EZH2 is implicated in tumorigenesis and correlates with poor prognosis in multiple tumor types. Recent reports have identified somatic heterozygous mutations of Y641 and A677 residues within the catalytic SET domain of EZH2 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The Y641 residue is the most frequently mutated residue, with 22% of GCB (Germinal Cell B-cell) DLBCL and FL harboring mutations at this site. These lymphomas exhibit increased H3K27 tri-methylation (H3K27me3) due to altered substrate preferences of the mutant enzymes. However, it is unknown whether direct inhibition of EZH2 methyltransferase activity alone will be effective in treating lymphomas carrying activating EZH2 mutations. Herein, we demonstrate that GSK126, a potent, highly-selective, SAM-competitive, small molecule inhibitor of EZH2 methyltransferase activity, decreases global H3K27me3 levels and reactivates silenced PRC2 target genes. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and dramatically inhibits the growth of EZH2 mutant DLBCL xenografts in mice. Together, these data demonstrate that pharmacological inhibition of EZH2 activity may provide a promising treatment for EZH2 mutant lymphoma.
Nonsense-mediated RNA decay (NMD) is regulated by a variety of cellular stresses. We expose U2OS cells to several stresses and assess RNA expression in the absence of transcription (i.e. stability). These studies identify transcripts that are stabilized by the physiological inhibition of NMD.
Compare the gene expression profile among human CD34+ cord blood cells infected with MIGR1 MIGR1-AML1-ETO or MIGR1-AML1-ETO∆NHR1 AML1-ETO promotes the self-renewal of human hematopoietic stem/progenitor cells (HSPCs). We found deletion of NHR1 domain abrogates AML1-ETO induced expasion of HSPCs.
With the aging population there is a growing focus on dementia, especially Alzheimer’s disease (AD). The molecular basis underlying the pathogenesis of AD is gradually being elucidated. Increasing evidence has shown that the immunological function of leukocytes plays a crucial role in the development of neurodegenerative disorders. However, there have been few studies among the Taiwanese population. The aim of this study was to investigate potential biomarkers for early diagnosis of Alzheimer’s disease from blood leukocytes.
We established two clones of induced pluripotent stem cells (iPSC) with the presenilin 2 mutation N141 (PS2-1 iPSC and PS2-2 iPSC) by retroviral transduction of primary human fibroblasts. To show the similarity among 201B7 iPSC, PD01-25 iPSC(Sporadic Parkinson's disease patient derived iPSC), PS2-1 iPSC, PS2-2 iPSC, this experiment was designed.
Micro-inflammation and gut dysfunction are features of diarrhea-irritable bowel syndrome (d-IBS) patients although the underlying interacting molecular mechanisms remain mostly unknown. Therefore, we aimed to identify critical networks and signaling pathways active in chronic diarrhea-associated inflammation. Keywords: Comparison of gene expression
The environmental carcinogen (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), causes bulky-adduct DNA damages, triggers certain signaling pathways, and elicits gene expression changes. Here, we focused on the temporal gene expression changes induced by a low concentration (0.05 µM) BPDE in human amnion epithelial FL cells. Differential gene expression profiles at 1, 10 and 22 h post BPDE treatment were obtained using Affymetrix HG-U133 Plus 2.0 oligonucleotide microarrays. A cohort of gene expression changes related to cell cycle progression, cell growth or apoptosis, stress response, and post-transcriptional regulation was validated with quantitative real-time RT-PCR. The alteration of several cell cycle-related genes was correlated and possibly contributed to the cell cycle arrest phenotype. Paradoxical transcriptional regulations regarding cell growth or apoptosis emerged in response to BPDE treatment, which indicated that cell fate was determined by integrated signals. The temporal transcriptional changes would be of help to clarify the molecular mechanism of cellular response to BPDE. Keywords: time course
Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Keywords: comparison of two phenotypes, resolver and pustule former
Parental MM6 cells as an additional control, were treated with LPS and PMA. Genes affected by the treatments were identified. HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4+ T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4+ T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that are Cyclin T1-dependent for their induction in activated CD4+ T cells and during macrophage differentiation and activation. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value < 0.00021). SiRNA depletions of two CTDGs identified here, CDK11 and Casein kinase1gamma1, suggest that these genes are also involved in HIV-1 replication. It is therefore likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors. Keywords: knockdown
Background and Purpose Aneurysmal subarachnoid hemorrhage almost always from saccular intracranial aneurysm (sIA), is a devastating form of stroke that affects working age population. Cellular and molecular mechanisms predisposing to the rupture of the sIA wall are largely unknown. Such knowledge would facilitate the design of novel diagnostic tools and therapies for the sIA disease. Methods We compared the whole genome expression profile of eleven ruptured sIA wall samples, resected at a median of 15 hours after rupture, to that of eight unruptured ones. Signaling pathways enriched in the ruptured sIA walls were identified with bioinformatic analyses. Their transcriptional control was predicted in silico by seeking the enrichment of conserved transcription factor binding sites in the promoter regions of differentially expressed genes. Results Overall, 686 genes were significantly upregulated and 740 downregulated in the ruptured sIA walls. Significantly upregulated biological processes included: response to turbulent blood flow; chemotaxis; leukocyte migration; oxidative stress; vascular remodelling; and extracellular matrix degradation. Toll like receptor (TLR) signalling and NF-κB, HIF1A and ETS transcription factor binding sites were significantly enriched among the upregulated genes. Conclusions We identified pathways and candidate genes associated to the rupture of human sIA wall. These results provide a molecular basis analysis (a) to identify rupture-prone sIAs and (b) to prevent their rupture. Novel measures to prevent the rupture of sIA wall may include inhibition of response to turbulent blood flow, leukocyte migration, TLR signalling, or blockade of NF-κB, HIF1A and ETS transcription factors.
B lymphoblastoid cell lines were obtained from Coriell Cell Repositories. Cell lines were grown according to Coriell guidelines and total RNA was extracted labeled, and hybridized to an Affymetrix Human Genome Focus Array as previously described. We found extensive variation in gene-expression levels and estimate that ~83% of genes are differentially expressed among individuals and that ~17% of genes are differentially expressed among populations. By decomposing total gene-expression variation into within- versus among-population components, we find that most expression variation is due to variation among individuals rather than among populations, which parallels observations of extant patterns of human genetic variation. Keywords: resting expression levels in B lymphoblastoid cell lines
This SuperSeries is composed of the SubSeries listed below.
TNF antagonists are routinely used in severe rheumatoid arthritis (RA) patients who failed conventional DMARD therapy. According to large clinical trials the three available drugs (adalimumab, infliximab and etanercept) display similar effects in terms of efficacy, tolerability and side effects. These studies also indicate that about 25% of RA patients treated with TNF-antagonists do not display any significant clinical improvement. The aim of this study was to investigate global molecular patterns in synovial biopsies from RA patients obtained 12 weeks after initiation of adalimumab therapy.
The pathogenesis of acne has been linked to multiple factors such as increased sebum production inflammation, follicular hyperkeratinization, and the action of Propionibacterium acnes within the follicle. 13-cis Retinoic Acid (13-cis RA, isotretinoin) is the most potent agent in acne treatment. Surprisingly, its mechanism of action in acne is still unknown. Gene expression profiling of skin from 8 patients treated with isotretinoin was performed to gain insights into its mechanism of action. Skin biopsies were obtained from the patients at baseline and at 8 weeks isotretinoin treatment. Gene array expression profiling was conducted using Affymetrix HG-U133A 2.0 arrays in order to examine changes in gene expression as a result of treatment. After treatment, 784 genes were significantly changed: 197 up-regulated and 587 down-regulated. The majority of genes that were up-regulated at 8 weeks encode structural proteins of the extracellular matrix such as collagens, fibulin and fibronectin. The preponderance of genes that were down-regulated at 8 weeks are involved in the metabolism of steroids, cholesterol and fatty acids. Keywords: drug treatment effects
The similarity in gene-expression profiles suggest that PGL2 like SDHD, is involved in the functionality of the SDH complex, and that tumor formation in these three subgroups involves the same pathways as in SDH linked paragangliomas. We were not able to clarify the identity of PGL2 on 11q13. The lack of differential gene-expression of chromosome 11 genes might indicate that chromosome 11 loss, as demonstrated in SDHD-linked paragangliomas, is an important feature in the formation of a paraganglioma regardless of the genetic background. Keywords: disease state analysis
Affymetrix U133A comparison of two groups (10 samples each): untreated (androgen-dependent) primary prostate cancer (Gleasons 5-9) and androgen-independent primary prostate cancer. All samples were microdissected for tumor cells only. Keywords = advanced prostate cancer Keywords = androgen-independence Keywords = laser capture microdissection Keywords = RNA amplification Keywords: other
To study characteristics of the orapharyngeal epithelia which may influence susceptibility or resistance to HIV we performed microarray analysis of the tonsil and gingival epithelium.Tonsil epithelium has been implicated in HIV pathogenesis, but its role in oral transmission remains controversial. We performed microarray analysis of Laser Capture Microdissected tonsil and gingival epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared to the epithelium of another oro-pharyngeal site, gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV binding molecules, FcRγIII, complement receptor 2, and various complement components. This increased expression of molecules involved in viral recognition, binding and entry may favor virus-epithelium interaction in an environment with reduced innate anti-viral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa. Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate anti-viral factors may render the tonsil a potential site for oral transmission. Keywords: Cross sectional
Identification of potential tumor suppressor genes using the GINI strategy in Mantle Cell Lymphoma cell lines Keywords: response to drug treatment
Imatinib has become the current standard therapy for patients with chronic myelogenous leukaemia (CML). For a better understanding of the Imatinib-related molecular effects in vivo we assessed gene expression profiles of Philadelphia Chromosome positive (Ph+) CD34+ cells from peripheral blood of 6 patients with de novo CML in chronic phase. After 7 days of treatment with Imatinib the Ph+ CD34+ cells were reassessed to look for changes in the transcriptome. The expression level of 303 genes was significantly different comparing the transcriptome of the Ph+ CD34+ cells before and after 7 days of Imatinib therapy (183 down-regulated, 120 up-regulated, lower bound ≥1.2-fold). For a substantial number of genes governing cell cycle and DNA replication, the level of expression significantly decreased (CDC2, RRM2, PCNA, MCM4). On the other hand, therapy with Imatinib was associated with an increase of genes related to adhesive interactions, such as L-selectin or CD44. A group of 8 genes with differential expression levels were confirmed using a gene specific quantitative real-time PCR. Thus, during the first week of treatment, Imatinib is preferentially counteracting the bcr-abl induced effects related to a disturbed cell cycle and defective adhesion of leukemic Ph+ CD34+ cells.
To determine whether enhanced self-renewal and tumorigenicity in UT2 cells (derived from the second human–mouse xenotransplantation of U2OS cell-formed osteosarcoma tissues) correlate with increased expression of stem/progenitor cell-associated genes we measured global gene expression in MSC, U2OS and UT2 cells by microarray analysis. Compared to U2OS and MSC, molecules involved in regulation of self-renewal signaling pathways of cancer stem cells were also up-regulated in UT2 cells, including those in the Notch, Wnt, and TGF-beta pathways. These data suggest a genetic basis for the enhanced self-renewal and tumorigenicity of osteosarcoma-initiating cell in UT2 cells.
We have studied the genes activated in human liver transplantation to identify potential target genes for the prevention or treatment of related injuries. In a first protocol in order to evaluate the effect of Ischemia-Reperfusion Injury (IRI) on gene expression profile, we compared gene expression levels in transplanted-reperfused livers versus basal values in donor livers, identifying 795 genes significantly modified in human liver after transplantation. Some genes are likely to be completely activated by IRI, as they are not expressed at all in basal livers. In a second protocol, in order to identify gene dysregulations already present in donor livers, which might affect gene expression profile after transplantation, gene expression evaluated in the first study was compared to control livers (espression data retrieved from ArrayExpress database). About 900 genes in donor livers are dysregulated if compared to the control condition. At least 400 of them remain dysregulated or become more and more dysregulated after transplantation.
We are studying signaling pathways and growth properties of cultured human ovarian cancer cells that are expressing the G protein-coupled receptor luteinizing hormone receptor (LHR),particularly interested in the changes that occur when the receptor is activated by its cognate ligand, gonadotropin (LH). To investigate these questions, we have employed the SKOV3 ovarian cancer cell line that has been stably transfected with LHR, and can then test the response of these cells in culture following exposure to LH.
Combined overexpression of miR-125b with miR-99a and/or miR-100 induced VCR resistance in ETV6-RUNX1-positive leukemic cells Reh. We used microarrays to detail the global changes in gene expression of Reh cells upon enforced expression of miR-125 per se compared with combination of overexpression of miR-125b miR-100 and/or miR-99a
Purpose: Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior resistance to androgen ablation, and frequent but short responses to chemotherapy. The model systems we report reflect the biology of the human disease and can be used to improve our understanding of SCPC and to develop new therapeutic strategies for it. Experimental Design: We developed a set of CRPC xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts and used a panel of 60 human tumors to validate our findings using immunohistochemistry. Results: We show that SCPC and LCNEC xenograft models retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other M-phase cell cycle genes in the absence of AR, retinoblastoma (RB1) and cyclin D1 (CCND1) expression and confirm these findings in a panel of CRPC patients’ samples. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts and that there is amplification of the UBE2C locus and microdeletions of RB1 in a subset of these, but no AR nor CCND1 deletions. Moreover, the AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts. Conclusion: Modeling human prostate cancer with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Futures studies in the xenografts will address the functional significance of the findings.
Chlamydia trachomatis is an obligate intracellular Gram-negative bacterium that frequently causes an asymptomatic genital tract infection gradually cleared by host immunity Transcriptome profiles were made of endometrial tissue from women with or without genital tract C. trachomatis infection, to characterize host responses to infection. Profiles showed that infection polarized host defense toward Type 2 immune responses. Responses included fibrin deposition, enhanced wound repair, and tissue remodeling.
We compared PC3 cells with or without harboring the wild-AR construct in the growth conditions of 1nM R1881 10nM R1881 and ethanol (the solvent for R1881). The MOCK control is PC3 cells transfected with the empty vectors.
From the result of comparative the gene expression analyses of human hepatoma cell line HepG2 following exposures of three heavy metals; arsenic, cadmium and nickel and three carcinogens; N-dimethylnitrosoamine (DMN), 12-O-tetradecanoylphorbol-13-acetate (TPA) and tetrachloroethylene (TCE), 31-55% of the genes altered by As, Cd and Ni exposure were overlapped with those by three model carcinogen exposures in our experiments. In particular, three heavy metals shared certain characteristics with TPA and TCE in remarkable up-regulations of the genes associated with progression of cell cycle, which might play a central role in heavy metal carcinogenesis. In addition, this characteristic of gene expressions alteration was counteracted by intracellular accumulation of vitamine C in As-exposed cells but not in Cd- and Ni-exposed cells. These results suggest that the cell proliferative responses are caused by reactive oxygen species mainly in As exposure, while other mechanisms would be involved in these responses in Cd and Ni exposures. Keywords: other
The species Staphylococcus (S.) aureus harbors 19 superantigen gene loci six of which are located in the enterotoxin gene cluster (egc). While these egc superantigens are far more prevalent in clinical S. aureus isolates than non-egc superantigens, they are not a prominent cause of toxic shock. Moreover, neutralizing antibodies against egc superantigens are very rare, even among carriers of egc-positive S. aureus strains. In search of an explanation we have tested two non-exclusive hypotheses: 1) egc and non-egc superantigens have unique intrinsic properties and drive the immune system into different directions; 2) egc and non-egc-superantigens are released by S. aureus under different conditions, which shape the immune response. A comparison of three egc (SEI, SElM and SElO) and three non-egc superantigens (SEB, SElQ, TSST-1) revealed that both induced proliferation of human PBMC with comparable potency and elicited similar Th1/Th2-cytokine signatures. This was supported by gene expression analysis of PBMC stimulated with one representative superantigen from each group (SEI and SEB). They induced very similar transcriptional changes, especially of inflammation-associated gene networks, corresponding to a very strong Th1- and Th17-dominated immune response. In contrast, the regulation of superantigen release differed markedly between both superantigen groups. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc superantigens were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc superantigens is not due to their intrinsic properties, which are very similar, but caused by their differential release by S. aureus.
Objective: Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases. Investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Methods: We developed a sensitive gene expression based method to overcome the challenges of measuring PC using flow cytometry. Whole genome microarray analysis of sorted cellular fractions identified a panel of genes IGHA, IGJ, IGKC, IGKV, and TNFRSF17, expressed predominantly in PC. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy; evaluating the relationship between PC and other autoimmune disease-related genes; and estimating PC levels in affected blood and tissue from multiple autoimmune diseases. Results: The PC signature was highly sensitive and capable of detecting as few as 300 PCs. The PC signature was reduced over 90% in scleroderma patients following anti-CD19 treatment and this reduction was highly correlated (r = 0.77) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed 30-35% of lupus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusion: This newly developed PC signature provides a robust and accurate method to measure PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases that may benefit from PC depleting therapy.
DUSP1 is involved in different cellular pathways including cancer cell proliferation angiogenesis, invasion and resistance to chemotherapy. To understand more about the cellular responses regulated by DUSP1 in NSCLC cells, we interfered DUSP1 expression in the NSCLC cell line H460 and studied the changes in gene expression differentially regulated by this phosphatase. Keywords: Expression profiling by array in cells with genetic modification
A cohort of distinct thyroid neoplasias was hybridized onto the Affymetrix U95 GeneChip
To identify candidate genes involved in enhanced tumorigenicity of CD133+ liver tumor-initiating cells Affymetrix Human Genome U133 Plus GeneChip 2.0
Shikonin is a active component isolated from the roots of the traditional Chinese herb Lithospermum erythrorhizon and exhibits multiple pharmacological properties such as anti-oxidant, anti-inflammatory, and anti-tumor effects. Here, the effects of shikonin on the gene expression of human lymphoma U937 cells were investigated using an Affymetrix GeneChip system. The cells were treated with 100 nM shikonin, and followed by incubation for 3h at 37°C. Flow cytometry analysis with Annexin-V and propidium iodide demonstrated that no cell death was observed in the cells at 100 nM shikonin. Of approximately 47,000 probe sets analyzed, many genes that were differentially expressed by a factor 2.0 or greater were identified in the cells treated with the compound.
MicroRNAs are small non-coding RNA species some of which are playing important roles in cell differentiation. However, the level of participations of microRNAs in epithelial cell differentiation is largely unknown. Here, we found that expression levels of four microRNAs (miR-210, miR-338-3p, miR-33a and miR-451) were significantly increased in differentiated stage of T84 cells, compared with undifferentiated stage. Additionally, we demonstrate that miR-338-3p and miR-451 contribute to the formation of epithelial basolateral polarity by facilitating translocalization of beta1 integrin to the basolateral membrane. However, candidate target mRNAs of miR-338-3p and miR-451 and the mechanism behind observed phenomena is uncertain. Then, we performed comprehensive gene expression analysis to identify candidate target mRNAs and understand their mechanisms.
Rheumatoid arthritis (RA) a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patient with RA by oligonucleotide microarrays. Gene expression profiles reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress. Keywords: cell type comparison, disease state analysis
Left ventricular mass (LVM) and cardiac gene expression are complex traits regulated by factors both intrinsic and extrinsic to the heart. To dissect the major determinants of LVM we combined expression quantitative trait locus1 and quantitative trait transcript (QTT) analyses of the cardiac transcriptome in the rat. Using these methods and in vitro functional assays, we identified osteoglycin (Ogn) as a major candidate regulator of rat LVM, with increased Ogn protein expression associated with elevated LVM. We also applied genome-wide QTT analysis to the human heart and observed that, out of 22,000 transcripts, OGN transcript abundance had the highest correlation with LVM. We further confirmed a role for Ogn in the in vivo regulation of LVM in Ogn knockout mice. Taken together, these data implicate Ogn as a key regulator of LVM in rats, mice and humans, and suggest that Ogn modifies the hypertrophic response to extrinsic factors such as hypertension and aortic stenosis.
B19V NS1 is known to be cytotoxic and involved in the pathogenesis of B19V infection. Our previous data demonstrated that NS1 impaired the cell-cycle progression of the CD36+ EPCs by inducing a stable G2 arrest. Microarray analysis was used to identify genes whose expressions were associated with the NS1-induced G2 arrest. A total of 1045 genes displayed a more than 1.5-fold differential expression in the NS1-transduced cells. Out of 1045 differentially expressed genes 177 were involved in cell-cycle regulation and 51 were involved in the regulation of cell differentiation. Keywords: RNA
The identification of a marker that is expressed in the conjunctival epithelium but not in the corneal epithelium has been a growing need. A more specific marker of limbal and conjunctival epithelia would be necessary to detect non-corneal epithelial cells on the corneal surface. To search for conjunctival specific marker(s) we first performed preferential gene profiling in the conjunctiva in direct comparison to that in the cornea using microarray technique.
The exit of antigen-presenting cells (APC) and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels is however unknown. Here we show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LEC) leading to expression of the key leukocyte adhesion receptors ICAM-1 VCAM-1 and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of DC via afferent lymphatics. Lastly, we show that TNF_-stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking mAbs. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for anti-inflammatory therapy. Keywords: TNFalpha, Lymphatic endothelium, induction, Inflammation
The trophoblast cell lineage is specified as early as the blastocyst stage leading to the individualization of trophectoderm from pluripotent cells of the inner cell mass. We used a double in vitro transcription mRNA amplification technique and compared trophectoderm with pluripotent stem cells.
We used gene expression profiling of human DCIS and IBC to discover uniquely expressed genes that may also regulate progression.
Traditional Chinese medicines (TCM) usually composed of a mixture of components, may simultaneously target multiple genes/pathways and thus achieve superior efficacy for complex diseases such as cancer. To identify novel mechanisms of action and potential health benefits for a TCM formula Si-Wu-Tang (SWT) widely used for women’s health, we obtained the DNA microarray expression profiles for SWT, its active component ferulic acid, and estradiol in human breast cancer cell line MCF-7 and analyzed the gene expression signatures associated with each treatment using the “Connectivity Map” (cMAP). This study indicates that DNA microarray profiling analysis and cMAP data mining provide a powerful approach to discover unknown mechanisms of actions and identify potential new health benefits for TCM.
We previously found that a native lipoprotein mix with a high VLDL+LDL/HDL ratio causes a global de novoDNA methylation in THP-1 macrophages. In the present experiment we assessed the consequences of global lipoprotein-induced de novo DNA methylation on global gene expression in the same cells. Moreover we sought to use gene expression array data to measure RNA expression levels for candidate factors mediating the epigenetic effects of lipoproteins. Keywords: comparison treated vs. control
Gene expression was compared between four B-cell derived HL cell lines (L428 L1236, L591, KMH2) and GC B cells from three different patients. Keywords: Cell type comparison
Expression patterns of Dendritic cells co cultured with cord blood MSC were compared with cord blood MSC (USSC). Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSC themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second party T-cells evoked abundant proliferation that was inhibited by third party cord blood USSC. Optimal inhibition was seen with one cord blood USSC for every dendritic cell. Blocking HLA-G only saw partial recovery of proliferation. Several cytokines prostaglandins, gangliosides, enzymes like arginase, NO synthase and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition based on the microarrays and functional tests. Although the mechanism by which it does so remains partially undefined and subject to further study, cord blood multipotent stromal cells are strong inhibitors of the immune response and therefore allow their use in tissue regeneration settings in an allogeneic setting. Keywords: Adult stem cells, developmental biology, gene expression, genomics, human cord blood, mesenchymal stem cells, cel type comparison
SOX genes encode a family of high-mobility group transcription factors that play critical roles in organogenesis. Virtually all members of SOX family have been found to be deregulated in tumors of various origins. However little is known about the cellular and molecular behaviours involved in the oncogenic potential of SOX proteins. Cell culture experiments, tissue analysis, and molecular profiling revealed that SOX2 promotes cell proliferation and tumorigenesis through its transcription regulation of cell cycle related genes in breast cancer cells Keywords: Gene Transfection
Gene network of erythropoietic cells Special attention has recently been drawn to the molecular network of different genes that are responsible for the development of erythroid cells. The aim of our study was to establish in detail the phenotype of early erythroid cells and to compare the gene expression profile of separated early erythroid precursors with the CD34-positive compartment. Multicolor flow cytometric analyses of human bone marrow mononuclear cell fractions (n=20) defined three distinct early erythroid stages. Sorted populations were analyzed for their gene expression profile using Affymetrix array technology. In 4524 genes, a differential regulation was found in CD105-positive erythroid cells as compared to the CD34 common progenitor compartment (2,362 upregulated genes). A highly significant difference was observed in the expression level of genes with known functions, e.g. transcription factors, heme synthesis, iron and mitochondrial metabolism and transforming growth factor-ß signaling. A comparison with recently published data showed over 800* genes which as yet have not been reported to be upregulated in the early erythroid lineage as compared to the CD34 compartment . The gene expression level within distinct pathways can be illustrated directly by applying the Ingenuity software program. The results of the gene expression analyses can be seen at http://www.ncbi.nlm.nih.gov/geo/info/submission.
The MYC oncogene which is commonly mutated/amplified in tumors, represents an important regulator of cell growth owing to its ability to induce both proliferation and apoptosis. Recent evidence links MYC to altered miRNA expression, thereby suggesting that MYC-regulated miRNAs might contribute to tumorigenesis. To further analyze the impact of MYC-regulated miRNAs we investigated a murine lymphoma model harboring the MYC transgene in a Tet-off system in order to control its expression. Microarray-based miRNA expression profiling revealed both known and novel MYC targets. Among the miRNAs repressed by MYC we identified the potential tumor suppressor miR-26a, which possessed the ability to attenuate proliferation in MYC-dependent cells. Interestingly, miR-26a was also found to be deregulated in primary human Burkitt lymphoma samples, thereby likely being of clinical relevance. While today only few miRNA targets have been identified in human disease, we could show that ectopic expression of miR-26a influenced cell cycle progression by targeting the bona fide oncogene EZH2, a Polycomb protein and global regulator of gene expression yet unknown to be regulated by miRNAs. Thus, in addition to directly targeting protein-coding genes, MYC modulates genes important to oncogenesis via deregulation of miRNAs, thereby vitally contributing to MYC-induced lymphomagenesis.
We investigated the transcriptional effects of p63 binding by analyzing ME180 cells depleted for all p63 isoforms via expression of a small hairpin RNA (shRNA) targeting the p63 oligomerization domain. Keywords: differential expression profiling from transcription factor depletion
Peripheral blood samples were collected before (0 hour) and at 24 hours after exposure from healthy subjects who participated in previous controlled exposures to ultrafine carbon particles (UFP 50 microg/m3) or filtered air (FA)(n = 3 each). The exposure time was 2 hours. RNA from mononuclear cell fraction (>85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. We used microarray to explore significantly altered genes after ultrafine carbon particle exposure.
Study was carried out to examine how E2 and TNFa together influence gene expression in breast cancer cells compared to either factor alone. Keywords: drug response
We decompose the genome-wide expression patterns in 38 embryonic human lung (53-154 days post conception/dpc) into their independent dominant directions of transcriptomic sample variation in order togain global insight of the developing human lung transcriptome.The characteristic genes and their corresponding bio–ontologic attribute profile for the latter were identified. We noted the over–representation of lung specific attributes (e.g., surfactant proteins) traditionally associated with later developmental stages, and highly ranked attributes (e.g., chemokine–immunologic processes) not previously reported nor immediately apparent in an early lung development context. We defined the 3,223–gene union of the characteristic genes of the 3 most dominant sources of variation as the developing lung characteristic sub–transcriptome (DLCS). It may be regarded as the minimal gene set describing the essential biology of this process. The developing lung series in this transcriptomic variation perspectiveform a contiguous trajectory with critical time points that both correlate with the 2 traditional morphologic stages overlapping -154 dpc and suggest the existence of 2 novel phases within the pseudoglandular stage. To demonstrate that this characterization is robust, we showed that the model could be used to estimate the gestational age of independent human lung tissue samples with a median absolute error of 5 days, based on the DLCS of their lung profile alone. Repeating this procedure on the homologous transcriptome profiles of developing mouse lung 14–19 dpc, we were able to recover their correct developmental chronology. Whole human fetal lung gene expression profiling from estimated gestational ages 53 to 154 days post conception. Keywords: Whole lung gene expression profiling, lung development, human fetus.
Three nasopharyngeal carcinoma cell lines (CNE1 CNE2, and HONE1) expression patterns against an immortalized nasopharyngeal epithelial cell line NP69.
Time-course and concentration-effect experiments with multiple time points and drug concentrations provide far more valuable information than experiments with just two design-points (treated vs. control) as commonly performed in most microarray studies. Analysis of the data from such complex experiments, however, remains a challenge. Here we present a semi-automated method for fitting time profiles and concentration-effect patterns, simultaneously, to gene expression data. The submodels for time-course included exponential increase and decrease models with parameters such as initial expression level, maximum effect, and rate-constant (or half-time). The submodel for concentration-effect was a 4-parameter Hill model. The method was applied to an Affymetrix HG-U95Av2 dataset consisting of 51 arrays. The specific study focused on the effects of two platinum drugs, cisplatin and oxaliplatin, on A2780 human ovarian carcinoma cells. Replicates were available at most time points and concentrations. Eighteen genes were selected; time-course and concentration-effect were modeled simultaneously. Comparisons of model parameters helped distinguish genes with different expression patterns between the two drug treatments. This overall paradigm can help in understanding the molecular mechanisms of the agents, and the timing of their actions. Keywords: time course , dose response, cisplatin, oxaliplatin, Human ovarian carcinoma cells
Title: Transcriptome analysis of human endometrial tissues from healthy post-menoupausal women reflecting the endometrial response to 3-weeks treatment with tibolone E2 and E2+MPA. In an observational, open, non-randomized, controlled study uterine tissues were collected in order to generate endometrial gene expression profiles. healthy postmenopausal women were enrolled into the following treatment groups: Control-group; Tibolone-group, 2,5 mg of tibolone (administered orally) every day, starting 21 days prior to surgery; Estradiol-group, 2 mg of estradiol (administered orally) every day, starting 21 days prior to surgery; Estradiol+progestagen-group, 2 mg of estradiol (administered orally) and 5 mg of MPA (Medroxy Progesteroneacetate, administered orally) every day, starting 21 days prior to surgery. Pure (100%) endometrium was isolated from the snap-frozen uterine tissues and used for RNA isolation. RNA was labeled and hybridized to whole genome Affymetrix U133plus2 GeneChips containing 54,614 probe sets, representing approximately 47,000 transcripts. Relative to the control group, 940 genes are regulated in the endometrium of E2 treated patients, whereas only 198 genes are significantly regulated in endometria from tibolone or E2+MPA treated patients. Furthermore, only 9% of E2 regulated genes are also regulated by tibolone (85 out of 940), only 5% are also regulated by E2+MPA treatment and the overlap between tibolone and E2+MPA treatment is about 10%. This indicated that tibolone-treatment results in a weak endometrial profile similarity to E2 treatment and no profile similarity to E2+MPA treatment. A more detailed analysis showed that down stream processes, such as regulation of the cell cycle, angiogenesis and cell proliferation are almost not affected by tibolone treatment but, in contrast, are significantly affected by E2. For example, upon staining with Ki67, a marker for mitotic activity, significantly increased stromal as well as glandular cell proliferation was observed in the endometria from the E2-only treated group, while tibolone treatment resulted only in a slight increase in stromal cell proliferation (and no increase in glandular cell proliferation). These results indicate that in contrast to long-term tibolone use, short-term (21-days) use results in some estrogenic stimulation of the endometrium, which is clearly far less and rather different from what is observed during E2 treatment. References: Klaassens et al., 2006 Hanifi-Moghaddam et al., 2007 Verheul et al., 2007
The goal of the microarray experiment was to do a head-to-head comparison of the U1 Adaptor technology with siRNA in terms of specificity at the genome-wide level. U1 Adaptors represent a novel gene silencing method that employs a mechanism of action distinct from antisense and RNA interference (RNAi). The U1 Adaptor is a bifunctional oligonucleotide having a “Target Domain” that is complementary to a site in the target gene's terminal exon and a “U1 Domain” that binds to the U1 small nuclear RNA (snRNA) component of the U1 small nuclear ribonucleoprotein (U1 snRNP) splicing factor. Tethering of U1 snRNP to the target pre-mRNA inhibits 3' end processing (i.e. polyA tail addition) leading to degradation of that RNA species within the nucleus thereby reducing mRNA levels. We demonstrate that U1 Adaptors can specifically inhibit both reporter and endogenous genes. Further, targeting the same gene either with multiple U1 Adaptors or with U1 Adaptors and small interfering RNAs (siRNAs), strongly enhances gene silencing, the latter as predicted from their distinct mechanisms of action. Such combinatorial targeting requires lower amounts of oligonucleotides to achieve potent silencing.
Human microvascular endothelial cells (HMVEC) treated with vascular endothelial growth factor (VEGF) Antrhax Edema Toxin (ET), or the Epac activator, 8-pCPT-2'-O-Me-cAMP (8CPT) Human microvascular endothelial cells (HMVEC) were treated with VEGF alone or VEGF in combination with either the the Epac-specific cAMP-mimetic, 8-pCPT-2'-O-Me-cAMP (8CPT), or anthrax edema toxin (ET), an adenylyl cyclase. ET or 8CPT can inhibit VEGF-mediated chemotaxis and angiogenesis. The goal of the study was to identify genes regulated by cAMP production (ET) or by activation of Epac/Rap (8CPT) that may mitigate the effects of VEGF treatment.
Activation of Signal Transducer and Activator of Transcription 3 (STAT3) is common in prostate cancers. STAT3 may induce cell proliferation and resistance to apoptosis as well as promote tumor angiogenesis, invasion, and migration by activating gene expression. Many STAT3-dependent transcriptional responses are mediated through protein-protein interactions that involve the amino-terminal domain (N-domain). In this study, we found that inhibition of the STAT3 N-domain using novel inhibitor ST3-Hel2A-2 induces apoptotic death in prostate cancer cells. The cell death was accomponied by robust activation of pro-apoptotic gene. Using chromatin immunoprecipitation and tiling human promoter arrays (ChIP-chip), we have defined genome-wide targets of STAT3 in DU145 prostate cancer cells. We found that upregulated pro-apoptotic genes were bound by STAT3 in prostate cancer cells, and that STAT3 binding was decreased following inhibition of the STAT3 N-domain.
JS-K a NO donor which capable to induce cancer apopotosis, it's killing mechanism was investigated in this study. We found out that JS-K induce apopotosis via deregulating the GSH/GSSG redox couple.
An Hodgkin Lymphoma cell line have been treated with an LNA inhibitor for miR-9 or with a scramble LNA to identify miR-9 regulated pathways that could be important for Hodgkin Lymphoma pathogenesis.
In this study we have investigated the effect of lysine-specific demethylase 6B (KDM6B) on gene expression in normal human germinal center (GC) B cells using a non-viral vector-based system.
Interstitial cystitis (IC) a chronic bladder disease with an increasing incidence, is diagnosed using subjective symptoms in combination with cystoscopic and histological evidence. The ultimate goal is the development of a diagnostic assay for IC on a molecular level. By cystoscopic examination, IC can be classified into an ulcerative and a non-ulcerative subtype. To better understand this debilitating disease on a molecular level, a comparative gene expression profile of bladder biopsies from patients with ulcerative IC and control patients has been performed. Candidate marker genes for ulcerative IC were identified.
Presently there is a deficiency of effective therapies designed to target clear cell renal cell carcinoma (ccRCC), with poor prognosis resulting in patients with advanced disease. Additionally, there is a lack of molecular factors which can be remedially targeted resulting in tumor specific inhibition, and therefore current therapeutic approaches often produce adverse side effects in patients. We identified that Stearoyl-CoA desaturase 1 (SCD1) was consistently overexpressed in patient ccRCC samples, and further investigation of SCD1 as a potential molecular target for ccRCC intervention utilizing a SCD1 inhibitor (A939572) resulted in tumor specific growth inhibition and induction of cell death. In order to understand the mechanism by which the SCD1 inhibitor mediated its anti-tumor effects, we performed gene array analysis and compared expression patterns between treated and untreated samples.
We used microarrays to detail the global programme of gene expression by circulating TCRVgamma9+ gamma delta T cells isolated from healthy individualstested either as resting cells or cells activated by phosphoantigen BrHPP and IL-2at an early(+6hrs) and a late (+7days) timepoint. We find that with more “NK cell” genes than alphabeta T cells and more “T cell” genes than NK cells, the circulating TCRVgamma9+ gamma delta T cells cells have a hybrid transcriptome. The gene signature of the activated cells recapitulates their physiological functions: Th1 cytokine, chemokine and cytotoxic activities at first and mitotic activity at later time points. The gene expression pattern of activated normal gamma delta T cells is nevertheless clearly distinctive from that of NK/T and peripheral T cell lymphomas of the gamma delta subtype.
D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment we engineered mouse strains which allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D associated kinase activity in mice bearing ErbB2-driven mammary carcinomas halted cancer progression and triggered tumor-specific senescence, without compromising the animals' health. Ablation of cyclin D3 in mice bearing T-cell acute lymphoblastic leukemias (T-ALL) triggered tumorspecific apoptosis. Such selective killing of leukemic cells can be also achieved by inhibiting cyclin D associated kinase activity in mouse and human T-ALL models. Hence, contrary to what one might expect from ablation of a cell cycle protein, acute shutdown of a D-cyclin leads not only to cell cycle arrest, but it also triggers tumor cell senescence or apoptosis, and it affects different tumor types through distinct cellular mechanisms. Inhibiting cyclin D-activity represents a highly-selective anticancer strategy which specifically targets cancer cells without significantly affecting normal tissues.
This SuperSeries is composed of the SubSeries listed below.
Genomic profiling of RNA from cultured human fibroblasts of donor samples in the 10-14th passage was carried out to determine expression changes in the fibroblasts of individual with different degrees of pulmonary fibrosis. Donors consisted of individuals with rapid progressing pulmonary fibrosis slow progressing pulmonary fibrosis, or no fibrosis.
The human liver plays a vital role in meeting the body's metabolic needs and maintaining homeostasis. To address the molecular mechanisms of liver function we integrated multiple gene expression datasets from microarray, MPSS, SAGE and EST platforms to generate a transcriptome atlas of the normal human liver. The integrated liver transcriptome data should provide a valuable resource for the in-depth understanding of human liver biology and liver disease. Microarray is one of these platforms. In this part, adult human liver tissues from 10 adult human were surgically resected due to hemangioma in the liver. The gene expression of normal human liver was detected by Microarray platform.
Drugs directly targeting Hepatitis C (HCV) are often rendered useless by the high mutation rate of the virus. Thus we deduce that targeting of host factor that affect HCV replication may provide enhanced therapy fort HCV infection. Hepatocyte cell line Huh7 is known to be non-permissive for Hepatits C (HCV) replication. Through a method developed by the Rice laboratory (Blight, K.J., et al., J Virol, 2002), selection of a small subset of permissive hepatocytes is possible. The Rice laboratory generated the first permissive cell line, Huh7.5, using this method. We generated another permissive cell line, HRP1, using the same method. With microarray, we compared the expression of host mRNAs in non-permissive Huh7 to both Huh7.5 and HRP1 searching for host factors lost in the cell lines permisive for HCV replication.
Time course comparison to tissue origin and with control cell line HT29 derived from colorectal adenocarcinoma. Status of expression pattern is different in adenocarcinoma of each patient. Human tumor cells extensively changes their gene- and protein expression patterns during their cultivation clonal selection and expansion, thereby loosing many of the characteristics of their primary origin. In this study we analyzed if these expression changes could be circumvented by using short-term primary cell culture models derived from colorectal cancer patients. We compared several primary cells from tumor tissues using a standardized protocol which yielded similar cell populations. For monitoring the gene expression changes induced by cell preparation and cultivation we collected the tissues immediately after resection and isolated cells before seeding, and after 24 and 72 hours of cultivation from each patient. Keywords: time course
Carbon monoxide (CO) is an endogenous messenger that suppresses inflammation modulates apoptosis and promotes vascular remodeling. Here, microarrays were employed to globally characterize the CO (250 ppm) suppression of early (1 h) LPS-induced inflammation in human monocytic THP-1 cells. CO suppressed 79 of 101 immediate-early genes induced by LPS; 19% (15/79) were transcription factors and most others were cytokines, chemokines and immune response genes. The prototypic effects of CO on transcription and protein production occurred early but decreased rapidly. CO activated p38 MAPK, ERK1/2 and Akt and caused an early and transitory delay in LPS-induced JNK activation. However, selective inhibitors of these kinases failed to block CO suppression of LPS-induced IL-1beta, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-kappaB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IkappaBalpha in human monocytes, thereby inhibiting NF-kappaB signal transduction. CO broadly suppresses the initial inflammatory response of human monocytes to LPS by reshaping proximal events in TLR4 signal transduction such as stress kinase responses and early NF-kappaB activation. These rapid, but transient effects of CO may have therapeutic applications in acute pulmonary and vascular injury.
Gene expression profiles of malignant carcinomas surgically removed from ovarian cancer patients pre-treated with chemotherapy (neo-adjuvant) prior to surgery group into two distinct clusters. One group clusters with carcinomas from patients not pre-treated with chemotherapy prior to surgery (C-L) while the other clusters with non-malignant adenomas (A-L). Although the C-L cluster is preferentially associated with p53 loss-of-function (LOF) mutations the C-L cluster cancer patients display a more favorable clinical response to chemotherapy as evidenced by enhanced long-term survivorships. Keywords: Patient Tumor Samples
Primary cultures of patient tumor cells (PCPTC) were used in a cell-based cytotoxicity screen. Microarray-based mRNA profiling was used to identify the mechanism-of-action for the small molecule VLX 50.
It has been found that fat oxidation is reduced in the skeletal muscle of obese humans. This study aims to identify the mRNA of proteins involved in fat oxidation that may be reduced in obese and morbidly obese individuals. Information gathered will help in understanding how obesity contributes to cardiovascular disease via insulin resistance. Keywords: other
Ventilator induced lung injury can lead to serious conditions like ARDS which are associated with a high mortality (around 30% Stapleton et al., Chest, 2005). We hypothesized that changes of expression levels of different genes would lead us to the identification of critical target genes, which might influence the inflammation and outcome associated with this condition. We used human whole genome U133 Plus 2.0 microarrays to detail the changes of gene expression and identified distinct classes of up-regulated genes during this process.
Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS).
Lactic acidosis and hypoxia are two prominent tumor microenvironmental stresses that are both known to exert important influences on gene expression and phenotypes of cancer cells. But very little is known about the cross-talk and interaction between these two stresses. We performed gene expression analysis of MCF7 cells exposed to lactic acidosis hypoxia and combined lactic acidosis and hypoxia. We found the hypoxia response elicited under hypoxia was mostly abolished upon simultaneous exposure to lactic acidosis. The repression effects are due to loss of HIF-1α protein synthesis under lactic acidosis. In addition, we showed lactic acidosis strongly synergizes with hypoxia to activate the unfold protein response (UPR) and inflammation response which are highly similar to amino acid deprivation responses (AAR). The statistical factor analysis of hypoxia and lactic acidosis responses indicated that ATF4 locus, an important activator in the UPR/AAR pathway, is amplified in subsets of breast tumors and cancer cell lines. Varying ATF4 levels dramatically affect the ability to survive the post-stress recovery from hypoxia and lactic acidosis and may suggest its selection of ATF4 amplification in human cancers. These data suggest that lactic acidosis interacts with hypoxia by both inhibiting the canonical hypoxia response and while activating the UPR and inflammation response. Gain of ATF4 locus may offer survival advantages to allow successful adaptation to frequent fluctuations of oxygen and acidity in tumor microenvironment. Collectively, our studies have provided linkage between the short-term transcriptional responses to the long term selection of the DNA copy number alterations (CNAs) under tumor microenvironmental stresses.
A long form (tRNase ZL) of tRNA 3' processing endoribonuclease (tRNase Z or 3' tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA). We discovered in human kidney 293 cell extracts various new small noncoding RNAs (ncRNAs) including 5'-half-tRNAs and 28S rRNA fragments, co-immunoprecipitated with tRNase ZL, and demonstrated that two of these ncRNAs work as sgRNAs for tRNase ZL in vivo as well as in vitro. In order to find genuine mRNA targets of tRNase ZL guided by ncRNAs, we performed DNA microarray analysis for mRNAs from the 293 cells transfected with the tRNase ZL expression plasmid, and found that PPM1F and DYNC1H1 mRNAs are its genuine targets.
To identify molecules to serve as diagnostic markers for high-grade prostate cancer (PC) and targets for novel therapeutic drugs we investigated the gene expression profiles of high-grade PCs using a cDNA microarray combined with laser microbeam microdissection.
PDE4 inhibitors which activate cAMP signaling by reducing cAMP catabolism, are known to induce apoptosis in B lineage chronic lymphocytic leukemia (CLL) cells but not normal human T cells. The explanation for such differential sensitivity remains unknown. Here, we report studies contrasting the response to PDE4 inhibitor treatment in CLL cells and normal human T and B cells. Affymetrix gene chip analysis in the three cell populations following treatment with the PDE4 inhibitor rolipram identified a set of up-regulated transcripts with unusually high fold-changes in the CLL samples, several of which are likely part of compensatory negative feedback loops. The high fold-change were due to low basal transcript levels in CLL cells, suggesting that cAMP-mediated signaling may be unusually tightly regulated in this cell type. Keywords: drug response
Expression profiling of Xenografts of Hepatocellular Carcinoma Keywords: Human Cancer
The improvement of Ewing's sarcoma (EWS) therapy is currently linked to find strategies to select patients with poor and good prognosis at diagnosis and to generate modified treatment regimens. In this study we analyze the molecular factors governing EWS response to chemotherapy in order to identify genetic signatures that may be used for risk-adapted therapy. Keywords: Gene Expression Analysis.
Invasion into deep myometrium and/or lymphovascular space is a well-known risk factor for endometrial cancer metastasis resulting in poor prognosis. It is therefore clinically important to identify novel molecules that suppress tumor invasion. Reduced expression of the metastasis suppressor, KISS1 (kisspeptin), and its endogenous receptor, GPR54, has been reported in several cancers, but the significance of the KISS1/GPR54 axis in endometrial cancer metastasis has not been clarified. Metastin-10 is the minimal bioactive sequence of genetic products of KISS1. Clinicopathological analysis of 92 endometrial cancers revealed overall survival is improved in cancers with high expression of GPR54. Through RNAi and mousemodel analyses, metastin-10 was predicted to suppress invasion and metastasis of GPR54-expressing endometrial cancers. These data suggest that metastin-10 may induce genetic changes in the metastatic character of endometrial cancers.
Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human keratinocytes untreated and treated with dexamethasone (DEX) for 1, 4, 24, 48 and 72 hours using large-scale microarray analyses. The majority of genes were found regulated only after 24 hours and remained regulated throughout the treatment. In addition to expected anti-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation and metabolism. GCs not only effectively block signaling by TNF-alpha and IL-1 but also by IFN-gamma, which was not previously known. Specifically, GCs suppress the expression of essentially all IFN-gamma-regulated genes, including IFN-gamma receptor and STAT-1. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs have anti-apoptotic effects in keratinocytes by inducing the expression of anti-apoptotic and repressing pro-apoptotic genes. Consequently, GCs treatment blocked UV-induced apoptosis of keratinocytes. GCs have a profound effect on wound healing by inhibiting cell motility and the expression of pro-angiogenic factor VEGF. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGF-beta-1 and -2, MMP1, 2, 9 and 10 and inducing TIMP-2. Finally, GCs promote terminal stages of epidermal differentiation while simultaneously inhibiting the early stages. These results provide new insights into the beneficial and adverse effects of GCs in epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.
Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T cell lymphoma graft versus host disease and allografted organ rejection. Its clinical and experimental efficacy in both cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the dendritic antigen presenting cell (DC) differentiation pathway, as determined by expression of relevant genes. The resulting DC are capable of processing and presentation of exogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of co-stimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome indicates that activation or suppression of more than 3500 genes produces a reproducible distinctive molecular signature. Pathway analysis suggests that DC maturation may be triggered by transient adherence of passaged monocytes to plasma proteins coating the ECP plastic ultraviolet exposure plate. Co-incubation with lymphocytes, simultaneously induced by ECP to undergo apoptosis, may accelerate conversion of monocytes to DC. The efficiency with which ECP induces new functional DC supports the possibility that these cells participate prominently in the clinical successes of the treatment. ECP may offer a practical source of DC for use in a spectrum of immunotherapeutic trials. We have used microarrays to analyze the expression of genes modulated by ECP treatment.
Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4
Expression of a constitutively active Notch-1 intracellular domain (NICD) in MCF-10A cells was found to induce two distinct types of 3D structures: large hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. These heterogeneous phenotypes reflect differences in Notch pathway activation levels. High Notch activity caused loss of cell adhesion and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. In order to gain insight into the dosage-dependent transcriptional events triggered by Notch1 activation, gene expression profiles induced 48 hours after infection of MCF-10A cells with retroviral vectors expressing full-length Notch-1, L1601P+∆P, or NICD were compared. Full-length Notch-1 induced the weakest effect, L1601P+∆P induced an intermediate effect and NICD induced the strongest effect. Results provide insight into the dichotomous activites of Notch during development and tumorigenesis.
In order to define the inhibitory activity of PMN-Ectosomes we investigated the early gene expression profiles of resting and zymosan A-stimulated human monocyte-derived macrophages in the absence or presence of PMN-Ectosomes.
To identify CD24 signaling pathway Affymetrix Human Genome U133 Plus GeneChip 2.0
Cellular responses to carcinogens are typically studied in transformed cell lines which do not reflect the physiological status of normal tissues. To address this question, we have characterized the transcriptional program and cellular responses of normal human lung WI-38 fibroblasts upon exposure to the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE). Exposure to BPDE induces a strong inflammatory response in WI-38 primary fibroblasts. Whole-genome microarray analysis shows induction of several genes related to the production of inflammatory factors, including those that encode interleukins (ILs), growth factors, and enzymes related to prostaglandin synthesis and signaling. This is the first demonstration that a strong inflammatory response is triggered in primary fibroblasts in response to a reactive diol epoxide derived from a polycyclic aromatic hydrocarbon.
The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin by way of the production of secreted proteins, termed hepatokines. To identify hepatic secretory proteins involved in insulin resistance, we performed liver biopsies in humans with or without type 2 diabetes and conducted a comprehensive analysis of gene expression profiles.
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II) which both signal through type I IGF receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-IIbinding and by inducing cell surface receptor down-regulation via internalization and degradation. In vitro, h10H5 exhibits anti-proliferative effects on cancer cell lines. In vivo, h10H5 demonstrates single-agent anti-tumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models, and even greater efficacy in combination with the chemotherapeutic agent Docetaxel or an anti-VEGF antibody. Anti-tumor activity of h10H5 is associated with decreased AKT activation and glucose uptake, and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors. Keywords: Gene expression changes as markers for drug activity
The placenta is considered one of the candidate cell sources in cellular therapeutics because of a large number of cells and heterogenous cell population with myogenic potentials. We first analyzed myogenic potential of cells obtained from six parts of the placenta i.e., umbilical cord, amniotic epithelium, amniotic mesoderm, chorionic plate, villous chorion (chorion frondosum), , and decidua basalis. Implantation of placenta-derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of human dystrophin. Co-existence of human and murine nuclei in one myotube and presence of human dystrophin in murine myotube suggests that human dystrophin expression is due to cell fusion between host murine myocytes and implanted human cells. In vitro analysis revealed that cells derived from amniotic mesoderm, chorionic plate, ,and villous chorion efficiently transdifferentiate into myotubes. These cells fused to C2C12 murine myoblasts by in vitro co-culturing, and murine myoblasts start to express human dystrophin after fusion. These results demonstrate that placenta-derived cells, especially extraembryonic mesodermal cells, have a myogenic potential and regenerative capacity of skeletal muscle. Determination of cell specification with the gene chip analysis revealed that each placental cell has a distinct expression pattern. Keywords: Determination of cell specification
Estrogen deprivation using aromatase inhibitors is currently the standard of care for patients with estrogen-receptor (ER)-positive breast cancer. Unfortunately prolonged estrogen deprivation leads to drug resistance (i.e. hormone-independent growth). We therefore used DNA microarray analysis to study the gene expression profiles of wild-type MCF-7 cells (which are sensitive to antihormone therapy) and long-term estrogen deprived MCF-7:5C and MCF-7:2A breast cancer cells (which are resistance to estrogen-deprivation; aromatase inhibitor resistant). Transcriptional profiling of wild-type MCF-7 cells and estrogen deprived MCF-7:5C and MCF-7:2A cells was performed using Affymetrix Human Genome U133 Plus 2.0 Array. Keywords: breast cancer cells, estrogen
8-oxoguanine is excised from DNA by 8-oxoguanine DNA glycosylase-1 during DNA base excision repair. We used microarrays to gain insight into the potential biological function of the excised free 8-oxoguanine base.
We are investigating the transcriptional response of newborns in response to prenatal arsenic exposure We used microarrays to detail the global programme of gene expression response due to prenatal arsenic exposure Keywords: dose (arsenic)
Breast cancer is a molecularly biologically and clinically heterogeneous group of disorders. Understanding this diversity is essential to improving diagnosis and optimising treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but information is scant on the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease. Here we used the Infinium Methylation Platform to profile at single-CpG resolution (over 14,000 genes interrogated) the methylomes of 119 breast tumours. It emerges that many genes whose expression is linked to the ER status are epigenetically controlled (or/ we show that the two major phenotypes of breast cancers determined by ER status are widely involving epigenetic regulatory mechanisms), offering the prospect of a novel approach to treating ER-positive tumours. We have distinguished methylation-profile-based tumour clusters, some coinciding with known “expression subtypes” but also new entities that may provide a meaningful basis for refining breast tumour typology. We show that methylation patterns may reflect the cellular origins of tumours. Having highlighted an unexpectedly strong epigenetic component in the regulation of key immune pathways, we show that a set of immune genes have high prognostic value in specific tumour categories. By laying the ground for better understanding of breast cancer heterogeneity and improved tumour taxonomy, the precise epigenetic portraits drawn here should contribute to better management of breast cancer patients.
To identify patterns of change in gene expression that correlated with drug treatment (saline control ADH-1 alone, melphalan alone and ADH-1 plus melphalan) in our animal model of regional therapy for extremity melanoma we evaluated gene expression using microarray genechips and a Pearson correlation analysis. Genes were ranked according to the degree to which expression correlated with systemic ADH-1 treatment alone or in combination with regional melphalan. Tissue samples were harvested at 4, 24 and 48 hours after treatment with melphalan. Specific cellular pathways associated with drug treatment were identified using gene set enrichment analysis (GSEA). Keywords: time course; chemotherapy; melanoma; melphalan; N-cadherin; ADH-1
G-protein coupled receptors (GPCRs) have diverse roles in physiological processes including immunity. Gs-coupled GPCRs increase while Gi-coupled ones decrease intracellular cAMP. Previous studies suggest that, in epithelial cells, Gs-coupled GPCRs enhance whereas Gi-coupled GPCRs suppress pro-inflammatory immune responses. In order to examine the issue, we chose beta2 adrenergic receptor and GPR40 as representatives of Gs- and Gi- coupled GPCRs, respectively, and examined their effects on TNF-alpha and IFN-gamma-(TNF-alpha + IFN-gamma) induced gene expression by HaCaT. We used microarrays to detail the global changes of gene expression induced by a beta2 adrenergic receptor agonist terbutaline or GPR40 agonist GW9508 pre-treatment in TNF-alpha + IFN-gamma - stimulated HaCaT cells.
Transcriptional profile of PCSC spheres in SCM-1% KO (stem-like cells) vs adherent cultures in PCSC-Celprogen medium (differentiated-like cells) Two-condition experiment: Sphere vs. Parental/adherent cells. Biological replicates: 2 sphere replicates 2 adherent replicates.
An evaluation of biopsies from patients with in-transit extremity melanoma who have been treated with melphalan in the setting of isolated limb infusion or isolated limb perfusion. Gene expression profiles were obtained from 21 lesions across 19 patients and evaluated for expression that correlated with response to melphalan isolated limb infusion.
Leishmania major infected human dendritic cells (DCs) exhibit a marked induction of IL-12 ultimately promoting a robust Th1-mediated response associated with parasite killing and protective immunity. In this study we utilized Affymetrix Genechips to globally assess the host cell genes and pathways associated with L. major infection during early infection (2, 4, 8, and 24 hrs) in human myeloid-derived DCs. Bioinformatic analyses of the hybridized microarray chips identified 728 genes, represented by 848 unique probe sets, which, when compared to uninfected samples were observed to be significantly differentially expressed by one-way ANOVA. Altogether, the data provide a genome-wide perspective on the transcriptional influences Leishmania species exert within human DCs during early infection, and provides a platform for further investigations toward functionally characterizing candidate genes of importance to the IL-12 based immune response to infections. In the current study, we further investigate the L. major infected DC transcriptional during early time points after infection via microarray analysis.
Primary cilium serves as a cellular “antenna” to sense environmental signals. Ciliogenesis requires the removal of CP110 to convert the mother centriole into the basal body. Actin dynamics is also critical for cilia formation. How these distinct processes are properly regulated remains unknown. Here we show that miR-129-3p a microRNA conserved in the vertebrates, controlled cilia assembly by down-regulating both CP110 and four proteins critical for actin dynamics, Arp2, Toca1, abLIM1, and abLIM3. Consistently, blocking miR-129-3p repressed cilia formation in cultured mammalian cells, whereas its overexpression potently induced ciliogenesis in proliferating cells and extraordinary cilia elongation. Moreover, inhibition of miR-129-3p in zebrafish embryos suppressed cilia assembly in the Kupffer’s vesicle and pronephric duct, leading to developmental abnormalities including curved body, pericardial oedema, and randomised left-right patterning. Our results thus unravel a novel mechanism that orchestrates both the centriole-to-basal body transition and subsequent cilia assembly via microRNA-mediated posttranscriptional regulations. We want to find the targets of miR-129-3p by overexpressing miR-129-3p oligo or control oligo in hTERT-RPE1 cells. Through microarray analysis we could check the downregulated genes and these genes might be the targets of miR-129-3p.
Trovafloxacin is a broad spectrum antibiotic that inhibits the uncoiling of supercoiled DNA in various bacteria by blocking the activity of DNA gyrase and topoisomerase IV. Specific members of this drug family display high activity against eukaryotic type II topoisomerase as well as cultured mammalian cells and in vivo tumor models. Trovafloxacin seems to have a higher affinity for eukaryotic polymerase II system than the other quinolone agents tested. This effect coupled with other factors, such as an inflammatory response, might result in a hepatotoxic reaction seen with drug. In this study we found genes regulated by trovafloxacin - induced and repressed - to be located much closer to each other than genes distributed randomly all over the genome (< 100 kbp). Keywords: Trovafloxacin-treated human hepatocytes versus non-treated human hepatocytes
The study was completed to compare expression profiles of primary human beta cells (in the form of adult human islets) to the expression profile of hESC-derived beta-like cells. A HES3 line modified by homologous recombination to express GFP under the insulin promoter allowed us to FACS sort the hESC-derived cells into purified insulin-positive (presumably beta-like cells), and insulin-negative populations.
The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. Corresponding ChIP-Seq data are available in NCBI's Short Read Archive at ftp://ftp.ncbi.nlm.nih.gov/sra/Studies/SRP000/SRP000105 We find that nucleosome phasing relative to the transcription start sites (TSSs) is directly correlated to RNA polymerase II binding. Furthermore, the first nucleosome downstream of TSSs exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the -1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome. This microarray dataset is the gene expression data from resting and activated CD4+ T cells. We used this data to look at nucleosome positioning, Pol II and a few chromatin modifications at genes that are silent and activated in both resting and activated T cells as well as genes that are induced or repressed with T cell activation. Keywords: nucleosome mapping, expression data complementary to Solexa ChIP-Seq data
In addition to their role in the development and function of the reproductive system estrogens have significant anti-inflammatory properties. Although both estrogen receptors (ERs) can mediate anti-inflammatory actions, ERbeta is a more desirable therapeutic target because ERalpha mediates the proliferative effects of estrogens on the mammary gland and uterus. In fact, selective ERbeta agonists have beneficial effects in preclinical models involving inflammation without causing growth-promoting effects on the uterus or mammary gland. However, their mechanism of action is unclear. The purpose of this study was to use microarray analysis to determine whether ERbeta-selective compounds produce their anti-inflammatory effects by repressing transcription of proinflammatory genes. We identified 49 genes that were activated by TNF-alpha in human osteosarcoma U2OS cells expressing ERbeta. Estradiol treatment significantly reduced the activation by TNF-alpha on 18 genes via ERbeta or ERalpha. Most repressed genes were inflammatory genes, such as TNF-alpha, IL-6, and CSF2. Three ERbeta-selective compounds, ERB-041, WAY-202196, and WAY-214156, repressed the expression of these and other inflammatory genes. ERB-041 was the most ERbeta-selective compound, whereas WAY-202196 and WAY-214156 were the most potent. The ERbeta-selective compounds repressed inflammatory genes by recruiting the coactivator, SRC-2. ERB-041 also repressed cytokine genes in PBMCs, demonstrating that ERbeta-selective estrogens have anti-inflammatory properties in immune cells. Our study suggests that the anti-inflammatory effects of ERB-041 and other ERbeta-selective estrogens in animal models are due to transcriptional repression of proinflammatory genes. These compounds might represent a new class of drugs to treat inflammatory disorders. Keywords: estrogen receptor, gene regulation, TNF
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Keywords: siRNA, transfection, chemical modification, off-targets
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. Originally NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. We have now systematically analyzed the molecular mechanism of variable NMD efficiency and used different HeLa cell strains as a model system. The results of this analysis show that NMD efficiency can be remarkably variable and represents a stable characteristic of these strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the HeLa strain analyzed. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. We conclude that cellular concentrations of RNPS1 modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that controls NMD. Keywords: NMD UPF1 knock down
Expression data from HT-29 human colon adenocarcinoma cells treated with IFN-γ for 24 hr
Ependymomas are glial tumors that share morphologic similarities with ependymal cells. Here we laser microdissected ependymoma cells of 15 infratentorial tumors and generated gene expression profiles. These profiles were compared to those of 7 laser microdissected ependymal tissues.
BACKGROUND: Cadmium is implicated in prostate carcinogenesis but its oncogenic action remains unclear. OBJECTIVES: In this study we aimed to decipher changes in cell growth and the transcriptome in an immortalized human normal prostate epithelial cell line (NPrEC) following exposure to low-dose Cd. METHODS: Synchronized NPrEC cells were exposed to different doses of Cd and assayed for cell viability and cell-cycle progression. We investigated changes in transcriptome by global profiling and used Ingenuity Pathways Analysis software to develop propositions about functional connections among differentially expressed genes. A neutralizing antibody was used to negate the effect of Cd-induced up-regulation of tumor necrosis factor (TNF) in NPrEC cells. RESULTS: Exposure of NPrEC to 2.5 μM Cd enhanced cell viability and accelerated cell-cycle progression. Global expression profiling identified 48 genes that exhibited ≥ 1.5-fold changes in expression after 4, 8, 16, and 32 hr of Cd treatment. Pathway analyses inferred a functional connection among 35 of these genes in one major network, with TNF as the most prominent node. Fourteen of the 35 genes are related to TNF, and 11 exhibited an average of > 2-fold changes in gene expression. Real-time reverse transcriptase-polymerase chain reaction confirmed the up-regulation of 7 of the 11 genes (ADAM8, EDN1, IL8, IL24, IL13RA2, COX2/PTGS2, and SERPINB2) and uncovered a 28-fold transient increase in TNF expression in Cd-treated NPrEC cells. A TNF-neutralizing antibody effectively blocked Cd-induced elevations in the expression of these genes. CONCLUSIONS: Noncytotoxic, low-dose Cd has growth-promoting effects on NPrEC cells and induces transient overexpression of TNF, leading to up-regulation of genes with oncogenic and immunomodulation functions. KEY WORDS: carcinogenesis, cytokine, global expression profiling, heavy metals, immune response, inflammation, Ingenuity Pathway Analysis, knowledge-based analysis, prostate cancer.
Oculo-facio-cardio-dental syndrome (OFCD) is a rare genetic disorder characterized by teeth with extremely long roots (radiculomegaly) and craniofacial, eye and cardiac abnormalities. The mutation of the transcriptional co-repressor BCOR has been identified as being responsible for oculo-facio-cardio-dental (OFCD) syndrome. Mesenchymal stem cells (MSCs) is isolated from the root apical papilla of an OFCD patient. Gene expression profiling is performed and compared between mutant MSCs and wild type MSCs.
Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90 % of patients can be cured but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested deregulation of the retinoic acid (RA) pathway in high-risk WT. This could be validated in a large independent tumor set. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/intermediate risk, suggesting a beneficial impact of RA on advanced WT. To investigate the possible mode of action of retinoids as a novel therapeutic agent we treated primary tumor cell cultures with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid / HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. Although some of the effects appear to be reversible, these findings provide further evidence of a potential utility of retinoids in Wilms tumor treatment.
Epithelial ovarian cancer is the leading cause of death among gynecologic malignancies. Diagnosis usually occurs after metastatic spread largely reflecting vague symptoms of early disease combined with lack of an effective screening strategy. Epigenetic mechanisms of gene regulation, including DNA methylation, are fundamental to normal cellular function and also play a major role in carcinogenesis. To elucidate the biological and clinical relevance of DNA methylation in ovarian cancer, we conducted expression microarray analysis of 39 cell lines and 17 primary culture specimens grown in the presence or absence of DNA methyltransferase (DNMT) inhibitors. Two parameters, induction of expression and standard deviation among untreated samples, identified 378 candidate methylated genes, many relevant to TGF-beta signaling. We analyzed 43 of these genes and they all exhibited methylation. Treatment with DNMT inhibitors increased TGF-beta pathway activity. Hierarchical clustering of ovarian cancers using the 378 genes reproducibly generated a distinct gene cluster strongly correlated with TGF-beta pathway activity that discriminates patients based on age. These data suggest that accumulation of age-related epigenetic modifications leads to suppression of TGF-beta signaling and contributes to ovarian carcinogenesis.
The expression level for 15 887 transcripts in lymphoblastoid cell lines from 19 monozygotic twin pairs (10 male 9 female) were analysed for the effects of genotype and sex. On an average, the effect of twin pairs explained 31% of the variance in normalized gene expression levels, consistent with previous broad sense heritability estimates. The effect of sex on gene expression levels was most noticeable on the X chromosome, which contained 15 of the 20 significantly differentially expressed genes. A high concordance was observed between the sex difference test statistics and surveys of genes escaping X chromosome inactivation. Notably, several autosomal genes showed significant differences in gene expression between the sexes despite much of the cellular environment differences being effectively removed in the cell lines. A publicly available gene expression data set from the CEPH families was used to validate the results. The heritability of gene expression levels as estimated from the two data sets showed a highly significant positive correlation, particularly when both estimates were close to one and thus had the smallest standard error. There was a large concordance between the genes significantly differentially expressed between the sexes in the two data sets. Analysis of the variability of probe binding intensities within a probe set indicated that results are robust to the possible presence of polymorphisms in the target sequences. Keywords: Monozygotic twin pair Expression Profiles
To study effects of IFNalpha treatment on monocyte-derived macrophages which may influence susceptibility or resistance to HIV.
From the parental breast cancer cell line MCF7 (weakly invasive) we progressively selected hyperinvasive subclones. We compared the gene expression between the parental MCF7-I0 and the hyper-invasive cells MCF7-I6. We used Affymetrix U133 Plus 2 microarrays to detail the global programme of gene expression underlying weakly and highly invasive breast cancer cells derived from the human breast cancer cell line MCF7.
Large-scale cancer genomics projects are profiling hundreds of tumors at multiple molecular layers including copy number, mRNA and miRNA expression, but the mechanistic relationships between these layers are often excluded from computational models. We developed a supervised learning framework for integrating molecular profiles with regulatory sequence information to reveal regulatory programs in cancer, including miRNA-mediated regulation. We applied our approach to 320 glioblastoma profiles and identified key miRNAs and transcription factors as common or subtype-specific drivers of expression changes. We confirmed that predicted gene expression signatures for proneural subtype regulators were consistent with in vivo expression changes in a PDGF-driven mouse model. We tested two predicted proneural drivers, miR-124 and miR-132, both underexpressed in proneural tumors, by overexpression in neurospheres and observed a partial reversal of corresponding tumor expression changes. Computationally dissecting the role of miRNAs in cancer may ultimately lead to small RNA therapeutics tailored to subtype or individual.
Our group recently described a population of antigen presenting cells that appear to be critical in psoriasis pathogenesis termed inflammatory myeloid dendritic cells (CD11c+ BDCA1-). Triggering receptor expressed on myeloid cells type-1 (TREM-1) signaling was a major canonical pathway in the published transcriptome of these cells. TREM-1 is a member of the immunoglobulin superfamily, active through the DAP12 signaling pathway, with an unknown ligand. Activation through TREM-1 induces inflammatory cytokines including IL-8, MCP/CCL2 and TNF. We now show that TREM-1 was expressed in the skin of healthy and psoriatic patients, and there was increased soluble TREM-1 in the circulation of psoriasis patients. In psoriasis lesions, TREM-1 was co-localized with dendritic cells as well as CD31+ endothelial cells. TREM-1 expression was reduced with successful NB-UVB, etanercept and anti-IL-17 treatments. An in vitro model of PGN-activated monocytes as inflammatory myeloid DCs was developed to study TREM-1 blockade, and treatment with a TREM-1 blocking chimera decreased allogeneic Th17 activation, as well as IL-17 production. Furthermore, TREM-1 blockade of ex vivo psoriatic dendritic cells in an alloMLR also showed a decrease in IL-17. Together, these data suggest that the TREM-1 signaling pathway offers a novel therapeutic target to prevent the effects of inflammatory myeloid DCs in psoriasis.
ABSTRACT Background: The expression of BCL11B has been reported in normal and transformed cells derived from T-lymphocytes neurons, keratinocytes and recently in a subset of squamous cell carcinomas. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open. Methodology/Principal Findings: Here, by employing an overexpression strategy and cells endogenously expressing BCL11B we revealed formerly unidentified features of Bcl11b which shed some light on the potential involvement of the protein in tumor maintenance. Two different T cell lines were forced to overexpress BCL11B which resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A. Conclusions: The data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells.
Investigation of malignancy-associated expression of gene sets based on ovarian tumour histone modification status.
MDAMB231 cells express the SV40TAg signature. The normal mammary epithelial tissue does not express the signature. CK0082 Empty1 and Gata3 samples are not reported in this manuscript
The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation,
Knockdown of Sox2 in SW620 colorectal cancer cells decrease their growth rates in vitro and in vivo in xenograft models. We used microarrays to detail the global programme of gene expression in Sox2 Knockdown sw620 cells compared with mock knockdown sw620 cells
Background. Vaginal atrophy (VA) is the thinning of the vaginal epithelial lining typically the result of lowered estrogen levels during menopause. Some of the consequences of VA include increased susceptibility to bacterial infection, pain during sexual intercourse, and vaginal burning or itching. Although estrogen treatment is highly effective, alternative therapies are also desired for women who are not candidates for hormone replacement therapy (HRT). The ovariectomized (OVX) rat is widely accepted as an appropriate animal model for many estrogen-dependent responses in humans; however, since reproductive biology can vary significantly between mammalian systems, this study examined how well the OVX rat recapitulates human biology at the transcriptional level. This report describes an analysis of expression profiling data, comparing the responses of rat and human vaginae to estrogen treatment. Results. The level of differential expression between pre- vs. post- estrogen treatment was calculated for each of the human and OVX rat datasets. Probe sets corresponding to orthologous rat and human genes were mapped to each other using NCBI Homologene. A positive correlation was observed between the rat and human responses to estrogen. Genes belonging to several biological pathways and GO categories were similarly differentially expressed in rat and human. A large number of the coordinately regulated biological processes are already known to be involved in human VA, such as inflammation, epithelial development, and EGF pathway activation. Conclusions. At the transcriptional level, there is evidence of significant overlap of the effects of estrogen treatment between the OVX rat and human VA samples. Keywords: Disease State Analysis: Animal Model Validation
Infection with Chlamydia pneumoniae a human respiratory pathogen, has been associated with various chronic diseases such as asthma, coronary heart disease and importantly atherosclerosis. Possibly because the pathogen can exist in a persistent form. TNF-a has been reported to induce chlamydial persitence in epithelial cell lines, however the mechanism of TNF-a-induced persistence has not been reported. Moreover, C. pneumoniae persistently infect human dendritic cells (DCs) and activate DCs to produce cytokines including TNF-a. Induction of chlamydial persistence by other cytokines such as IFN-g is known to be due to indoleamine 2,3-dioxygenase (IDO) activity. The present study therefore, investigated whether C. pneumoniae infection can induce IDO activity in dendritic cells, and whether the restriction of chlamydial growth in the DCs by TNF-a is IDO-dependent. Our data indicate that infection of DCs with C. pneumoniae resulted in the induction of IDO expression. Reporting on our use of anti-TNF-a antibody adalimumab and varying concentrations of TNF-a, we further demonstrate that IDO induction following infection of DCs with C. pneumoniae is TNF-a-dependent. The anti-chlamydial activity induced by TNF-a and the expression of chlamydial 16S rRNA gene, euo, groEL1, ftsk and tal genes was correlated with the induction of IDO. Addition of excess amounts of tryptophan to the DC cultures resulted in abrogation of the TNF-a-mediated chlamydial growth restriction. These findings suggest that infection of DCs by C. pneumoniae induces production of functional IDO, which subsequently causes depletion of tryptophan. This may represent a potential mechanism for DCs to restrict bacterial growth in chlamydial infections. Keywords: Chlamydia pneumoniae, Dendritic cells, TNF-a, Indoleamine 2,3-dioxygenase
Growth factor signaling and angiogenesis may promote endocrine-resistance in breast cancer and blocking these pathways can overcome resistance in preclinical models. We conducted a phase-II study of adding the VEGFR/Ras/Raf/MAPK inhibitor sorafenib to endocrine therapy in metastatic ER-positive breast cancer either upon progression or after maximal response with measurable residual disease. Tumor biopsies and serum were collected on days 1 and 28. Primary endpoint was response by RECIST after 3 months and secondary endpoints included safety, time to progression (TTP), and biomarker assessment. Planned sample size was 43 patients but the study closed after 11 patients because of slow accrual. 8 patients had progressive disease (PD) on entry and 3 had stable disease (SD). One patient with SD discontinued sorafenib after 2-weeks because of grade 3 rash. Of the 10 remaining patients after adding sorafenib, 7 had SD (70%), 3 had PD (30%) and median TTP was 6.1-months. Of the 8 patients who entered the study with PD on endocrine therapy, 5 converted to SD (62%) with a median TTP of 6.4-months. Notably, patients on tamoxifen had a median TTP of 8.4-months. The most common adverse events were hypophosphatemia, hypokalemia, and rash, and the majority were grade 1&2 with no grade 4 toxicities. There was a significant reduction in serum VEGFR2 and PDGFR-α on day-28 (p-values 0.0035 and 0.017, respectively). Both serum VEGF and sVEGFR-1 were increased on day-28, but the differences were not statistically significant (p-values 0.3223 and 0.084, respectively). Microarray analysis identified 32 suppressed genes with an FDR of <0.20 and at least a 2-fold change with no induced genes and 29 KEGG pathways were enriched on day-28. Our study suggests that sorafenib can restore endocrine sensitivity, particularly tamoxifen, and this strategy of adding novel agents in patients progressing on endocrine therapy should be examined in future trials.
In multiple myeloma (MM) endothelial progenitor cells (EPCs) regulate tumor angiogenesis and disease progression. EPCs from 20 newly diagnosed patients with advanced MM were examined for genomic instability by RNA microarrays to assess changes in gene expression.
Endothelial cell activation and dysfunction underlie many vascular disorders including atherosclerosis and inflammation. Here, we show that interleukin (IL)-4 markedly induced vascular cell adhesion molecule (VCAM)-1, both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor (TNF)- alpha resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor, STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep-sequencing (ChIP-seq) in endothelial cells revealed regions of transient and sustained transcription factor binding. By combining DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6-dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within -16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease (ChIP-seq data not submitted to GEO).
Human CD14 positive monocytes were purified from healthy volunteers’ blood and cultured in vitro for 4 12, 24, 72 hours. While culturing, macrophages were activated alternatively with interleukin-4 (IL-4 100 ng/ml) or classically with interferon-gamma (IFNg 100 ng/ml)+tumor necrosis factor (TNF 50 ng/ml) or left without activation. Simultaneously, macrophages were also treated with vehicle (DMSO:ethanol) or 1mM synthetic PPARg agonist, Rosiglitazone. We used Affymetrix microarrays (U133Plus 2.0) to analyze activation and PPARg-induced gene expression changes.
Abnormal function of genes is at the root of most cancers but heritable cancer syndromes account for a very small minority of all tumors in humans and domestic animals. The majority of cancers are “sporadic,” that is, they are not heritable in the strictest sense. Instead, sporadic cancers occur due to interactions of unknown intrinsic (heritable) and environmental factors that lead to malignant transformation and uncontrolled growth. Identification of heritable risk factors in sporadic human cancers is difficult because individual genetic backgrounds are very heterogeneous. To this end, individual genetic backgrounds of purebred dogs are more homogeneous, and dog breeds show different predilection to develop specific cancers. Here, we used genomic screens based on gene expression profiling to identify sets of genes that may contribute to the development of canine hemangiosarcoma, a relatively common endothelial sarcoma. Specific genes in a single breed (Golden Retrievers) are modulated by (or with) heritable risk traits, showing functional features that appear to modulate tumor behavior. Our results suggest these methods are suitable to identify genes that will enhance our understanding of how these cancers happen, as well as possible treatment targets that will improve outcomes of both human and canine cancer patients. 24 samples were analysed. 12 samples with ATII cells with without influenza virus infection at 4h and 24h; 12 samples with AMs with without influenza virus infection at 4h and 24h
Pancreatic cancer is a devastating disease with both local invasion and distant metastasis. Identifying the genes expressed in liver metastases and signatures of metastatic progression would therefore be of particular importance as they could aid in both recurrence prediction as well as representing novel therapeutic targets. Keywords: Gene expression profiling
Ficolled AML-M0 sample gene expression profiles on Affymetrix HGU133Plus2.0 GeneChips. Acute myeloid leukemia (AML) classified as FAB-M0 is defined as a subtype with minimally differentiated morphology. Here we investigated by gene expression (GEP) profiling whether AML-M0 cases should be considered as one or more unique molecular subgroups that discriminates them from other AML patients. By applying GEP and subsequent unsupervised analysis of 35 AML-M0 samples and 253 previously reported AML cases we demonstrate that AML-M0 cases express a unique signature. Hematological transcription regulators such as CEBPA, CEBPD, PU.1 and ETV6 and the differentiation associated gene MPO appeared strongly down-regulated, in line with the very primitive state of this type of leukemia. Moreover, AML M0 cases appeared to have a strong positive correlation with a previously defined immature AML subgroup with adverse prognosis. AML-M0 leukemias frequently carry loss-of-function RUNX-1 mutation and unsupervised analyses revealed a striking distinction between cases with and without mutations. RUNX1 mutant AML-M0 samples showed a distinct up-regulation of B-cell-related genes, e.g. members of the B-cell receptor complex, transcriptions regulators RUNX3, ETS2, IRF8 or PRDM1 and major histocompatibility complex class II genes. Importantly, expression of one single gene, i.e. BLNK, enabled prediction of RUNX1 mutations in AML-M0 with high accuracy. We propose that RUNX1 mutations in this subgroup of AML cause lineage infidelity, leading to aberrant co-expression of myeloid and B-lymphoid genes in the same cells.
To gain insight into the etiopathogenesis of Multiple sclerosis (MS) we investigated gene expression changes in CD4+ and CD8+ T lymphocytes from monozygotic twins (MZ) discordant for relapsing remitting MS. We studied 4 monozygotic twin pairs discordant for disease with the affected co-twin free of disease modifying therapies (F/M = 3/1, mean age 36.25±3.9). Following leukapheresis, CD4+ and CD8+ T cells were separated and studied by Affymetrix GeneChip®
During the human B cell (Bc) recall response rapid cell division results in multiple Bc subpopulations. The TLR-9 agonist CpG oligodeoxynucleotide, combined with cytokines, causes Bc activation and division in vitro and increased CD27 surface expression in a sub-population of Bc. We hypothesized that the proliferating CD27lo subpopulation, which has a lower frequency of antibody-secreting cells (ASC) than CD27hi plasmablasts, provides alternative functions such as cytokine secretion, costimulation, or antigen presentation. We performed genome-wide transcriptional analysis of CpG activated Bc sorted into undivided, proliferating CD27lo and proliferating CD27hi subpopulations. Our data supported an alternative hypothesis, that CD27lo cells are a transient pre-plasmablast population, expressing genes associated with Bc receptor editing. Undivided cells had an active transcriptional program of non-ASC B cell functions, including cytokine secretion and costimulation, suggesting a link between innate and adaptive Bc responses. Transcriptome analysis suggested a gene regulatory network for CD27lo and CD27hi Bc differentiation.
Directing differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a perquisite for the clinical use of hESC in regenerative medicine protocols. Here we report the generation of mesodermal cells with differentiation potential to myocytes, osteoblasts, chondrocytes and adipocytes. We demonstrate that during hESC differentiation as embryoid bodies (EB), inhibition of TGF-b/Activin/Nodal signaling using SB-431542 (SB) markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), early myogenic transcriptional factors (Myf5, Pax7) as well as myocyte committed markers (NCAM, CD34, Desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT). Establishing EB outgrowth cultures (SB-OG) in the presence of SB (1 uM) led to further enrichment of cells expressing markers for myocyte progenitor cell: CD34+ (33%), NCAM+ (CD56) (73%), PAX7 (25%) and mature myocyte proteins (MYOD1, tropomyocin, fast MHC an d SERCA1). Further analysis using DNA microarray revealed differential up-regulation of 117 genes (>2-fold compared to control cells) annotated to myogenic development and function. During ex vivo culture, contracting myocytes were observed (80% of the population) and the cells formed myofibres when implanted intramuscularly in vivo. Furthermore, in the presence of fetal bovine serum (10% FBS), SB-OG cells developed morphologically and phenotypically into a homogeneous stromal (mesenchymal) stem cell (MSC)-like population expressing characteristic MSC CD markers: CD44 (100%), CD73 (98%), CD146 (96%) and CD166 (88%). They were karyotypically normal and were able to differentiate ex vivo and in vivo into osteoblasts, adipocytes and chondrocytes.
Osteoblasts are key players in bone remodeling. The accessibility of human primary osteoblast-like cells (HOb) from bone explants render them a lucrative model for studying molecular physiology of bone turnover discovery of novel anabolic therapeutics and mesenchymal cell biology in general. Relatively little is known about resting and dynamic expression profiles of HObs and no studies have been conducted to date to systematically assess the osteoblast transcriptome. The aim of this study was to characterize HObs and investigate signaling cascades and gene networks using genomewide expression profiling in resting and Bone Morphogenic Protein (BMP)-2 and Dexamethasone induced cells. Our data showed a vast number of genes and networks expressed predominantly in HObs as compared to closely related cells such as fibroblasts or chondrocytes. For instance, genes in the insulin-like growth factor (IGF) signaling pathway were enriched in HObs (p=0.003) and included the binding proteins (IGFBP1, 2, 5) and IGF-2 and its receptor. Another HOb specific expression pattern included leptin and its receptor (p<10-8). Furthermore, after stimulating HObs with Dexamethasone or BMP-2, the expression of several interesting genes and pathways were observed where data supported the role of peripheral leptin signaling in bone cell function. In conclusion, we provide the landscape of tissue-specific and dynamic gene expression in HObs, a resource, which will allow utilization of osteoblasts as a model to study specific gene networks and gene families related to human bone physiology and diseases. Keywords: cell type comparison
The establishment of induced pluripotent stem (iPS) cells methodologies has widened the possibilities of stem cell related clinical therapies. However safety concerns over current reprogramming technologies as well as lack of efficient protocols for differentiation raise serious questions over their usage in clinical settings. Direct lineage conversion may represent a safer technology for cell-therapy. Here, we identify Sox2 as a factor able to transdetermine human mesenchymal stem cells (MSCs) to hematopoietic progenitor cells (HPCs), recapitulating the molecular events during in vivo hematopoiesis. This technology offers an efficiency of up to 70% HPC generation in less than 8 days under feeder-free conditions. With the goal of devising ways for transplantation into diseased patients we developed two different methodologies that avoid exogenous DNA integration or viral infections. The first, by transduction of recombinant Sox2 protein, and the second by functional replacement of Sox2 by inhibition of TGFß signaling. The DNA integration-, virus-, and feeder-free transdetermination system here described may complement and enhance current approaches for autologous as well as heterologous HPC transplantation in humans.
Genes responses in A549 and H460 cells after GSI (RO4929097-001-003 2 uM) treatment. Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of gamma-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.
This SuperSeries is composed of the SubSeries listed below.
analyzed changes in cytokine/chemokine production and gene expression levels in human peripheral blood mononuclear cells upon teratment with 15µM,2,4-benzenetriol Keywords: treatment with chemical compound
Human fetal liver fibroblast were reprogrammed to chiPS without exogenous DNA integration using a single episomal vector. The chiPS were then transplanted into SCID mice and HuSCID to form teratomas. We used microarrays to profile the gene expression differences between the teratomas formed by chiPS in SCID and HuSCID mice.
screenning the differentially expressed genes of Jurkat-FF3 Jurkat-miR146a, Jurkat-miR146a-sponge cell lines.
We defined the major transcriptional responses in primary human bronchial epithelial cells (HBECs) after either infection with influenza or treatment with relevant ligands. We used four different strategies each highlighting distinct aspects of the response. (1) cells were infected with the wild-type PR8 influenza virus that can mount a complete replicative cycle. (2) cells were transfected with viral RNA (‘vRNA’) isolated from influenza particles. This does not result in the production of viral proteins or particles and identifies the effect of RNA-sensing pathways (e.g., RIG-I.). (3) Cells were treated with interferon beta (IFNb), to distinguish the portion of the response which is mediated through Type I IFNs. (4) Cells were infected with a PR8 virus lacking the NS1 gene (‘DNS1’). The NS1 protein normally inhibits vRNA- or IFNb-induced pathways, and its deletion can reveal an expanded response to infection.
The insulin-like growth factor-I (IGF-IR) and androgen (AR) receptors are important players in prostate cancer biology. Functional interactions between the IGF-I and androgen signaling pathways seem to have crucial roles in the progression of prostate cancer from early (benign) to advanced (metastatic) stages. DNA methylation is a major epigenetic alteration affecting gene expression. Hypermethylation of tumor suppressor promoters is a frequent event in human cancer leading to inactivation and repression of specific genes. The aim of the present study was to identify the entire set of methylated genes (“methylome”) in a cellular model that replicates prostate cancer progression.
Inflammatory mediators play a role in the pathogenesis/progression of chronic heart failure (CHF). The aim of the present study was to identify diagnostic/prognostic markers and gene expression profiles of CHF vs control. Keywords: CHF patients profiling vs controls.
Using the surface marker EPHB2 we have FACS-purified and profiled stem cell-enriched cell fractions from normal human mucosa, crypt proliferative progenitors and late transient amplifying cells to define a gene expression program specific for normal human colon epithelial stem cells
We identified 1700 differentially expressed probesets in DKD glomeruli and 1,831 in diabetic tubuli; 330 probesets were commonly differentially expressed in both compartments. The canonical complement signaling pathway was determined to be statistically differentially regulated in both DKD glomeruli and tubuli and was associated with increased glomerulosclerosis even in an additional set of DKD samples.
Expression profiling according to differentiation was analysed in 34 neuroblastomas
AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4+CD7- Sezary cells from patients with Sezary syndrome (SS). Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1-mediated transformation microarray analysis was performed to identify differentially expressed genes in AHI-1 suppressed CTCL cells. Fifteen up-regulated and six down-regulated genes were identified and confirmed by Q-RT-PCR. Seven were further confirmed in a microarray analysis of CD4+CD7- Sezary cells from SS patients. HCK and BIN1 emerged as new candidate cooperative genes, with differential protein expression which correlates with observed transcript changes. Interestingly, changes in HCK phosphorylation and biological response to its inhibitor, dasatinib, were observed in AHI-1 suppressed or overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells, which also exhibit differential MYC protein expression. In addition, aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells. Keywords: Cell type comparison
Bone marrow (BM) has been considered so far the reference source for stromal cells (SC) originally called mesenchymal stem cells. Recently human adult adipose tissue (AT) has been reported as a valuable source for the isolation of cells exerting a mesenchymal-like phenotype. Though both BM- and AT- derived stromal cells (BMSC and ATSC) share similar immuno-phenotype and exhibit multi-lineage potential in vitro a consensual panel of specific markers is still debated and the precise molecular mechanisms governing their differentiated fate are not fully understood. The aim of this study was to compare the genome wide expression profiles of stromal cells isolated from AT and BM and to emphasize the core of MSC stemness properties. Moreover we focused on the molecular characteristics of ATSC, attempting to reveal their specific features.We identified an overlapping dataset of 190 genes commonly regulated by ATSC and BMSC. Among these, we were able to categorize 6 key biological families that could be regarded as a stemness signature that underlie the self-renewal potential and the ability to generate progenitor cells. In particular, a pivotal role of signalling pathways along with the expression of numerous transcription regulators emerged from this study. Genes specifically modulated in ATSC, suggested that these cells posses anti-oxidative and neuroprotective properties. Taken together, these results provide new hints towards the understanding of the molecular basis of MSC maintenance and suggest that ATSC have interesting properties which could be useful for several potential clinical applications. Keywords: genomic profiling
During human pregnancy a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) ± fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, CTBs isolated from PE and control placentas were cultured for 48 h, enabling differentiation/invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation that resolved in culture. The upregulated genes included SEMA3B. Adding this protein to normal CTBs inhibited invasion and re-created aspects of the phenotype of these cells in PE. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression, the autocrine actions of the upregulated molecules, including SEMA3B, impair differentiation/invasion/signaling and patient-specific factors determine the signs.
The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define the role of c-Myb in human hematopoietic lineage commitment we studied the effects of its silencing during the commitment of human CD34+ Hematopoietic stem/progenitor cells. In CD34+ cells c-Myb silencing determined a cell cycle arrest in G0/G1 phase which strongly decreased the clonogenic efficiency, togheter with a reduction of erythroid colonies coupled with an increase of the macrophage and megakaryocyte ones. Moreover, morphological and flow cytometry data supported the preferential macrophage and megakaryocyte differentiation of c-Myb-silenced CD34+ cells. Taken together our data indicate that c-Myb is essential for the commitment along the erythroid and granulocyte lineages but not for the macrophage and megakaryocyte differentiation. Gene expression profiling of c-Myb-silenced CD34+ cells identified some potential c-Myb targets which can account for these effects, to study by Chromatin Immunoprecipitation and Luciferase Reporter Assay.
MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers but knowledge of its targets in colon cancer remains limited. To investigate the role of miR-145 in colon cancer, we have employed a microarray based approach to identify miR-145 targets. Based on seed site enrichment analyses and unbiased word analyses, we found a significant enrichment of miRNA binding sites in the 3’-untranslated regions (UTRs) of transcripts down-regulated upon miRNA overexpression, which represent potential miR-145 targets. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, gene expression, cancer, cell cycle, DNA replication, recombination and repair. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets based on 3’UTR luciferase assays and western blots for endogenous proteins.
The NOTCH1 signaling pathway directly links extracellular signals with transcriptional responses in the cell nucleus and plays a critical role during T-cell development and in the pathogenesis over 50% of human T-cell lymphoblastic leukemia (T-ALL) cases. However little is known about the transcriptional programs activated by NOTCH1. Using an integrative systems biology approach we show that NOTCH1 controls a feed-forward loop transcriptional network that promotes cell growth. Inhibition of NOTCH1 signaling in T-ALL cells led to a reduction in cell size and elicited a gene expression signature dominated by downregulated biosynthetic pathway genes. By integrating gene expression array and ChIP-on-chip data, we show that NOTCH1 directly activates multiple biosynthetic routes and induces c-MYC gene expression. Reverse engineering of regulatory networks from expression profiles showed that NOTCH1 and c-MYC govern two directly interconnected transcriptional programs containing common target genes that together regulate the growth of primary T-ALL cells. These results identify c-MYC as an essential mediator of NOTCH1 signaling and integrate NOTCH1 activation with oncogenic signaling pathways upstream of c-MYC. Keywords: Drug treatment
Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.
MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression by binding to 3’UTRs of target mRNAs. Kaposi’s sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including primary effusion lymphoma (PEL), encodes 12 miRNA genes, but only a few regulatory targets are known. We found that KSHV-miR-K12-11 shares 100% seed-sequence homology with hsa-miR-155, a miRNA frequently found up-regulated in lymphomas and critically important for B cell development. Based on this seed-sequence homology, we hypothesized that both miRNAs regulate a common set of target genes and as a result, could have similar biological activities. Examination of five PEL lines showed that PELs do not express miR-155, but do express high levels of miR-K12-11. Bioinformatics tools predicted the transcriptional repressor BACH-1 to be targeted by both miRNAs and ectopic expression of either miR-155 or miR-K12-11 inhibited a BACH-1 3'UTR containing reporter. . Furthermore, BACH-1 protein levels are low in cells expressing either miRNA. Gene expression profiling of miRNA-expressing stable cell lines revealed 66 genes that were commonly down-regulated. For select genes, miRNA targeting was confirmed by reporter assays. Thus, based on our in silico predictions, reporter assays, and expression profiling data, miR-K12-11 and miR-155 regulate a common set of cellular targets. Given the role of miR-155 during B cell maturation, we speculate that miR-K12-11 may contribute to the distinct developmental phenotype of PEL cells, which are blocked in a late stage of B cell development. Together, these findings indicate that KSHV miR-K12-11 is an ortholog of miR-155. Keywords: comparison, experiemental versus control
Gene expression microarray profile for human embryonic kidney cells (HEK293T CRL-11268) under untreated conditions.
Cancer stem cells are believed to be responsible for tumor initiation and development. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma stem cells. Anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for maintaining the stem-like properties and tumorigenicity of glioblastoma stem cells.
The 3-Deazaneplanocin A (DZNep) one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by quantitative RT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Ectopic expression of ALOX5 reduced sensitivity to DZNep in H929 cells. Furthermore, down-regulation of ALOX5 by RNA interference could also induce apoptosis in H929. Gene expression analysis on MM patient dataset indicated ALOX5 expression was significantly higher in MM patients compared to normal plasma cells. We also found that Bcl-2 was overexpressed in DZNep insensitive cells, and cotreatment with DZNep and ABT-737, a Bcl-2 family inhibitor, synergistically inhibited growth and induced apoptosis of DZNep insensitive MM cells. Taken together, this study shows one of mechanisms of the DZNep efficacy on MM correlates with its ability to down-regulate the ALOX5 levels. In addition, DZNep insensitivity might be associated with overexpression of Bcl-2, and the combination of ABT-737 and DZNep could synergistically induced apoptosis. These results suggest that DZNep may be exploited therapeutically for a subset of MM.
Tumor tissue heterogeneity is a well known feature of several solid tumors. Neuroblastic Tumors (NTs) is a group of paediatric cancers with a great tissue heterogeneity. Most of NTs are composed of undifferentiated poorly differentiated or differentiating neuroblastic (Nb) cells with very few or absent Schwannian stromal (SS) cells: these tumors are grouped as Neuroblastoma (Schwannian stroma-poor). The remaining NTs are composed of abundant SS cells and classified as Ganglioneuroblastoma (Schwannian stroma-rich) intermixed or nodular and Ganglioneuroma. The importance to understand Nb and SS gene signatures in NTs, is to clarify the complex network mechanism of tumor growth and progression. In order to identify the Nb and SS cells gene signatures, we analyzed the gene expression profiling of 19 cases of neuroblastic tumors: 10 stroma poor (NTs-SP) and 9 stroma rich (NTs-SR), by high density oligonucleotide microarrays. Moreover, the analysis was performed in parallel on both whole and laser microdissected tumor samples: from 4 of 19 cases, was isolated different areas all composed of pure cellular populations. We performed genome wide expression analysis by using Affymetrix technology and we used two different approaches for data analysis: SAM (Significance Analysis of Microarrays) and a method based on Game Theory (GT), to identify genes differently expressed in SS and Nb cells. Differently from the SAM method, the analysis based on GT (Moretti et al. (2006)) gives the advantage of selecting relevant genes not only according to the expression profile of each single gene, but considering also gene interaction. Keywords: Gene signature of Neuroblastic and Schwannian stromal cells.
The FHIT gene at 3p14.2, encompasses the common fragile site FRA3B and is frequently inactivated in primary tumors and cell lines of lung, head and neck, stomach, esophagus, cervix and breast cancer. In particular, loss of the FHIT protein is one of the most frequent alteration in lung tumors and pre-invasive lung lesions, suggesting a role for this gene in the early stages of lung carcinogenesis. Adenoviral-mediated restoration of FHIT expression in FHIT-negative cell lines results in cell cycle alteration, induction of apoptosis (through activation of the cytoplasmic apoptotic pathway) and regression of the tumorigenic phenotype. Taken together these observations support the hypothesis of a role for FHIT in human carcinogenesis, but little is known about its mechanism of action. Identification of transcriptional targets of FHIT is therefore critical to understand the pathways by which FHIT promotes growth arrest and apoptosis.
The clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversial discussed. The aim of this study was to clarify the significance of aberrant CEBPA PM with regard to clinical features in a cohort of 572 de novo AML with wildtype CEBPA and normal karyotype. The distal promoter was methylated in 54/572 cases (9.41%) whereas proximal PM was never detected. Methylation of the core promoter was detected in only 8 of 326 cases (2.45%) and thus seems to be a rare event in AML. There was no correlation between CEBPA distal PM age, sex, white blood cell (WBC) count or Hb levels at diagnosis. We also were not able to detect a significant correlation between the presence of CEBPA distal PM and molecular mutations such as FLT3-ITD, NPM1, AML1, MLL-PTD and IDH1. Solely the frequency of IDH2R140 mutations was significantly reduced in CEBPA distal PM positive compared to CEBPA distal PM negative cases (p=0.01). Furthermore, analysis of CEBPA mRNA expression level revealed no difference between CEBPA distal PM positive and CEBPA distal PM negative cases, suggesting that CEBPA distal PM has no influence on CEBPA expression. CEBPA distal PM did not show impact on overall survival (OS), event free survival (EFS) or incidence of relapse. Also when other mutations were taken into regard no prognostic impact of CEBPA distal PM could be shown. In contrast, a distinct expression profile of CEBPA distal PM positive cases compared to CEBPA mutated and CEBPA distal PM negative cases was observed. In addition, a significantly higher frequency of CEBPA distal PM was detected in RUNX1-RUNX1T1 positive AML compared to the CEBPA witdtype cases. We conclude that the presence of aberrant CEBPA PM has no clinical relevance and is therefore a negligible prognostic marker in de novo AML with normal karyotype.
Goals/objectives: to identify various gene expression in B cell subsets derived from human PBMC and cord blood
Chromosomal rearrangements involving ETS factors ERG and ETV1, occur frequently in prostate cancer. We here examine human prostate cancer cells control VCaP and LNCaP cells with ERG- or ETV1-silenced VCaP or LNCaP cells, respectively, in hormone deprived and stimulated conditions.
Investigation of expression profile in XDP brains. Keywords: an XDP patient and neurologically normal control
Four healthy human volunteers underwent an acute bout of resistance exercise with the right leg at 2 pm. Biopsies were removed from the Vastus Lateralis muscle 6 h (8 pm) and 18 h (8 am) after exericise Keywords = Human skeletal muscle Keywords = resistance exerise Keywords = diurnal Keywords = circadian Keywords: time-course
The tissue-specific pattern of mRNA expression can indicate important clues about gene function. High-density oligonucleotide arrays offer the opportunity to examine patterns of gene expression on a genome scale. Toward this end we have designed custom arrays that interrogate the expression of the vast majority of protein-encoding human and mouse genes and have used them to profile a panel of 79 human and 61 mouse tissues. The resulting data set provides the expression patterns for thousands of predicted genes, as well as known and poorly characterized genes, from mice and humans. We have explored this data set for global trends in gene expression, evaluated commonly used lines of evidence in gene prediction methodologies, and investigated patterns indicative of chromosomal organization of transcription. We describe hundreds of regions of correlated transcription and show that some are subject to both tissue and parental allele-specific expression, suggesting a link between spatial expression and imprinting. Keywords: different tissues
Estrogen receptors (ERs) which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and more than 1500 putative secondary target genes of estradiol in MCF7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Keywords: drug treatment
Homodimerization of Mpl can also be accomplished in the absence of Tpo by binding of a synthetic ligand (Chemical inducer of dimerization, CID) to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. In contrast to Tpo stimulation, F36VMpl dimerization in human CD34+ progenitor cells generates robust erythropoiesis. Microarray gene expression profiling of progenitors demonstrated that F36VMpl dimerization, but not Tpo, results in upregulation of critical erythroid genes.
Lipid rafts are cholesterol-rich cell signaling platforms and their physiological role can be explored by cholesterol depletion. To dress a global picture of transcriptional changes ongoing after lipid raft disruption we performed whole-genome expression profiling in epidermal keratinocytes, a cell type which synthesizes its cholesterol in situ. We used microarrays to identify transcriptional changes in gene expression of cholesterol-depleted keratinocytes. Cholesterol depletion by methyl-beta-cyclodextrin disrupts the organization of lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains.
A weakly bone metastatic variant of the breast cancer cell line MDA-MB-231 SCP6, gave rise to highly bone metastatic sublines (PD1, PD2A-E) after long time dormancy in vivo. These cell lines were subjected to microarray analysis with data drawn from previous studies (Kang et al., 2003; Minn et al. 2005; Lu and Kang 2009; Lu and Kang 2010). Keywords: Cell type comparison
The goal of this project was to characterize changes in gene expression in response to the anti-cancer agent sapphyrin PCI-2050. Cultured A549 human lung cancer cells were treated with sapphyrin PCI-2050 or actinomycin D a known transcripitonal inhibitor. The gene expression profiles of drug-treated and control A549 cultures were determined using Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA). Further details are provided in our published manuscript: . Keywords: responses to treatments with anti-cancer agents
Expression profiles of 17 melanoma cell lines were analysed to identify genes differentially expressed between cell lines harbouring wild-type or mutant p16INK4A. Relevant paper: Pavey et al. (2007). Note: all of these cell lines contained wild-type p14ARF so that the transcriptional effects of p16INk4A could be determined without interference from p14ARF. Keywords: Affymetrix Hu133_Plus microarrays
Ossifying fibroma (OF) is a common benign fibro-osseous neoplasm of orofacial bones showing progressive enlargement of the affected jaw with deficiency in bone formation. Our aim is to discover the molecular difference between mesenchymal stem cells from OF and normal jaw bone to provide basis for development of clinical therapy. Global gene expression comparison and gene ontology analysis found that several singaling pathways are involved in the process, including TGFb signaling and Notch signaling. Inhibition of TGFb signaling in mesenchymal stem cells of OF can increase the osteogenesis and decrease proliferation, shedding light on developing strategies for therapy.
The pituitary tumor-transforming gene (PTTG1) is a recently discovered oncogene implicated in the malignant progression of a number of neoplasms. It has been shown to drive both endocrine and non-endocrine malignancies but has not yet been studied in the context of renal cell carcinoma (RCC). Clear cell RCC (ccRCC) is cytogenetically characterized by deletion of chromosome 3p, harboring the von-Hippel Lindau tumor suppressor gene, and amplification of chromosome 5q. The significance of copy number gain of chromosome 5 is not clear, but is presumed to be the location of oncogenes that influence ccRCC development or progression. The PTTG1 oncogene maps to chromosome 5q, and here we show that PTTG1 is amplified in clear cell RCC, is overexpressed in tumor tissue relative to adjacent normal kidney, and expression is associated with high grade, high stage, and poor prognosis. Furthermore, we establish a functional role for PTTG1 in ccRCC tumorigenesis and progression. PTTG1 ablation reduces both the tumorigenic ability of ccRCC cells in vitro and in vivo and the invasive ability of these cells in vitro. An analysis of genes whose transcription is regulated by PTTG1 was supportive of an association with invasive and metastatic disease. PTTG1-dependent expression of the Rho-GEF ECT2, another proto-oncogene, is observed in a number of ccRCC cell lines, and ECT2 expression correlates with PTTG1 expression, high stage, high grade, and poor prognosis ccRCC. As GEF's have been promoted as potential drug targets for targeted cancer therapeutics, the relationship between the PTTG1 and ECT2 oncogenes may be able to be exploited for the treatment of this disease.
Objective: The etiology of PCOS is mostly unknown. Existing data support both genetic and environmental factors in its pathogenesis. Design: Prospective case - control study. Setting: University Hospital. Patients: 25 patients undergoing IVF-ICSI treatment. Intervention: Genome-wide oligonucleotide microarray technology was used to study differential gene-expression patterns of cultured human cumulus cells from IVF patients divided into 4 groups according to disease state (PCOS vs. Control) and BMI (Obese vs. Lean). Results: Two differential PCOS gene expression profiles were established: Lean-Type was formed by comparing PCOS lean (PL) vs. non-PCOS lean (NL) individuals; Obese-Type was formed by comparing PCOS obese (PO) vs. non-PCOS (NO) obese patients. Conclusions: Different molecular pathways are associated with PCOS in Lean and Obese individuals as demonstrated by gene expression profiling of cumulus cells. Our findings provide insights into the molecular pathogenesis of PCOS. We used microarrays to study the gene expression of human cultured cumulus cells. We compared the genes expression of lean PCOS, Obese PCOS, lean controls and obese controls. Different molecular pathways are associated with PCOS in Lean and Obese patients. Keywords: disease state analysis
Mediator complex has been known as pivotal regulator of RNA polymerase II. Mediator complex has two CDK subunits in vertebrates named CDK8 and CDK19. To elucidate functional difference between CDK8 and CDK19 in human cell, we employ siRNA mediate knockdown assay using HeLa S3 cell line. According to this assay these CDKs possess highly redundancy in HeLa S3 cell transcription regulation mechanism but in several genes, each CDK shows gene specific regulatory function.
We investigated the clinical implications of lung developmental transcription factors (TTF-1 NKX2-8, and PAX9) which we recently discovered as cooperating oncogenes activated by way of gene amplification at chromosome 14q13 in lung cancer. Using stable transfectants of human bronchial epithelial cells, RNA expression profiles (signatures) representing activation of the biological pathways defined by each of the three genes were determined and used to risk stratify a non-small cell lung cancer (NSCLC) clinical dataset consisting of ninety-one early stage tumors. Co-activation of the TTF-1 and NKX2-8 pathways identified a cluster of patients with poor survival, representing approximately 20% of patients with early stage NSCLC, whereas activation of individual pathways did not reveal significant prognostic power. Importantly, the poor prognosis associated with co-activation of TTF-1 and NKX2-8 was validated in two other independent clinical datasets. Further, lung cancer cell lines showing co-activation of the TTF-1 and NKX2-8 pathways were shown to exhibit resistance to cisplatin, the standard of care for the treatment of NSCLC. Since TTF-1 and NKX2-8 lack specific inhibitors at the current time, we explored an alternative therapeutic strategy. Using signatures of signaling pathway activation, we identified deregulation of specific oncogenic pathways (Ras and Myc) in the TTF-1/NKX2-8 co-activated cohort. In vitro experiments demonstrated the ability of a Ras pathway-specific therapy to inhibit tumor cell growth in TTF-1/NKX-2 activated cells, thus, suggesting that modulation of the Ras pathway is a rational strategy to targeted therapy in high risk NSCLC patients with co-activation of specific lung developmental pathways. Keywords: Transcription factor expression analysis
Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients by viral vector-mediated factor transduction represent a powerful tool for biomedical research and may provide a source for cell replacement therapies. However the proviruses encoding the reprogramming factors represent a major limitation of the current technology because even low vector expression may alter the differentiation potential of the iPSCs and induce malignant transformation. Here we show that fibroblasts from five patients with idiopathic Parkinson’s disease (PD) can be efficiently reprogrammed into hiPSCs and subsequently differentiated into dopaminergic neurons. Moreover, we derived PD specific hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Upon factor deletion these cells maintain a pluripotent state and intact karyotype. Importantly, these factor-free hiPSCs show a global gene expression profile, which is more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in conventional virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from patients with schizophrenia and bipolar disorder and healthy controls. RNA was isolated from these cell populations amplified, and analysed using Affymetrix HG133plus2.0 GeneChips. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. Keywords: cell type comparison, laser capture microdissection
The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis differentiation, remodeling and repair. However, little is known about their function in the adult lung. We use Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 for 48h when the transepithelial electrical resistance (TER) reached a threshold level. The goal is to identify direct target genes of GRHL2 and early events in the uncoupling of junctional interactions, including those regulating transepithelial resistance.
Mechanical unloading by ventricular assist devices (VAD) leads to significant gene-expression changes often summarized as reverse remodeling. However little is known on individual transcriptome changes during VAD-support and its relationship to non-failing hearts (NF). In addition no data are available for the transcriptome regulation during non-pulsatile VAD-support. Therefore we analysed the gene-expression patterns of 30 paired samples from VAD-supported (including 8 non-pulsatile VADs) and 8 non-failing control hearts (NF) using the first total human genome-array available. Transmural myocardial samples were collected for RNA-isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data was analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). 352 transcripts were differentially regulated between samples from VAD-implantation and NF, whereas 510 were significantly regulated between VAD-transplantation and NF (paired t-test p<0.001, fold change >=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD-support. Unsupervised hierarchical clustering of paired VAD- and NF-samples revealed separation of HF- and NF- samples, however individual differentiation of VAD-implantation and VAD-transplantation was not accomplished. Clustering of pulsatile and non-pulsatile VAD did not lead to robust separation of gene expression patterns. During VAD-support myocardial gene expression changes do not indicate reversal of the HF-phenotype, but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and non-pulsatile VAD-supported hearts did not provide evidence for a pump-mode specific transcriptome pattern.
To investigate molecular mechanisms of resistance we used two different in vivo xenograft models of estrogen receptor-positive (ER+) breast cancer, with or without HER2 over-expression (MCF7/HER2-18 and MCF7 wt, respectively). Mice with established tumors were assigned to the following treatment groups: continued estrogen supplementation (E2), estrogen deprivation (ED), ED plus tamoxifen (Tam), all with or without the EGFR tyrosine kinase inhibitor gefinitinib (G). Another group received ED plus the antiestrogen fulvestrant (MCF7 wt only). Tumors with acquired or de novo resistance to these endocrine therapies were profiled for mRNA expression using Affymetrix Genechip arrays. Keywords: multiple group comparison
Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation development and DNA damage responses. Although two homologous Drosophila YY family members (pleiohomeotic (pho)) and pleiohomeotic-like (phol)) are redundant, the functional significance of a recently described mammalian YY1-like gene (YY2) is unknown. Using microarray and gene set enrichment analysis (GSEA), we found that lentiviral constructs containing short hairpin loop YY1- and YY2-specific inhibitory RNAs (shYY1 and shYY2) caused significant changes in both redundant and distinguishable expression patterns. Ribosomal protein genes were the most significant gene set up-regulated by both shYY1 and shYY2, although combined shYY1/shYY2 knockdowns were not additive. In contrast, shYY2 reversed anti-proliferative effects of shYY1 on E2F target genes, and shYY2 particularly altered UV damage response, platelet-specific genes and mitochondrial function genes. The most YY2-specific gene was the platelet glycoprotein CD36 whose ligand is thrombospondin - a key UV response gene. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Taken together, our studies show that YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously thought to uniquely respond to YY1. Functions of thrombospondin and CD36 in inflammation, atherogenesis, innate immunity and malaria pathogenesis reveal new potential regulatory roles for YY1 and YY2.
A large proportion of patients suffering from the malignant pediatric tumor neuroblastoma die of progressive disease despite intensive therapy. Neuroblastomas belong to the group of neuroblastic tumors together with the more benign, differentiated ganglioneuroblastomas and ganglioneuromas. Little is known of the genes driving the differentiation processes in these tumor types. A search for the transcription factors differentially expressed between ganglioneuromas, ganglioneuroblastomas, and neuroblastomas in a series of 110 neuroblastic tumors (NB110) identified a large number of HOX- and TALE (Three Amino acid Loop Extension)-class homeobox transcription factor genes. The MEIS1-3, PBX1 and -3, and PKNOX1 TALE genes showed highest expression in neuroblastomas and lowest in ganglioneuromas and ganglioneuroblastomas. The PKNOX2 and TGIF1-2 genes showed the opposite expression pattern. This suggests an involvement of TALE genes in neuroblastoma differentiation. Expression of MEIS1, a known oncogene in haematopoietic tumors, was high in all neuroblastomas, and strongly correlated with undifferentiated histology. Consequently, we generated IMR-32 neuroblastoma cells capable of inducible shRNA-mediated MEIS1 knockdown. We observed differentiation, growth arrest and induction of apoptosis upon MEIS1 down-regulation. Affymetrix profiling of time-course experiments using these cells allowed the identification of MEIS1 target genes. Analysis of the target genes in the NB110 series showed that 323 of these were also significantly correlated to MEIS1 expression and to tumor differentiation in neuroblastic tumors. Genes involved in the cell cycle and in developmental pathways were over-represented in this gene set. We conclude that MEIS1 governs several of the signal transduction routes important for neuroblastoma survival and differentiation.
Gene expression profiling in arterial tissue from type 2 diabetic patients
An evaluation of multifocal lesions from patients with in-transit extremity melanoma to determine if all lesions from a patient harbor homogeneous patterns of gene expression Gene expression profiling studies can help guide treatment for cancer patients by providing tools in the form of gene-expression signatures to characterize a tumor in terms of underlying biology predicted response to therapy, metastatic progression and/or recurrence. The utility of gene signatures for defining therapeutic strategies in the treatment of extremity in-transit melanoma will be dependent on the genetic relationship between the multifocal lesions typically present in this disease and the extent to which a single lesion is representative of residual tumor burden. Using microarray-based gene expression profiling we examined 43 in-transit melanoma lesions across 17 patients with multifocal disease to determine whether one lesion could accurately characterize the underlying biology and genetic profile of a patient's tumor. Principal component analysis, unsupervised hierarchical clustering, one-way analysis of variance (ANOVA) and gene signatures predictive of chemosensitivity and oncogenic pathway activation showed gene expression patterns to be highly similar (p-values: <0.006; average r = 0.979) between lesions from a single patient but to be significantly different across patients (p<0.05). These findings demonstrate that individual melanoma tumor nodules in patients with multifocal disease are genetically similar and a single lesion can be used to predict response to chemotherapy, evaluate the activation status of oncogenic signaling pathways and characterize other aspects of the biology of an individual patient's disease. These results will facilitate the utilization of gene expression profiling in clinical trials of targeted therapy in melanoma allowing for more rational identification of candidates for specific therapies. Keywords: Disease state analysis
We used microarrays to detail gene expression changes in Hs 294T human melanoma cells after treatment with elesclomol alone or in combination with NAC, to aide in identifing the mechnism of action of elesclomol. Keywords: treatment
Full title: Three-dimensional culture of AIDS-NHL cells influences gene expression related to B-cell development proliferation and survival The AIDS-NHL-derived cell line, UMCL01-101, representing diffuse large B-cell lymphoma of immunoblastic morphology (AIDS-IBL), was grown in conventional, static suspension culture or three-dimensionally (3D) in the Rotating Wall Vessel (RWV) bioreactor. The objective was to assess the impact on gene expression of growth as a three-dimensional tissue assembly. Global gene expression analysis was performed on UMCL01-101 cells grown under either condition using Affymetrix microarray.
To analyze the impact of Aire on gene expression profile in a model cell line we used 293T cells and transfected them either with an Aire expression plasmid pCMV-Aire (where mAire is driven by CMV promoter) or with a control plasmid pCMV2B. Total RNA was extracted 48 hours post transfection, processed and used for gene expression profiling by Affymetrix. The data demonstrate that Aire has a very broad impact, effecting (upregulating and downregulating) hundreds of differents genes, however these genes differ dramatically from its targets in medullary epithelial cells. Keywords: transfection
The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However many of the mechanisms governing intestinal epithelial cell migration and the coordination of interactions with adjacent cells and the extracellular matrix are not fully understood. We have evaluated in vivo gene expression patterns of ileal epithelial cells in healthy human subjects, isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of the small intestinal mucosa. Expression profiles in villus epithelium and Paneth cell lineages were determined by quantitative real-time PCR, DNA microarray, and immunohistochemistry based methods. Relative expression levels of selected epithelial biomarkers were compared between the ileum, jejunum, duodenum, colon, stomach, and esophagus. Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations. Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel, most notably in the ileum, in comparison to other sites along the gastrointestinal tract. Our findings provide new and important insights regarding the molecular machinery employed by small intestinal epithelial cells to mediate their function and spatial organization in vivo. Keywords: analysis of epithelial cells from crypt or upper villus regions
This SuperSeries is composed of the SubSeries listed below.
Interferon-induced transmembrane protein 1 (IFITM1) is one of the three members of the interferon-induced transmembrane family and has recently been identified as a new molecular marker in human colorectal cancer. However its functional roles in colorectal cancer are still elusive. In this study, we investigate the gene expression profiling of HT-29 cells with IFITM1 knockdown. We revealed that several invasive- and carcinogenesis-related genes were differentially expressed.
Negative immunomagnetic selection has become the method of choice for isolating T cell subsets for functional studies due to concerns that directly binding antibody to the surface of a cell as occurs with positive selection, results in cross-linking of surface antigens, altered gene transcription and subsequent cellular activation. However there is little data to support this. We therefore examined the impact of the method of immunomagnetic cell selection on the gene expression profile of healthy human CD4 and CD8 T cells in a total of 21 cases. Keywords: Methodology
M21 or M21L cells were grown either in a 2-dimensional culture (on plastic) or in a 3-dimensional-collagen model.
Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, new small drugs were designed to block AKT activity for cancer treatment. Here we characterize the biological effects of the phosphatidyl inositol phosphate analogs SH-5 and SH-6 in colorectal cancer cell lines. We demonstrate that the two compounds did not reduce AKT phosphorylation significantly in the presence of growth factors, but induce a broad range of morphological and transcriptional alterations. Transcriptomic profiling in inhibitor-treated SW480 cells revealed a cluster of down- regulated genes associated with mitosis. Moreover, the treatment of SW480 cells with either SH-5 or SH-6 caused the formation of binucleated cells as a result of a specific abscission defect.
The pathophysiology of endometriotic lesion development remains unclear but involves a complex interaction between ectopic endometrium and host peritoneal tissues. We hypothesised that disruption of this interaction was likely to suppress endometriotic lesion formation. We hoped to delineate the molecular and cellular dialogue between ectopic human endometrium and peritoneal tissues in nude mice as a first step towards testing this hypothesis. Human endometrium was xenografted into nude mice and the resulting lesions were analysed using microarrays. A novel technique was developed that unambiguously determined whether RNA transcripts identified by the microarray analyses originated from human cells (endometrium) or mouse cells (stroma). Four key pathways (ubiquitin/proteosome, inflammation, tissue remodelling/repair and ras-mediated oncogenesis) were revealed, that demonstrated communication between host stromal cells and ectopic endometrium. Keywords: Disease state analysis
Gene expression profiles during the differentiation of HUVEC on matrigel were analyzed.
Transient expression of two factors or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.
Dysregulation of pyramidal cell network function by the soma- and axon-targeting inhibitory neurons that contain the calcium-binding protein parvalbumin (PV) represents a core pathophysiological feature of schizophrenia. In order to gain insight into the molecular basis of their functional impairment we used laser capture microdissection (LCM) to isolate PV-immunolabeled neurons from layer 3 of Brodmann’s area 42 of the superior temporal gyrus (STG) from postmortem schizophrenia and normal control brains. We then extracted ribonucleic acid (RNA) from these neurons and determined their messenger RNA (mRNA) expression profile using the Affymetrix platform of microarray technology. 739 mRNA transcripts were found to be differentially expressed in PV neurons in subjects with schizophrenia, including genes associated with WNT (wingless-type), NOTCH and PGE2 (prostaglandin E2) signaling, in addition to genes that regulate cell cycle and apoptosis. Of these 739 genes, only 89 (12%) were also differentially expressed in pyramidal neurons as found in the accompanying study, suggesting that the molecular pathophysiology of schizophrenia appears to be predominantly neuronal type-specific. Taken together, findings of this study provide a neurobiological framework within which hypotheses of the molecular mechanisms that underlie the dysfunction of PV neurons in schizophrenia can be generated and experimentally explored and, as such, may ultimately inform the conceptualization of targeted molecular intervention.
To broaden the appeal of the NIH Stem Cell Database we analyzed a subset of undifferentiated human embryonic stem cell lines (5 lines in duplicate) on the Affymetrix platform. One standard culture protocol was used in conjunction with rigorous quality control. Expanded description of methods used and are available at: http://stemcelldb.nih.gov.
This study provides a comparison of genes expressed in reconstructed cultured epidermis derived from four different donors. GeneChips were used to compare reconstructed human EpiDerm™ EPI-200 tissues prepared from four different neonatal foreskin donors (254 1188, 219 and 926). This work will assist in providing a deeper understanding of genes expressed in cultured skin equivalents. Keywords: Cell type/donor comparison
Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognisable by its distinctive red skin lesions. The lesions are KSHV-infected spindle cells most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The KSHV virus expresses multiple microRNA in a single cluster. Here we test the effects of this KSHV microRNA cluster in LEC cells using Affymetrix hgu133plus2 chips.
In polygenic disorders we do not know exactly how many genes are involved in the pathomechanism, but the analysis of fetal gene expression can get us closer to the solution. In our study we were searching for the genetic background of the polygenic neural tube defect, which is the second most common birth defect in the world (1 in 1000 live births). Our data revealed novel candidate genes, like SLAP, LST1 and BENE, which can play an important role in the pathogenesis of neural tube defects. We created a data warehouse from the results, suitable for further analysis. This study also demonstrates that a routinely collected amount of amniotic fluid (as small as 6 mL) is enough to successfully hybridize isolated RNA to expression arrays, making the ability to use the technique from normally collected amniotic fluid samples. Keywords: Prenatal gene expression signature
RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets Affymetrix HG-U133 arrays were hybridized with probe from total RNA isolated from blood sampled from 14 umbilical cords and 14 healthy adult humans. Keywords: Adult vs. fetal reticulocyte transcriptome comparison
Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Keywords: time course, cell type comparison, tissue engineered cartilage; osteoarthritis; Hyaff-11 scaffold; human chondrocytes; gene expression profiling; regenerative medicine; differentiation potential
MicroRNAs (miRNAs) have been globally profiled in cancers but there tends to be poor agreement between studies including in the same cancers. Additionally few putative miRNA targets have been validated. To overcome the lack of reproducibility, we profiled miRNAs by next generation sequencing and locked nucleic acid miRNA microarrays, and we verified concordant changes by quantitative RT-PCR. Notably, miR-125b and the miR-99 family members miR-99a, -99b, -100 were down-regulated in all assays in advanced prostate cancer cell lines relative to the parental cell lines from which they were derived. All four miRNAs were also down-regulated in human prostate tumor tissue compared to normal prostate. Transfection of miR-99a, -99b or -100 inhibited the growth of prostate cancer cells and decreased the expression of prostate-specific antigen (PSA), suggesting potential roles as tumor suppressors in this setting. To identify targets of these miRNAs, we combined computational prediction of potential targets with experimental validation by microarray and polyribosomal loading analysis. Three direct targets of the miR-99 family that were validated in this manner were the chromatin remodeling factors SMARCA5 and SMARCD1 and the growth regulatory kinase mTOR. We determined that PSA is post-transcriptionally regulated by the miR-99 family members at least partially by repression of SMARCA5. Together, our findings suggest key functions and targets of miR-99 family members in prostate cancer suppression and prognosis.
Total 23 samples were derived from [1] HUVEC treated in the absence (0h) or presence of hypoxia (1 2, 4, 8, 12, and 24 hrs) to determine hypoxia-regulated gene in endothelial cells, [2] control siRNA or HIF1α siRNA transfected HUVEC cells treated in the absence or presence of hypoxia, [3] control siRNA or KDM3A siRNA transfected HUVEC cells treated in the absence or presence of hypoxia, [4] ChIP-seq data for HIF1 binding sites and histone modifications under normoxia and hypoxia in endothelial cells.
The whole-genome oligonucleotide microarray analysis of laser microdissected human colonic epithelial cells can contribute to determination of disease-specific expression alterations in colonic epithelial cells and to localize the origin the expression changes measured in whole biopsy samples. Keywords: whole genomic expression
The androgen receptor (AR) is the principal target for treatment of non-organ confined prostate cancer (PCa). Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes and point towards a role for secondary transcription factors (TFs) therein. We identified a novel indirect mechanism of androgen action in which effects of androgens on PCa cells are mediated by Serum Response Factor (SRF). To identify and characterize genes and cellular processes that are androgen-regulated in an SRF-dependent manner in PCa Affymetrix HG-U133 Plus 2.0 GeneChip Array analysis was performed starting from RNA obtained from LNCaP cells in which androgen stimulation was combined with siRNA-mediated SRF silencing. To this end, LNCaP cells were seeded in 60 mm dishes at a density of 550,000 cells per dish in antibiotic-free medium. The next day, cells were transfected with siGenome SmartPool siRNA targeting SRF (Dharmacon, Lafayette, CO) or a custom-made control SmartPool targeting luciferase (LUC condition) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Forty-two hours after transfection, cells were treated with 5nM R1881 or ethanol vehicle. 3 biological triplicates were included per treatment group. Forty-eight hours later, cells were harvested in Trizol reagent (Invitrogen). RNA was isolated, purified on RNeasy columns (Qiagen, Germantown, MD) and checked for integrity by Agilent testing (Affymetrix, Santa Clara, CA). cDNA was generated and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the manufacturer’s instructions at the Mayo Clinic Advanced Genomics Technology Microarray Shared Resource core facility.
The three-dimensional (3D) folding of the chromosomal fibre in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Especially basic principles of 3D chromatin and chromosome organisation are still elusive. In this paper, we quantitatively analyse the 3D structure of large parts of chromosomes 1 and 11 in the G1 nucleus of human cells and relate it to the human transcriptome map (HTM). Despite a considerable cell-to-cell variation, our results show that subchromosomal domains, which are highly expressed, are more decondensed, have a more irregular shape and are located in the nuclear interior compared to clusters of low expressed genes. These aspects of chromosome structure are shared by six different cell lines and therefore are independent of cell type specific differences in gene expression within the investigated domains. Systematic measurements show that there is little to no intermingling of chromatin from different parts of the same chromosome, indicating that the chromosomal fibre itself is a compact structure. Together, our results reveal several basic aspects of 3D chromosome architecture, which are related to genome function. Keywords: cell line comparison
Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of SCID/Beige and NOD/SCID/IL2γ-receptor null (NSG) mice, under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (~23% and ~19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing unique 25 patients. Most tumors yielding xenografts were “triple-negative” (ER-PR-HER2+) (n=19). However, we established lines from three ER-PR-HER2+ tumors, one ER+PR-HER2-, one ER+PR+HER2- and one “triple-positive” (ER+PR+HER2+) tumor. Serially passaged xenografts show biological consistency with the tumor of origin, are phenotypic stability across multiple transplant generations at the histological, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses. Xenografts representing 12 patients, including two ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis.
T-cell/histiocyte rich B cell lymphoma (THRBL) and nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) share some morphological characteristics including a prominent stromal reaction, but display a markedly different prognosis. To investigate the difference between the stromal reactions of these lymphomas at the molecular level, we performed microarray expression profiling on a series of THRBL and NLPHL cases. Keywords: lymphoma, NLPHL, THRBL, stromal reaction
We identified recurrent NOTCH1 mutations in 12% of MCLs. 2 out of 10 tested MCL cell lines (Rec-1 and SP-49) were sensitive to inhibition of the NOTCH pathway by gamma-secretase inhibition. The aim of this study was to identify transcriptional targets of NOTCH signaling in MCL.
Induced pluripotent stem (iPS) cells have generated interest for regenerative medicine as they allow for producing patient-specific progenitors in vitro with potential value for cell therapy. In many instances however, an off-the-shelf approach would be desirable, such as for cell therapy of acute conditions or when the patient's somatic cells are altered as a consequence of chronic disease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are newborn, immunologically immature cells with minimal genetic and epigenetic alterations, and several hundred thousand immunotyped CB units are readily available through a worldwide network of CB banks. Here, we show that CB stem cells can be reprogrammed to pluripotency by retroviral transduction with OCT4, SOX2, KLF4, and c-MYC, in a process that is extremely efficient and fast. The resulting CB-derived iPS (CBiPS) cells are phenotypically and molecularly indistinguishable from human embryonic stem (hES) cells. Furthermore, we show that generation of CBiPS can be efficiently achieved without the use of the c-MYC and KLF4 oncogenes and just by overexpression of OCT4 and SOX2. Our studies set the basis for the creation of a comprehensive bank of HLA-matched CBiPS cells for off-the-shelf applications.
Atherosclerosis (AT) is a chronic inflammatory disease characterized by the accumulation of inflammatory cells lipoproteins and fibrous tissue in the walls of arteries. AT is the primary cause of heart attacks and stroke and the leading cause of death in westernized countries. To date, the pathogenesis of AT is not well-defined. Studies have shown that the dedifferentiation of contractile and quiescent vascular smooth muscle cells (SMC) to the proliferative, migratory and synthetic phenotype in the intima is pivotal for the onset and progression of AT. To further delineate the mechanisms underlying the pathogenesis of AT, we have analyzed the early molecular pathways and networks of SMC phenotype transformation as induced by the presence of minimally-oxidized LDL (moxLDL).
The involvment of bile acids such as deoxycholic acid (DCA) in gastro-esophageal reflux disease and subsequent Barrett’s metaplsia has been postulated. This study examines gene expression induced by exposure to DCA in esophageal cells and may be utilised in cross-comparisons with data derived from gene expression studies of Barrett’s esophagus and associated adenocarcinoma.
This SuperSeries is composed of the SubSeries listed below.
HT29 cells were transsfected with siRNA (siHX) which is targeting the human endogeous retrovirus HERV-HX. Cells were harvested 72 h after transfection and knock-down of HERV-HX was evaluated by quantitative RT-PCR. Keywords: Genetic modification
Transcriptional expression data for bioactive small molecules for mechanism identification. Keywords: Expression profiling by array
The response of human neutrophils to the emerging pathogen Mycobacterium abscessus has not been described. However M. abscessus infections are frequently associated with neutrophil-rich abscesses. To better understand the reponse of neutrophils to M. abscessus we performed gene expression analysis using Affymetrix HG-U133A Plus 2.0 microarrays. Human neutrophils from healthy donors were stimulated with isogenic rough and smooth morphotypes of M. abscessus. Staphylococcus aureus was used as a control. Gene expression was compared to neutrophils left unstimulated. Neutrophils from four individual donors were isolated on separate days and stimulated with freshly prepared bacteria. Neutrophils (stimulated and control) were left for 2 hours before total RNA was isolated, and biotinylated cRNA was prepared by standard methods. Analysis indicates that M. abscessus morphotypes induce a limited number of genes, when compared to S. aureus, which are enriched in genes for cytokines and chemokines, including neutrophil-specific chemokines. These data suggest that neutrophils have a limited response to M. abscessus, which may contribute to neutrophil-rich abscess formation.!Series_overall_design = Human neutrophils from healthy donors were exposed to rough Mab (ATCC 19977T), smooth Mab (ATCC 19977T) and S. aureus (CF clinical strain) for two hours; control cells were exposed to saline.
The dermal papilla (DP) of the hair follicle plays crucial roles in the hair follcie morphogenesis and cycling. Thus the elucication of human DP molecular signature is of great interest. DP cell culture by conventional method impairs intrinsic properties of DP cells. Isolatoion of human DP is hampered by the lack of specific cell surface markers. Thus, it still depends on manual microdissection, with which the removal of minor contamination is unfeasible. Cultured DP cells are mostly pure. Aggregation of cultured DP cells was shown to restore some intrinsic properties in cultured DP cells. Fibroblasts are distinct dermal cell population and provide baseline for DP arrays.
Advanced ovarian cancers are initially responsive to chemotherapy with platinum drugs but develop drug resistance in most cases. We showed recently that hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and that this effect is mediated by the p38 mitogen-activated protein kinase. In this work we integrated genome-wide expression profiling, in silico data survey, and functional assays to identify transcripts regulated in SK-OV-3 ovarian cancer cells made more responsive to CDDP by HGF. Using oligonucleotide microarrays, we found that HGF pretreatment changes the transcriptional response to CDDP. Quantitative reverse transcription-PCR not only validated all the 15 most differentially expressed genes but also confirmed that they were primarily modulated by the combined treatment with HGF and CDDP and reversed by suppressing p38 mitogen-activated protein kinase activity. Among the differentially expressed genes, we focused functional analysis on two regulatory subunits of the protein phosphatase 2A, which were down-modulated by HGF plus CDDP. Decrease of each subunit by RNA interference made ovarian cancer cells more responsive to CDDP, mimicking the effect of HGF. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or revert chemotherapy resistance.
Medulloblastoma (MB) is the most common malignant brain tumor in children among whom overexpression or amplification of MYC oncogenes has been associated with poor clinical outcome. Although the MYC functions during normal development and oncogenesis in various systems have been extensively investigated, the transcriptional targets mediating MYC effects in MB are still elusive. Their identification and roles during MB onset and progression are important and will ultimately suggest novel potential therapeutic targets. cDNA microarray analysis was used to compare the effects of overexpressing and silencing MYC on the transcriptome of a MB-derived cell line. We identified 209 genes with potential relevance to MYC-dependent cellular responses in MB. Among the MYC-responsive genes, we found members of the bone morphogenetic protein (BMP) signaling pathway, which plays a crucial role during the development of the cerebellum. In particular, the cytokine gene BMP7 was identified as a direct target of MYC in MB cells. Similar to the effect induced by BMP7 silencing by siRNA, the use of a small-molecule inhibitor of the BMP/SMAD signaling pathway reduced cell viability in a panel of MB cells. Altogether, our findings indicate that high MYC levels drive BMP7 expression in MB to induce pro-survival and pro-proliferative cellular pathways. This observation suggests that targeting the BMP/SMAD pathway may be a new therapeutic concept for the treatment of childhood MB.
Purpose and Experimental Design: The purpose of this study is to find a methylation-related gene that could become a biomarker or therapeutic target in colorectal carcinoma (CRC). We screened candidate genes suspected to be silenced by DNA methylation using oligonucleotide microarray analysis. To investigate the clinical significance of one candidate gene (UNC5B) we analyzed the correlation between mRNA expression and clinicopathological features using clinical tissue samples. Finally, methylation specific PCR analysis was performed to reveal whether the promoter region was methylated in CRC cell lines. Results: We found 75 candidate genes that were potentially suppressed by DNA methylation in CRC. We focused on UNC5B, a possible tumor suppressor gene and regulator of apoptosis known to be inactivated in CRC. The mRNA expression analysis using tissue samples revealed that UNC5B mRNA was down-expressed in about 20% of CRC patients, and the patients with low-UNC5B-expression tumors showed a significantly higher recurrence rate after curative surgery. According to the univariate and multivariate analysis, low UNC5B expression was an independent risk factor for postoperative recurrence in stage I, II, and III CRC patients. Furthermore, patients with low expression of UNC5B in tumors had significantly poorer prognosis than those with high expression of UNC5B. Although UNC5B mRNA expression was restored by the demethylation treatment in CRC cell lines, the promoter region of UNC5B was not methylated. Conclusion: UNC5B is a potential biomarker for the selection of patients with high risk of postoperative recurrence and worse prognosis in CRC.
Inflammation is common to many disorders and responsible for tissue and organ damage. However the associated peripheral cytokine milieu is frequently dilute and difficult to measure, necessitating development of more sensitive and informative biomarkers for mechanistic studies, earlier diagnosis, and monitoring therapeutic interventions. Previously, we have shown that sera from type 1 diabetes (T1D) patients induces a unique disease-specific pro-inflammatory transcriptional profile in fresh peripheral blood mononuclear cells (PBMCs) compared to sera of healthy controls. To address the potential variance introduced by heterogeneity in responsiveness of PBMCs from different donors, we evaluated human leukemia cell lines as surrogates for fresh PBMCs. Expression signatures of 7 different cell lines were 1) tested in their power to differentiate sera of T1D patients from healthy controls; and 2) compared to the signature obtained with fresh PBMCs.
Introduction: Amplification at chromosome 8q24 is one of the most frequent genomic abnormalities in human cancers and is associated with reduced survival duration in breast and ovarian cancers. The minimal amplified region encodes c-MYC and the non-coding RNA PVT1 including miR-1204 encoded in exon 1b. Here we analyzed the genomic changes at chromosome 8q24.21 in breast cancer and the functional roles of miR-1204 in breast and ovarian cancer progression. Methods: The genomic changes at chromosome 8q24.21 were detected in 997 breast cancer tumors and 40 breast cancer cell lines. Expression of miR-1204 in breast and ovarian cancer cell lines was investigated by qRT-PCR method. The role of miR-1204 in the tumorigenesis of breast and ovarian cancer was explored using both knockdown and overexpression of miR-1204 in vitro. Candidate miR-1204 target genes from two independent expression microarray datasets and computational predict programs were identified and further validated by qRT-PCR and western blot methods. The role of inhibition of miR-1204 on tamoxifen sensitivity in breast cancer cells was also investigated. Results: MiR-1204 is frequently co-amplified with MYC and expression of miR-1204 is strongly correlated with the expression and amplification of the noncoding PVT1 transcript and less so with MYC in human breast and ovarian cancer cells. Inhibition of miR-1204 decreases cell proliferation and increased apoptosis in breast and ovarian cancer cell lines with 8q24 amplification, but not in lines without amplification and so may be involved in Myc-induced apoptosis. Additionally, overexpression of miR-1204 enhances both breast and ovarian cancer cell growth and Myc-initiated Rat1A cell transformation. Computational and experimental analyses 30 promising candidate miR-1204 target genes. mRNA levels for these genes were assessed after over expression and knockdown of miR-1204 as were protein levels for 10 genes for which antibodies were available. These studies implicated VDR and ESR1 as miR-1204 targets. Inhibition of miR-1204 increased response to tamoxifen in Estrogen Receptor negative breast cancer cell lines. Conclusions: We conclude that amplification of miR-1204 contributes to breast and ovarian pathophysiology at least in part, by increasing proliferation and down regulating apoptosis and by decreasing expression of VDR and ESR1.
Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation. Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362-672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP, ACSL, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8. Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes. Keywords: Analysis of target gene regulation by using microarrays
Our previous studies have shown that bone morphogenetic protein 2 (BMP2) a morphogen belonging to the TGFβ superfamily, is markedly induced in human primary endometrial stromal cells (HESC) as they undergo differentiation in response to steroid hormones and cAMP. WNT4 is a downstream target of BMP2 regulation in these cells. To identify the common downstream targets of BMP2 and WNT4 in human endometrial stromal cells, we performed gene expression profling of human ensometrial stromal cell transduced with BMP2 or WNT4 adenovirus. Gene expression profiling revealed that FOXO1, a forkhead family transcription factor and a known regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. These studies uncovered a linear pathway involving BMP2, WNT4, and FOXO1 that operates in human endometrium to critically control decidualization.
The immortalized human urothelial cell line UROtsa, was transformed in six parallel cultures with continual passaging in1 µM Cd+2 until the cells were able to attain the ability to form colonies in soft agar and subcutaneous tumors in nude mice. The gene expression profiles between cadmium-transformed and control samples were compared and the differentially expressed genes were identified.
Comparisons among breast cancer metastases at different organs revealed distinct microenvironments as characterized by cytokine content. Such microenvironment distinction might be important to dictate how the cancer cells adapt to survival before they successfully colonize. Keywords: Disease state analyses
The 8p11 myeloproliferative syndrome (EMS) also referred to as the stem cell leukemia/lymphoma syndrome, is a chronic myeloproliferative disorder that rapidly progresses into an acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinase activity within FGFR1. The two most common fusion genes in human EMS are ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) and BCR/FGFR1. To study the transcriptional programs becoming deregulated by the FGFR1 fusion genes, global gene expression analysis on human CD34+ cord blood cells expressing either of the fusion oncogenes ZMYM2/FGFR1 and BCR/FGFR1 was performed. As a reference gene we also included the more studied BCR/ABL1 fusion oncogene associated with chronic myeloid leukemia. We found that the 3 different fusion oncogenes had in common the upregulation of several genes involved in the JAK/STAT signalling pathway and also other sets of genes. However, the gene expression profiles were not identical, suggesting that both the tyrosine kinase containing gene and the partner gene would affect the transcription of downstream target genes.
We have examined the biological effect of EWS/ETS in human MPCs using UET-13 cells that are obtained by prolonging the lifespan of human bone marrow stromal cells using the retroviral transgenes hTERT and E7. By exploiting tetracycline-inducible systems for expressing EWS/ETS (EWS/FLI1 and EWS/ERG) we investigated candidates for genes whose expression is regulated by EWS/ETS in human MPCs. Keywords: Time course
To determine how aging impacts gene expression in hematopoietic stem cells (HSCs) human CD34+ cells from bone marrow (34BM) and mobilized stem cell products (34P38NPBSC) were examined using microarray-based expression profiling. Differential expression changes were confirmed by microarray comparisons of younger and older expanded T-cell populations.
This series represents the rehydration series of the human 293h media depleted storage on agarose / rehydration condition course analysis. Samples include Control Monolayer 0 hr desiccation, 0 hr rehydration, 6 hr rehydration, 24 hr rehydration, and 72 hr rehydration. Keywords = 293h cells Keywords = desiccation Keywords = rehydration Keywords = spheroid Keywords = stabilization Keywords = ambient temperature Keywords: other
Nonmalignant human mammary epithelial cells (HMEC) seeded in laminin-rich extracellular matrix (lrECM) form polarized acini and in doing so, transit from a disorganized proliferating state to an organized growth-arrested state. We hypothesized that the gene expression pattern of organized and growth-arrested HMECs would share similarities with breast tumors with good prognoses. Using Affymetrix HG-U133A microarrays, we analyzed the expression of 22,283 gene transcripts in 184 (finite life span) and HMT3522 S1 (immortal nonmalignant) HMECs on successive days after seeding in a lrECM assay. Both HMECs underwent growth arrest in G0-G1 and differentiated into polarized acini between days 5 and 7. We identified gene expression changes with the same temporal pattern in both lines and examined the expression of these genes in a previously published panel of microarray data for 295 breast cancer samples. We show that genes that are significantly lower in the organized, growth-arrested HMEC than in their proliferating counterparts can be used to classify breast cancer patients into poor and good prognosis groups with high accuracy. This study represents a novel unsupervised approach to identifying breast cancer markers that may be of use clinically. Keywords: time course
The head and neck / oral squamous cell carcinoma (HNOSCC) is a diverse group of cancers which develop from many different anatomic sites and are associated with different risk factors and genetic characteristics. The oral tongue squamous cell carcinoma (OTSCC) is one of the most common types of HNOSCC. It is significantly more aggressive than other forms of HNOSCC, in terms of local invasion and spread. In this study, we aim to identify specific transcriptomic signatures that associated with OTSCC. Keywords: Disease/Control analysis
Behçet’s disease (BD) is a multisystemic immuno-inflammatory disorder characterized by a generalized vasculitis particularly at the orogenital mucosa and eye. It is a complex disease with unclear pathogenesis. To better understand BD´s etiology, we performed genomic expression profiling of patients and controls.
This study utilise the examination of normal gastro-intestinal tissues to determine a tissue specific signal for use in deriving the intestinal signature of intestinal metaplasias of the oesophagus. Normal oesophageal colonic and duodenal tissue biopsies were taken after informed consent and RNA was extracted following histological examination of adjacent tissues for normal aperaing mucosa.
Human abdominal adipose tissue was obtained with informed consent from a 33-year old Caucasian female (BMI = 32.96 Kg/m2) undergoing lipoaspiration. Adipose stromal cells (hASCs) were isolated and differentiated into adipocytes in vitro.
In vivo changes of gene expression profiles (GEP) of tumor cells 48hr after single agent therapy may vary by treatment and provide added predictive power over baseline GEP information. In newly diagnosed patients with multiple myeloma (MM) GEP data were obtained on tumor cells prior to and 48hr after dexamethasone (n=45) or thalidomide treatment (n=42); in case of relapsed MM, GEP data were obtained prior to (n=36) and after (n=19) lenalidomide administration. Dexamethasone and thalidomide induced both common and unique GEP changes. Combined baseline and 48hr changes of GEP in a subset of genes that were discovered in newly diagnosed MM also predicted event-free and overall survival in relapsed patients receiving lenalidomide. Combined with baseline molecular features, changes in GEP following short-term single agent treatment may help guide treatment decisions for patients with MM. The genes whose altered expression is related to eventual survival may also point to mechanisms of action and resistance to different classes of drugs. Keywords: drug response
The mission of expO is to build on the technologies and outcomes of the Human Genome Project to accelerate improved clinical management of cancer patients. IGC's Expression Project for Oncology (expO) seeks to integrate longitudinal clinical annotation with gene expression data for a unique and powerful portrait of human malignancies providing critical perspective on diagnostic markers, prognostic indicators, and therapeutic targets. The goal of expO and its consortium supporters is to procure tissue samples under standard conditions and perform gene expression analyses on a clinically annotated set of deidentified tumor samples. The tumor data is updated with clinical outcomes and is released into the public domain without intellectual property restriction. Series-matrices are available at ftp://ftp.ncbi.nlm.nih.gov/pub/geo/DATA/SeriesMatrix/GSE2109/. For more information, see http://www.intgen.org/ Keywords: cancer portraits
PGC-1 transcription factor was customized to limit its interations to ERRalpha. This mutant (2x9) was used to dissect the transcription activation patterns that are attributable to the PGC1-ERR interaction and PGC-1 actions that are independent of ERR. Inactive mutant with the deleted LLXXL motifs (L2L3) and wt PGC-1 were used as negative and positive controls respectively. BGAL-expressing construct was used to control for non-specific effects of adenoviral infection. Keywords: ERRalpha PGC-1alpha, nuclear receptor, orphan nuclear receptor, coactivator, transcription factors
The aim of this study was to describe the gene expression patterns related to the differentiation and mineralization of bone-forming cells including activation and/or repression of osteogenic or non-osteogenic pathways, remodeling of cell architecture, cell adhesion, cell communication, and assembly of extracellular matrix. The study implied patient selection, tissue collection, isolation and culture of human marrow stromal cells (hMSC) and osteoblasts (hOB), and characterization of bone-forming cells. RNA samples were collected at defined time points, in order to understand the regulation of gene expression during the processes of cell differentiation/mineralization that occur during bone repair. Transcriptome analysis was performed by using the Affymetrix GeneChip microarray technology platform and GeneChip® Human Genome U133 Plus 2.0 Array. Our results help to design a gene expression profile of bone-forming cells during specific steps of osteogenic differentiation. These findings offer an useful tool to monitor the behaviour of osteogenic precursors cultured in presence of exogenous stimuli, i.e. growth factors, or onto 3D scaffolds for bone engineering. Moreover, they can contribute to identify and clarify the role of new genes for a better understanding of the molecular mechanisms regulating osteogenesis. Keywords: time course
Comparison of mRNA expression showed widespread changes in the circulating CD8+ but not CD4+ T-cells from patients with severe asthma. No changes were observed in the CD4+ and CD8+ T-cells in non-severe asthmatics versus healthy controls. Bioinformatics analysis showed that the changes in CD8+ T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs we also observed widespread changes in expression of non-coding RNA species including natural antisense, pseudogenes, intronic long ncRNAs and long intergenic long ncRNAs in CD8+ T-cells from severe asthmatics. Measurement of the miRNA expression profile showed selective down-regulation of miR-28-5p in CD8+ T-cells and reduction of miR-146a and miR-146b in both CD4+ and CD8+ T-cells.
There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program by reprogramming of a more differentiated cell type by oncogenic insults or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. Here we discribe Myoepithelial Progenitor Cells (MPCs) that show properties of EMT and claudin low subtype of breast cancers. Through microarray analysis, we have found that these K5-/K19- cells show similar gene expression pattens of the claudin-low subtype of breast cancer.
Gene expression profiling has demonstrated clinical utility as a predictive tool in clinical oncology. We have identified genes associated with invasion of pancreatic cancer and with potential for identifying early recurrence. We used Affymetrix Human U133 Plus 2.0 microarrays to identifiy specific predictive profiles in pancreatic cancer, and the evolution of gene expression. We identified distinct classes of up-regulated genes during this process.
63 melanoma cell lines hybridized to Affymetrix Hu133_Plus 2 oligo arrays. The aim of this study was to identify potential downstream targets of key oncogenes and TSGs in melanoma (including p14ARF p16INK4A, BRAF etc). Publications relevant to this series include: Johansson et al. Pigment Cell Res 2007. Keywords: melanoma
Human neuronal differentiation alters responsiveness to innate immune stimuli and virus infections. We used microarrays to examine the transcriptional responses of the human BE(2)-C neuroblastoma cell line to retinoic acid-induced differentiation and type I IFN stimulation.
A phase I trial of a SRC kinase Inhibitor dasatinib, in combination with paclitaxel and carboplatin in patients with advanced or recurrent ovarian cancer. Background: We conducted a phase I study of dasatinib, an oral SRC tyrosine kinase inhibitor, in combination with paclitaxel and carboplatin in advanced and recurrent epithelial ovarian cancer (EOC). Methods: The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included toxicity, response rate (RR), pharmacokinetics and pharmacodynamics. Based on the 3+3 design, cohorts of 3-6 pts received paclitaxel 175 mg/m2 and carboplatin AUC 6 every three weeks with escalating doses of dasatinib (100, 120, 150 mg daily), followed by an 8 patient expansion cohort. Results: Twenty patients were enrolled between 06/07 and 12/09. The median age was 61 yrs (42-82) with a median of 2 prior regimens (0-6), and 71% had platinum-sensitive disease. There were 3-6 pts in each cohort, and 8 in the expansion cohort. Pharmacokinetics were observed over the first 2 cycles of therapy. One DLT was observed in the 100 mg dasatinib cohort (grade 3 myalgia. Other toxicities in all cycles included neutropenia (95% grade 3-4), thrombocytopenia (35% grade 3-4), and fatigue (10% grade 3). The RR was 45% (complete responses, 3/18(17%); partial responses, 5/18(28%)) and 56% (10/18) had stable disease. The PFS6-month actuarial estimate was 86%. The median PFS and OS were 7.8 and 16.2 months, respectively. Conclusions: Due to the high incidence of myelosuppression with subsequent cycles the recommended phase II dose is 150 mg daily of dasatinib in combination with paclitaxel and carboplatin. The combination was safe with evidence of clinical activity in advanced EOC.
HIV-1 Tat induces the expression of interferon (IFN)-inducible genes in immature dendritic cells (iDC) in the absence of IFN production. We evaluated how three alleles of Tat and some Tat mutants differ in cellular gene modulation and whether a similar gene induction pattern could be detected by treating cells with IFN’s. The three alleles and mutants with the exception of mutants TatSF21-47 and TatSF2G48-R57A that do not localize in the nucleous, modulated to different degrees IFN-inducible genes without concomitant induction of IFN’s. The first exon TatSF21-72 and the minimal transactivator TatSF21-58, all induced genes to a significantly greater extent than full-length Tat. The 2nd exon appears to diminish the gene modulation that can be observed when the first exon alone is expressed. To investigate what domain of Tat are critical to the host-pathogen interactions that are Tat-dependent during HIV infection, we evaluated a variety of Tat-mutants and found that in antigen presenting cells, blood-derived myeloid iDC and macrophages, the second exon of Tat reduces innate immunological responses which are maximal when a Single exon Tat is expressed.
Normal human tissue samples from ten post-mortem donors were processed to generate total RNA which was subsequently analyzed for gene expression using Affymetrix U133 plus 2.0 arrays. Donor information: Donor 1 - 25 year old male; donor 2 - 38 year old male; donor 3 - 39 year old female; donor 4 - 30 year old male; donor 5 - 35 year old male; donor 6 - 52 year old male; donor 7 - 50 year old female; donor 8 - 48 year old female; donor 9 - 53 year old female; donor 10 - 23 year old female Keywords: normal human tissue comparison
Elevated levels of androgen receptor (AR) in prostate cancer confer resistance to current antiandrogens and play a causal role in disease progression due to persistent target gene activation. Through pharmacologic and genetic approaches we show that half of all direct AR target genes, including TMPRSS2, the primary driver of ETS fusion transcripts in 70 percent of human prostate cancers, require histone deacetylase (HDAC) activity for transcriptional activation by AR. Surprisingly, the HDAC3-NCoR complex, which typically functions to repress gene expression by nuclear receptors, is required for AR target gene activation. Prostate cancer cells treated with HDAC inhibitors have reduced AR protein levels, but we show that the mechanism of blockade of AR activity is through failure to assemble a coactivator/RNA polymerase II complex after AR binds to the enhancers of target genes. Failed complex assembly is associated with a phase shift in the cyclical wave of AR recruitment that typically occurs in response to ligand treatment. HDAC inhibitors retain the ability to block AR activity in hormone refractory prostate cancer models and therefore merit clinical investigation in this setting. HDAC-regulated AR target genes defined here can serve as biomarkers to ensure sufficient levels of HDAC inhibition. Keywords: Androgen receptor, histone deacetylase, prostate cancer, dose response
Background Accurate assessment of treatment efficacy would facilitate clinical trials of new anti-tuberculosis drugs. TB patients exhibit altered peripheral immunity which reverts during successful treatment. We hypothesised that these changes could be observed in whole blood transcriptome profiles. Methods Ex vivo blood samples from 27 pulmonary TB patients were assayed at diagnosis and during conventional treatment. RNA was processed and hybridised to Affymetrix GeneChips to determine expression of over 47,000 transcripts. Findings There were significant changes in expression of over 4,000 genes during treatment. Rapid, large scale changes were detected, with down-regulated expression of ~1,000 genes within the first week, including inflammatory markers such as the complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B cell markers, transcription factors and signalling molecules. Interpretation The expression of many genes is drastically altered during TB disease, with components of the humoral immune response being markedly affected. The treatment-induced restoration reflects the simultaneous suppression and activation of different immune responses in TB. The rapid initial down-regulation of expression of inflammatory mediators coincides with rapid killing of actively dividing bacilli, whereas slower delayed changes occur as drugs act on dormant bacilli and as lung pathology resolves. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity.
In previous studies human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this manuscript, we tried characterization of hDPSCs isolated from an earlier developmental stage to evaluate potential usage of these cells for tissue regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at the later stage. When the cells from either group were cultured in medium promoting differentiation towards cells of the osteo/odontoblastic lineage, both became alkaline phosphatase positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of a number of genes, such as WNT16, was markedly changed with increasing number of passages, which might have caused the loss of their characteristics as hDPSCs. Keywords: gene expression study, long-term cultures
The presence of senescent cells in the aging/degenerating human disc is now well-recognized. Senescent cells are viable cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue, however, offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells. Here we use a novel experimental design using laser capture microdissection to selectively separately harvest senescent and non-senescent annulus cells in paraffin-embedded tissue, and then compare their gene expression with microarray analysis. An initial in vitro study using cultured human annulus cells was first performed to test whether there was any difference in identification of senescent cells using the accepted histochemical methodology vs. the immunofluoresent identification of cells positive for senescence-associated-ß-galactosidase in control cells and cells induced into stress-induced premature senescence via hydrogen peroxide exposure. No statistically significant difference was found between the 2 methods. Laser capture microdissection was used to separately harvest senescent and non-senescent cells from 11 human annulus specimens, and microarray analysis was used to determine gene expression levels. Genes with established relationships to senescence were found to be significantly upregulated in senescent cells vs. non-senescent cells. Additional genes related to cytokines, cell proliferation, and other cell processes were also identified.
The Cohesin apparatus has a canonical role in sister chromatid cohesion. Heterozygous mutations in Nipped B-like (NIPBL) SMC1A, and SMC3 have been found in 60% of probands with Cornelia de Lange Syndrome (CdLS), a dominant multi-system genetic disorder with variable expression. We have performed a genome-wide transcription assessment as well as cohesin binding analysis using human lymphoblastoid cell lines (LCLs) from probands with CdLS and controls. Here, we report a unique profile of genes dysregulated in CdLS that correlates with different clinical presentations. Genome-wide analysis of cohesin binding demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of an insulator. However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS probands, indicating an alternative role of cohesin as a classic transcription factor. Cohesin also co-localizes with CTCF at the boundary elements affecting neighboring gene expression in CdLS probands. We propose that the CdLS phenotype is the result of dysregulated gene expression rather than defective sister chromatid cohesion. Phenotype specific expression profiles are also described. Keywords: Disease state analysis, Genetic modification
A549 and H1299 cell lines were transfected with hMOF specific siRNA or control siRNA and Affymetrix oligonucleotide microarray was conducted to systematically determine downstream targets regulated by hMOF.
The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regu- latory events from gene activation to heterochromatinization. In yeast the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p. We used microarrays to detail the global programme of gene expression after knockdown of HIRA and CABIN1 in 3 replicates
BACKGROUND: Cervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH) and microsatellite marker analysis for the detection of loss of heterozygosity (LOH). Currently high throughput methods such as array comparative genomic hybridization (array CGH), single nucleotide polymorphism array (SNP array) and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression. RESULTS: Genome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA) was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH). Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR). CONCLUSION: This study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene expression is useful for the identification of gene specific targets that could be relevant for the development and progression in cervical cancer. Keywords: DNA copynumber RNA expression correlation
The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. Β-catenin a cytoplasmic component, plays a major role in the transduction of the canonical wnt/ β-catenin signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma. We selected and expanded isogenic clones from hepatocellular carcinoma-derived Huh7 cells with high and low β-catenin/TCF activities. We showed that, high TCF activity Huh7 cells lead to bigger and more aggressive tumors when xenografted into nude mice. We used SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis in parallel, to compare gene expression between low (basal) and high (transfected) β-catenin/TCF activity clones, those had been xenografted into nude mice. We compared and contrasted SAGE and genome-wide microarray data, in parallel. Finally; after combined analysis, we identified BRI3 and HSF2 as novel targets of Wnt/β-catenin signaling in hepatocellular carcinoma.
Transcriptional profiling of RWPE-1 cells stably expressing human androgen receptor (as described in Altintas et al. Mol Cell Endocrinol 2011) treated with a non-metabolisable androgen, R1881
Mature NK and T-cell lymphomas are occasionally encountered in Asia but are very rare in Western populations. In part due to its rarity little is known about this group of neoplasms, and despite being rather different disease entities, they are all treated similarly but with diverse clinical outcomes. Novel biomarkers (at both the genetic and protein levels) are needed to resolve diagnostic difficulties, improve prognostication and develop targeted therapies. To rectify this deficiency, we interrogated the transcriptome of several NK and mature T-cell lymphomas by whole-genome expression profiling for new markers that may further stratify this diverse group of conditions. Our initial efforts have identified a promising candidate marker that appears to differentiate NKTL lymphoma from other forms of T-cell neoplasms, and this finding has been validated by immunohistochemistry on archival material in a large number of patient cases.
Background and aims: Dysregulation of intestinal epithelial cells performance associates with an array of pathologies whose onset mechanisms are incompletely understood. The aim of the present study was to provide a map of gene expresssion patterns along the human healthy adult gastro-intestinal tract and to implement a new procedure for microarray data noise filtering that would allow their use as a reference when screening for pathological deviations such as inflammatory bowel disease (IBD). Methods: Gene expression profiles in antrum, duodenum, jejunum, ileum and transverse colon biopsies were measured with the Affymetrix U133A array and principal component analysis was used to identify region-selective biomarkers. These data were intersected with highly variable genes from a public dataset of gene expression in the ileal and colonic healthy regions of UC and Crohn’s disease patients. Moreover, gene sets covering gut functions not entirely accounted for by the available public tools were constructed to monitor their expression along the GI tract. Results: 166 genes were found to be responsible for distinguishing the five regions considered. Fourteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion, and six other novel genes. Similar analysis of the IBD datasets revealed that samples stratify based on disease rather than on the intestinal region. This withstanding, eleven genes were identified as possible early predictors of Crohn’s and/or UC in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. Conclusions: This novel approach, validated by retrieving known gene profiles, allowed the identification of promising new leads both in health and IBD state. Keywords: gastro-intestinal tract comparison
Copper-based chemotherapeutic compounds Casiopeinas have been presented as able to promote selective programmed cell death in cancer cells, thus being proper candidates for targeted cancer therapy. DNA fragmentation and apoptosis -in a process mediated by reactive oxygen species- for a number of tumor cells, have been argued to be the main mechanisms. However, a detailed functional mechanism (a model) is still to be defined and interrogated for a wide variety of cellular conditions; before establishing settings and parameters needed for their wide clinical application. Microarrays were used to determine the expression profile from HeLa cells in order to propose a model for the role played by intrinsic apoptosis triggered by the oxidative stress caused by Cas-II-gly.
Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC) which is associated with a high mortality rate that is related to a propensity for invasion and metastasis. We report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain of and loss of function experiments. Inhibition of EGFR signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting interdependence of two common genetic alterations with periostin function. Our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (3D) culture can offer an important tool to discover novel biologic effectors in cancer.
The transcription factors PAX3 and MITF are required for the development of the neural crest and melanocyte lineage and both proteins play important roles in melanoma cell growth and survival. PAX3 transcriptionally activates MITF expression during neural crest development, but the relationship between these transcription factors during melanocyte development and in melanoma cells is currently poorly understood. This study aimed to further our understanding of the interaction between transcriptional networks controlled by PAX3 and MITF by assessing the effect of siRNA-mediated knockdown of PAX3 and MITF in metastatic melanoma cell lines. The goals of this study were to determine (i) if PAX3 is required for maintaining expression of MITF in melanoma and melanocyte cell lines; (ii) whether PAX3 and MITF independently, or redundantly, influence growth and survival in melanoma cell lines; and (iii) to investigate the respective roles of PAX3 and MITF expression in melanoma cell differentiation. Microarrays were used to measure global changes in transcript expression in response to siRNA-mediated knockdown of PAX3 or MITF compared to non-targeting controls in two metastatic melanoma cells lines.
Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biological characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes including HOXA9. Here we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias utilizing RNAi. Gene expression profiling after HOXA9 suppression demonstrated co-downregulation of a program highly expressed in human MLL-AML (this study) and murine MLL-leukemia (Krivtsov et al. 2006) stem cells including HOXA10, MEIS1, PBX3 and MEF2C. Our data indicates an important role for HOXA9 in human MLL-rearranged leukemias, and suggests targeting HOXA9 or downstream programs may be a novel therapeutic option.
Transcription factor complexes bind to regulatory sequences of genes providing a system of individual expression regulation. Targets of distinct transcription factors usually map throughout the genome, without clustering. Nevertheless, highly and weakly expressed genes do cluster in separate chromosomal domains with an average size of 80 to 90 genes. We therefore asked whether, besides transcription factors, an additional level of gene expression regulation exists that acts on chromosomal domains. Here we show that identical green fluorescent protein (GFP) reporter constructs integrated at 90 different chromosomal positions determined by sequencing, obtain expression levels that correspond to the activity of the domains of integration. These domains are about 80 genes long and can exert an effect of up to 8-fold on the expression of integrated genes. 3D-FISH shows that active domains of integration have a more open chromatin structure than integration domains with weak activity. These results reveal a novel domain-wide regulatory mechanism that, together with transcription factors, exerts a dual control over gene transcription. Keywords: HEK293 cells
Skeletal muscle (rectus femoris) gene expression was analyzed from diet-resistant and diet-sensitive obese women undergoing clinically supervised weight-loss at a weight management clinic The goal of the study was to characterize global gene expression profiles in skeletal muscle from obese women prior to their participation in a clinically supervised, low-calorie diet, weight management program. Following entry into the weight-loss program, subjects can be categorized as being 'diet-sensitive' or 'diet-resistant' depending on the rates of weight loss achieved. In the current study, we selected an equal number of diet-sensitive and diet-resistant subjects for comparative expression profiling
In order to find the difference between human lung tissue-derived fibroblasts and human vascular adventitial fibroblasts for enhancing tumor formation ablity of human lung adenocarcinoma cell line A549 we found that human vascular adventitial fibroblasts enhance A549 tumor formation in vivo compared to human lung tissue-derived fibroblasts. To find the responsible genes for this phenomena, we used microarray analysis to find the expression difference between lung tissue-derived fibroblasts and vascular adventitial fibroblas
The hepatitis E virus (HEV) a non enveloped RNA virus, causes viral hepatitis. The viral open reading frame 2 (ORF2) protein represents the capsid protein of HEV which is known to cause endoplasmic reticulum stress in ORF2 expressing cells. The initiation of endoplasmic reticulum stress induced apoptosis mainly involves the transcriptional activation of pro-apoptotic gene CHOP which will further trigger the major apoptotic pathways. However, the activation of CHOP by ORF2 protein in this study does not induce apoptotic markers such as Bax translocation to mitochondria. We have used the Affymetrix microarray platform to screen the pro-apoptotic effects induced by the expression of ORF2 protein in human hepatic cell lines (Huh7). The Huh7 cells were transduced either with recombinant adenovirus encoding the HEV ORF2 (Ad-ORF2) or an adenovirus encoding the green fluorescent protein (Ad-GFP). The array results consistently showed an ORF2 specific induction of mRNA corresponding to the chaperones Hsp72, Hsp70B’ and co-chaperone Hsp40. These studies provide further mechanisms of the ER stress mediated pro apoptotic effects caused by the ORF2 protein and its potential role for the activation of anti-apoptotic activity of the host cell.
ERG (Ets Related Gene) is an ETS transcription factor that was originally described for its role in a number of human cancers. Our preliminary data demonstrate that ERG exhibits a highly EC restricted pattern of expression in cultured primary cells and several adult tissues including the heart lung, and brain. In response to inflammatory stimuli, such as TNF-alpha, we observed a marked reduction of ERG expression in EC. To further define the role of ERG in the regulation of normal EC function we used RNA interference to knockdown ERG. Knockdown of ERG in human umbilical vein EC (HUVEC) using siRNA was associated with the reduction of a number known ERG targets. Keywords: SIRNA Functional Role
Biopsies (lymph nodes ascites or hydrothorax) from 60 patients with cancer of unknown primary origin were analyzed. Results provide insight into the molecular pathogenesis of CUP.
Highly pathogenic avian influenza viruses (HPAIV) induce severe inflammation in poultry and men. There is still an ongoing threat that these viruses may acquire the capability to freely spread as novel pandemic virus strains that may cause major morbidity and mortality. One characteristic of HPAIV infections is the induction of a cytokine burst that strongly contributes to viral pathogenicity. It has been suggested that this cytokine overexpression is an intrinsic feature of infected cells and involves hyperinduction of p38 mitogen activated protein kinase (MAPK). Here we investigate the role of MAPK p38 signaling in the antiviral response against HPAIV in mice as well as in endothelial cells, the latter a primary source for cytokines during systemic infections. Global gene expression profiling of HPAIV infected endothelial cells in the presence of the MAP kinase p38-specific inhibitor SB202190 revealed, that inhibition of MAPK p38 leads to reduced expression of interferon (IFN) and other cytokines after A/Thailand/1(KAN-1)/2004 (H5N1) and A/FPV/Bratislava/79 (H7N7) infection. Furthermore, the expression of interferon stimulated genes (ISGs) after treatment with IFN or conditioned media from HPAIV infected cells was decreased when the target cells were preincubated with SB202190. Finally, promoter analysis confirmed a direct impact of p38 MAPK on the IFN-enhanceosome and ISG-promoter activity. In vivo inhibition of MAP kinase p38 greatly diminishes virus induced cytokine expression concomitant with reduced viral titers, thereby protecting mice from lethal infection. These observations show, that MAPK p38 acts on two levels of the antiviral IFN response: Initially the kinase regulates IFN induction and at a later stage MAPK p38 controls IFN signaling and thereby expression of IFN-stimulated genes. Thus, inhibition of MAP kinase p38 may be an antiviral strategy that significantly protects mice from lethal influenza via suppression of overshooting cytokine expression.
The patients who underwent surgery for primary lesions were examined. All patients had metastatic or recurrent CRC and received modified FOLFOX6. Responders and nonresponders were determined based on the best observed response at the end of the first-line treatment mFOLFOX6. Gene-expression profiles of primary CRC were determined using Human Genome GeneChip arrays U133.
compare the gene expression profile between cep701 treated HEL cells with shPRMT5 knockingdown HEL cells. HEL cells contain homologous alells with mutation Jak2V617F. We found JAK2V617F can inactivate PRMT5 activity by directly phosphorylating PRMT5 through histone methylation.
Resident stem/progenitor cells in lungs are important for tissue homeostasis and repair. We isolated human lung progenitor cells and named alveolar epithelial progenitor cells (AEPCs)(Fujino N et al. 2011. Lab Invest. 91:363). AEPCs have phenotypes of both alveolar epithelial type II (ATII) cells and mesenchymal stem cells. AEPCs had the potential to generate ATII-like cells in vitro. ATII-like cells derived from AEPCs expressed protein and mRNA of pulmonary surfactant, and displayed lamellar bodies containing the surfactants. However, it has not been evaluated whether global gene expression of the ATII-like cells from AEPCs was similar to that of mature ATII cells isolated from human lung tissues. This study demonstrated gene expression profiles of ATII-like cells from AEPCs. In addition, transcriptomes in AEPCs and mature ATII cells were deposited in the GEO website (GSE21095 and GSE29133, respectively).
Langerhans cell histiocytosis (LCH) is a disease characterized by the accumulation of eponymous CD1a+ Langerin+ Langerhans-cell (LC)-like dendritic cells (DC) of largely unknown origin. Here we have performed comparative transcriptome analysis of highly purified CD207+/CD1a+ Langerhans cell histiocytosis (LCH) cells derived from different locations and disease courses and three major human dendritic cell lineages: epidermal Langerhans cells myeloid dendritic cells (mDC1) and plasmacytoid dendritic cells (pDC) in order to investigate the relationship between LCH cells and naturally occurring dendritic cells. Data obtained indicate that LCH cells form a distinct DC entity. Furthermore, we have identified transcripts that are uniquely expressed by LCH cells in comparison to LC, mDC1, and pDC, and induce LCH-specific features in human DC.
The purpose of this experiment was to identify oestrogen regulated genes in human primary cell cultures of neuronal cells modelling the developing human nervous system. We were especially interested in genes involved in proliferation differentiation and migration of neuronal cells and genes involved in or linked to neurodegenerative diseases. We have therefore assessed gene expression changes, using Affymetrix GeneChips (HG-U133A), of oestrogen treated human neuronal cell cultures.
Quercetin is a flavonol modifying numerous cell processes with potent antiproliferative effects on cancer cell-lines. The aim of this study was to explore by gene-array analysis the effect of quercetin on cancer-related gene expression in HepG2 cells followed by verification with RT-PCR and analysis of the expected phenotypic changes (migration, cell cycle, cell proliferation). Quercetin induces significant changes on cell-adhesion related genes, leading to reduced migratory capacity and disorganization of the actin cytoskeleton. Several genes related to DNA functions, cellular metabolism and signal-transducer activities were also modified, while an early effect on G–protein related cascades possibly via protease-activated receptor 2 and phospholipase C-γ1 was identified. Cyclin-D associated events in G1 and ubiquitin-dependent degradation of cyclin-D1 were also affected, resulting in cell-cycle arrest without activation of apoptosis pathways. In conclusion quercetin (3μM) exerts its cellular effects by modifying numerous genes related to mechanisms involved in cancer initiation and promotion.
Treatment of MCF7 breast cancer cells by cisplatin leads to a very specific metabolic response and an onset of cell death about 10-11 h after beginning of treatment. For more detailed understanding of the molecular processes underlying the specific metabolic response mRNA was isolated from MCF7 cells when the specific changes, (i) induction of glycolysis and (ii) onset of cell death, were detected during online measurement in the cell biosensor system.
Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) frequently complicates combined anti-retroviral therapy (ART) and anti-tubercular therapy in HIV-1 co-infected tuberculosis (TB) patients. The immunopathological mechanism underlying TB-IRIS is incompletely defined. Differential transcript abundance in PBMC from IRIS and control patients stimulated with heat killed H37Rv was determined by microarray
Ulcerative colitis (UC) and Crohn’s disease (CD) are inflammatory bowel diseases (IBD) with variable overlapping clinical features and complex pathophysiologies. To identify pathogenic processes underlying these disease subtypes, using single endoscopic pinch biopsies to estabolish 36 expression profiles, we elucidated gene expression patterns of active and inactive areas of UC and CD, and compared these to infectious colitis and healthy controls. Keywords: RNA
In this experiments different treatments were applied to lung cancer cell lines 1)H720 cell line suspension was treated by 1 micromole of 5-lipoxygenase activating protein inhibitor MK886 serum-free in TIS medium for 24 h. 2) A549 cell line was treated as H720 by MK886 3) A549 was treated by 100 nM of silencing cocktail (siRNA) prepared against the 4-th exon of the gene and generated by RNASEIII digestion of double stranded RNA. The cocktail was prepared by Ambion technology see the manufacturer's protocol. The observed silencing was >90% 4) Based on Oligoengine vectors, silencing constructs were created against HRPA2B1 transcript(HNRPA2B1 is a subunit of spliceosome). Stable transfectants were raised harboring the construct. Keywords: drug treatment
Cancer stem cells (CSCs) are plastic in nature a characteristic that hampers cancer therapeutics. Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and half of the cases are highly aggressive. By treating NB cell lines (SKNAS, SKNBE(2)C, CHP134, SY5Y) with epigenetic modifiers for a short time followed by sphere-forming culture conditions, we have established stem cell-like NB cells that are phenotypically stable for over a year. These cells are characterized by their high expression of stemness factors, stem cell markers, and open chromatin structure. We referred to these cells as induced CSC (iCSC). SKNAS iCSC and SKNBE(2)C iCSC clones (as few as 100 cells) injected subcutaneously into SCID/Beige mice formed tumors, and in one case, SKNBE(2)C iCSC metastasized to the adrenal gland, suggesting their increased metastatic potential. SKNAS iCSC xenografts showed the histologic appearance of totally undifferentiated “large-cell” NBs (LCNs), the most aggressive and deadly form of NB in humans. Immunohistochemical analyses showed that SKNAS iCSC xenografts expressed high levels of the stem cell marker CXCR4, while the SKNAS monolayer cell xenografts did not. The patterns of CXCR4 and MYC expression in SKNAS iCSC xenografts resembled those in the LCNs. The xenografts established from the NB iCSCs shared two common features: the LCN phenotype and high-level MYC/MYCN expression. These observations suggest that NB cells with large and vesicular nuclei, representing their open chromatin structure, are indicative of stem cell-like tumor cells, and that epigenetic changes may have contributed to the development of these most malignant NB cells.
IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation
Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSI) which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Analysis of gene expression in GSI-responsive and GSI-resistant cell lines treated with Compound E identifies differential resopnses to GSI. Keywords: Drug response
Purpose: Evaluate gene expression profiles after inducing differentiation in cultured interstitial cystitis (IC) and control urothelial cells. Materials and Methods: Bladder biopsies were taken from IC patients and controls (women having surgery for stress incontinence). Primary cultures were grown in Keratinocyte Growth Medium with supplements. To induce differentiation in some plates the medium was changed to DMEM-F12 with supplements. RNA was analyzed with Affymetrix chips. Three nonulcer IC patients were compared with three controls. Results: After inducing differentiation, 302 genes with a described function were altered at least 3-fold with p <0.01 in both IC and control cells. Functions of the162 upregulated genes included cell adhesion (e.g. claudins, occludin, cingulin); urothelial differentiation, retinoic acid pathway and keratinocyte differentiation (e.g. skin cornified envelope components). The 140 downregulated genes included genes associated with basal urothelium (e.g. p63, integrins ?4, ?5 and ?6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins and members of the Wnt and Notch pathways. Comparing IC vs. control cells after differentiation, only seven genes with a described function were altered at least 3-fold with p <0.01. PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased in IC; AKR1C2 and MKNK1 were increased in IC. Conclusions: Differentiation-associated changes occurred in both IC and control cells. Comparing IC vs. control revealed very few differences. This study may have included IC patients with minimal urothelial deficiency and/or selected the cells that were most robust in culture. Also, the abnormal urothelium in IC may be due to post-translational changes and/or the bladder environment.
We found microRNA miR-23b was down-regulated in local inflammatory tissues of autoimmune disease such as RA SLE and related mouse models such as CIA, lpr, EAE. Re-expression of miR-23b significantly inhibits autoimmune pathogenesis of CIA, Lpr and EAE. To identify potential targets of miR-23b, we use microarray gene-expression analysis to identify transcripts which could be repressed by miR-23b. RA: rheumatoid arthritis, CIA: Collagen-induced arthritis, SLE: systemic lupus erythematosus, EAE: experimental autoimmune encephalomyelitis
Premature progesterone (P) rise during GnRH antagonist cycles for IVF is a frequent phenomenon and has been associated with lower pregnancy and implantation rates. Different thresholds of progesterone have been used so far to define its premature rise during the follicular phase of an IVF stimulated cycle. In this study we evaluated endometrial gene expression on the day of oocyte retrieval according to the level of serum progesterone on the day of hCG administration in GnRH antagonist cycles.Endometrial biopsies from eleven patients were taken with a Pipelle de Cornier (Prodimed, Neuilly-en-Thelle, France) on the day of oocyte retrieval in a GnRH antagonist/rec-FSH stimulated IVF cycle with fresh embryo transfer. Biopsies were analysed for gene expression with Affymetrix Human Genome (HG) U133 Plus 2.0 Arrays and GCOS software (Affymetrix, Santa Clara, CA, USA). Patients were divided into three different groups according to their progesterone serum concentration on the day of hCG administration (A) P <= 0.9 ng/mL, (B) 1 < P < 1.5 ng/mL, and (C) P > 1.5 ng/mL. Serum P was measured with the automated Elecsys immunoanalyser (Roche Diagnostics, Mannheim, Germany). Selected differentially expressed genes were validated with quantitative real-time PCR (QPCR) with TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Keywords: gene expression analysis, premature progesterone rise
Approximately KSHV vIRF4 deregulate 284 genes by two-folds
Investigation of Gene Expression Profiling in Unstaged Head Hair Follicles Plucked from Men and Women Keywords: Gene Expression Profiling of Normal Hair Follicles
Expression profiling was used to identify genes differentially expressed in MSS (microsatellite stable) and MSI (microsatellite unstable) colon cancer cell lines. Data submitted in support of manuscript entitled Villin expression is frequently lost in poorly differentiated colon cancer Diego Arango, Sheren Al-Obaidi, David S. Williams, Jose Dopeso, Rocco Mazzolini, Georgia Corner, Do-Sun Byun, Carmel Murone, Lars Tögel, Nikolajs Zeps, Lauri A. Aaltonen, Barry Iacopetta and John M. Mariadason, American Journal of Pathology, 2012.
MEK5 is activated by shear stress in large vessel endothelial cells (ECs) and contributes to the suppression of pro-inflammatory changes in the arterial wall. We used microarray analyses of total RNA from MEK5/CA-transduced HDMECs compared to LacZ control-transduced HDMECs to identify distinct classes of several regulated genes including KLF4, eNOS, and ICAM. We conclude that MEK5 activation by shear stress may modulate inflammatory responses in the microvasculature, and these actions are partly mediated by KLF4.
Inflammatory hepatocellular adenomas (IHCA) are benign liver tumours defined by the presence of inflammatory infiltrates and by the elevated expression of inflammatory proteins in tumour hepatocytes12. Here we show a striking activation of the IL6 signalling pathway in this tumour type, and sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene that encodes the signalling co-receptor gp130. Indeed, ~70% of IHCA harbour small in-frame deletions that target the binding site of gp130 for IL6, and expression of the most frequent gp130 mutant, Delta-STVY190, in hepatocellular cells activates STAT3 in absence of ligand. Further, analysis of hepatocellular carcinomas revealed rare gp130 alterations always accompanied by ß-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas explains their inflammatory phenotype, and suggest that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.
To begin to identify genes involved in the transdifferentiation process we analyzed Barrett’s esophagus (with no dysplasia) normal esophagus and small intestine biopsy samples by Affymetrix microarray. Keywords: microarray expression analysis
Microarray experiments were carried out to ascertain whether TOP2β is required for DHT induced androgen receptor target gene expression. We investigated the effect of pharmacological inhibition or RNA interference-mediated depletion of TOP2β on gene expression in androgen-dependent LNCaP prostate cancer cells.
The periodontium are the tissues supporting and investing the tooth and consists of the periodontal ligament the gingiva, the root cementum, and the alveolar bone. The functions of the cell populations in health and disease regarding the host-mediated tissue destruction are not well understood. To get a first idea, of which genes might play a distinct role in chronic periodontal disease in vivo, we compared the genom-wide gene expressions of chronic inflamed and healthy periodontal ligament cells by microarray analysis and validated the data by real-time RT-PCR. The expression rates of 14.239 genes were investigated and 3.018 of them were found differentially expressed by at least two-fold, the expression rates of 1.451 genes were significantly up-regulated and the expression rates of 1.567 genes were significantly down-regulated in inflamed PDL cells. We focused on mainly structural components, for example, laminins and integrins, as well as degrading enzymes, for example, MMPs and cathepsins. The molecular composition of the laminin network varies in chronic inflamed compared to healthy PDL cells in vivo. Furthermore, integrin alpha6beta4, together with laminin-332, might be involved in chronic periodontal inflammation. Findings that diverse keratins were upregulated in chronic disease indicate that the epithelial cell rests of Malassez might also be involved in chronic periodontal inflammation. Also cathepsin B and cathepsin C might participate in the connective tissue destruction. The microarray analysis has identified a profile of genes potentially involved in chronic periodontal inflammation in vivo. Further studies are needed to entirely understand cellular activities during chronic periodontal inflammation in vivo. Keywords: expression analysis
Enzalutamide (formerly MDV3100 and available commercially as Xtandi) a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration-resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: (1) binding of androgens to AR, (2) AR nuclear translocation, and (3) association of AR with DNA. Here we used Affymetrix human genome microarray technology to investigate the global programme of gene expression of LNCaP cells in response to enzalutamide alone and in the context of DHT-stimulated androgen receptor gene expression.
This SuperSeries is composed of the SubSeries listed below.
gd T cells recognize unprocessed or non-peptide antigens respond rapidly to infection, and localize to mucosal surfaces. We have hypothesized that the innate functions of gd T cells may be more similar to those of cells of the myeloid lineage than to other T cells. To begin to test this assumption, we have analyzed the direct response of cultured human and peripheral blood bovine gd T cells to pathogen associated molecular patterns (PAMPs) in the absence of APCs using microarray, real time RT-PCR, proteome array, and chemotaxis assays. Our results indicate that purified gd T cells respond directly to PAMPs by increasing expression of chemokine and activation related genes. The response was distinct from that to known gd T cell antigens and different from the response of myeloid cells to PAMPs. In addition, we have analyzed the expression of a variety of PAMP receptors in gd T cells. Freshly purified bovine gd T cells responded more robustly to PAMPs than did cultured human cells and expressed measurable mRNA encoding a variety of PAMP receptors. Our results suggest that rapid response to PAMPs through the expression of PAMP receptors may be another innate role of gd T cells. Keywords: parallel sample
Gene expression patterns were assessed in normal human bronchial epithelial (NHBE) cells exposed to cigarette smoke (CS) from a typical "full flavor" American brand of cigarettes in order to develop a better understanding of the genomic impact of tobacco exposure which can ultimately define biomarkers that discriminate tobacco-related effects and outcomes in a clinical setting. NHBE cells were treated with CS for 15 minutes and alterations to the transcriptome assessed at 1,2,4 and 24 hours post-CS-exposure using high-density oligonucleotide microarrays. Keywords: Time course
Distinct processes govern the transition from myometrial quiescence to activation during both term and preterm labor. We sought the specific gene sets responsible for initiating term and preterm labor along with a core set of effector genes necessary for labor independent of gestational age and the underlying trigger. The Effector Gene Set consisted of 49 genes present in both preterm and term labor but absent from non-labor samples. 122 genes were specific to preterm labor (Preterm Initiator Set) and 229 to term labor (Term Initiator Set). The Term Initiator and the Effector Sets reflected predominantly inflammatory processes. Surprisingly, the Preterm Initiator Gene Set reflected molecular and biological events almost exclusive of inflammation. Preterm and term labor differ dramatically in their unique, initiator gene profiles, suggesting alternative pathways underlie these events. Inflammatory processes are ubiquitous to the Term Initiator and the Effector Gene Sets, supporting the idea term parturition is an inflammatory process. The absence of inflammatory processes in the Preterm Initiator Set suggests inflammation is secondary to processes triggering spontaneous preterm birth, and could explain the lack of therapeutic efficacy associated with anti inflammatory/antibiotic regimens. Keywords: myometrial gene expression, preterm versus term labor
Coenzyme Q10 (CoQ10) is an obligatory element in the respiratory chain and functions as a potent antioxidant of lipid membranes. More recently anti-inflammatory effects as well as an impact of CoQ10 on gene expression have been observed. To reveal putative effects of Q10 on LPS-induced gene expression, whole genome expression analysis was performed in the monocytic cell line THP-1. 1129 probe sets have been identified to be significantly up-regulated (p < 0.05) in LPS-treated cells when compared to controls. Text mining analysis of the top 50 LPS up-regulated genes revealed a functional connection in the NFκB pathway and confirmed our applied in vitro stimulation model. Moreover, 33 LPS-sensitive genes have been identified to be significantly down-regulated by Q10-treatment between a factor of 1.32 and 1.85. GeneOntology (GO) analysis revealed for the Q10-sensitve genes a primary involvement in protein metabolism, cell proliferation and transcriptional processes. Three genes were either related to NFκB transcription factor activity, cytokinesis or modulation of oxidative stress. In conclusion, our data provide evidence that Q10 down-regulates LPS-inducible genes in the monocytic cell line THP-1. Thus, the previously described effects of Q10 on the reduction of pro-inflammatory mediators might be due to its impact on gene expression.
Analysis of umbilical vein endothelial cells (HUVEC) treated with Egr-3 siRNA under the VEGF treatment for 01, and 4 h. Egr-3, a member of early growth response family, is immediately and dramatically induced by VEGF in HUVEC, which regulates expression of many genes related to endothelial activation.
The purpose of the dataset is to analyze expression of genes induced by KRAS and regulated by TBK1 The proto-oncogene KRAS is mutated in a wide array of human cancers most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. An alternative strategy for targeting KRAS is to identify gene products that, when suppressed or inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference (RNAi) to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkB kinase, TBK1, was selectively essential in cells that harbor mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF- B anti-apoptotic signals involving cREL and BCL-XL that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations identify TBK1 as a potential therapeutic target in KRAS mutant tumors and establish a general approach for the rational identification of co-dependent pathways in cancer.
Translocations of ETS transcription factors are driver mutations in diverse cancers. We investigated the genomic network of the ETS fusion EWS/FLI1 in Ewing's sarcoma (ESFT) as a model of ETS-driven tumorigenesis. ChIP-Seq and transcriptional analysis identified E2F3 as a principle co-factor of EWSFLI1 defining functionally distinct gene sets. While EWS/FLI1 binding independent of E2F3 predominantly associated with repressed differentiation genes significant co-localization with E2F3 was discovered at proximal promoters of activated growth-related genes. Thus, EWS/FLI1 promotes oncogenesis by simultaneously perturbing differentiation state and augmenting the expression of genes co-regulated by E2F3. Integration of additional E2F3 and ERG localization data from prostate cancer containing TMPRSS2/ERG verified that the ETS-E2F module is also found in prostate cancer and may be of general relevance to ETS driven cancers.
Objective: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors with a major impact on erythroid cell development. Here we investigated TR activity on red cell gene expression and identified TR target genes. The impact of the TR target gene GAR22 (growth arrest specific 2 [GAS2]-related gene on chromosome 22) on red cell differentiation was determined. Methods: SCF/Epo dependent red cell progenitors were differentiated in vitro in the presence or absence of thyroid hormone. Hormone-induced changes in gene expression were measured by a genome-wide approach with DNA microarrays. Ectopic expression of the TR target gene GAR22 was used to determine its impact on red cell differentiation. Results: Ligand-activated TR effectively accelerated red cell progenitor differentiation in-vitro concomitantly with inducing growth arrest. We demonstrate that activated TR induced specific gene expression patterns of up- or down-regulated genes including distinct clusters associated with accelerated differentiation in response to treatment. Mining for T3 induced genes identified BTEB1 (basic transcription element binding protein 1/Krüppel-like factor 9) and GAR22 as TR target genes. BTEB1/KLF9 is a known TR target gene while GAR22, initially identified as a putative tumor suppressor, represents a novel TR target gene. We demonstrate that ectopic GAR22 expression in red cell progenitors lengthens the cell cycle and causes growth inhibition, but leaves red cell gene expression unaffected. Conclusion: This study identifies GAR22 as a novel and direct TR target gene. Our results suggest that hormone-induced GAR22 might represent an important trigger of growth inhibition induced by thyroid hormone in red cell progenitors.
In this study we performed gene expression profiling of 14 cases of grade II gliomas. The results of these analysis were used in unsupervised analyses to compare correlations between the histological subtype of grade II gliomas and gene expression profiles
Extremely variable clinic and genetic features characterize Mitochondrial Encephalomyopathy Disorders (MED). Pathogenic mitochondrial DNA (mtDNA) defects can be divided into large-scale rearrangements and single point mutations. Clinical manifestations become evident when a threshold percentage of the total mtDNA is mutated. In some MED the "mutant load" in an affected tissue is directly related to the severity of the phenotype. However, the clinical phenotype is not simply a direct consequence of the relative abundance of mutated mtDNA. Other factors, such as nuclear background, can contribute to the disease process, resulting in a wide range of phenotypes caused by the same mutation. Using Affymetrix oligonucleotide cDNA microarrays (HG-U133A), we studied the gene expression profile of muscle tissue biopsies obtained from 12 MED patients (4 common 4977-bp deleted mtDNA and 8 A3243G: 4 PEO and 4 MELAS phenotypes) compared with age-matched healthy individuals. Keywords = mtDNA mutation Keywords = muscle biopsy Keywords = human Keywords = mitochondrial disease Keywords: other
Inadequate protein intake initiates an accommodative response with adverse changes in skeletal muscle function and structure. mRNA level changes due to short-term inadequate dietary protein might be an early indicator of accommodation. The aims of this study were to assess the effects of dietary protein and the diet-by-age interaction on the skeletal muscle transcript profile. Self-organizing maps were used to determine expression patterns across protein trials. 958 transcripts were differentially expressed (P<0.05) with diet and 853 had a diet-by-age interaction (P<0.05) using ANOVA. The results for diet alone revealed that P63 was associated with up-regulation of transcripts related to ubiquitin-dependent protein catabolism and muscle contraction and P63 and P94 resulted in up-regulation of transcripts related to apoptosis and down-regulation of transcripts related to cell differentiation; muscle and organ development; extracellular space; and responses to stimuli and stress. The diet-by-age expression patterns demonstrated that across the three protein trials transcripts related to protein metabolism were affected by age. Changes in skeletal muscle mRNA levels in the younger and older males to protein intake near or below the RDA are indicative of an early accommodative response. 5052 transcripts were determined as differentially expressed (P<0.05) between the younger and older males of which 2556 met the False Discovery Rate correction (P=0.0081). The age-related changes in the transcript profile were consistent with aging skeletal muscle phenotypes including; mitochondrial dysfunction (UP- and DOWN-regulation), RNA splicing (UP), oxidative stress (UP), apoptosis (UP), and energy metabolism (DOWN). Keywords: Age and dietary protein response
This study used microarray expression analysis to identify global changes in transcript alteration in response to MEK inhibition. Genes under ERK control were identified in a panel of V600E BRAF and RTK-activated tumor cells and xenografts using short-term inhibition of ERK activity using the MEK inhibitor PD0325901 (Pfizer). Keywords: paired treatment and control
This SuperSeries is composed of the SubSeries listed below.
A sensitive assay to identify biomarkers that can accurately diagnose the onset of breast cancer using non-invasively collected clinical specimens is ideal for early detection. In this study we have conducted a prospective sample collection and retrospective blinded validation (PRoBE design) to evaluate the performance and translational utilities of salivary transcriptomic and proteomic biomarkers for the non-invasive detection of breast cancer. The Affymetrix HG U133 Plus 2.0 Array and 2D-DIGE were used to profile transcriptomes and proteomes in saliva supernatants respectively. Significant variations of salivary transcriptomic and proteomic profiles were observed between breast cancer patients and healthy controls. Eleven transcriptomic biomarker candidates and two proteomic biomarker candidates were selected for a preclinical validation using an independent sample set. Transcriptomic biomarkers were validated by RT-qPCR and proteomic biomarkers were validated by quantitative protein immunoblot. Eight mRNA biomarkers and one protein biomarker have been validated for breast cancer detection, yielding ROC-plot AUC values between 0.665 and 0.959. This report provides proof of concept of salivary biomarkers for the non-invasive detection of breast cancer. The salivary biomarkers’ discriminatory power paves the way for a PRoBE-design definitive validation study. Keywords: Salivary biomarker, Breast cancer, Early detection, Salivary transcriptome, Salivary proteome
Spontaneous intracerebral hemorrhage (ICH) represents about 15% of all strokes and is associated with high mortality rates. Our aim was to identify the gene expression changes and biological pathways altered in the brain following ICH. Twelve brain samples were obtained from four deceased patients who suffered an ICH including perihematomal tissue (PH) and the corresponding contralateral white (CW) and grey (CG) matter.
U2OS-ERa U2OS-ERb, and U2OS-ERab cells treated with 4HT or E2 Keywords: other
Effects of the pan-anti-apoptotic BCL-2 family small molecule inhibitor obatoclax mesylate (GeminX Pharmaceuticals), on gene expression were evaluated by microarray analysis in order to gain insights into the killing mechanism by this compound in two human MLL-AF4 cell lines. The results of the gene expression profiling substantiated other lines of evidence derived from genetic and chemical cell death pathway inhibition, Western blot analysis, flow cytometric apoptosis assays, and electron microscopic analyses, showing triple apoptosis, autophagy, and necroptosis death pathway activation by this agent. The results also demonstrated modulation of a number of novel targets of obatoclax encoding various cell death factors at the gene expression level.
Samples were taken from colorectal cancers in surgically resected specimens in 35 colorectal cancer patients. The expression profiles were determined using Affymetrix Human Genome U133 Plus 2.0 arrays. Comparison between the sample groups allow to identify a set of discriminating genes that can be used for molecular markers for CIN phynotype.
Treating unselected cancer patients with new drugs dilutes proof of efficacy when only a fraction of patients respond to therapy. We conducted a meta-analysis on eight primary breast cancer microarray datasets representing diverse breast cancer phenotypes. We present a high-throughput protocol which incorporates drug sensitivity signatures to guide preclinical testing for effective therapeutic agents. Specifically we focus on drug classes currently undergoing early phase clinical testing. Our genomic and experimental results suggest that the majority of basal-like breast cancers should respond to inhibitors of the phosphatidylinositol-3-kinase pathway, and that a relatively low toxicity histone deacetylase inhibitor, valproic acid, may target aggressive breast cancers. For a subset of drugs, prediction of sensitivity associates with tumor recurrence, suggesting clinical relevance. Preclinical studies using both cell lines and patient tumors grown in 3-dimensional in vitro and orthotopic in vivo preclinical models provide an efficient and highly relevant assessment of drug sensitivity in tumor phenotypes, and validate our genomic analyses. Together, our results show that high-throughput transcriptional profiling can significantly impact drug selection for breast cancer patients. Pre-identification of patient response may not only improve therapeutic response rates, it can also assist in quickly identifying the optimal inclusion criteria for clinical trials. Our model facilitates personalized drug therapy for cancer patients and may be generalized for study of drug efficacy in other diseases.
To delineate the role of hypoxia in esophageal epithelial biology we carried out gene array experiments using a non-transformed immortalized diploid human esophageal cell line, EPC2-hTERT (Mol Cancer Res. 2003;1:729-38). Unlike cancer cell lines, EPC2-hTERT has no genetic alterations at early passages that may affect the cellular response to hypoxia.
The effects of the CDK inhibitor PHA-848125 (referred to as CDK-125) on the MCF7 human adenocarcinoma mammary cell line were analysed by gene expression profiling.
The identification of genes and deduced pathways from the mature human oocyte can help us better understand oogenesis folliculogenesis, fertilization, and embryonic development. Human metaphase II oocytes were used within minutes after removal from the ovary, and its transcriptome was compared with a reference sample consisting of a mixture of total RNA from 10 different normal human tissues not including the ovary. RNA amplification was performed by using a unique protocol. Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays were used for hybridizations. Compared with reference samples, there were 5,331 transcripts significantly up-regulated and 7,074 transcripts significantly down-regulated in the oocyte. Of the oocyte up-regulated probe sets, 1,430 have unknown function. A core group of 66 transcripts was identified by intersecting significantly up-regulated genes of the human oocyte with those from the mouse oocyte and from human and mouse embryonic stem cells. GeneChip array results were validated using RT-PCR in a selected set of oocyte-specific genes. Within the up-regulated probe sets, the top overrepresented categories were related to RNA and protein metabolism, followed by DNA metabolism and chromatin modification. This report provides a comprehensive expression baseline of genes expressed in in vivo matured human oocytes. Further understanding of the biological role of these genes may expand our knowledge on meiotic cell cycle, fertilization, chromatin remodeling, lineage commitment, pluripotency, tissue regeneration, and morphogenesis.
This SuperSeries is composed of the SubSeries listed below.
In Alzheimer’s disease (AD) early deficits in learning and memory are a consequence of synaptic modification which are likely induced by toxic beta-amyloid oligomers (oAβ). To identify molecular targets downstream of oAβ binding we prepared synaptoneurosomes from frontal cortex of control and IAD patients, and isolated mRNAs for comparison of gene expression. This approach elevated synaptic mRNAs above the threshold necessary for expression changes to be discriminated and also reduced other cellular mRNAs. In patients with minimal cognitive impairment (MCI) termed incipient AD (IAD) global measures of cognition declined with increasing levels of dimeric Aβ (dAβ). These patients also showed increased expression of neuroplasticity related genes, many encoding 3' UTR consensus sequences that regulate local translation in the synapse. One such gene, GluR2, displayed elevated mRNA and protein expression in IAD. Other neurotransmitter-related genes were also upregulated. Overexpressed genes may induce compensatory as well as negative effects on cognition and provide targets for intervention to moderate the response to dAβ.
The proinflammatory cytokine TNFalpha is critical in maintaining liver homeostasis since it is a major determiner of hepatocyte life and death. Considering this, gene transcription profiling was examined in control and TNFalpha treated HepG2 cells. Results indicated that TNFalpha could significantly alter the expression of a significant number of genes; most of them were functionally distributed among molecular functions like catalytic activity, binding, molecular transducer activity, transporter activity, translation and transcription regulator activities or enzyme regulator activity. Also, within genes up-regulated by TNFalpha, several GO terms related to lipid and fat metabolism were significantly overrepresented indicating global dysregulation of fat metabolism within the hepatocyte and those within the down-regulated dataset included genes involved in immunoglobulin receptor activity and IgE binding thereby indicating a compromise in immune defense mechanism(s) apart from those involved the DNA binding and protein binding categories. The interacting network of “lipid metabolism, small molecule biochemistry” was derived to be significantly affected that correlated well with the top canonical pathway of “biosynthesis of steroids” and molecular and cellular function of “lipid metabolism”. All these indicate TNFalpha to be significantly altering the transcriptome profiling within HepG2 cells with genes involved in lipid and steroid metabolism being the most favoured. This study suitably addresses the genes that determine TNFalpha mediated alterations within the hepatocyte mainly the phenotypes of hepatic steatosis and fatty liver that are associated with several hepatic pathological states.
Neurotrophin receptors play a pivotal role in the biology and prognosis of neuroblastoma (NB). While expression of TrkA/NTRK1 confers a good prognosis to NB patients expression of TrkB/NTRK2 is associated with unfavorable outcome. We have transfected the neurotrophin-receptor null cell line SY5Y with either full-length TrkA or TrkB and performed transcriptional profiling to analyse the effects of Trk-expression without activation and in a time course after activation.
Immature cell populations including stem cells and progenitor cells, can be found in “side-population (SP)” cells. Although SP cells isolated from some adult tissues have been reported elsewhere, isolation and characterization of human trophoblast SP cells remained to be reported. We used microarrays to detail the global program of gene expression underlying cell differentiation and identified up-regulated genes of SP cells.
Study of a metastatic progression of an ependimoma. Chromosomal imbalances metaolic and gene expression profiles. Keywords: comparative genomics
Previously we constructed a coculture model to analyze the effect of macrophages on intestinal epithelial cells, and found that TNF-a secreted from human macrophage-like THP-1 cells induced cell damage to intestinal epithelial Caco-2 cells (Exp.Cell.Res. 2006, 312(19):3909-19). In this study, we present activation of NF-kB in Caco-2 cells within 15 min after coculturing. To reveal how TNF-a secreted from THP-1 cells affects Caco-2 cells in an early stage of coculture, we exhaustively analyzed the changes of gene expression in Caco-2 cells cocultured with THP-1 cells over the time periods of 0, 1, 3, 6, 24, and 48 h by using a DNA microarray. Differentially expressed genes extracted with maSigPro demonstrated that IEX-1 was the lowest p-value gene, that is, the most significantly changed gene among the up-regulated genes. The genes expressed in a similar pattern to IEX-1 involved immunity, apoptosis, and protein kinase cascade. These findings suggest that the stimuli of TNF-a from THP-1 cells activates NF-kB, leading induction of various gene expression. This pattern of gene expression indicates that not only early defense response but also cell death occurs at the same time, causing inflammatory condition.
Smallpox is a highly communicable often fatal diseae. There is currently no licensed treatment for smallpox and vaccinia virus (VV) is currently used for immunization. While immunization with VV can provide good protection against exposure to the smallpox virus, the current vaccine is far from optimal. Complications occur in 1/1,000-1/10,000 vaccinees, with at least one death per million vaccinees. We have constructed recombinant VV strains which are less pathogenic, yet provide a protective immune response. These viruses contain various mutations in the E3L which is known to block the host antiviral response. Identifying the host genes involved in producing a strong protective immunological response to these attenuated viruses would not only increase our understanding of the proteins and pathways involved in effective smallpox vaccination, but aid in the development of alternative vaccine strains which enhance these specific immune responses. We will determine gene expression patterns in HeLa cells at various times following infection with wtVV and several VV constructs containing mutations in the E3L gene. The VV E3L gene product blocks the host antiviral response by sequestering viral danger signals, including double-stranded RNA and Z-DNA. VV constructs containing mutations in E3L which allow host cell recognition of either of these danger signals leads to a decrease in viral pathogensis. In this project we will dissect the cellular inflammatory response to infection with wtVV in comparison to VV containing mutations in the E3L gene. By understanding why certain strains of VV are non-pathogenic, yet highly immunogenic, it is possible to gain a better understanding on the mechanisms of poxvirus pathogenesis and the host response. We will examine three times points following infection with VV: 2 HPI, 6 HPI and 9 HPI. These times points represent keys points in the virus replication cycle. Several VV constructs will be used which contain mutations in the E3L gene. These constructs alter the ability of E3L to sequester double-stranded RNA and/or Z-DNA and therefore have a direct effect on viral pathogenesis. Fourteen constructs will be used including: mock, wtVV, VVdelE3L, VVE3Ldel83N, VVE3Ldel37N, VVE3Ldel26C, VVE3Ldel7C, VVE3L Y48A, VVE3L P63A, VVE3L K167T, VV-ATV, VV-ADAR/E3L, VVdelK3L, VVdelK3L-E3Ldel37N. Cells will be infected at an MOI of 5 to allow infection of all cells. At each time point, cells will be harvested by scraping. RNA will be isolated using a Trizol RNA extraction protocol (Invitrogen) followed by RNA purification using the RNeasy cleanup kit available from Qiagen. Keywords: time-course
We established a novel model to assess the function of proteins under in vivo conditions. The model relies on the expansion of HEK293 cells in immunodeficient NOD.Scid mice. To validate the novel model we performed microarray gene expression profiling of NOD.Scid-expanded HEK293 cells relative to conventionally cultivated cells. Microarray analysis revealed that cell expansion in NOD.Scid mice restored an imbalanced chaperone system without inducing a major upregulation of the entire protein folding machinery.
The aim of this study was to describe the gene expression patterns related to the differentiation and mineralization of bone-forming cells including activation and/or repression of osteogenic or non-osteogenic pathways, remodeling of cell architecture, cell adhesion, cell communication, and assembly of extracellular matrix. The study implied patient selection, tissue collection, isolation and culture of human marrow stromal cells (hMSC) and osteoblasts (hOB), and characterization of bone-forming cells. RNA samples were collected at defined time points, in order to understand the regulation of gene expression during the processes of cell differentiation/mineralization that occur during bone repair. Transcriptome analysis was performed by using the Affymetrix GeneChip microarray technology platform and GeneChip® Human Genome U133 Plus 2.0 Array. Our results help to design a gene expression profile of bone-forming cells during specific steps of osteogenic differentiation. These findings offer an useful tool to monitor the behaviour of osteogenic precursors cultured in presence of exogenous stimuli, i.e. growth factors, or onto 3D scaffolds for bone engineering. Moreover, they can contribute to identify and clarify the role of new genes for a better understanding of the molecular mechanisms regulating osteogenesis. Keywords: time course
We developed an experimentally derived molecular signature from mouse tumor models that is closely associated with survival of colorectal cancer (CRC) patients.
To identify the gene signature accounting for the distinct clinical outcomes in ovarian clear cell cancer patients Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. Arrays analyzed using BRB ArrayTools and PathwayStudio software was used to identify the signaling pathways.
Pericytes derived from skin dermis can substantially enhance the short-term tissue-regenerative capacity of human epidermal cells already committed to differentiation; they also display both phenotypic and functional properties of mesenchymal stem cells. In this microarray analysis we compared the gene expression profile of dermal pericytes to that of the remaining dermal cells of neonatal human foreskin.
20 patients with unstable angina were divided into patient group by using coronary angiography. The sex- and age-matched healthy individuals were enrolled as control group. Venous bloods were collected for extracting RNA.
We used Affymetrix HG U133 Plus 2.0 GeneChips to compare the transcriptome of MDA-MB-231 cells transfected with SDC1-siRNA against negative control siRNA-transfected cells.
Introduction: Sepsis is a complex immunological response to infection characterized by early hyperinflammation followed by severe and protracted immunosuppression suggesting that a multi-marker approach has the greatest clinical utility for early detection, within a clinical environment focused on SIRS differentiation. Pre-clinical research using an equine sepsis model identified a panel of gene expression biomarkers that define the early aberrant immune activation. Thus, the primary objective was to apply these gene expression biomarkers to distinguish patients with sepsis from those who had undergone major open surgery and had clinical outcomes consistent with systemic inflammation due to physical trauma and wound healing. Methods: This was a multi-centre, prospective clinical trial conducted across 4 tertiary critical care settings in Australia. Sepsis patients were recruited if they met the 1992 Consensus Statement criteria and had clinical evidence of systemic infection based on microbiology diagnoses (n=27). Participants in the post-surgical (PS) group were recruited pre-operatively and blood samples collected within 24 hours following surgery (n=38). Healthy controls (HC) included hospital staff with no known concurrent illnesses (n=20). Each participant had minimally 5ml of PAXgene blood collected for leucocyte RNA isolation and gene expression analyses. Affymetrix array and multiplex tandem (MT)-PCR studies were conducted to evaluate transcriptional profiles in circulating white blood cells applying a set of 42 molecular markers that had been identified a priori. A LogitBoost algorithm was used to create a machine learning diagnostic rule to predict sepsis outcomes. Results: Based on preliminary microarray analyses comparing HC and sepsis groups. A panel of 42-gene expression markers were identified that represented key innate and adaptive immune function, cell cycling, WBC differentiation, extracellular remodelling and immune modulation pathways. Comparisons against GEO data confirmed the definitive separation of the sepsis cohort. Quantitative PCR results suggest the capacity for this test to differentiate severe systemic inflammation from HC is 92%. AUC ROC curve findings demonstrated sepsis prediction within a mixed inflammatory population, was between 86 - 92%. Conclusions: This novel molecular biomarker test has a clinically relevant sensitivity and specificity profile, and has the capacity for early detection of sepsis via the monitoring of critical care patients. GEO Note: Data made available represents the preliminary microarray investigation performed on Human U133 Plus 2.0 GeneChips (Affymetrix), assaying 41 patient samples (Sepsis n=10, Post-Surgical n=11, Control n=20).
The hallmark of human cancer is heterogeneity mirroring the complexity of genetic and epigenetic alterations acquired during oncogenesis. We extracted RNA of 34 cultured human ovarian carcinoma cell lines and performed expression microarrays so that cultured cell lines can represent in vivo human tumors.
Our results suggest that 48h of immobilization increases expression of mRNA for components of the UPP and MT function while decreasing mRNA and protein for components involved in ECM integrity. We hypothesized that 48h of immobilization would increase gene expression and respective protein products for components of the ubiquitin proteasome pathway (UPP). Keywords: stimulus or stress design
Female BRCA1 mutation carriers have a nearly 80% probability of developing breast cancer during their life-time. We hypothesized that the breast epithelium at risk in BRCA1 mutation carriers harbors mammary epithelial cells (MECs) with altered proliferation and differentiation properties. Microarray studies revealed that PMEC colonies from BRCA1 mutation carriers anticipate expression profiles found in BRCA1-related tumors and that the EGFR pathway is upregulated in BRCA1 mutation carriers compared ton non BRCA1 mutation carriers. Keywords: Class comparison and pathway analysis
Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in normal eukaryotic cellular function. Here we characterized the metabolic and transcriptional properties of A549 lung cancer cells and their isogenic mitochondrial DNA (mtDNA)-depleted rho zero counterparts grown in cell culture and as tumor xenografts in immune-deficient mice. A manuscript summarizing our conclusions is under review. Keywords: genetic modification and culture conditions
Comparison of Chronic Lymphocytic Leukemia patients expressing high or low levels of ZAP70 mRNA: prognostic factors and interaction with the microenvironment. Zeta-associated protein 70 (ZAP70) is a widely recognized prognostic factor in chronic lymphocytic leukemia (CLL) but mechanisms by which its higher expression leads to a poor outcome remain to be fully explained. In an attempt to unveil unfavorable cellular properties linked to high ZAP70 expression, we used gene expression profiling to identify genes associated with disparities in B-cells from CLL patients expressing high versus low ZAP70 mRNA, measured by quantitative real-time PCR. Keywords: comparison of poor and good prognosis CLL patient transcriptome regarding ZAP70 expression
Expression profiling of MRC5 IFN gamma treated MRC5 and PGF cells. Keywords: cell type comparison, IFN gamma treatment.
The aim of this experiment is to determine the similarities and differences between gene expression profiles in HepaRG cells versus primary human hepatocytes human liver, and the commonly used HepG2 cell.
Kasumi-1 AML cells that were transfected in triplicate with AML1-ETO or luciferase siRNA constructs by either Amaxa nucleofection or Biorad siLentFect and incubated for 96 hours. Microarrays used to discover an AML1-ETO signature for a GE-HTS screen to identify AML1-ETO modulators.
Epidemiological studies indicate that adverse intrauterine and postnatal environment has a long-lasting role in chronic kidney disease (CKD) development. Epigenetic information can represent a plausible carrier for mediating this programming effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and CKD tubule samples obtained from patients show significant differences. Cytosine methylation changes showed high concordance (98%) with a large (n=87) replication dataset. We rarely observed differentially methylated regions (DMR) on promoters. Histone modification-based kidney specific genome-wide gene regulatory region annotation maps (promoters enhancers, transcribed and repressed regions) were generated. DMRs mostly overlapped with putative enhancer regions and were enriched in consensus binding sequences for important renal transcription factors, indicating their importance in gene expression regulation. A core set of genes, including transforming growth factors and collagens, showed cytosine methylation changes correlating with downstream transcript levels. Our report raises the possibility that epigenetic dysregulation plays a role in CKD development via influencing core profibrotic pathways. We used microarrays to detail the differences of gene expression of human tubule epithelial cells between chronic kidney disease and normal.
Side populations have recently been identified in ovarian cancers and may play an important role in post treatment relapse and resistance to chemotherapeutic drugs. In this study we aimed to identify the differential expression between IGROV1 SP and NSP on Affymetrix HG-U133plus2 microarrays. We found ovarian tumour SP cells frequently over-express the multi-drug resistance associated P-glycoprotein (ABCB1) by Rank Product (FDR<0.05), and by geneset enrichment analysis, embryonic stem cell-associated ‘NOS’ signature (Notch/Oct4/Sox2 regulated genes) and Polycomb Repressive Complex 2 (PRC2) genes were over-expressed, while PRC2-repressed target genes were significantly under-expressed in the SP from ovarian cell lines compared to non-SP (FDR<10-4).
Transient transfection of a Ewing's Sarcoma cell line expressing type I EWS-FLI1 fusion and doxycycline-inducible short hairpin RNA against EWS-FLI1 (A673sh)
Relative contribution of sequence and structural features to the mRNA-binding of Argonaute/miRNA complexes and the degradation of miRNA targets How miRNAs recognize their target sites is a puzzle that many experimental and computational studies aimed to solve. Several features such as perfect pairing of the miRNA seed additional pairing in the 3’ region of the miRNA, relative position in the 3’ UTR and the A/U content of the environment of the putative site have been found to be relevant. Here we have used a large number of previously published data sets to assess the power that various sequence and structure features have in distinguishing between putative sites that do and those that do not appear to be functional. We found that although different data sets can give widely different answers when it comes to ranking the relative importance of these features, the sites inferred from most transcriptomics experiments as well as from comparative genomics appear similar at this level. This suggests that miRNA target sites have been selected in evolution on their ability to trigger mRNA degradation. To understand at what step in the miRNA induced response individual features play a role, we further transfected human HEK293 cells with miRNAs and analyzed the association of Argonaute/miRNA complexes with target mRNAs and the degradation of these messages. We found that structural features are only important for Argonaute binding, while sequence features such as the A/U content of the 3’ UTR are important for mRNA degradation. Keywords: IP-array analysis
Gene expression on peripheral blood mononuclear cells (PBMC) from SPARKS CHARMS juvenile idiopathic arthritis (JIA) cohort pre and post methotrexate therapy. This is the first study to our knowledge to evaluate gene expression profiles in children with JIA before and after MTX, and to analyze genetic variation in differentially expressed genes. We have identified a gene, which may contribute to genetic variability in MTX response in JIA.
Actomyosin contractility regulates cell morphology and movement. The objective of this study was to identify whether actomyosin contractility regulates gene expression in tumour cells and whether such genes are involved in cell morphology and movement. Gene expression analysis was carried out on highly contractile melanoma cell line A375M2 plated on a deformable collagen matrix under conditions where actomyosin contractility could be altered following treatment with blebbistatin a direct inhibitor of myosin II, or Rho-kinase inhibitors Y27632 or H1152 that interfere with signalling to myosin II.
This SuperSeries is composed of the SubSeries listed below.
Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4 SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem (hiPS) cells. hiPS cells generated with this system were molecularly and functionally similar to human embryonic stem (hES) cells, demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes, suggesting that the kinetics of reprogramming are cell-type dependent. Using our inducible system, we developed a strategy to induce hiPS cell formation at high frequency by generating differentiated cells that contain the viral transgenes in a pattern that enables successful induction of pluripotency. Upon addition of doxycycline to differentiated hiPS-derived cells, we obtained “secondary” hiPS cells at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells with high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and LPS. Data were analyzed using a rigorous combinatorial approach with three separate statistical algorithms. Thirty-six genes met the criteria of up-regulated expression in response to Stx1 treatment with 14 genes uniquely up-regulated by Stx1. Microarray data were validated by real time RT-PCR for genes encoding Egr-1 (transcriptional regulator), COX-2 (inflammation), and DUSP1, 5 and 10 (regulation of MAPK signaling). Stx1-mediated signaling through ERK1/2 and Egr-1 appears to be involved in the increased expression of the proinflammatory mediator TNF-α. Activation of COX-2 expression is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1