Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling
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ABSTRACT:
ORGANISM(S): Rattus norvegicus Homo sapiens
PROVIDER: GSE3299 | GEO | 2005/09/13
SECONDARY ACCESSION(S): PRJNA92809
REPOSITORIES: GEO
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Project description:Extensive efforts are underway to identify anti-angiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial cell growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGFR-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neo-angiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells, and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for anti-angiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors. Total RNA isolated from cultured cells or tumor tissue samples was used to make fluorescently labeled cRNA that was hybridized to DNA oligonucleotide microarrays. Briefly, 4 µg of total RNA from an individual tumor sample or endothelial cell culture were used to synthesize dsDNA through RT. cRNA was produced by in vitro transcription and labeled postsynthetically with Cy3 or Cy5. Two populations of labeled cRNA, a reference population and an experimental population, were compared with each other by competitive hybridization to microarrays. For animal tumor studies, reference cRNA pools were made by pooling equal amounts of cRNA from each tumor in the appropriate vehicle-dosed group. Species-specific microarrays were used throughout this study. Human microarrays contained 23,916 oligonucleotide probes corresponding to individual genes or expressed sequence tags. Rat microarrays contained probes to 22,592 genes or expressed sequence tags. Oligonucleotide probe sequences were chosen to maximize gene specificity and minimize the 3' replication bias inherent in RT of mRNA. In addition, both microarray formats contained 1,000 control probes for quality control purposes. All oligonucleotide probes on the microarrays were synthesized in situ with inkjet technology (Agilent Technologies, Palo Alto, CA). After hybridization, arrays were scanned and fluorescence intensities for each probe were recorded. Ratios of transcript abundance (experimental to control) were obtained following normalization and correction of the array intensity data. Gene expression data analysis was done with the Rosetta Resolver gene expression analysis software (version 3.2, Rosetta Biosoftware, Seattle, WA). For each gene sequence present on the microarrays, statistical significance of differential gene expression was determined by calculating P values.
2005-09-13 | E-GEOD-3299 | biostudies-arrayexpress
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Project description:Although differentiation of mice embryonic stem cells into vascular endothelial cells (ECs) gives a model for investigating molecular mechanisms of vascular development in vivo, temporal dynamics of gene expressions and chromatin modifications have not been studied until now. Here, we interrogated transcriptome and two histone modifications, H3K4me3 and H3K27me3, with a genome-wide scale during ECs differentiation and elucidated epigenetic switch peculiar to ECs. We find Gata2, Fli1, Sox7, and Sox18 are master regulators from genetic and epigenetic data, these genes were induced after Etv2 activation. These genes have specific histone modification pattern which is repressed by H3K27me3 modification at Flk-sorted mesoderm and changed to the bivalent (H3K4me3 and H3K27me3 both positive) state rapidly after vascular endothelial cells growth factor (VEGF) stimuli. Using a previously reported ECs differentiation model, we demonstrate that four transcription factors are critical for ECs specific gene expressions and efficient differentiation. Moreover, from knockdown experiments using si-RNA, we discovered these factors inhibited not only TGFβ signaling pathway, that is endothelial mesenchymal transition pathway, but also other near lineage commitment, including blood cells, skeletal muscle cells, vascular smooth muscle cells, and cardiomyocytes. We further identify each factor specific target genes during ECs differentiation by microarray, including both activating and repressing genes. Together, our findings from a detailed epigenetic approach provide a basic understanding temporal regulated chromatin signatures and resulting gene expression profile during ECs commitment, which is applicable to other models of differentiation and production of mature and long lasting ECs for regenerative medicine. Total 17 samples were derived from [1] ES cells, Flk-sorted mesoderm cells, and in the absense or presence of VEGF (6, 12, 24, and 48h) to determine VEGF activated genes during endothelial cells differentiation, [2] control si-RNA, si-Gata2, si-Fli1, si-Sox7, or si-Sox18 transfected cells under VEGF stimuli, [3] control si-RNA or si-Mix (si-Gata2, si-Fli1, si-Sox7, and si-Sox18) transfected cells under VEGF stimuli for the identification of each transcription factor dependent genes during endothelial cells differentiation.
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