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Transcription profiling by array of human MCF7 cells after treatment with epidermal growth factor or heregulin

ABSTRACT: ErbB receptor ligands, epidermal growth factor (EGF) and heregulin (HRG), induce dose-dependent transient and sustained intracellular signaling, proliferation and differentiation of MCF-7 breast cancer cells, respectively. In an effort to delineate the ligand-specific cell determination mechanism, we investigated time-course gene expressions induced by EGF and HRG that induce distinct cellular phenotypes in MCF-7 cells. To analyze the effects of ligand dosage and time for the gene expression independently, we developed a statistical method for decomposing the expression profiles into the two effects. Our results indicated that signal transduction pathways devotedly convey quantitative properties of the dose-dependent activation of ErbB receptor to early transcription. The results also implied that moderate changes in the expression levels of numbers of genes, not the predominant regulation of a few specific genes, might cooperatively work at the early stage of the transcription for determining the cell fate. However, the EGF- and HRG-induced distinct signal durations resulted in the ligand-oriented biphasic induction of proteins after 20 min. The selected gene list and HRG-induced prolonged signaling suggested that transcriptional feedback to the intracellular signaling results in a graded to biphasic response in the cell determination process, and that each ErbB receptor is inextricably responsible for the control of amplitude and duration of cellular biochemical reactions. MCF-7 human breast cancer cells were incubated from 5min to 90min after administration of different concentrations of the growth hormones (epidermal growth factor (EGF) and heregulin (HRG)). Control was set as growth hormone non-treated cells. For each growth hormone, one control was used.

ORGANISM(S): Homo sapiens

SUBMITTER: Nagashima T

PROVIDER: S-ECPF-GEOD-6462 | biostudies-other | 2007 Feb

REPOSITORIES: biostudies-other

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Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation. Experiment Overall Design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturer’s protocols.

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Transcription profiling by array of human MCF7 cells after treatment with epidermal growth factor or heregulin

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