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Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray.

ABSTRACT: To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed approximately 3-5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced approximately 1.5-2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, transforming growth factor alpha and stromal cell-derived factor 1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, depend on the E2 concentration, especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions.

SUBMITTER: Coser KR

PROVIDER: S-EPMC283534 | biostudies-literature | 2003 Nov

REPOSITORIES: biostudies-literature

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Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray.

Coser Kathryn R KR Chesnes Jessica J Hur Jingyung J Ray Sandip S Isselbacher Kurt J KJ Shioda Toshi T

Proceedings of the National Academy of Sciences of the United States of America 20031110 24

To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes by using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics. We used it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growt ...[more]

PMID: 14610279

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Project description:"To obtain comprehensive information on 17beta-estradiol (E2) sensitivity of genes that are inducible or suppressible by this hormone, we designed a method that determines ligand sensitivities of large numbers of genes using DNA microarray and a set of simple Perl computer scripts implementing the standard metric statistics, and employed it to characterize effects of low (0-100 pM) concentrations of E2 on the transcriptome profile of MCF7/BUS human breast cancer cells, whose E2 dose-dependent growth curve saturated with 100 pM E2. Evaluation of changes in mRNA expression for all genes covered by the DNA microarray indicated that, at a very low concentration (10 pM), E2 suppressed 3~5 times larger numbers of genes than it induced, whereas at higher concentrations (30-100 pM) it induced 1.5~2 times more genes than it suppressed. Using clearly defined statistical criteria, E2-inducible genes were categorized into several classes based on their E2 sensitivities. This approach of hormone sensitivity analysis revealed that expression of two previously reported E2-inducible autocrine growth factors, TGF-? and SDF-1, was not affected by 100 pM and lower concentrations of E2 but strongly enhanced by 10 nM E2, which was far higher than the concentration that saturated the E2 dose-dependent growth curve of MCF7/BUS cells. These observations suggested that biological actions of E2 are derived from expression of multiple genes whose E2 sensitivities differ significantly and, hence, dependent on the E2 concentration especially when it is lower than the saturating level, emphasizing the importance of characterizing the ligand dose-dependent aspects of E2 actions. (paper abstract) Keywords: Dose Response, Time Course MCF7/BUS cells were exposed to varying concentrations of 17beta-estradiol (E2) for 48 hours. Experiments were repeated five times independently. In another experiment, MCF7/BUS cells were subjected to hormone starvation. At day 0, 1, and 2, RNA samples were isolated from cell culture. Experiments were repeated three times independently."

Project description:Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring potential underlying molecular mechanisms in human MCF7 breast cancer cells. Principal Findings: Gene expression profiling revealed that the expression of approximately 150 genes was influenced by both 17β-estradiol (E2) and a hypomethylating agent 5-aza-2’-deoxycytidine (DAC). Based on gene ontology (GO), CpG island prediction analysis and previously reported estrogen receptor (ER) binding regions, we selected six genes for further analysis (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2). GO analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis, while CpG island prediction of promoter regions reveals that the promoters of these genes contain at least one CpG island. Using chromatin immunoprecipitation, we show that ERα is recruited to CpG islands in promoters, but neither in an E2- nor in a DAC-dependent fashion. DAC treatment reactivates the expression of all selected genes although only the promoters of BTG3 and FHL2 genes are methylated, with E2 treatment showing no effect on the methylation status of these promoters. Conclusions: We identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes. The aim of the study was to identify genes regulated by estrogen signaling and DNA methylation, using microarray approach. We compared the effects of E2 and DAC on global gene expression profiles in MCF7 cells. By comparing C_E2 (MP5-MP8 average) with C_EtOh samples (MP1-MP4 average), 802 genes were identified as up-regulated by E2, while 851 genes were identified as down-regulated by E2. 1017 genes were identified as up-regulated by DAC, by comparing A_EtOh (MP9-MP12 average) samples with C_EtOh, suggesting that DNA methylation is involved in their regulation. To identify possible common targets, we have compared the DAC up-regulated genes with E2-regulated genes. 88 annotated genes are found to be up-regulated by both E2 and DAC, suggesting that E2 has a hypomethylation-like effect on the regulation of these genes. 58 annotated genes are found to be down-regulated by E2 and up-regulated by DAC, suggesting that E2 has a hypermethylation-like effect on the regulation of these genes.

Dataset Information

Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray.

Publications

Global analysis of ligand sensitivity of estrogen inducible and suppressible genes in MCF7/BUS breast cancer cells by DNA microarray.

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