Progesterone is a key reproductive hormone that binds to intranuclear progesterone receptors (PRs) which regulate transcription. There are two PR isoforms, PR-A and PR-B, that have very different biological effects. Both isoforms are coexpressed in breast cancers. Hypothesis:the effects of progesterone in breast cancers are influenced the A:B ratio present in the tumor.
Aim 1. To construct breast cancer cell lines in which the PR-A to PR-B ratio can be varied, and to study progesterone regulation of endogenous C/EBPbeta, Stat5a and bcl-xL genes as the PR isoform ratio is varied. T47D Breast cancer cell lines in which one PR isoform is fixed, and the other can be varied from low to high levels will be created using the ecdysone inducible system to control the A:B ratio. These cells will be used to study endogenous expression of three genes differentially regulated by the two PRs: C/EBPbeta and Stat5a, which are regulated by PR-B but not PR-A, and bcl-xL, which is regulated by PR-A but not PR- B. These studies represent the first attempt to understand how the A:B ratio regulates expression of endogenous, progesterone-regulated genes.
Aim 2. To elucidate the mechanisms by which PRA and PR-B differentially regulate target genes, by cloning and characterizing the promoters of three genes differentially regulated by the PR isoforms. The hypothesis that the structure of the promoters of C/EBPbeta, Stat5a and bcl-xL is responsible for their differential regulation by PR-A or PR-B will be tested by cloning the promoters of these genes. The promoter fragments will be subcloned into luciferase reporter vectors which will be transiently transfected into cells expressing only PR-A or PR-B to study their regulation. The cis-acting PR-regulated elements will be mapped by mutagenesis. These studies represent the first attempts to determine whether promoter-specific factors explain differential transcriptional regulation by PR-A vs. PR-B. Since PRs are prognostic indicators in breast cancer, these studies may require revision of clinical assays to include measurement of the two isoforms.
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