ROLE OF PROGESTERONE RECEPTOR RATIO IN BREAST CANCER

孕酮是一种关键的生殖激素，它与调节转录的核内孕酮受体(PR)结合。有两种PR亚型，PR-A和PR-B，具有非常不同的生物学效应。这两种异构体在乳腺癌中共同表达。假设：黄体酮在乳腺癌中的作用受到肿瘤中A/B比率的影响。目的：1.构建PR-A/PR-B比值可变的乳腺癌细胞系，研究PR异构体比值变化对内源性C/EBPbeta、Stat5a和bclxl基因孕激素的调节作用。T47D乳腺癌细胞系，其中一种PR亚型是固定的，另一种可以从低水平到高水平变化，将使用蜕皮激素诱导系统来控制A：B比率。这些细胞将被用来研究由两个PR差异调节的三个基因的内源性表达：受PR-B但不受PR-A调节的C/EBPbeta和Stat5a，以及受PR-A但不受PR-B调节的bclxl。这些研究首次试图了解A：B比率如何调节内源性孕激素调节基因的表达。目的2.通过克隆和鉴定PR异构体差异调控基因启动子，阐明PRA和PR-B差异调控靶基因的机制。C/EBPbeta、Stat5a和bc1-xl基因启动子的结构是PR-A或PR-B差异调控的原因，这一假说将通过克隆这些基因的启动子来检验。启动子片段将被亚克隆到荧光素酶报告载体中，该载体将被瞬时导入只表达PR-A或PR-B的细胞中，以研究它们的调控。顺式作用的PR调节元件将通过突变来定位。这些研究首次尝试确定启动子特异性因素是否能解释PR-A和PR-B转录调控的差异。由于PR是乳腺癌的预后指标，这些研究可能需要修订临床分析以包括这两种异构体的测量。

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.