ROLE OF PROGESTERONE RECEPTOR RATIO IN BREAST CANCER

孕酮是一种关键的生殖激素，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。这两种亚型在乳腺癌中共表达。假设：孕激素在乳腺癌中的作用受到肿瘤中A：B比例的影响。目标1.构建PR-A/PR-B比值可变的乳腺癌细胞系，研究孕激素对内源性C/EBP β、Stat 5a和bcl-xL基因的调节作用。T47 D使用蜕皮激素诱导系统控制A：B比率，将产生其中一种PR同种型固定而另一种可从低水平变化到高水平的乳腺癌细胞系。这些细胞将用于研究由两种PR差异调节的三种基因的内源性表达：C/EBP β和Stat 5a，其由PR-B调节但不受PR-A调节，以及bcl-xL，其由PR-A调节但不受PR- B调节。这些研究代表了第一次试图了解A：B比例如何调节内源性胆固醇调节基因的表达。 目标二。通过克隆和鉴定三个受PR异构体差异调节的基因的启动子，阐明PRA和PR-B差异调节靶基因的机制。将通过克隆这些基因的启动子来测试C/EBP β、Stat 5a和bcl-xL的启动子的结构负责PR-A或PR-B对它们的差异调节的假设。将启动子片段亚克隆到荧光素酶报告载体中，将其瞬时转染到仅表达PR-A或PR-B的细胞中以研究其调节。顺式作用PR调节元件将通过诱变作图。这些研究代表了第一次尝试，以确定是否启动子特异性因子解释差异转录调节PR-A与PR-B。由于PR是乳腺癌的预后指标，这些研究可能需要修改临床测定，以包括两种亚型的测量。

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.