PROGESTERONE RECEPTORS IN BREAST CANCER

黄体酮在正常的乳房和乳房中有增殖作用。 乳腺癌和黄体酮拮抗剂对生长有抑制作用。 孕激素效应是由孕激素受体(PR)和孕激素受体介导的。 乳腺癌细胞，有两种自然存在的亚型-- 120 kDa B受体(PRB)和N端截短94 kDa A-受体- 受体(PRA)。因为同二聚体和异二聚体都可以在 其中，三类受体二聚体(A：A，A：B，B：B)可与DNA结合， 每一种都具有潜在的不同转录效应。要分析 PR二聚化机制与功能异质性 在黄体酮的背景下，由三个二聚体类别传递的 在乳腺癌中的激动剂和拮抗剂作用，目的如下 建议：目标1：通过分析来表征孕激素激动剂的作用 PR二聚化、DNA结合和转录。我们将使用DNA绑定 PR、野生型PRA和PRB的结构域(DBD)突变体，以及体外和 体内功能测定，以表征PR二聚化，分析 DNA结构对二聚化的影响，并分析了 PR同源和杂二聚体被占据时的功能容量 激动剂R5020。我们还将构建一种“亮氨酸拉链”的突变体 可能包括PR的二聚化结构域的模块，映射其 边界和关键氨基酸，并分析其在PR二聚化中的作用。 目的2：确定两类黄体酮的分子机制 对抗者。三种不同的拮抗剂通过 R5020，以及一个阻断PR与DNA的结合。我们将分析机制 PRA和PRB的同源和异源二聚，DNA结合和 在拮抗剂的影响下激活。我们将分析 孕酮反应元件(Pre)结构、启动子的作用 复杂性和细胞特异性在转录调控中的作用 抗孕激素。我们将解决DUAL中涉及的机制 抗孕激素的激动剂/拮抗剂作用 CAMP在这一功能转换中的作用。目标3：分析乳腺癌 孕激素激动剂和拮抗剂对细胞增殖的影响 并分析一个可能的天然PR突变体。我们将鉴定细胞系 对孕激素激动剂有不同的表型反应 用双参数流式细胞仪(FCM)定量 细胞亚群的增殖效应及异质性分析 因为它们单独受到孕激素的影响，或者当它们与孕激素联合使用时 雌二醇和他莫昔芬。激素对细胞周期参数的影响将 被测量，并将开发改进的FCM方法来定量S- 分期、非增殖性G0群体和孕激素标记物。一个 将产生PR阴性的T47D株，用于转基因和AS 核扩散研究的控制点。因为突变的受体可能是 参与荷尔蒙抵抗的形成，我们将描述一种 推定为天然PR突变体。我们预计这些研究将开始。 明确孕激素在人乳房中作用的分子机制 癌症。

Progesterone has a proliferative effect in the normal breast and in breast cancers, and progesterone antagonists are growth inhibitory. Progestin effects are mediated by progesterone receptors (PR) and in breast cancer cells, there are two naturally occurring isoforms -- the 120 kDa B-receptors (PRB) and the N-terminally truncated 94 kDa A- receptors (PRA). since both homo- and heterodimers can form between them, three classes of receptor dimers (A:A, A:B, B:B) can bind to DNA, each having a potentially different transcriptional effect. To analyze the mechanisms of PR dimerization and the functional heterogeneity imparted by the three dimeric classes, in the context of progesterone agonist and antagonist actions in breast cancer, the following aims are proposed: Aim 1: To characterize progestin agonist action by analyzing PR dimerization, DNA binding, and transcription. We will use DNA binding domain (DBD) mutants of PR, and wild-type PRA and PRB, plus in vitro and in vivo functional assays, to characterize PR dimerization, to analyze the influence of DNA structure on dimerization, and to analyze the functional capacity of PR homo- and heterodimers when occupied by the agonist R5020. We will also construct mutants of a "leucine zipper" module likely to include the dimerization domain of PR, map its boundaries and key amino acids, and analyze its role in PR dimerization. Aim 2: To define the molecular mechanisms of two classes of progesterone antagonists. Three different antagonists inhibit transactivation by R5020, and one blocks binding of PR to DNA. We will analyze mechanisms of PRA and PRB homo- and heterodimerization, DNA binding and transactivation under the influence of the antagonists. We will analyze the role of progesterone response element (PRE) structure, promoter complexity, and cell specificity on transcriptional regulation by antiprogestins. We will address mechanisms involved in the dual agonist/antagonist actions of antiprogestins focusing on the possible role of cAMP in this functional switch. Aim 3: To analyze breast cancer cell proliferation as influenced by progestin agonists and antagonists, and to analyze a possible natural PR mutant. We will identify cell lines having phenotypically different responses to progestin agonists and antagonists, and use dual parameter flow cytometry (FCM) to quantitate proliferative effects and analyze heterogeneity of cell subpopulations as they are influenced by the progestins alone, or when combined with estradiol and tamoxifen. Hormone effects on cell cycle parameters will be measured, and improved FCM methods will be developed to quantitate S- phase, the non-proliferative G0 population, and a progestin marker. A PR-negative T47D line will be generated for use in transfection and as a control for the proliferation studies. Since mutant receptors may be involved in development of hormone resistance, we will characterize a putative natural PR mutant. We anticipate that these studies will begin to define molecular mechanisms of progestin actions in human breast cancers.