REGULATION OF BREAST CANCER CELL CYCLE BY ESTRADIOL

雌二醇对乳腺癌细胞周期的调节

• 批准号: 2429869
• 负责人: JAY wimalasena WIMALASENA
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2429869
• 关键词: MCF7 cell; RNase protection assay; breast neoplasms; cell cycle; cell cycle proteins; cyclins; enzyme activity; enzyme biosynthesis; enzyme complex; enzyme inhibitors; estradiol; estrogen inhibitor; estrogen receptors; female; gene expression; genetic promoter element; hormone regulation /control mechanism; immunoprecipitation; mitogen activated protein kinase; northern blottings; phosphorylation; protein kinase; protein metabolism; transcription factor; western blottings

Breast Cancer (breast Ca) is a leading cause of death for women in the U.S. and will affect approximately 105 of the present female population. Many studies have demonstrated that 17beta-estradiol (E2) is a critical factor in the genesis of breast Ca and can promote breast Ca cell growth in vivo as well as in vitro in the presence of growth factors. However, the mechanisms underlying regulation of the breast Ca cell cycle by E2 are poorly, if at all, understood. Our recent data suggests that E2 (in the absence of exogenous growth factors) can directly activate cyclin dependent kinase 2 (Cdk2) activity in MCF-7 cells, leading to hyperphosphorylation of retinoblastoma protein (pRb) and induction of S- phase entry. Furthermore, E2 appears to inactivate a Cdk inhibitor (CKI) present in untreated G1 cells. These regulatory effects of E2 in the nanomolar concentration range are inhibited by an excess of steroidal antiestrogen (ICI l82,780). Thus, we hypothesize that E2 directly regulates one or more of the following critical steps in the MCF-7 cell cycle (a) Expression of D cyclin gene(s) and synthesis of D cyclin(s); (b) Activation of Cdk4 kinase activity; (c) Inhibition of a CKI active in G1 phase; and (d) Activation of Cdk2 kinase activity (perhaps via activation of Cdk activating kinase - CAK) leading to pRb hyperphosphorylation and entry into S-phase. We have proposed 3 specific aims to directly test facets of this hypothesis. They are: (1) Studies to investigate whether E2 regulates D- type cyclin synthesis/degradation, in particular that of cyclin D1, and whether Cdk4 kinase activity and/or steady-state protein levels are regulated by E2. (2) We will investigate the possibility that E2 treatment inactivates a Cdk inhibitor present in quiescent MCF-7 cells resulting in Cdk activation and G1 transit. We propose that E2 will be unable to bring about inactivation of putative inhibitors present in ER negative breast Ca cells or other cells lacking ER. (3) Studies under Specific Aim 3 will explore the possibility that E2, directly or indirectly, regulates cyclin D1 promoter activity. Cyclin D1 promoter reporter gene constructs will be expressed in MCF-7 cells and ER negative breast Ca cells. Specific activation of these promoter constructs in response to E2 will be measured, and specific response elements required for this activation will be determined by deletion and mutation experiments. The results of these studies will, for the first time, directly demonstrate how E2 regulates early events in the cell cycle of E2 responsive breast Ca cells.

乳腺癌（乳腺癌）是女性的主要死亡原因 美国将影响当前的女性大约105个。 许多研究表明，17beta-雌二醇（E2）是关键 乳腺CA的起源的因素，可以促进乳腺CA细胞生长 在存在生长因子的情况下，体内以及体外。 然而， E2对乳腺CA细胞周期调节的基础机制为 理解的很差，如果有的话。 我们最近的数据表明E2（在 缺乏外源生长因子）可以直接激活细胞周期蛋白 MCF-7细胞中依赖性激酶2（CDK2）活性，导致 视网膜细胞瘤蛋白（PRB）的高磷酸化和S-的诱导 阶段输入。 此外，E2似乎使CDK抑制剂（CKI）失活 存在于未处理的G1细胞中。 E2在 纳摩尔浓度范围被过量的类固醇抑制 抗雌激素（ICI L82,780）。 因此，我们假设E2直接 调节MCF-7单元中以下一个或多个关键步骤 循环（a）d cyclin基因的表达和d Cyclin（S）的合成； （b） CDK4激酶活性的激活； （c）抑制活跃在G1中的CKI 阶段; （d）CDK2激酶活性的激活（也许是通过激活 CDK激活激酶-CAK）导致PRB高磷酸化和 进入S期。 我们提出了3个特定目标，以直接测试该方面 假设。 它们是：（1）研究E2是否调节D-的研究 类型的细胞周期蛋白合成/降解，特别是细胞周期蛋白D1的合成，而 CDK4激酶活性和/或稳态蛋白水平是否是 由E2调节。 （2）我们将研究E2治疗的可能性 灭活静态MCF-7细胞中存在的CDK抑制剂，导致 CDK激活和G1传输。 我们建议E2无法带来 关于ER负乳房Ca中存在的推定抑制剂的失活 细胞或其他缺乏ER的细胞。 （3）在特定目标下的研究3将 探索直接或间接调节细胞周期蛋白的E2的可能性 D1启动子活性。 Cyclin D1启动子报告基因构建体将是 在MCF-7细胞和ER负乳腺CA细胞中表达。 具体的 这些启动子构建体对E2的激活将是 测量，此激活所需的特定响应元素将 通过缺失和突变实验确定。 这些研究的结果将首次直接 证明E2如何调节E2细胞周期中的早期事件 反应性的乳房CA细胞。