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）是女性死亡的主要原因 美国目前约有 105 名女性人口受到影响。 许多研究表明，17β-雌二醇 (E2) 是一种关键的 乳腺钙生成的因素，可促进乳腺钙细胞生长 在生长因子存在的情况下体内和体外。 然而， E2 调节乳腺 Ca 细胞周期的机制是 如果有的话，也很难理解。 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 细胞中依赖激酶 2 (Cdk2) 活性，导致 视网膜母细胞瘤蛋白 (pRb) 的过度磷酸化和 S- 的诱导 相进入。 此外，E2 似乎可以灭活 Cdk 抑制剂 (CKI) 存在于未经处理的 G1 细胞中。 E2的这些调节作用 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 细胞中的一个或多个以下关键步骤 周期 (a) 细胞周期蛋白 D 基因的表达和细胞周期蛋白 D 的合成； (二) Cdk4激酶活性的激活； (c) 抑制 G1 期活跃的 CKI 阶段; (d) Cdk2 激酶活性的激活（可能通过激活 Cdk 激活激酶 - CAK）导致 pRb 过度磷酸化和 进入S期。 我们提出了 3 个具体目标来直接测试这一点 假设。 它们是： (1) 研究 E2 是否调节 D- 类型细胞周期蛋白合成/降解，特别是细胞周期蛋白 D1 的合成/降解，以及 Cdk4 激酶活性和/或稳态蛋白水平是否 受E2监管。 (2)我们将调查E2处理的可能性 使静态 MCF-7 细胞中存在的 Cdk 抑制剂失活，从而导致 Cdk 激活和 G1 转运。 我们建议E2将无法带来 关于 ER 阴性乳腺 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) 具体目标 3 下的研究将 探索E2直接或间接调节细胞周期蛋白的可能性 D1 启动子活性。 细胞周期蛋白 D1 启动子报告基因构建体将是 在 MCF-7 细胞和 ER 阴性乳腺 Ca 细胞中表达。 具体的 这些启动子构建体响应 E2 的激活将是 测量，并且此激活所需的特定响应元素将 通过缺失和突变实验来确定。 这些研究的结果将首次直接 展示 E2 如何调节 E2 细胞周期的早期事件 反应性乳腺钙细胞。