DOMINANT NEGATIVE ESTROGEN RECEPTORS AND BREAST CANCER

显性负雌激素受体与乳腺癌

• 批准号: 3204030
• 负责人: BENITA S KATZENELLENBOGEN
• 金额: $ 17.61万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3204030
• 关键词: DNA binding protein; MCF7 cell; athymic mouse; cell growth regulation; dimer; estrogen receptors; estrogens; gel mobility shift assay; gene expression; genetic regulatory element; genetic transcription; growth inhibitors; hormone regulation /control mechanism; hormone related neoplasm /cancer; molecular oncology; mutant; neoplasm /cancer genetics; neoplastic growth; protein engineering; protein structure function; receptor binding; site directed mutagenesis; transcription factor; transfection; western blottings

We will develop and investigate a novel method for functional inactivation of estrogen receptor (ER) in estrogen-dependent human breast cancer cells. Our approach is based on dominant negative mutants. These are ER mutants which are inactive on their own, but are able to suppress the activity of wild-type ER when they are co-expressed in the same cells. Our Specific Aims are: (1) To generate more powerful dominant negative mutants of ER; (2) To determine the molecular mechanisms responsible for the effectiveness of the mutants; (3) To use the dominant negative mutants to block estrogen-dependent growth of breast cancer cells in vitro and in vivo in nude mice. In preliminary studies we generated three effective and ER-specific dominant negative mutants. These mutants appear to function by inactivation of the C-terminal transactivation domain of ER. We will generate even more potent mutants by replacing both the C- and N-terminal transactivation domains. We will use the P22 challenge phage system to generate ER mutants with enhanced binding to the estrogen response element. These mutations, which lead to increased ability to compete for binding to the estrogen response element, will be introduced into dominant negative mutants with inactive transactivation domains to produce extremely potent dominant negative mutants. We will determine the relative contributions of competition for binding to the estrogen response element, formation of heterodimers, and interference with ER-specific components of the transcription apparatus to the activity of our most effective dominant negative mutants. The ability of our most powerful dominant negative mutants to block estrogen-dependent growth of MCF-7 human breast cancer cells will be evaluated. Stably transfected cells producing the dominant negative mutant ERs will be tested for suppression of estrogen-stimulated growth in and for suppression of the expression of some growth factors and proteins thought to be important in ER-stimulated growth and tumorigenicity. We will use stably transfected MCF-7 cells expressing a potent dominant negative mutant from a regulated promoter to initiate estrogen-dependent growth of MCF-7 tumors in athymic nude mice. Then we will activate production of the dominant negative mutant, and determine if growth of the tumors is blocked or reversed. These studies should provide new insights into the molecular mechanism of ER action and the proliferation and tumorigenicity of breast cancer cells, and allow evaluation of a novel approach for the suppression of estrogen-dependent breast cancer growth.

我们将开发和研究一种新的方法， 雌激素依赖性乳腺癌雌激素受体失活 癌细胞 我们的方法是基于显性负突变体。 这些 是ER突变体，其本身无活性，但能够抑制 当它们在相同的细胞中共表达时， 细胞 我们的具体目标是：（1）产生更强大的主导力量 雌激素受体阴性突变体;（2）探讨雌激素受体的分子机制 （3）使用显性基因， 阻断乳腺癌雌激素依赖性生长的阴性突变体 细胞在体外和裸鼠体内。 在初步研究中，我们产生了三种有效的ER特异性 显性负突变体 这些突变体似乎通过 ER的C-末端反式激活结构域的失活。 我们将 通过替换C-和N-末端产生更有效的突变体 反式激活结构域 我们将使用P22挑战噬菌体系统， 产生与雌激素反应结合增强的ER突变体 元素 这些突变，导致竞争能力的增加， 与雌激素反应元件结合，将被引入到 具有失活反式激活结构域的显性负突变体， 产生非常强大的显性负突变体。 我们将确定 竞争结合雌激素的相对贡献 反应元件、异二聚体的形成和干扰 转录器的ER特异性组分对 我们最有效的显性负突变体 我们最大的能力 强大的显性负突变体，以阻止雌激素依赖的生长， 将评价MCF-7人乳腺癌细胞。 稳定转染 将检测产生显性阴性突变ER的细胞， 抑制雌激素刺激的生长和用于抑制 一些生长因子和蛋白质的表达被认为是重要的 ER刺激的生长和致瘤性。 我们将稳定地使用 转染的MCF-7细胞表达一种有效的显性负突变体， 启动MCF-7雌激素依赖性生长的受调控启动子 无胸腺裸鼠中的肿瘤。 然后我们将启动生产 显性阴性突变体，并确定肿瘤的生长是否 阻挡或逆转。 这些研究应该为分子机制提供新的见解 雌激素受体作用与乳腺癌的增殖和致瘤性 细胞，并允许评价一种新的方法，用于抑制 雌激素依赖性乳腺癌的生长。