DOMINANT NEGATIVE ESTROGEN RECEPTORS AND BREAST CANCER

我们将开发和研究一种新的泛函方法 雌激素依赖人乳房雌激素受体失活的研究 癌细胞。我们的方法是基于显性负突变。这些 ER突变体本身是不活跃的，但能够抑制 野生型内质网共表达时的活性 细胞。我们的具体目标是：(1)产生更强大的支配力 内质网负性突变体；(2)确定分子机制 负责突变体的有效性；(3)使用显性 负性突变体阻断乳腺癌雌激素依赖性生长 裸鼠体内和体外细胞。 在初步研究中，我们产生了三个有效的和特定于ER的 显性负性突变体。这些突变体似乎通过 ER的C末端反式激活结构域失活。我们会 通过替换C-端和N-端产生更强大的突变体 转录激活域。我们将使用P22挑战噬菌体系统来 产生与雌激素反应结合增强的ER突变体 元素。这些突变导致了竞争能力的增强 结合雌激素反应元件，将被引入 具有非活性反式激活结构域的显性负性突变体 产生极强的显性负突变。我们将决定 结合竞争对雌激素的相对贡献 响应元件、杂二聚体的形成和干扰 转录装置的内质网特异性成分对酶活性的影响 我们最有效的显性负性突变体。我们大多数人的能力 强大的显性负性突变体阻断雌激素依赖的卵巢癌生长 将对MCF-7人乳腺癌细胞进行评估。稳定转染体 产生显性负突变ER的细胞将被检测 抑制雌激素刺激的生长，并用于抑制 一些被认为重要的生长因子和蛋白质的表达 在雌激素受体刺激的生长和致瘤性方面。我们将稳定使用 表达强效显性负性突变体的MCF-7细胞 启动MCF-7细胞雌激素依赖性生长的调控启动子 裸鼠体内的肿瘤。然后我们将激活生产 显性负性突变体，并确定肿瘤的生长是否 受阻或倒转。 这些研究应该为分子机制提供新的见解。 雌激素受体的作用与乳腺癌的增殖和致瘤性 细胞，并允许评估一种新的方法来抑制 雌激素依赖性乳腺癌的生长。

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.