DOMINANT NEGATIVE ESTROGEN RECEPTORS AND BREAST CANCER

别名：The PI正在研究一种新的方法 雌激素受体（ER）的功能失活， 依赖性人乳腺癌细胞的基础上使用有效的显性 阴性（DN）ER突变体。私家侦探已经产生了几种有效的DN-ER 并显示它们抑制雌激素刺激的基因表达， 乳腺癌细胞的增殖。 本建议的重点是 在这项工作中取得了两个重要进展：1. 2-一种新的辅阻遏蛋白REA的杂交相互作用克隆 雌激素作用的抑制物。 REA选择性增强效力 的DN-ER，而对野生型ER的影响非常小， 其他核受体。 2)开发一个系统来 产生以高亲和力和选择性结合的靶向DN-ER 特定的激素反应元素。 具体目的是：1）分析其分子机制， 辅阻遏物REA被DN-ER募集，并增强其 活动DN-ER/REA相互作用的物理和功能映射将 使用GST下拉方法进行，哺乳动物2-杂交 反式激活测定和突变分析。 使用抗体 REA和反义方法，细胞内RE将是 中和/消除和DN-ER/REA的功能重要性 将在完整细胞中定义相互作用。私家侦探将确定 可能增强辅阻遏物的其他REA相互作用伴侣 活性，并表征REA对ER细胞的作用 分布 2)为了寻找其他显性负辅阻遏物， 私家侦探将使用双杂交相互作用克隆两个最有效的 DN-ER。 3)为了优化受体-辅阻遏物相互作用，2-杂交 用REA筛选将用于筛选ER突变体文库， 表现出辅阻遏物结合增强的突变体。 4)为了评估角色， c-myc、TGF α和组织蛋白酶D基因在增殖中的作用， ER阳性乳腺癌细胞的侵袭性，修饰的P22 攻击噬菌体系统将用于产生结合 对不同的非共有ERE具有高亲和力 在每一个基因中。 私家侦探会将基因导入- 使用她的高效腺病毒系统将选择性DN-ER导入细胞， 确定它们对基因表达的影响，并检查它们的重要性， ER调节乳腺癌细胞增殖和侵袭。

DESCRIPTION: The P.I. is investigating a novel method for the functional inactivation of estrogen receptors (ERs) in estrogen- dependent human breast cancer cells based on the use of potent dominant negative (DN) ER mutants. The P.I. has generated several potent DN-ERs and shown that they inhibit estrogen-stimulated gene expression and proliferation of breast cancer cells. The present proposal focuses on two critical advances made during this work: 1 the identification by 2-hybrid interaction cloning of a novel corepressor protein, denoted REA for repressor of estrogen action. REA selectively enhance the potency of DN-ERs, while having very little effect on wild type ER and no effect on other nuclear receptors. 2) The development of a system for generating targeted DN-ERs which bind with high affinity and selectivity to specific hormone response elements. The Specific Aims are: 1) To analyze the molecular mechanisms by which the corepressor REA is recruited by DN-ERs and potentiates their activity. Physica and functional mapping of DN-ER/REA interaction will be carried out using GST pull-down methods, mammalian 2-hybrid transactivation assays and mutational analyses. Using antibodies to REA, and antisense methodology, intracellular RE will be neutralized/eliminated and the functional importance of the DN-ER/REA interaction will be defined in intact cells. The P.I. will identify additional REA interaction partners which may potentiate corepressor activity, and characterize the effect of REA on ER cellular distribution. 2) To search for other dominant negative corepressors, the P.I. will use 2-hybrid interaction cloning with the two most potent DN-ERs. 3) To optimize receptor-corepressor interaction, 2-hybrid screening with REA will be used to screen ER mutant libraries for mutants exhibiting enhanced corepressor binding. 4) To assess the roles of the c-myc, TGFa and cathepsin D genes in the proliferation and invasiveness of ER positive breast cancer cells, the modified P22 challenge phage system will be used to create DN-ERs that bind selectively and with high affinity to the different non-consensus EREs found in each of these genes. The P.I. will introduce the gene- selective DN-ERs into cells using her efficient adenovirus system, determine their effects on gene expression, and examine their importance in ER regulated breast cancer cell proliferation and invasiveness.