DOMINANT NEGATIVE ESTROGEN RECEPTORS AND BREAST CANCER

描述：私家侦探正在调查一种新的方法 雌激素中雌激素受体(ER)的功能失活 基于强势优势利用的依赖性人乳腺癌细胞 阴性(DN)ER突变体。私家侦探已经产生了几个有效的DN-ER 结果表明，它们抑制雌激素刺激的基因表达，并 乳腺癌细胞的增殖。本提案的重点是 在这项工作中取得的两个关键进展：1 一种新的辅阻遏子蛋白REA的2-杂交相互作用克隆 用来抑制雌激素的作用。REA有选择地增强效力 而对野生型ER的影响很小，没有影响 在其他核受体上。2)系统的开发 产生具有高亲和力和选择性结合的靶向dN-ER 对特定的荷尔蒙反应元件。 其具体目的是：1)分析分子机制 辅阻遏子REA被dN-ER招募并增强其 活动。Dn-ER/REA相互作用的Physica和功能图谱 使用GST下拉方法进行，哺乳动物2-杂交 反式激活分析和突变分析。使用抗体来 ReA，和反义方法学，细胞内RE将是 中和/取消和DN-ER/REA的功能重要性 相互作用将在完好的细胞中定义。私家侦探会确认 可能增强辅阻遏子的额外REA相互作用伙伴 活性，并表征REA对ER细胞的影响 分发。2)寻找其他显性负抑制因子， P.I.将使用双杂交相互作用克隆，其中最有效的两种 DN-ERS。3)优化受体-辅阻遏子相互作用，2-杂交 REA筛选将用于筛选ER突变体库 突变体表现出增强的辅阻遏子结合。4)评估角色 C-myc、TGFa和组织蛋白酶D基因在肿瘤细胞增殖和转移中的作用 修饰的P22基因对ER阳性乳腺癌细胞侵袭力的影响 挑战噬菌体系统将用于创建绑定的DN-ER 对不同的非共识ERE具有选择性和高亲和力 在这些基因中的每一个都有发现。私家侦探会引入这种基因- 利用她高效的腺病毒系统选择性地将dN-ERs送入细胞， 确定它们对基因表达的影响，并检查它们的重要性 ER调控乳腺癌细胞的增殖和侵袭力。

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.