MOLECULAR BASIS OF ESTROGEN ACTION IN OVARIAN CANCER

目标是定义与雌激素(E)相对应的分子事件 卵巢癌的反应性生长和侵袭性。而雌激素 受体(ER)在大多数卵巢癌中表达，分子 E和抗雌激素的作用还没有被研究过。建议数 研究可能有助于制定治疗策略和 诊断标记物。 为了确定E和抗雌激素的作用，我们将使用卵巢模型 在生长和生长过程中表现出不同ER功能的癌细胞株 转录调控和肿瘤组织中孕酮所在位置 受体(PR)的表达似乎与ER的表达是解偶联的。这个 具体目标是：(1)使用模型细胞系来确定效果 用Matrigel侵袭试验检测E和抗雌激素对侵袭力的影响 并分泌蛋白酶、组织蛋白酶D和120 kDa的标志物。 Northern印迹、免疫沉淀和Western印迹。(2) 鉴定卵巢癌细胞和肿瘤中的ER变异体和突变 显示内质网功能改变的组织。我们将使用聚合酶链式反应 聚合酶链式反应(PCR)和寡核苷酸引物扩增出的序列 编码已知的ER功能结构域。直接测序，化学 错配切割与单链构象多态 对聚合酶链式反应产物的分析将用于检测内质网变异和 突变。(3)确定ER磷酸化模式是否发生改变 在对雌激素反应不同的卵巢癌细胞中 免疫沉淀和免疫印迹32P标记的ER。

The goal is to define molecular events that correspond to estrogen (E) responsive growth and invasiveness in ovarian cancer. While estrogen receptor (ER) is expressed in most ovarian carcinomas, the molecular effects of E and antiestrogens have not been investigated. The proposed studies may facilitate the development of treatment strategies and diagnostic markers. To define the effects of E and antiestrogens, we will use model ovarian carcinoma cell lines that exhibit varied ER function in growth and in transcriptional regulation and in tumor tissue where progesterone receptor (PR) expression appears to be uncoupled from ER expression. The specific aims are: (1) Use the model cell lines to determine the effects of E and antiestrogens on invasiveness by using a Matrigel invasion assay and on secretion of the protease, Cathepsin D and the 120kDa marker by Northern blotting, immunoprecipitations and Western blotting. (2) Identify ER variants and mutations in ovarian carcinoma cells and tumor tissue that exhibit altered ER function. We will use polymerase chain reaction, (PCR) and oligonucleotide primers to amplify sequences that encode known functional domains of ER. Direct sequencing, chemical mismatch cleavage and single stranded conformational polymorphism analysis of PCR products will be used to detect ER variants and mutations. (3) Determine whether ER phosphorylation patterns are altered in ovarian carcinoma cells that differ in their response to estrogen by immunoprecipitations and Western blotting 32P-labelled ER.