COVALENT INTERACTIONS OF ESTROGENS IN TARGET TISSUES

目标组织中雌激素的共价相互作用

• 批准号: 3317426
• 负责人: JACK FISHMAN
• 金额: $ 20.61万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3317426
• 关键词: estradiol; estrogens; estrone; high performance liquid chromatography; hormone metabolism; hormone receptor; hormone regulation /control mechanism; laboratory rat; progesterone; radioimmunoassay

The long-term objective of this application is to determine whether two estradiol (E2) physiological metabolites, 2-hydroxyestrone (2- 0HEl) and 16 alpha-hydroxyestrone (16 alpha-0HEl) able to form covalent bonds with cellular proteins and estrogen receptors (ER) are involved in tumorigenesis. In rat uterus in vivo, rat endometrium primary cultures and in human breast cancer cells in culture we intend to examine 2-0HEl and 16 alpha-0HEl: 1) Specific locus of covalent adduct formation; 2) Classical (non-covalent) and covalent-derived biological effects in target tissue phenotype; 3) Control of gene expression, identify mRNAs that codify for intracellular and secreted proteins and investigate the possibility that E2 metabolites might influence the activation of cellular oncogenes. We will use methods of cellular and subnuclear fractionation to localize steroid-protein complexes in rat uterus and in human breast cancer cells MCF-7 in culture, following short (1-6 hrs) and long-term exposure (4-6 wks) to radioinert and radiolabeled estrogens. Primary cultures of rat endometrium cells grown in the presence and absence of E2 and its metabolites will be examined. High performance liquid chromatography, polyacrylamide gel electrophoresis under denaturing and non-denaturing conditions will be used to characterize estrogen-nuclear protein complexes. Monoclonal antibodies to ER and polyclonal antibodies to 16 alpha- 0HEl-adduct complexes will be used to examine intranuclear binding sites, on Western blots and following peptide digestion of steroid- protein complexes. Several c-DNAs, for human ER, EGF and EGF-like proteins, human glucocorticoid and progesterone receptor, to measure expression of these natural genes and probes for cellular oncogenes will be used in hybridization experiments with poly(A+)RNA isolated from target tissues and by quantitation of the rate of transcription of specific genes in isolated nuclei from MCF-7 cells and rat uterus. Protein synthesis and putative activation of cellular oncogenes will be monitored in MCF-7 cells and primary cultures of rat endometrium by pulses of radiolabeled amino acids, immunoprecipitation and immunoblotting.

该应用程序的长期目标是确定是否 两种雌二醇（E2）生理代谢物，2-羟基雌酮（2- 0HEl)和16α-羟基雌酮(16α-0HEl)能够形成 与细胞蛋白和雌激素受体 (ER) 形成共价键 参与肿瘤发生。 在大鼠子宫体内、大鼠子宫内膜原代培养物和 我们打算检查培养物中的人类乳腺癌细胞 2-0HEl 和16α-0HE1：1)共价加合物形成的特定位点； 2) 经典（非共价）和共价衍生的生物效应 靶组织表型； 3) 基因表达的控制、鉴定 编码细胞内和分泌蛋白的 mRNA 研究 E2 代谢物可能影响的可能性 细胞癌基因的激活。 我们将使用细胞和亚核分级分离的方法 在大鼠子宫和人类中定位类固醇蛋白复合物 培养中的乳腺癌细胞 MCF-7，经过短暂（1-6 小时）和 长期暴露于放射性惰性和放射性标记的环境（4-6周） 雌激素。 大鼠子宫内膜细胞的原代培养 将检查 E2 及其代谢物是否存在。 高效液相色谱法、聚丙烯酰胺凝胶 变性和非变性条件下的电泳将 可用于表征雌激素-核蛋白复合物。 ER 单克隆抗体和 16 α- 多克隆抗体 0HEl-加合物复合物将用于检查核内结合 位点，在蛋白质印迹和类固醇肽消化后 蛋白质复合物。 多种 c-DNA，用于人类 ER、EGF 和 EGF 样蛋白、人类 糖皮质激素和孕激素受体，以测量 这些天然基因和细胞癌基因探针将被使用 在与从靶标分离的聚 (A+)RNA 的杂交实验中 组织并通过转录率定量 MCF-7 细胞和大鼠子宫分离细胞核中的特定基因。 蛋白质合成和细胞癌基因的假定激活 将在 MCF-7 细胞和大鼠原代培养物中进行监测 通过放射性标记的氨基酸脉冲来子宫内膜， 免疫沉淀和免疫印迹。