COVALENT INTERACTIONS OF ESTROGENS IN TARGET TISSUES

此应用程序的长期目标是确定是否 两种雌二醇(E_2)生理代谢物--2-羟基雌酮(2-羟基雌酮) 0HEL)和16α-羟雌酮(16α-0HEL)能够形成 与细胞蛋白和雌激素受体(ER)的共价键 都与肿瘤的发生有关。 在活体大鼠子宫、大鼠子宫内膜原代培养和 培养的人乳腺癌细胞我们打算检测2-0HEL 和16个α-0HEL：1)共价加合物形成的特异位置； 2)经典(非共价)和共价衍生的生物效应 在靶组织表型；3)基因表达调控，鉴定 编码细胞内和分泌的蛋白质和 研究E2代谢物可能会影响 细胞癌基因的激活。 我们将使用细胞和亚核分馏的方法 类固醇蛋白复合体在大鼠子宫和人子宫中的定位 乳腺癌细胞MCF-7在培养过程中短期(1-6小时)和 长期接触放射性惰性物质和放射性标记物质(4-6周) 雌激素。大鼠子宫内膜细胞的原代培养 将检查E2及其代谢物的存在和不存在。 高效液相色谱，聚丙烯酰胺凝胶 变性和非变性条件下的电泳会 用于表征雌激素-核蛋白复合体。 抗ER的单抗和抗16α的多克隆抗体 0HEL-加合物将用于检查核内结合 在蛋白质印迹和类固醇多肽消化后的位置- 蛋白质复合体。 人类ER、EGF和EGF样蛋白的几个c-DNA，人类 糖皮质激素和孕激素受体的表达 这些天然基因和细胞癌基因的探针将被使用 在与从靶标分离的聚(A+)RNA的杂交实验中 组织和通过对转录速率的量化 从MCF-7细胞和大鼠子宫中分离的细胞核中的特异性基因。 蛋白质合成与细胞癌基因的可能激活 将在MCF-7细胞和大鼠原代培养中进行监测 子宫内膜通过放射性标记氨基酸的脉冲， 免疫沉淀和免疫印迹。

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.