COVALENT INTERACTIONS OF ESTROGENS IN TARGET TISSUES

本申请的长期目标是确定 两种雌二醇（E2）生理代谢产物，2-羟基雌酮（2- OHEl）和16 α-羟基雌酮（16 α-OHEl）能够形成 与细胞蛋白和雌激素受体（ER）的共价键 与肿瘤发生有关。 在大鼠体内子宫中，大鼠子宫内膜原代培养物和 在培养的人乳腺癌细胞中，我们打算检测2- 0 HE 1 1）共价加合物形成的特定位点; 2)经典（非共价）和共价衍生生物效应 在靶组织表型中; 3）控制基因表达，鉴定 编码细胞内和分泌蛋白的mRNA， 研究E2代谢物可能影响 细胞癌基因的激活。 我们将使用细胞和亚核分离的方法， 在大鼠子宫和人子宫中定位类固醇-蛋白质复合物 培养的乳腺癌细胞MCF-7，在短时间（1-6小时）和 长期暴露（4-6周）于放射性惰性和放射性标记 雌激素 原代培养大鼠子宫内膜细胞， 检查是否存在E2及其代谢物。 高效液相色谱法，聚丙烯酰胺凝胶 在变性和非变性条件下的电泳将 用于表征雌激素-核蛋白复合物。 ER的单克隆抗体和16 α- 0 HE 1-加合物将用于检查核内结合 位点，在蛋白质印迹上和类固醇的肽消化后， 蛋白质复合物 几种c-DNA，用于人ER、EGF和EGF样蛋白，人 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的探针将被用于 在与分离自靶的poly（A+）RNA的杂交实验中， 组织和定量的转录率， MCF-7细胞和大鼠子宫的分离细胞核中的特异性基因。 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 子宫内膜通过放射性标记的氨基酸脉冲， 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.