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.
