2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds exhibit a broad spectrum of antiestrogenic activities in the female rat uterus and in human breast cancer cell lines. For example, in MCF-7 human breast cancer cell lines, TCDD inhibits 17beta-estradiol-induced cell proliferation, postconfluent foci formation, secretion of the 34-, 52- and 160-kDa proteins, progesterone receptor levels and tissue plasminogen activator activity. TCDD causes a decrease in the nuclear estrogen receptor in MCF-7 and in wild-type and mutant Hepa 1c1c7 cell in culture. This proposed study will investigate the molecular mechanisms of TCDD- mediated antiestrogenicity in MCF-7 human breast cancer cells and delineate the nature of the cross-talk between the aryl hydrocarbon (Ah) and estrogen receptor-mediated endocrine systems. The first experiment will investigate the mechanism of TCDD-induced downregulation of the nuclear estrogen receptor (ER) in MCF-7 cells. The concentration- and time-dependent effects of TCDD and related compounds and the effects of TCDD on ER mRNA levels and half-life as well as ER protein stability will be determined; the results will be compared to those obtained for phorbol ester (TPA) and ICI 164,384 (an ER antagonist) which elicit similar responses but through different cellular pathways. Experiments 2 to 4 will investigate the mechanism of action of TCDD as an inhibitor of three estrogen-induced genes, namely the progesterone receptor (PR), the 52-kDa protein (procathepsin D) and a plasmid containing the estrogen-responsive 5'-flanking region of the Vitellogenin A2 gene linked to a bacterial chloramphenicol acetyl transferase (CAT) reporter gene (pA2(-821/+ 14)- CAT). The effects of TCDD on estrogen-induced PR and procathepsin D gene transcription and translation will be characterized. In addition, the possible interactions between the Ah receptor and the mannose-6-phosphate receptor as a pathway for inhibition the E2-induced secretion of the 52- kDa protein will also be investigated. It is hypothesized that the mechanism of TCDD-mediated antiestrogenicity may involve the induction of specific gene(s) and gene product(s) (M) which bind to specific cis- genomic elements in the 5'-flanking regions of estrogen-induced genes and thereby inhibit gene transcription. Plasmid constructs containing 5'- flanking regions from the PR gene (-2762/+ 788)-CAT), the procathepsin D gene [(-296/+ 54)SEAP] and the Vitellogenin A2 gene [pA2(-821/+ 14)-CAT] have been obtained and using molecular biology techniques, the cis-genomic regions of these 5'-flanking regions which are required for TCDD-induced antiestrogenicity will be identified and sequenced. In a parallel study (Experiment 5), the TCDD-induced genes in MCF-7 cells will be cloned, the TCDD inducibility of the specific clones will be determined and the clones which code for the TCDD-induced trans-acting factor(s) (M) will be specifically targeted for further characterization. The results of this study will not only enhance our understanding of the interaction between two or more endocrine systems but also identify induced proteins which may have some clinical prognostic value for breast cancer in women.
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