Covalent binding of the endogenous estrogen 16 alpha-hydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization.

Abstract

The interactions of 16a-hydroxyestrone (16a-OHE1), a metabolite of estradiol (E2), with estrogen receptors (ERs) were compared in this study to the classic E2-receptor mechanism in human breast cancer cells MCF-7 in culture. When MCF-7 cells were incubated with radioinert 16a-OHE1 or its 3H-labeled form for 4 weeks, the estrogen bound extensively and irreversibly in a time-dependent fashion to nuclear protein species that correspond to the ER. Here weshow that the interactions of 16a-OHE, with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16ci-OHE,-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16a-OHE, to the ER corresponds to two different forms: a classical noncovalent interaction similar to that of E2, and a covalent adduct formation between the metabolite and the ER. These complexes localized preferentially in nuclear matrix components as revealed by cell fractionation and probing with a monoclonal anti-ER antibody.[3H]16a-OHEj-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at %66 kDa that was absent when the exposure of cells was done in the presence of E2 in competition and that was also absent in PH]E2incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16a-hydroxylase, the enzyme responsible for the formation of 16a-OHE1, in breast cancer patients and in women at enhanced risk for the disease, suggest that covalent modification of the ER may be one mechanism of malignant transformation in estrogen target tissues.The pathway of receptor-mediated estrogen actions in target tissues has been studied extensively (1)(2)(3)(4)(5). Evidence that target tissues for estradiol (E2) respond variably to its metabolites raises questions whether the estrogen receptor (ER) bound to these metabolites might exert unusual functions at the nuclear level. We have previously shown that the natural estrogen metabolite 16a-hydroxyestrone (16a-OHE1), whose role in biological processes has not been established, binds covalently to primary amino groups of proteins by a nonenzymatic process. In vitro and in vivo covalent interactions of 16a-OHE1 with proteins occur between the steroid carbonyl and the E-amino group of lysine residues in many human tissues (6-9). Our group has been studying the role of steroid metabolites and their covalent interactions in the induction of human breast cancer, since we have found that the activity of estrogen 16a-hydroxylase, the enzyme involved in the formation of 16a-OHE,, is increased in a population of women with breast cancer and in women at risk for the disease (10, 11).The objective of the present study has been to test directly the hypothesis that 16a-OHE, binds covalently to nuclear regulatory proteins, specifically the ER, in estrogen target cells. We now report that covalent binding of 16a-OHE1 to specific nuclear proteins is extensive and that ERs at ...

Estrone/17β‐estradiol conversion to, and tumor necrosis factor inhibition by, estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

“…The 16␣-hydroxylated estrogens covalently bind to estrogen receptors and thereby induce growth of breast cancer cells in vitro (27).…”

Inhibition of in vitro angiogenesis by 2‐methoxy‐ and 2‐ethyl‐estrogen sulfamates

“…2-Hydroxyestrogens and their 2-methoxy derivatives are non-estrogenic whereas 16␣-hydroxy metabolites are estrogenic due, in part, to their ability to form adducts with proteins. 1,2 Research carried out by Bradlow and others 3,4 has indicated that although the 2-hydroxylase pathway is associated with a reduced risk of breast cancer, increased formation of 16␣-hydroxy metabolites may increase breast cancer risk. It was suggested recently that the major derivative of 2-hydroxyestrogens, 2-methoxyestradiol (2-MeOE2, Fig.…”

Az ösztrogénmetabolom biológiai és klinikai jelentősége lokális folyamatokban

“…A 16αOH-E1 az ösztrogénreceptor-fehérjével kovalens és másodlagos kötést egyaránt teremt. A komplex főként a nukleáris mátrixra lokalizálódik, és ott részt vesz az ösztrogénre-ceptor mediálta malignus folyamatokban [52]. Pellino és mtsai kapcsolatot találtak az autoimmun kórképek és a malignus folyamatok kialakulása között: Crohn-betegségben szenvedő nőket és nőnemű rokonaikat vizsgálva pozitív korrelációt találtak az emlőkarcinóma kialakulá-sának esélyével [53].…”