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

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Abstract
The interactions of 16 .alpha.-hydroxyestrone (16.alpha.-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 16.alpha.-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 we show that the interactions of 16.alpha.-OHE1 with the ER are different from those of E2 with the receptor. Dissociation of tritiated E2-ER or 16.alpha.-OHE1-ER complexes, salt extraction, DNase and proteinase K digestion, and ethanol treatment demonstrated that the binding of 16.alpha.-OHE1 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]16.alpha.-OHE1-ER complexes analyzed by polyacrylamide gel electrophoresis demonstrated a radiolabeled band at .simeq.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 [3H]E2 incubations. The present results when considered together with our previous findings of elevated activities of estrogen 16.alpha.-hydroxylase, the enzyme responsible for the formation of 16.alpha.-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.