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 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 approximately 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.

• Authors: G E Swaneck, J Fishman
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 85 Issue 21 Pg. 7831-5 (Nov 1988) ISSN: 0027-8424 [Print] United States
• PMID: 3186693 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Hydroxyestrones
• Receptors, Estradiol
• 16-hydroxyestrone
• Estrone
• Estradiol
• Steroid 16-alpha-Hydroxylase

Topics

• Antibodies, Monoclonal
• Breast Neoplasms (metabolism)
• Cell Division
• Cell Line
• Cell Nucleus (metabolism)
• Electrophoresis, Polyacrylamide Gel
• Estradiol (metabolism)
• Estrone (analogs & derivatives)
• Humans
• Hydroxyestrones (metabolism)
• Receptors, Estradiol (metabolism)
• Steroid 16-alpha-Hydroxylase