Identification and Androgen Regulation of Two Proteins Released by T47D Human Breast Cancer Cells

Three [35S]methionine-labeled polypeptides released by T47D human breast cancer cells have been identified as corresponding to two proteins previously described in breast gross cystic disease fluid. A Mr 43,000 protein was immunoprecipitated by polyclonal antibodies to the Zn-alpha 2-glycoprotein. A Mr 18,000 and a Mr 13,000 polypeptide were both immunoprecipitated by four monoclonal antibodies directed against four separate epitopes of Mr 15,000 gross cystic disease fluid protein, a major protein that is characteristic of apocrine gland secretions. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified Mr 15,000 gross cystic disease fluid protein migrated at the level of the Mr 18,000 protein of T47D cells. The proteins were regulated by androgens and progestins. In addition to a general stimulation of protein secretion, 5 alpha-dihydrotestosterone (DHT) specifically increased, by 2- to 20-fold, the release of the Mr 43,000 and Mr 18,000 proteins into the medium. DHT also increased the cellular level of the Mr 18,000 protein, as shown by immunoprecipitation with a Mr 15,000 gross cystic disease fluid protein antibody, which suggests a stimulation of protein synthesis. The progestin 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione also increased the release in the medium of the Mr 43,000 and Mr 18,000 proteins, but higher molar concentrations were required than in the case of DHT. The induction of these proteins by DHT and 17,21-dimethyl-19nor,4,9-pregnadiene-3,20-dione was specifically inhibited by the antiandrogen flutamide which has no effect on other progestin-regulated proteins. This suggests an effect mediated by the androgen receptor. This is the first report on the identification of two proteins induced by androgens in a human breast cancer cell line. These proteins should be useful in studying the role of androgens in human mammary tumors and their mechanism of action in cell culture.