[CANCER RESEARCH 47, 2787-2792, June 1, 1987] Identification and Androgen Regulation of Two Proteins Released by T47D Human Breast Cancer Cells1 D. Chalbos, D. Haagensen, T. Parish, and H. Rochefort2 1NSERM U148, Unitéd'Endocrinologie Cellulaire et Moléculaire,34100 Montpellier, France [D. C., H. R.]; Department of Surgery, The Jewish Hospital of St. Louis, Washington University Medical Center, St. Louis, Missouri 63110 [D. H.J; and Cambridge Research Laboratory, Cambridge, Massachusetts 02139 [T. P.] ABSTRACT scribed several glycoproteins isolated from breast gross cystic Three [35S|methionine-labeled polypeptides released by T47D human disease fluids, three of which have been identified and charac terized. They include a progesterone-binding protein (M, breast cancer cells have been identified as corresponding to two proteins 24,000), the Zn-a2-glycoprotein (M, 44,000), and a glycoprotein previously described in breast gross cystic disease fluid. A M, 43,000 protein was immunoprecipitated by polyclonal antibodies to the 7,n-«2- with a molecular weight of approximately 15,000, termed GCDFP-153 (8). These proteins have been purified to homo glycoprotein. A M, 18,000 and a M, 13,000 polypeptide were both immunoprecipitated by four monoclonal antibodies directed against four geneity, and antibodies (8-10) and cDNA (11, 12) probes for separate epitopes of M, 15,000 gross cystic disease fluid protein, a major GCDFP-15 have been prepared. protein that is characteristic of apocrine gland secretions. In sodium Specific immunoprecipitation was used here to identify two dodecyl sulfate-polyacrylamide gel electrophoresis, purified M, 15,000 proteins secreted by T47D cells as the Zn-a2-glycoprotein and gross cystic disease fluid protein migrated at the level of the M, 18,000 the GCDFP-15 protein of breast gross cystic disease fluid. The protein of T47Dcells. androgen-induced increase in the accumulation of GCDFP-15 The proteins were regulated by androgens and progestins. In addition to a general stimulation of protein secretion, 5a-dihydrotestosterone was confirmed by immunoprecipitation of cellular proteins. (DHT) specifically increased, by 2- to 20-fold, the release of the M, The regulation of these two proteins by progestin and androgen is probably mediated by the androgen receptor, based on dose- 43,000 and M, 18,000 proteins into the medium. DHT also increased the cellular level of the M, 18,000 protein, as shown by immunoprecipitation response experiments and inhibition by flutamide. with a M, 15,000 gross cystic disease fluid protein antibody, which suggests a stimulation of protein synthesis. The progestin 17,21-di- methyl-19-nor-4,9-pregnadiene-3,20-dione also increased the release in MATERIALS AND METHODS the medium of the M, 43,000 and M, 18,000 proteins, but higher molar Cell Culture. Clone 11 (passage 57) is a subline of the T47D cell line concentrations were required than in the case of DHT. The induction of (13), which was obtained by the agar method (14) in the laboratory of these proteins by DHT and 17,21-dimethyl-19nor,4,9-pregnadiene-3,20- Dr. I. Keydar (15). Single cells were grown in monolayers and recloned dione was specifically inhibited by the antiandrogen flutamide which has by the same method. Several single isogenic cell sublines were obtained, no effect on other progestin-regulated proteins. This suggests an effect including clone 11, which retains the marker chromosome of the mediated by the androgen receptor. parental line and contains high levels of progesterone receptors, as well This is the first report on the identification of two proteins induced by as estrogen, androgen, and glucocorticoid receptors (16).4 T47D clone androgens in a human breast cancer cell line. These proteins should be 11 is routinely cultured in RPMI 1640 supplemented with 10% fetal useful in studying the role of androgens in human mammary tumors and calf serum and 0.6 ¿tg/rnlbovine insulin (Sigma Chemical Co., St. their mechanism of action in cell culture. Louis, MO). The protocol used to test the effects of steroids on protein secretion INTRODUCTION has been described previously (2, 4). Briefly, cells were cultured with 10% (3 days) and then 3% (2 days) FCS-DCC to remove steroids. They Human mammary cancer cell lines have been found to con were then plated out in 0.8-cm-diameter microwells (15,000 cells/well) tain sex steroid receptors and to respond to sex steroid hor in medium plus 3% FCS-DCC. Two days later, steroids were added in mones with respect to specific protein synthesis and the control ethanol solution (final ethanol concentration, 0.2%) and solvent was of cell proliferation. Proteins induced by steroids in these in added to control cells. Flutamide was kindly donated by Schering vitro systems provide new potential markers of hormone re Laboratories (Bloomfield, NJ) and hormones were given by Roussel Uclaf (Romainville, France). The compounds were 99% pure as shown sponsiveness. These systems can also be used to study the by thin layer chromatography. After hormonal stimulation, cells were mechanisms by which steroid hormones modulate gene expres labeled for 6 h with 200 /iCi/ml [35S]methionine or [35S]cysteine (Ra- sion and cancer cell proliferation. In MCF7 cells, several estro diochemical Center, Amcrsham. England; specific activity, 600 Ci/ gen-regulated proteins have been reported (1,2). More recently, mmol) in 50 u\ of medium containing one-tenth the normal concentra progestin-regulated proteins have been found within cells (T47D tion of methionine or cysteine, respectively. For analysis of cellular or MCF7 cells) (3) or secreted by cells (T47D cells) (4). These proteins, cells were labeled for 4 h with [35S]methionine and washed proteins are defined according to their molecular weight under twice with phosphate-buffered saline. They were then dissolved in 50 ul of lysis buffer A, described by O'Farrell (17), containing 0.5% (w/v) denaturing conditions. sodium dodecyl sulfate. Cell lysis was completed by freezing and Among the progestin-regulated proteins secreted by T47D thawing 3 times (-20°C). cells, the M, 43,000, 22,000, and 18,000 proteins were also Immunoprecipitations. Radioactivity incorporated into proteins re found to be regulated by androgens (4, 5) and appear to be leased into the medium was determined by TCA precipitation of ali- related to the synandrogenic effect of progestins (6). quots of the media (5 ul); 60,000 cpm of TCA-precipitable material or Working separately, Haagensen and Mazoujian (7) have de- 'The common names and abbreviations used are: GCDFP-15, gross cystic Received1/3/86; revised10/20/86, 3/5/87; accepted3/10/87. disease fluid protein (M, 15,000); R5020 or promegestone, 17,21-dimethyl-19- The costs of publicationof this article were defrayedin part by the payment nor-4,9-pregnadiene-3,20-dione; DHT, Sa-dihydrotestosterone, 17j3-hydroxy-5o- of page charges. This article must therefore be hereby marked advertisementin androstane-3-one; RU486, 17/3-hydroxy-11¡3-(4-dimethylaminophenyl-1)-17a- accordancewith 18U.S.C. Section 1734solelyto indicatethis fact. (prop-l-ynil)estra-4,9-dien-3-one; flutamide, a,a,a-trifluoro-2-methyl-4'-nitro-m- 1This work was funded by the Institut National de la Santéetde la Recherche propionotoluidide; FCS-DCC, fetal calf serum treated with dextran-coated char Médicale,theUniversity of Montpellier I, and the Association pour la Recherche coal; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; cDNA, comple sur le Cancer. mentary DNA. 2To whom requests for reprints should be addressed. 4 D. Chalbos and F. Vignon, unpublished experiments. 2787 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1987 American Association for Cancer Research. ANDROGEN-INDUCED PROTEINS IN BREAST CANCER 60 ni of culture medium, as specified in the figure legends, were methionine-labeled proteins secreted by T47D cells (clone 11) incubated with antibodies for l h at 20"< in 100 HIMTris (pH 8), 100 has been reported previously (4). Cells treated for 5 days with IHMNaCl, 0.5% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, DHT secreted more proteins than control cells, as seen by and 0.5% (w/v) bovine serum albumin (final volume, 100 (¿1).The analyzing the same volume of medium on SDS-polyacrylamide antibodies were 10 n\ of rabbit antiserum to Zn-«2-glycoprotein or nonimmune rabbit serum; 2 ¿ilofmouse monoclonal antibodies A5, gel (Fig. 1, a and b). Three polypeptides, Mr 43,000, 18,000, D6, B1, or B15, directed against GCDFP-15 (10); or 4 /¿Iofnonimmune and 13,000, accumulated more specifically, according to anal mouse IgG (1 mg/ml). Protein A-Sepharose (100 pi; 6% w/v in the ysis of the same amounts of TCA-precipitable material (Fig. 1, same buffer) was then added. Tubes were agitated for 2 h at 4°Cand c and d) which have been shown to be induced by R5020 and then centrifuged at 10,000 x g for 2 min. The protein A-Sepharose gel DHT (4). The different molecular weights of the two smaller was washed 4 times with 1.2 ml of Tris-LiCl buffer (100 mM Tris-HCl, peptides (M, 18,000 and 13,000) were obtained using more pH 8.6-500 mM LiCl-0.5% Nonidet P-40) and then once with 50 mM suitable 15% or 20% polyacrylamide gels. Tris-HCl, pH 6.8. The antigen-immunoglobulin complexes were dis In an attempt to identify these androgen-regulated polypep sociated from the protein A-Sepharose gel by heating for 10 min at 90°Cin 60 pi of electrophoretic sample buffer. Tubes were centrifuged tides, several antibodies to proteins of similar molecular weight at 10,000 x g for 2 min and the supernatant was loaded onto SDS- were tested.