[CANCER RESEARCH 36, 1886-1893, June 1976] Characteristics of Progesterone-binding Components in Neoplastic Mammary Tissues of the Rat1 James E. Goral2 and James L. Wittliff3 University of Rochester Cancer Center and Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 SUMMARY glucocorticoid-bmnding components of mammary tissues have been well characterized (8-10, 13, 18, 36, 37), little is Characteristics of [3H]progesterone-bmnding components known of the intracellular entities associated with proges were studied in cell-free preparations of two hormonally terone. responsive tumors: the R3230AG mammary adenocarci The detailed study of Lawson and Pearlman (20) first noma and 9,10-dimethyl-1 ,2-benzanthracene-induced mam suggested that the mammary gland of the pregnant rat mary tumor of the rat. Progesterone-binding macromole possessed the biological capacity to retain progesterone in cules from cytosols of both mammary neoplasms exhibited Vivo. Later studies demonstrated that the DMBA4-induced sedimentation coefficients of 3.5 to 4.0 S on sucrose mammary tumor of the rat (30, 39), as well as certain human gradients of either low or high ionic strength. From Scat mammary carcinomas (39), contained specific progester chard analyses of titration data, apparent dissociation con one-binding sites. Using organ cultures of mammary gland stants of 4 to 6 x 10@M were determined for ligand-binding from virgin mice, Mehta (26) reported that [3H]progesterone protein complexes from either tumor. Specific progester was associated with sites exhibiting high affinity. Atger et one-binding capacities varied considerably, ranging from al. (1) demonstrated that the cytoplasm of guinea pig mam 150 to 650 fmoles/mg of cytosol protein. Optimal binding of mary gland contained a progesterone-binding macromole [3H]progesterone was reached by 2 to 3 hr at 3°,pH7.4, and cule that was excluded by Sephadex G-25. However, be then decreased rapidly. Specificity studies indicated that cause of the low binding capacity, no further characteriza cortisol, corticosterone, and triamcinolone acetonide com tionstudieswereattempted. peted effectively for [3H]progesterone-binding. This sug In @dditionto normal mammary gland, several mammary gested that [3Hjprogesterone was bound largely to a macro tumors have been reported to be responsive to progester molecule distinct from transcortin, which does not bind one administration. Pharmacological doses of the steroid glucocorticoids containing 9a-fluoro groups. Aldosterone, hormone depressed the growth rate of the R3230AG mam as well as several androgens and estrogens, were weak mary adenocarcinoma (14) and increased the incidence of competitors of binding except at high concentrations. The DMBA-induced mammary tumors (15, 23). Since these car nature of the inhibition of progesterone-binding sites by cinomas responded differently to progesterone treatment, it triamcinolone acetonide and corticosterone was competi was suggested that these effects may be related to differ tive. Goncurrent titrations of [3Hjprogesterone and ences in the characteristics of progesterone-binding sites. [3H]triamcinolone acetonide-binding sites demonstrated This study determined some of the kinetic and molecular that their binding capacities were similar, considering the properties of specific [3H]progesterone-binding compo relative stabilities of the complexes. These results, which nents in these hormonally responsive mammary tumors of indicated that progesterone and glucocorticoids compete the rat. Additionally, in view of our previous observation for the same binding site, suggest that these hormones may that progesterone competed for glucocorticoid-binding influence mammary gland differentiation and development sites (9, 13), it was necessary to explore the binding of by a common mechanism. [3Hjprogesterone directly. A portion of the data reported here was presented elsewhere (12). I INTRODUCTION MATERIALS AND METHODS Mammary gland differentiation, development, and func tion are influenced by several steroid hormones, including Chemicals. All chemicals were reagent grade unless oth cortisol, estrogens, and progestogens (17, 19, 22, 26, 31, erwise specified. 1,2,6,7-[3H]Progesterone (96 to 105 Gi/ 40, 42). The initiation of these effects is thought to be mmole) was purchased from New England Nuclear Corp., mediated by specific steroid-binding components that mod Boston, Mass. , and 1,2,4-[3H]triamcinolone acetonide (9a- ulate intracellular events (16, 44). Although estrogen and fluoro-1 11j,16a ,17a ,21-tetrahydroxypregna-1 ,4-diene-3 ,20- dione-16,17-acetonide) (10.7 Gi/mmole) was obtained from I This research was supported by USPHS Grant CA-12836 and CA-i 1198 Schwarz/Mann, Orangeburg, N. V. Unlabeled triamci from the National Cancer Institute. a Medical student Research Fellow supported by USPHS Grant T1-07-AM 01004-10. 4 The abbreviations used are: DMBA, 9,10-dimethyl-1 ,2-benzanthracene; 3 To whom requests for reprints should be addressed. l@,concentration of unlabeled steroid at which [3Hlprogesterone binding was Received November 11, 1975; accepted February 16, 1976. inhibited by 50%. 1886 CANCERRESEARCHVOL. 36 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Progesterone-binding Components in Mammary Tissue nolone acetonide and aldosterone were obtained from described previously was added to each vial and incubated Sigma Chemical Go. , St. Louis Mo. , and corticosterone, for 2 to 3 hr. Following the incubation period, cytosols were hydrocortisone, progesterone, and 17f3-estradiol were sup mixed with a pellet of dextran-coated charcoal, which was plied by Galbiochem, San Diego, Calif. Unlabeled dihydro prepared earlier by centrifuging 3 ml of the charcoal sus testosterone was purchased from Steraloids, Inc., Wilton, pension as suggested by Boylan and Wittliff (2). After an N. H. Tris-HGI (Trizma base) and DMBA were products of incubation of 10 mm, samples were centrifuged at 500 x g Sigma. Schwarz/Mann supplied the RNase-free sucrose, for 10 mm at 3°tosediment the charcoal. Aliquots (0.2 ml) of and Norit A was obtained from Matheson Coleman and Bell, the clear supernatant were removed and layered onto linear Norwood, Ohio. Omnifluor, from New England Nuclear 5 to 20% sucrose gradients prepared in Tris-HGI buffer, pH Corp., and Triton X-100, from Beckman Instruments, Inc., 7.4, containing 1.5 mM EDTA. Human serum albumin (4.6 S) Palo Alto, Calif. , were used in the preparation of a scintilla was used as a marker protein of known sedimentation ye tion cocktail. locity. Source and Maintenance of Animals. All animals were Calculations of Resufts. Computer programs described purchased from the Charles River Breeding Laboratories, earlier (3) for the Olivetti Programma 101 were used to Inc., Wilmington, Mass., and housed in the vivarium of the determine counting efficiencies and to convert radioactivity University of Rochester Medical Center. Female Fischer 344 data to steroid concentrations. Estimates of specific bind rats were used as hosts for the transplantable R3230AG ing by the dextran-coated charcoal assay were determined tumor, while the other mammary tumors studied were in as the difference between total and nonspecific binding. In duced by the carcinogen, DMBA, in young female Sprague the sucrose gradient procedure , steroid-binding compo Dawley rats. Transplantation of the R3230AC tumor and nents were identified by isotopic profiles. Specific binding induction of the DMBA-induced mammary tumor were per capacity was expressed as fmoles (10'@ mole) per mg of formed as described earlier (13). cytosol protein. Protein concentrations were estimated by Preparation of Cytosols. All procedures were performed the Geiger and Bessman (11) procedure when sulfhydryl at 0-3°unless otherwise noted. Mammary tumors were ex reagents were present, using bovine serum albumin as a cised quickly and placed in 10 mM Tris-HCI buffer, pH 7.4, standard. containing 1.5 mM EDTA, 250 mM sucrose, and 10 mM monothioglycerol. Tissues were minced on a Mcllwain tis sue chopper, stirred in the buffer to remove blood and milk RESULTS proteins, then blotted dry, and weighed. Using Duall ho mogenizers, mammary tissues were homogenized in buffer Time Courses of [3H]Progesterone Binding. Shown in using weight/volume ratios of 1/1 and 1/3 for R3230AC and Chart 1A, association of [3Hjprogesterone to specific sites in DMBA-induced mammary tumors, respectively. These ho cytosols from the R3230AG tumor occurred rapidly, reach mogenates were then centrifuged for 30 mm at 105,000 x g ing a maximum by -@2hr, which was followed by a decrease to prepare the cytosol fractions. in binding even in the presence of saturating concentra Dextran-coated Charcoal Procedure. This procedure is tions of labeled steroid. Specific binding capacity measured essentially an adaptation of the 3H-steroid-binding assays at 24 hr was only —25%ofthat determined at 2 hr. Similarly, that we reported earlier (10, 13). Constant volumes, 0.2 ml, binding of [3H]progesterone to specific sites in cytosols of the 105,000 x g supernatants were added to each of 0.5- from the DMBA-induced tumor was maximal by 2 to 3 hr dram glass shell vials (Fisher Scientific Go., Pittsburgh, (Chart 1B) at 3°,decreasing to undetectable levels by 24 hr. Pa.), containing [3H]progesterone alone, or in the presence Time courses of binding were comparable in 3 different of unlabeled progesterone,