GORAL AND WlTTLlFF
Comparison of Glucocorticoid-Binding Proteins in Normal and Neoplastic Mammary Tissues of the Rat? James E. Goral and James L. Wittliff*
ABSTRACT: Kinetic and molecular properties of components binding [3H]triamcinolone acetonide were studied using 105,OOOg supernatants of lactating mammary gland, R3230AC, and dimethylbenz[a]anthracene (DMBA) induced mammary tumors of the rat. Using a dextran-coated charcoal adsorption procedure, the relationship between specific glucocorticoid binding and protein concentration was linear in the range of 0.5-4.0 mg/reaction. These cytoplasmic macromolecules bound [3H]triamcinolone acetonide with limited capacity (50-400 fmol/mg of cytosol protein) and high affinity, Kd 10-8-10-9 M . Optimal binding was obtained when homogenizations were made in Tris buffers, at pH 7.4, containing monothioglycerol. Time course of association of [3H]triamcinolone acetonide and its binding sites showed maximal binding by 6-8 hr a t 3' which remained unchanged up to 24 hr. The rate constant of association a t 3' was in the range of 2-4 X IO' M - ' min-'. The rate constant of dissociation of bound [3H]triamcinolone acetonide could not be calculated accurately since the reaction was essentially irreversible for 5 hr at 3'. Estimation of the half-life of the steroid-binding protein
complexes from the Kd and the rate constant for association gave a value of 11-12 hr. From ligand specificity studies, the glucocorticoids, triamcinolone acetonide, corticosterone, cortisol, and dexamethasone competed well for [3H]triamcinolone acetonide binding sites. Progesterone, aldosterone, and the anti-glucocorticoid, cortexolone, were also good competitors while androgens and estrogens were weak inhibitors of binding. The binding components sedimented a t 7-8 S in sucrose gradients of low ionic strength and dissociated into lower molecular weight components sedimenting a t 4-5s in high ionic strength gradients. Studies in vivo using animals bearing the DMBA-induced tumor demonstrated that [3H]triamcinolone acetonide binding complexes were present in cytoplasmic and nuclear compartments. Sedimentation coefficients of the cytoplasmic and nuclear forms of these receptors labeled in vivo were 7-8s and 4-5S, respectively. These studies suggest that the molecular and kinetic binding properties of glucocorticoid receptors in neoplastic mammary tissues are similar to those of the normal mammary gland.
Glucocorticoids are known to influence development and differentiation of the mammary gland. Cellular differentiation, characterized histologically by an alveolar-secretory appearance and by increased rates of synthesis of milk proteins, has been observed in explants of pregnant mouse mammary gland cultivated in vitro in the presence of cortisol, insulin, and prolactin (Juergens et ai., 1965; Stockdale et ai., 1966). Evidence also indicates that the adrenal corticoids are required for lactation (Lyons et al., 1958; Nandi, 1959; Thatcher and Tucker, 1970). These effects have been related to the accumulation of rough endoplasmic reticulum in the epithelial cells of mammary tissue, presumably by a membrane-stabilizing effect of the glucocorticoids (Oka and Topper, 1971). Recently this laboratory demonstrated the presence of components in cytosol of lactating mammary gland of the rat which bound [)HI triamcinolone acetonide' specifically and with high affinity (Gardner and Wittliff, 1973a). In an-
other report (Gardner and Wittliff, 1973b), we presented evidence that the R3230AC mammary tumor, a well-differentiated adenocarcinoma of the rat whose growth is inhibited by the administration of hydrocortisone to the host (Hilf et ai., 1965), also contained [3H]triamcinolone acetonide binding components in the cytoplasm. Mammary tumors induced by dimethylbenz[a]anthracene2 represent another hormonally responsive neoplasm i n that the majority regress after ovariectomy-adrenalectomy of the host animal (Dao, 1964). This observation suggested that the mechanism of regression due to the administration of glucocorticoids may involve specific glucocorticoid-binding components similar to those we reported earlier (Gardner and Wittliff, 1973a,b). These early reports were concerned largely with the sedimentation properties of glucocorticoid-binding components and provided little quantitative information. In this paper we describe the application of a rapid, quantitative procedure for measurements of [)HI triamcinolone acetonide binding using dextran-coated charcoal to adsorb unbound steroid. Using this procedure we now present a more extensive characterization of the [)H]triamcinolone acetonide binding components of the mammary gland and the R3230AC mammary tumor and demonstrate for the first time their presence i n the DMBA-induced mammary tumor of the rat.
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From the University of Rochester Cancer Center and Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. Received December 31, 1974. A preliminary report of these studies was presented a t the 65th annual meeting of the American Association for Cancer Research. Houston. Texas, March 28-30, 1974 (Goral and Wittliff, 1974). This research was supported in part by Grants CA-12836 and CA-I 1198 and the Irwin Strasburger Memorial Medical Foundation. J.E.G. is a medical student Research Fellow supported by U.S. Public Health Service Grant T-07-AM-0 1004-8. 9u-Fluoro- 113.1 6rr.l7a,21-tetrahydroxypregna-1,4-diene-3,20dione- 16.17-acetonide. +
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2944
BIOCHEMISTRY, VOL.
14,
NO
13. 1915
Abbreviations used are: DMBA, 9,IO-dimethyl-l.2-benzanthracene; PCMB, p-chloromercuribenzoate.
GLUCOCORTICOID-BINDING PROTEINS IN MAMMARY TISSUE
m M Tris-HC1 buffer (pH 7.4) containing 1.5 m M EDTA and 10 m M monothioglycerol, then centrifuged at 4000 Chemicals. All chemicals were reagent grade unless othrpm in a 5-21 centrifuge (Beckman Instruments) and the erwise specified. [ 1,2,4-3H]Triamcinolone acetonide (10.7 supernatants discarded. Pellets were then placed in TenCi/mmol) was purchased from Schwarz/Mann and Broeck homogenizers and extracted (1 :1, w/v) with buffer checked periodically for purity by thin-layer chromatogra(pH 7.4) which contained KCI at a final concentration of phy. Unlabeled triamcinolone acetonide, d e ~ a m e t h a s o n e , ~ 0.4 M . This homogenization was continued intermittently and aldosterone were obtained from Sigma Chemical Co., for 1-1.5 hr at 3 O to provide optimal extraction before cenwhile corticosterone, hydrocortisone, progesterone, estratrifugation at 105,OOOg for 30 min at 3'. diol- 17p, estrone, and estriol were supplied by Calbiochem. Dextran-Coated Charcoal Procedure. This procedure is Unlabeled c o r t e ~ o l o n edihydrotestosterone, ,~ and testostersimilar to the [3H]estradiol-17/3 binding assay we reported one were purchased from Steraloids, Inc. Tris-HC1 (Trizma earlier (Gardner and Wittliff, 1 9 7 3 ~ ) .Constant volumes, base) and DMBA were products of Sigma Chemical c o . 0.2 ml, of the 105,OOOg supernatants were added to each of Schwarz/Mann supplied the RNase-free sucrose while 0.5-dram glass shell vials (Fischer Scientific) containing Norit A was obtained from Matheson Coleman and Bell. [3H]triamcinolone acetonide alone (total binding) or in the Omnifluor from New England Nuclear Corp. and Triton presence of unlabeled triamcinolone acetonide (nonspecific X-100 from Beckman were used in the preparation of scinbinding) which had been dried down previously under nitrotillation cocktail. gen. The concentration of the tritium-labeled and unlabeled Source and Maintenance of Animals. All animals were steroid varied with the experiment; in routine analyses a purchased from the Charles River Company, Wilmington, 100-fold excess of unlabeled triamcinolone acetonide was Mass., and housed in the vivarium of the University of used. Triplicate reactions were incubated for 16 hr at 3' Rochester Medical Center. Female Fischer 344 rats were unless noted. After the incubation period, 1 ml of a suspenused as hosts for R3230AC tumor transplants while sion of dextran-coated charcoal (10 m M Tris-HC1 buffer ,Sprague Dawley rats lactating 10-22 days were used as a (pH 7.4) containing 1.5 m M EDTA, 0.25 M sucrose, 0.05% source of normal mammary tissue. Mammary tumors were dextran, and 0.5-1.0% Norit A) was added. Following an induced by the carcinogen, dimethylbenz[a]anthracene in additional incubation of 10 min, the vials were centrifuged young Sprague Dawley female rats. at 1500 rpm for 10 min at 3O. Then an aliquot of each suTransplantation of the R3230AC Mammary Tumor. pernatant was removed and counted in toluene-based scinTissue implants (10-20 mg) which were obtained from 21tillation fluor containing Triton X-100 (4 g of Omnifluor in day-old tumors were transplanted by a sterile trocar tech300 ml of Triton X-100 and 700 ml of toluene). nique into the ventral subcutaneous tissue of female Fischer Sucrose Gradient Centrifugation of Steroid-Binding rats weighing approximately 150 g. Implants of tumor tisProteins. The assay was similar to that previously described sue were deposited along the axillary-inguinal line of the (Gardner and Wittliff, 1973a). In each of several 0.5-dram mammary bed where neovascularization occurred readily. glass shell vials, an appropriate quantity of [3H]triamciInduction of Mammary Tumors by DMBA. Virgin fenolone acetonide (usually 40-50 n M final concentration) male rats, 51-52 days of age, were each intubated intragasalone or in combination with an excess (usually 100-500trically each week for 5 weeks with 1 ml of sesame oil confold) of unlabeled triamcinolone acetonide was dried under taining 5 mg of DMBA. Approximately 4 weeks after the nitrogen just prior to the assay. An aliquot of cytosol (600 last intubation, animals were palpated for the development MI) prepared as described previously was added to each vial of mammary tumors. Tumors were obtained either by exciand incubated for -6 hr at 3'. Following the incubation pesion of the neoplasm from an ether-anesthetized animal or riod, cytosols were mixed with a pellet of dextran-coated from an animal which had been sacrificed by cervical dislocharcoal, which was prepared earlier by centrifuging 3 ml cation. Tissue specimens were routinely taken from the neoof the charcoal suspension as employed by Boylan and Witplasm and stained and evaluated histologically. tliff (1973). After an incubation of 10 min, samples were Preparation of Cytosols. All procedures were performed centrifuged at 500g for 10 min at 3O to sediment the charat 0-3O unless otherwise noted. Mammary glands or tumors coal. Aliquots (0.2 ml) of the clear supernatant were rewere excised quickly and placed in 10 m M Tris-HC1 buffer moved and layered onto linear sucrose gradients (usually (pH 7.4) containing 1.5 m M EDTA, 250 m M sucrose, and 5-40% or 5-20% sucrose prepared in Tris-HC1 buffer (pH 10 m M monothioglycerol. Tissues were minced with a 7.4) containing 1.5 m M EDTA and 10 m M monothioglyMcIlwain tissue chopper, stirred in buffer to remove blood cerol). For certain experiments, gradients were prepared and milk proteins, then blotted dry and weighed. Using with sucrose solutions containing 0.4 M KC1. Following Dual1 homogenizers, mammary tissues were disrupted in sample addition, gradients were centrifuged at 308,OOOg for buffer using weight/volume ratios of 1:4, 1:1, and 1:3 for 15-16 hr at 3' in an SW56Ti rotor in a Beckman L5-65 ulthe lactating mammary gland, R3230AC, and DMBA-intracentrifuge. Gradients were collected from the bottom duced mammary tumors, respectively. These homogenates into scintillation vials. Scintillation fluor was added to each were then centrifuged for 30 min at 105,OOOg to prepare the vial and the radioactivity was measured in
