Characteristics of Binding to Estrogen, Androgen, Progestin, and Glucocorticoid Receptors in 7,12-Dimethylbenz(A)

Home , Triamcinolone acetonide

[CANCER RESEARCH 40. 1612-1622, May 1980] 0008-5472/80/0040-OOOOS02.00 Characteristics of Binding to Estrogen, Androgen, Progestin, and Glucocorticoid Receptors in 7,12-Dimethylbenz(a)anthracene- induced Mammary Tumors and Their Hormonal Control1

Jacques Asselin,2 RÃ©jeanMelanÃ§on,Gilbert Moachon, and Alain BÃ©langer

Laboratory of Molecular Endocrinology, Le Centre Hospitalier de I UniversitÃ©Laval, Quebec G1V4G2, Quebec. Canada

ABSTRACT induced mammary tumors in the rat. Moreover, as revealed by hypophysectomy, adrenalectomy, and ovariectomy, the levels In order to perform simultaneous measurement of binding of of the progesterone and glucocorticoid receptors are under four classes of steroids and facilitate study of their mechanism different hormonal control. of action in 7,12-dimethylbenz(a)anthracene-induced mam mary tumors in the rat, we have investigated in detail the binding characteristics of 17/3-[2,4,6,7,16,17-3H]estradiol INTRODUCTION (17/8-[3H]estradiol), 17,21 -[6,7-3H]dimethyl-19-norpregna- 4,9-diene-3,20-dione ([3H]R5020), [3H]dexamethasone (DEX), Treatment of human breast cancer by endocrine manipula and 5a-[3H]dihydrotestosterone (DHT) in cytosol prepared from tion has shown that a certain proportion of these tumors are hormone dependent. This is supported by the presence of these tumors. Following assessment of optimal buffer compo steroid and peptide receptors in these tumors. In fact, estrogen sition, separation of bound and free steroids was achieved with (16, 23, 27, 41, 45), progesterone (38, 41, 45), and androgen dextran-coated charcoal, protamine sulfate, or hydroxylapatite. (31, 39) receptors have been reported in cytosol prepared Using the same buffer and the optimal method of separation from human breast cancer biopsies. Moreover, estrogen, pro for each tracer, we have found that the time course of binding gesterone, androgen, and glucocorticoid receptors have been of each labeled steroid was fast, the slowest rate of association demonstrated in the MCF-7 human breast cancer cell line (18). being obtained with [3H]DEX (t = 100 to 150 min) at 4Â°. In Binding of insulin, prolactin, and growth hormone has also addition, the level of specific binding of each tracer was stable for a period of at least 21 hr at 4Â°but decreased markedly at been described in human mammary carcinoma (17). Hormone-dependent mammary tumors developed in the rat 23Â°(except with 17/?-[3H]estradiol). Complete exchange of the are widely used as models of human breast cancer. Thus, the tracer from the binding component was achieved at 4Â° for growth and development of a large proportion of mammary [3H]R5020 and at 23Â°for 17/S-[3H]estradiol. Although [3H]DEX tumors induced in the rat by DMBA3 (21) are markedly influ dissociated from its binding component at 4Â°, the receptor enced by the endocrine status of the animal. Investigations at complex was unstable during long-term incubations necessary the molecular level have shown the presence of prolactin- (25, to achieve complete exchange. No significant exchange of 46), insulin- (25), and growth hormone- (25) binding compo [3H]DHT binding occurred at 4Â°.Using six increasing concen nents in this experimental mammary tumor. Moreover, estro trations of a large series of unlabeled steroids, each tracer gen- (23, 33), progesterone- (4, 19), glucocorticoid- (13), and showed a high degree of binding specificity. Sucrose gradient more recently androgen- (22) binding components have been analysis performed in low-ionic-strength buffer revealed spe found in the same tissue. The finding of specific hormone- cific and protamine sulfate-precipitable steroid-binding com binding components combined with the effect of hormone ponents migrating in the 8 to 9S area for 17/?-[3H]estradiol, 7S therapy on the growth and development of the DMBA-induced and 4 to 5S for [3H]R5020, and 7 to 8S for [3H]DEX and mammary tumor are good indicators that this neoplastic tissue [3H]DHT. Sucrose gradient analysis in a high-ionic-strength represents a good model of human hormone-dependent breast buffer (0.4 M KCI) gave only one labeled area (3 to 5S) for each cancer. tracer. Scatchard analysis revealed Kd's of 0.14 nw for 17/8- In order to further understand the mechanisms of steroid [3H]estradiol, 2.9 nM for [3H]R5020, 13 nM for [3H]DEX, and action in the development of this mammary tumor, it appears 0.45 nM for [3H]DHT binding. important to make a detailed comparison of the characteristics In a second series of experiments, while adrenalectomy had of steroid binding, as well as of the hormonal factors controlling no effect on the binding of any of the four classes of steroids, steroid-binding levels in the normal mammary tissue and the 17/8-[3H]estradiol, [3H]R5020, and [3H]DHT binding levels were DMBA-induced mammary tumors. Although the presence of decreased in 7,12-dimethylbenz(a)anthracene-induced mam the 4 types of steroid-binding components has been reported mary tumors after ovariectomy plus adrenalectomy or hypo- in cytosol from DMBA-induced mammary tumors, no detailed physectomy, while the binding of [3H]DEX remained un investigation of the binding properties of the different classes changed. The present data describe the optimal conditions for simul 3 The abbreviations used are: DMBA, 7,12-dimethylbenz(a)anthracene; taneous and specific measurement of binding of four classes R5020, 17.21 -[6,7-3H]dimethyl-19-norpregna-4,9-diene-3,20-dione; R1881, of steroids in cytosol from 7,12-dimethylbenz(a)anthracene- 17j8-[6,7-3H]hydroxy-17a-methylestra-4,9,11 -trien-3-one (methyltrienolone); 170-{3H]estradiol, 17/H2,4,6,7,16,17-3H]estradiol; [3H]DHT, [1,2,4,5,6,7-3H]- dihydrotestosterone; [3H]DEX, [6,7-3H]-dexamethasone; HAP, hydroxylapatite; ' Supported by a grant from the National Cancer Institute of Canada. Â¡.g.,intragastric; DES, diethylstilbestrol; DCC, dextran (0.05%)-coated charcoal 2 To whom requests for reprints should be addressed. (0.5%); R2858, 11/?-methoxyethinyl-17/3-estradiol; R2323, 13a-ethyl-17-hy- Received March 9, 1979; accepted January 25, 1980. droxy-18,19-dinor-1 7ÃŸ-pregna-4,9,11-triene-20yn-3-one.

1612 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Binding of Steroid Markers in DMBA Tumors of steroid receptors has been performed using uniform tech were removed 11 days after the surgical treatment except for niques. For example, the exchange rate of all steroid classes the hypophysectomized animals, from which the tumors were has not yet been reported, such information being of major removed 7 days after surgery. Steroid binding was always importance for optimal binding measurement. Moreover, only determined using fresh cytosol. [3H]triamcinolone acetonide has been used for the assay of the glucocorticoid-binding component (13), while it is well known Homogenization and Cytosol Preparation that this tracer binds also to the progesterone receptor (6). All procedures, except where indicated, were performed at Another relevant aspect is that intact animals were generally 4Â°. Routinely, tissue was minced and homogenized with two used for the characterization of steroid-binding components in 10-sec bursts of a Polytron PT 10-ST (Brinkmann Instruments) DMBA-induced mammary tumors, thus leaving the possibility in 3 volumes of Buffer A (25 mM Tris-HCI-1.5 mw EDTA-10 mw of interference by endogenous steroids. Consequently, as a a-monothioglycerol-10% glycerol, pH 7.4). The homogenate first step, we have investigated in detail the binding character was centrifuged at 39,000 x g for 20 min, and the resulting istics of 4 classes of steroids (estrogens, androgens, proges- supernatant was centrifuged at 105,000 x g for 90 min in a tins, and glucocorticoids) in cytosol prepared from this exper Beckman L5-65 centrifuge, using a 50 Ti or 60 Ti rotor. Protein imental mammary tumor, using animals deprived of the endog concentration was determined according to Lowry ef al. (30), enous steroids studied. Moreover, in a second series of exper using bovine serum albumin as standard. Correction was made iments, we have studied the effect of adrenalectomy, combined for the slight interference by a-monothioglycerol. adrenalectomy and ovariectomy, or hypophysectomy on ste roid binding levels. Binding Assays

After evaporation of the solvent under a stream of nitrogen, MATERIALS AND METHODS the labeled steroid was dissolved in Buffer B (10 mM Tris-HCI- Chemicals 1.5 mM EDTA-10 mM a-monothioglycerol, pH 7.4) and incu bated with the cytosol at the appropriate protein concentration. R5020 (51.4 Ci/mmol), R1881 (58.2 Ci/mmol), and the In order to measure nonspecific binding, a 200-fold excess of corresponding unlabeled steroids were synthesized at the the corresponding unlabeled steroid (except for 17/?-[3H]estra- Roussel Research Centre, Romainville, France, and were pro diol, where DES was used) was added to the corresponding vided by Dr. Jean-Pierre Raynaud. 17/H3H]Estradiol (152 Ci/ tubes, the incubation being performed in duplicate at 4Â°,gen mmol), [3H]DHT, (123 Ci/mmol), [3H]DEX (33 Ci/mmol), and erally using 100 Â¿ilofthe tracer solution and 200 /Â¿Iofcytosol. [6,7,-3H]triamcinolone acetonide (33.7 Ci/mmol) were pur For [3H]R5020-binding assays, a 25-fold excess of unlabeled chased from New England Nuclear. The corresponding unla DEX was added to the incubation mixture to mask the gluco beled steroids, DMBA, and protamine sulfate (from salmon, corticoid-binding component. Grade 1) were products of Sigma Chemical Co. Charcoal (Norit A) was obtained from Fisher Scientific Co., while dextran T-70 Separation of Bound and Free Steroids was a product of Pharmacia Fine Chemicals, Inc. HAP (DNA- grade Bio-Gel HTP) was obtained from Bio-Rad Laboratories. DCC Adsorption. At the end of incubation, 0.3 ml of DCC in Buffer B were added and, following an additional incubation for 10 to 60 min at 4Â°(depending upon the tracer used), the Tumor Induction and Animal Treatment mixture was centrifuged at 2000 x g for 10 min. Then, 0.3 ml Fifty-day-old female Sprague-Dawley rats, obtained from of the supernatant was removed and counted in 10 ml of Charles River Breeding Laboratories, received i.g. injections of Aquasol in a Beckman liquid scintillation system with a counting 1 ml of corn oil containing 20 mg of DMBA. Only tumors which efficiency of 35%. developed before 4 months after DMBA administration and Protamine Sulfate Precipitation. At the end of incubation, without necrosis were used throughout these experiments. In 0.3 ml of a protamine sulfate solution (2 mg/ml of Buffer B) order to eliminate the presence of endogenous steroids which was added, and, following an additional incubation for 10 min could interfere with the characterization of the steroid-binding at 4Â°, the precipitate was either centrifuged or filtered on components, ovariectomized or ovariectomized-adrenalecto- Gelman type A-E glass-fiber filters. The pellet was washed 3 mized animals were used. Cytosol prepared from tumors ob times with 1 ml of Buffer B, while the filters were washed 3 tained from bilaterally ovariectomized rats (24 hr previously) times with 5 ml of Buffer B or Buffer B containing 10% ethanol was used for 17/?-[3H]estradiol binding studies, while ovariec and 1% Triton X-100 (when using [3H]DHT as tracer). The tomized 17/8-[3H]-estradiol-treated rats (2 jug/day for 3 days) radioactivity present in the pellet was extracted with 2 volumes were used for experiments with [3H]R5020 binding. Animals of 1 ml ethanol. The filters or the ethanol extracts were counted adrenalectomized 4 to 7 days previously served as a source of in Aquasol. A lower level of nonspecific binding was generally material for [3H]DEX binding. Rats ovariectomized-adrenalec- found with the filtration method. tomized 24 hr previously were used for [3H]DHT binding meas HAP Assay. The HAP method used was essentially as pre urements. viously described (34, 49). In brief, 10 g of HAP were washed In a second series of experiments, the effect of adrenalec 5 times with 50 mM Tris-HCI-10 mM KH2POâ€ž,pH7.4 (Buffer tomy, combined adrenalectomy and ovariectomy, or hypophy C), until the supernatant reached a pH of 7.4, and, following sectomy on steroid binding was investigated, using cytosol dÃ©cantation, 37.5 ml of the same buffer were added. Prelimi prepared from tumors obtained from 4 groups of rats: Group nary experiments were performed in order to find the concen 1, intact; Group 2, adrenalectomized; Group 3, ovariectomized- tration of HAP needed to adsorb high levels of bound steroid. adrenalectomized; and Group 4, hypophysectomized. Tumors For this purpose, 25, 50, 100, 200, 300, or 500 fi\ of the HAP

MAY 1980 1613

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. J. Asselin et al. slurry were added to labeled cytosol and incubated for 40 min prepared from DMBA-induced mammary tumors. at 4Â°with agitation at 10-min intervals. The incubation mixture DCC Assay. The effect of time of incubation with DCC on the was then centrifuged, and the pellet was washed 4 times with extent of adsorption of free or weakly bound steroid from the 1 ml of Buffer C. After 2 extractions of the radioactivity with 1 incubation mixture was investigated. As illustrated in Chart 1, ml of ethanol at room temperature, 8 ml of Aguasol were added, incubation of the labeled cytosol with DCC rapidly removed and the mixture was counted. Although 100 jul of HAP slurry free hormones and lowered nonspecific binding. More impor could adsorb the high levels of bound steroid found in our tantly, no significant decrease in specific binding was observed binding assays, in order to be certain that complete adsorption up to 120 min of incubation with DCC when using 17/S-[3H]- of bound steroids was achieved, 300 n\ of the HAP slurry were estradiol (Chart 1A), [3H]DEX (Chart 1C), or [3H]DHT (Chart 1D) routinely added. as tracer. However, when using [3H]R5020 (Chart 1ÃŸ),incu bation with DCC led to a slow but progressive decrease in Sucrose Gradient Analysis specific binding, thus suggesting that [3H]R5020 dissociates Following incubation of the cytosol and tracer for 2 hr at 4Â°, rapidly from its receptor. This finding was in agreement with the mixture (200 /tl) was analyzed on 5 to 20% sucrose gra previous reports showing a high exchange rate of progestins dients prepared in Buffer A in 4-ml polyallomer tubes. Gradients from the progesterone receptor in rat or mouse tissues (12, were centrifuged in a Beckman Model L5-65 centrifuge using 37). In fact, this property has facilitated the development of an SW 56 rotor at 49,000 rpm for 19 hr. Fractions (5 drops) an exchange method between unlabeled progesterone and were then collected from the bottom of the tubes and counted [3H]R5020 at 4Â°(37) in these tissues. in 10 ml of Aguasol. Analysis on sucrose gradients was per In order to obtain maximal specific binding in the presence formed in buffer containing low or high (0.4 M KCI) ionic salt of low nonspecific binding, we chose for our subseguent ex concentrations. Moreover, the effect of treatment of the labeled periments to treat the labeled cytosol with DCC for periods of cytosol with DCC or protamine sulfate before sucrose gradient 10 min with [3H]R5020, 20 min for 17/?-[3H]estradiol, and 60 analysis was also investigated. For DCC treatment, the labeled min for [3H]DEX and [3H]DHT. cytosol was treated with a pellet of DCC (prepared from 300 Protamine Sulfate Precipitation. The removal of bound ste H\ of DCC) for 10 min at 4Â°.After centrifugation, 200 jul of the roids from the incubation mixture by precipitation with increas supernatant were layered on sucrose gradients. For protamine ing concentrations of protamine sulfate was next investigated. sulfate treatment, 200 /Â¿Iofthe supernatant obtained after DCC As illustrated in Chart 2, optimal precipitation of the steroid- treatment were incubated with 200 Â¿ilofthe protamine sulfate binding complexes was obtained at a final protamine sulfate solution (2 mg/ml of Buffer B) at 4Â°for 10 min. After centrifu concentration of 0.5 to 1 mg/ml. Similar concentrations of gation, 200 /il of the supernatant were analyzed on sucrose protamine sulfate have previously been found to be optimal for gradients. The results presented in Chart 6 were corrected for precipitation of the androgen-binding component prepared the dilution secondary to addition to an egual volume of the from the cytosol of rat ventral prostate (5, 7) and adrenals (5). protamine sulfate solution. The arrows represent migration of Conseguently, in all experiments described hereafter, precipi bovine serum albumin. tation of the labeled steroid-binding components was per formed at a final protamine sulfate concentration of 1 mg/ml. RESULTS AND DISCUSSION Comparison of Steroid-binding Levels as Measured by the 3 Separation Methods. Following assessment of the optimal The first experiments were aimed at finding the optimal conditions of the DCC, protamine sulfate, and HAP methods, it binding assay conditions for the 4 classes of steroids in cytosol was important to compare the absolute values of steroid bind-

Â¿ 6 en ÃŒ85 ~Ã¶ i 4 Â» e- < Chart 1. Effect of time of incubation with DCC on 17/H3H]estradiol (A), [3HlR5020 (B). [3H)DEX (C). and [3H]DHT (D) binding. The cytosol (200 nD was incu bated with 17/B-[3H]estradiol (5 nw), [3H]R5020 (10 nM). [3H]DEX (30 nM), or [3H]DHT (7 nM) in the pres ence or absence of a 200-fold excess of unlabeled steroid for 17 hr at 4Â°.After addition of 0.3 ml DCC, samples were removed at different time intervals and centrifuged, and the supernatant (0.3 ml) was counted in Aguasol. Total (â€¢),nonspecific (A), and specific (O) binding are presented. e- -e -e-

a. r 20 40 60 80 100 120 20 40 60 80 100 120

MIN WITH DCC

1614 CANCER RESEARCH VOL. 40

Chart 2. Effect of the protamine sulfate concentration on precipitation of bound 170-{3H]estradiol (A), [3H]R5020 (8), [3H]DEX (C), and [3H]DHT (O). The cytosol was incu bated as described in the legend to Chart 1. Different concentrations of protamine sulfate were then added, and the incubation mixture was treated as described in "Ma terials and Methods" for measurement of radioactivity. Total (â€¢),nonspecific (A), and specific (O) binding are represented. â€¢¿oâ€” A

05 10 15 20 25 30 0 05 10 15 2.0 25 30

PROTAMINE SULFATE CONG E NTRATION(mg/ml ) ing measured by these 3 techniques. As shown in Table 1, the Table 1 DCC and HAP methods gave approximately the same amount Comparison of 3 separation techniques on steroid binding levels of steroid binding for each tracer used. tissue)"Separation Specific binding (pmol/g However, for still undefined reasons, when using the prota 7/H3H]Es- mine sulfate method, lower levels of [3H]R5020 and [3H]DHT technique'DCC tradiol3.0 binding were obtained. Similar differences have also been Â±0.4C Â±1.6 Â±1.1 Â±0.1 observed using Scatchard data analysis. Protamine 3.4 Â±0.33.0 10.4 Â±1.812.7 6.6 Â±1.35.7 0.5 Â±0.10.8 Buffer Composition. As clearly shown in Table 2, in the sulfate absence of EDTA, low binding levels of [3H]DEX were obtained. HAP1 Â±0.5[3H]R502015.2Â±1.2[3HJDEX6.0Â±1.2[3H]DHT0.8Â±0.1 See "Materials and Methods" for details. The importance of a reducing agent for measuring glucocorti- 6 Specific binding levels were determined after incubation of cytosol with a coid binding in different rat tissues has been previously re saturating concentration of tracer in the presence or absence of a 200-fold excess of the unlabeled steroid for 17 hr at 4Â°. ported (13, 15) and is supported by the present findings of 0 Mean Â±S.E. of 3 experiments. reduced [3H]DEX binding in the absence of a-monothioglycerol (Buffer T). A similar result has been obtained by Scatchard data analysis (data not shown). It is also important to note that markedly decreased after 24 hr of incubation at this low tem the presence of EDTA and, to a lesser extent, glycerol was perature. This difference observed when using [3H]progester- essential for optimal binding of [3H]R5020. The effect of glyc one could possibly be explained by a high exchange rate of erol could be explained by a decrease in the exchange rate of progesterone from the receptor (12, 37) and the instability of [3H]R5020 as previously reported in rat uterus for progesterone free receptor sites. In fact, when using [3H]progesterone as (12). Approximately similar levels of 17/S-[3H]estradiol and tracer, the presence of glycerol in the buffer has been shown [3H]DHT binding were observed with the 5 buffers used. Con to be extremely important to protect binding sites (12). Such sequently, Buffer A was used for our subsequent experiments. data illustrate the importance of using [3H]R5020 as tracer for Time Course of Specific Binding. As illustrated in Chart 3, studies of the progesterone receptor (35-37). Following the the association of labeled steroids with the binding components present data, an incubation period of 17 to 21 hr at 4Â°was in cytosol prepared from DMBA-induced mammary tumors was chosen for our subsequent experiments. very rapid for 17/Ã®-[3H]estradiol(Chart 3-4), [3H]R5020 (Chart Exchange of Bound Steroids. As shown in Chart 4/4, addi 38), and [3H]DHT (Chart 3D). A slower rate of association was tion of a 200-fold excess of unlabeled DES to the prelabeled obtained with [3H]DEX (Chart 3C), a half-maximal binding being cytosol led only to a slight decrease in 17/Ã®-[3H]estradiolbind measured at approximately 2 hr for this particular preparation. ing at 4Â°.However, at 23Â°,in agreement with previous obser It can also be seen that binding was stable for each tracer for vations in other rat tissues and human breast cancer (41), a at least 21 hr of incubation at 4Â°.However, incubation at 23Â° rapid and complete exchange was obtained. It is thus possible led to a rapid decrease in [3H]R5020, [3H]DEX, and [3H]DHT to exchange completely 17/?-estradiol from its binding com binding, while for 17/Ã®-[3H]estradiola 10 to 25% decrease in ponent at 23Â°,although, asean be seen in Chart 4A, a relatively binding was generally observed after 21 hr of incubation. These small but significant decrease in 17/S-[3H]estradiol binding was results are in close agreement with previous observations using usually obtained under these conditions. cytosol obtained from other tissues (12, 32, 37, 41 ). However, Addition of a 200-fold excess of unlabeled R5020 (Chart 46) it should be mentioned that a previous report (14) has shown to the prelabeled cytosol rapidly decreased [3H]R5020 binding that the association of [3H]progesterone in cytosol prepared at 4Â°.This finding was in agreement with a rapid exchange rate from DMBA-induced mammary tumors was fast at 0Â°but was of progestin from their binding sites in rat tissues at low

MAY 1980 1615

Table 2 temperature (37). It is thus possible, without any significant Influence of butter composition on steroid binding levels degradation of [3H]R5020 binding sites, to obtain a complete Specific binding (pmol/g tissue)' exchange of progestins at 4Â°. Addition of a 200-fold excess of unlabeled DEX (Chart 4C) 7/H3H] [3H]DHT2.8Estradiol [3H]R5020 [3H]DEX slowly decreased [3H]DEX binding. However, during this period BuffercompositionBuffer of incubation at 4Â°,a significant loss of [3H]DEX binding was 0.22.7Â±0.2" 8.3 Â±0.9 0.2 Â±0.1 0.4 Â± T (10mMTris-HCI. obtained. Using [3H]DHT as tracer (Chart 4D), it can be seen 7.4)Buffer pH B (10mMTris-HCI-1 0.23.2+ 0.4 14.6 Â±3.3 4.7 Â±0.5 0.6 Â± that no significant exchange of [3H]DHT occurred at 4Â°in the ITIMEOT .5 presence of a 200-fold excess of the unlabeled steroid. o-monothioglycerol.pHA- 10 mM Effect of Cytosol Concentration. In order to measure steroid 7.4)Buffer binding under optimal conditions, we then studied the effect of A (25mMTris-HCI-1 0.32.9Â±0.3 15. 7 Â±3. 5 4.5 Â±0.5 0.6 Â± mmEOT .5 cytosol concentration on the binding of each tracer. Steroid a-monothioglycerol-10%glycerol,A- 10 mw binding was proportional to the cytosol protein concentration up to 8 mg protein per ml cytosol for each tracer used (data pH7.4)Buffer not shown). At higher protein concentrations, steroid binding S (10mMTris-HCI-250 0.12.2Â±0.1 7.9+1.2 1.4 Â±0.3 0.5 Â± levels were underestimated. A protein concentration ranging mwsucrose-10 a-monothioglycerol,pHmM between 6 to 8 mg/ml was thus chosen for our subsequent experiments, except for sucrose gradient experiments, where 7.4)Buffer a protein concentration of 16 to 24 mg/ml was used. mMTris-HCI-250Mg (10 Â±0.1 9.4 Â±2.0 1.7 Â±0.4 0.5 Â±0.1 mMsucrose-3 Specificity of Steroid Binding. In order to determine with mMMgCI2-1 precision the specificity of steroid binding, 6 increasing con a-monothioglycerol.pH0 mM centrations of a large series of the appropriate unlabeled

74)1 steroids were incubated in the presence of each tracer. As illustrated in Table 3, only estrogens or antiestrogens competed Specific binding levels were determined after incubation of cytosol with a for the binding of 17/?-{3H]estradiol with a displacing ability of saturating concentration of tracer in the presence or absence of a 200-fold excess of unlabeled steroid for 17 hr at 4Â°.Incubation was terminated by DCC. 1% or more with the following order of potency: 17/J-estradiol 0 Mean Â±S.E. of 3 experiments. > DES > R2858 = 11a-methoxyethinyl-1 7/Ã®-estradiols> na-

12 IB 24

MOUDS OF INCUBATION Chart 3. Time course of 17/i-{3H]estradiol (Ai, [3H]R5020 (8), [3H]DEX (C). and [3H]DHT (O) binding at 4Â° or 23Â°. The cytosol was incubated with the indicated tracers, as described in the legend to Chart 1. Bound and free hormones were separated by DCC for 17ÃŸ-{3H]estradiol, [3H]R5020, and [3H]DEX and by protamine sulfate for [3H]DHT, as described in "Materials and Methods." Total (â€¢),nonspecific (A), and specific (O) binding at 4Â°. as well as specific (A) binding at 23Â° are presented.

1616 CANCER RESEARCH VOL. 40

A B

40 80 120 0 12 18 24 HOURS OF INCUBATION Chart 4. Time course of dissociation of 170-{3H]estradiol (A), [3H]R5020 (B), [3H]DEX (C), and [3H]DHT (D) binding. The cytosol was incubated with a saturating concentration of tracer for 20 hr at 4Â°before addition of the corresponding unlabeled steroid (except for 17/i-{3H]estradiol for which DES was used) or the buffer alone. Steroid binding was then measured at the indicated time intervals, as described in the legend to Chart 3, and only specific binding is represented. O, stability at 4Â°;â€¢¿,dissociationat 4Â°;d. stability at 23Â°;â€¢¿,dissociationat 23Â°. foxidine = tamoxifen. These results clearly demonstrate the binding components) has permitted the specific determination high degree of specificity of 17/Ã®-[3H]estradiolbinding in cytosol of androgen-binding component in this tissue when [3H]R1881 prepared from DMBA-induced mammary tumors. It should, is used as tracer (6). The same approach has also been used however, be mentioned that the synthetic [3H]R2858 can also in DMBA-induced mammary tumors (data not shown). In agree be used to measure the estrogen-binding component in some ment with our previous data (4), [3H]R5020 binding in cytosol tissues of the rat or human (41). Since [3H]R2858 does not prepared from DMBA-induced mammary tumors shows a typi bind to sex-binding protein or a-fetoprotein, it presents advan cal progestin specificity. Since [3H]R5020 binds only weakly to tages over 17/?-{3H]estradiol when these plasma proteins are corticosteroid-binding globulin (10, 36), it is an advantageous present. We have found that the binding specificity of [3H]- tracer for study of the progesterone receptor. R2858 and 17/8-[3H]estradiol in cytosol prepared from DMBA- When [3H]DEX was used, the potent competitors were glu- induced mammary tumors was similar (data not shown). cocorticoids with the following order of potency: triamcinolone When [3H]R5020 was used as tracer, progestins were found acetonide > DEX > triamcinolone > cortisol and corticoster to be potent competitors with the following order of potency: one. In addition, cortexolone (11-deoxycortisol), R2323, R5020 :Â» progesterone > R2323 ;*> cyproterone acetate. R5020, and progesterone decreased [3H]DEX binding. This Other steroids led also to displacement of [3H]R5020 binding: competition by progestins on the glucocorticoid receptor is R1881 :Â»triamcinolone acetonide :Â» 17/8-estradiol > DHT. well documented in a variety of tissues. In fact, it has been This affinity of R1881 for the progesterone receptor is already postulated that progesterone and some other steroids could well documented (6, 20, 26, 32, 40). In fact, we have first increase the exchange rate of [3H]DEX from the glucocorticoid- observed that binding of [3H]R1881 in human benign prostatic binding component prepared from liver and kidney cytosol hyperplasia had a progestin-binding specificity (3), and we (44). In a more recent report, it was reported that a factor have shown that this tracer binds with high affinity to the present in the liver cytosol may play a role in the interaction of progesterone receptor in human uterus (3). It is of interest to progesterone with the glucocorticoid-binding component (11). mention the relatively high affinity of triamcinolone acetonide Since [3H]triamcinolone acetonide has been previously used for the progesterone receptor. The use of this steroid for to study the binding characteristics of the glucocorticoid re masking the progestin-binding component in benign prostatic ceptor in cytosol prepared from normal mammary gland and hyperplasia (which contains both androgen- and progestin- DMBA-induced mammary tumor (13), we have investigated in

MAY 1980 1617

Table 3 : 100 i Specificity of steroid binding (%)'Unlabeled Displacing ability

7/3-[3H]- steroid1 Estradiol10058 7/j-EstradiolDESR2858RU-16117'NafoxidineTamoxifenR5020ProgesteroneR2323DEXTriamcinoloneacetonideTriamcinoloneCortisolCortexoloneCorticosteroneDHTTestosteroneR1881R2956CyproteroneÂ±1"<0.5<0.5<0.5<0.5<0.52232 Â±413 Â±114 60 * Â±122<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5<0.5[3H]R50202<0.5<0.5<0.5<0.5<0.510031

Â±16 Â±223 Â±120 Â±2<0.57 Â±4100143 Â±4<0.5<0.5<0.5<0.5<0.5<110040

20 Â±1<0.5<0.5<0.5<11<0.580Â±1242

Â±411 Â±14 0 " 2.5x109 3X108 Â«'-7 3x10 7 10 6 Â±110 Â±1<0.5<0.54 STEROO CONCENTRATONSlMI Chart 5. Specificity of [3H)triamcinolone acetonide binding in cytosol pre Â±11178 pared from DMBA-induced mammary tumors of castrated rats treated with 10 Â±20<0.54 Â±2<0.525 Â±225 fig of 17/S-estradiol for 3 days. The cytosol was incubated with 25 nw [3H]- triamcinolone acetonide in the presence or absence of 6 increasing concentra acetateAldosteroneSpironolactoneDMBA1 Â±1<0.5<0.5<0.5[3H]DEX<0.5<0.51<0.5<0.5<0.55Â±1<0.55 tions of the indicated unlabeled steroid for 17 hr at 4Â°.Incubation was terminated by the addition of DCC for 10 min at 4Â°. After centrifugation. aliquots of the Â±31<0.5[3H]DHT3 supernatant were counted in Aguasol. O, R5020; â€¢¿,DEX;A, triamcinolone Â±1<0.5 acetonide; A. triamcinolone; D. progesterone: â€¢¿cortisol.

Cytosol was incubated with a saturating concentration of the tracer in the lone acetonide was used to mask the progestin-binding com presence or absence of 6 increasing concentrations of the indicated unlabeled steroids for 17 hr at 4Â°.Incubation was terminated by DCC treatment. The results ponent, the more stable [3H]R1881 can also be used. However, are expressed as a relative displacing ability (%) calculated as the molar concen since [3H]DHT is available at a very high specific activity and tration of the unlabeled steroid used as tracer required for 50% displacement of low androgen binding levels are present in cytosol from DMBA- the tracer, divided by the molar concentration of the radioinert steroid producing the same displacement. This value has been multiplied by 100 to represent induced mammary tumors, we have preferred [3H]DHT as an percentage of displacing ability. 6 Mean Â±S.E. of 3 experiments. androgen receptor tracer for our subsequent experiments. CRU-16117. 11a-methoxyethinyl-17/i-estradiol; R2956. 2o,2/?,17a-trime- It can be concluded from the data of Table 3 that 17/8-{3H]- thyl-17/3-hydroxy-4,9,1 l-estratriene-3-one. estradiol, [3H]R5020, [3H]DEX, and [3H]DHT can be used for specific investigation of estrogen-, progesterone-, glucocorti- detail the binding specificity of this tracer. As illustrated in coid-, and androgen-binding components in cytosol prepared Chart 5, DEX, triamcinolone, and cortisol, which have low from DMBA-induced mammary tumors. However, it is important affinity for the progesterone receptor, led to only partial dis to mention that the values of displacing abilities obtained could placement of the tracer. However, triamcinolone acetonide, be influenced by the time and temperature of incubation (3, progesterone, and R5020 were potent competitors and de 41, 42), as well as by the method of separation used. creased [3H]triamcinolone acetonide binding more than 90% at Sedimentation Characteristics of the Labeled Steroid- high concentrations. These data suggest that under our exper binding Components. The sedimentation properties of the imental conditions, about 50% of [3H]triamcinolone acetonide labeled steroid-binding macromolecules present in cytosol pre binding is due to its association with the progesterone-binding pared from DMBA-induced mammary tumors were investigated component. Following these observations, [3H]DEX was pre on sucrose gradients. Cytosol-binding components labeled in ferred to [3H]triamcinolone acetonide as a specific tracer for vitro by incubation for 2 hr at 4Â°with 17/Ã®-[3H]estradiol, [3H]- further investigation of the glucocorticoid-binding component R5020, [3H]DEX, or [3H]DHT alone, or with a 200-fold excess in cytosol prepared from DMBA-induced mammary tumors. of the corresponding unlabeled steroid (except for 17/S-[3H]- When [3H]DHT was used as tracer, unlabeled androgens estradiol, where DES was used), were analyzed directly on were potent competitors with the following order of potency: sucrose gradients or after treatment with DCC or DCC plus R1881 > DHT > testosterone. Antiandrogens such as cypro- protamine sulfate. Four types of labeled cytosol were analyzed: terone acetate and 2a,2/8,17a-trimethyl-17/}-hydroxy-4,9,11- (a) untreated labeled cytosol; (b) DCC-treated labeled cytosol; estratriene-3-one were less potent. In addition, R2323, spiron- (c) cytosol incubated with a 200-fold excess of the unlabeled olactone, 17/S-estradiol, progesterone, and R5020 had a dis corresponding steroid and treated with DCC; and (d) DCC- and placing ability of 1 to 5%. These data agree with previous protamine sulfate-treated labeled cytosol. reports on the specificity of androgen binding in cytosol pre The results illustrated in Chart 6 show that direct analysis of pared from the rat ventral prostate (5, 7-9). [3H]DHT can thus labeled cytosol on low-ionic-strength sucrose gradients yielded be adequately used for characterization of the androgen-bind- radioactive peaks migrating in the 8 to 9S area for 17/?-[3H]- ing component in cytosol prepared from DMBA-induced mam estradiol, 7S and 4 to 5S for [3H]R5020, and 7 to 8S for [3H]- mary tumors. When a 700-fold excess of unlabeled triamcino- DEX and [3H]DHT. When the labeled cytosol was treated with

1618 CANCER RESEARCH VOL. 40

04M KCI

Â¡' I-1 IÂ« 24 30 36 ~ 42 0 6 12 18 24 30 36 42 24 30 36 42 0 6 12 18 24 30 36 42 W IRAC1IONNUMBER Chart 6. Sedimentation characteristics of the labeled steroid-binding components. The labeled cytosol was analyzed under 4 different conditions: untreated labeled cytosol (O); DCC-treated labeled cytosol {â€¢);cytosol incubated with a 200-fold excess of unlabeled steroid and treated with DCC (A); and DCC plus protamine sulfate-treated labeled cytosol (A). Aliquots of the incubation medium were centrifuged on linear 5 to 20% sucrose gradients containing low or high (0.4 M KCI) ionic salt. Arrows, migration of bovine serum albumin.

DCC before layering onto sucrose gradients, radioactive peaks before treatment with DCC, or when the labeled cytosol was were found in 8 to 9S and 4 to 5S for 17/Ã®-[3H]estradiol, 7S treated with DCC plus protamine sulfate, the 3 to 5S peaks of and 4 to 5S for [3H]R5020, 7S for [3H]DEX, and 7S and 4 to 17/H3H]estradiol, [3H]R5020, and [3H]DEX binding were al 5S for [3H]DHT. However, the intensity of the peak was de most completely abolished, while that of [3H]DHT was 60 to creased for [3H]R5020 and [3H]DEX. The decrease in [3H]- 70% decreased. R5020 binding was not due to the adsorption of receptor on Binding Parameters. Increasing concentrations of labeled DCC, since following addition of [3H]R5020 after DCC treat steroids were incubated with cytosol prepared from DMBA- ment of the labeled cytosol, the 7 to 8S peak was found to induced mammary tumors in the presence or absence of an have the same intensity of the untreated labeled cytosol (data excess of the corresponding unlabeled steroids for 17 hr at 4Â° not shown). In different experiments, we have found that the (Chart 7). With 17ÃŸ-[3H]estradiol specific binding sites were relative importance of the 2 peaks labeled with [3H]R5020 is saturated at low concentrations of the tracer (mean value of somewhat variable. In fact, in some tumors, no 7S peak could 1.2 nw in 4 experiments). Scatchard analysis (43) of the data be seen. Further investigation could provide more information yielded an apparent Ka of 0.13 nM for this preparation, with a on the nature of the 4 to 5S peak, but it is possible that this mean value of 0.14 nw in various assays. It should, however, peak results from the exchange of [3H]R5020 from the fast- be mentioned that at low concentrations of the tracer, the moving binding component during the ultracentrifugation. bound/free values did not follow a straight line. This observa Specificity of steroid binding to the high-molecular-weight tion, obtained in all our assays, could possibly be explained by peaks is clearly suggested by the findings that preincubation degradation of the estrogen-binding component at low concen with a 200-fold excess of the corresponding unlabeled steroids trations of the tracer, thus leaving a large proportion of free led to a complete disappearance of tracer binding. Moreover, receptors, or by non-equilibrium conditions. Similar data have treatment of the labeled cytosol with DCC plus protamine also been previously obtained when 17/i-[3H]estradiol binding sulfate led to the disappearance of the 8 to 9S and 4 to 5S was studied in cytosol prepared from a transplantable mam peaks of 17/i-[3H]estradiol, the 7 to 8S and 4 to 5S peaks of mary tumor (48). [3H]R5020, and the 7 to 8S peaks of [3H]DEX and [3H]DHT Scatchard analysis of [3H]R5020 binding yielded a straight binding. line with an apparent Kd of 3 nw, a mean value of 2.9 r\M being found in several experiments. As illustrated in Chart 7, Kd's of We next studied the sedimentation profile of labeled cytosol in high-salt (0.4 M KCI) sucrose gradients. As shown in Chart 13 and 0.45 nM were measured for [3H]DEX and [3H]DHT 6, each tracer migrated as a single peak with a sedimentation binding, respectively. coefficient of 3 to 5S. Moreover, when the cytosol was incu Effect of Adrenalectomy, Adrenalectomy plus Ovariec- bated with an excess of the corresponding unlabeled steroids tomy, or Hypophysectomy on Steroid Binding. As illustrated

MAY 1980 1619

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. J. Asselin et al. in Table 4, no significant change in steroid binding was ob under different hormonal control. served in cytosol prepared from tumors of adrenalectomized 17/H3H]Estradiol binding was also markedly reduced after animals. However, when cytosol was obtained from tumors of hypophysectomy or combined ovariectomy and adrenalec- ovariectomized-adrenalectomized or hypophysectomized ani tomy. Moreover, injection of prolactin alone in hypophysecto mals, [3H]R5020-binding levels were markedly reduced to 2 to mized rats is known to stimulate 17/?-[3H]estradiol binding (2). 11% of control. Moreover, injection of 17/?-estradiol to hypo Such data are in agreement with previous observations indi physectomized animals led to a marked stimulation of [3H]- cating that prolactin deprivation can reduce 17/8-{3H]estradiol R5020 binding (data not shown). While it was well documented binding (1, 2, 24, 29, 47). However, it is important to mention that estrogen deprivation decreases [3H]R5020 binding in that after long-term castration (3 months), some tumors which uterus and in DMBA-induced mammary tumors (1, 2, 19, 24, initially showed regression can increase in size. The analysis 26), the present observation of maintained [3H]DEX-binding of steroid binding in these tumors has revealed relatively high levels after ovariectomy-adrenalectomy or hypophysectomy levels of 17/?-[3H]estradiol, [3H]DHT, and [3H]DEX binding with clearly shows that progestin and glucocorticoid receptors are low levels of [3H]R5020 binding (data not shown). It can also

O 12 24 36 48 UNBOUNDPH|De>nM Chart 7. Effect of increasing concentrations of 17/H3H]estradiol, [3H]R5020, [3H]DEX, or [3H]DHT on steroid binding. Constant volumes of cytosol (200 fil) were incubated for 17 hr at 4Â°with increasing concentrations of the indicated labeled steroids in the presence or absence of a 200-fold excess of the corresponding unlabeled steroids (except with 17/3-[3H]estradiol, which used DES). Measurement of binding was performed with DCC for 17/3-(3H]estradiol and [3H]R5020 and with protamine sulfate for [3H]DEX and [3H]DHT. Total (â€¢),nonspecific (A), and specific (O O) binding. Scatchard analyses of specific binding are also presented (O O).

Table 4 Effect of adrenalectomy. adrenalectomy plus ovariectomy, or hypophysectomy on estrogen, progestin, glucocorticoid, and androgen binding in cytosol from DMBA-induced mammary tumors in the rat. Statistical significance was measured according to the multiple-range test of Kramer (28). Value of each group was compared with value of the intact group. tissue)TreatmentIntact Steroid binding levels (pmol/g

7/H3H]Estradiol3.72

Â±0.30* (32)" Â±1.65(42) Â±0.25 (46) Â±0.06 (47) Adrenalectomy 4.49 Â±0.46 (23) 15.1 Â±1.06(22) 4.32 Â±0.25(12) 0.62 Â±0.08(22) Adrenalectomy + 1.45 Â±0.38C(15)2.29 0.37 Â±0.12C(12)1.54 4.49 Â±0.57(12)5.20 0.38(22)0.32 Â±0.05Â° ovariectomy Hypophysectomy1(9)" Â±0.31 "(9)[3H]R502017.8 Â±0.34C(8)[3H]DEX4.29 Â±0.86 (9)[3H]DHT0.60Â±0.04" Mean Â±S.E. Numbers in parentheses, number of tumors/group. cp<0.01. "p<0.05.

1620 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Binding of Steroid Markers in DMBA Tumors be seen in Table 4 that [3H]DHT binding is reduced after 10. Chan, D. W.. and Slaunwhite. W. R., Jr. The binding of a synthetic progestin. R5020. to transcortin and serum albumin. J. Clin. Endocrinol. Metab., 44: adrenalectomy plus ovariectomy or hypophysectomy. 983-985. 1977. Numerous studies have indicated that DMBA-induced mam 11. DiSorbo. D. M.. Milholland. R. J.. McPartland. R. P., and Rosen. F. Evidence mary tumors provide a useful model for studies of hormone- for a factor in liver cytosol which controls the competition between proges terone and triamcinolone acetonide for the glucocorticoid receptor. Life Sci.. dependent human breast cancer, the most convincing evidence 20. 1593-1598. 1977. pertaining to the finding in both tissues of a close correlation 12. Feil, P. D., Glasser. S. R.. Toft, D. O.. and O Malley. B. W. Progesterone binding in the mouse and rat uterus. Endocrinology, 91: 738-746. 1972. between hormone receptor levels and hormone dependency of 13. Goral, J. E., and Wittliff, J. L. Comparison of glucocorticoid-binding proteins tumor growth and development. While most previous studies in normal and neoplastic mammary tissues of the rat. Biochemistry, 14: have been performed with estrogen, progesterone, and, to 2944-2951, 1975. 14. Goral. J. E.. and Wittliff, J. L. Characteristics of progesterone-binding some extent, prolactin receptors, few data are available on components in neoplastic mammary tissues of the rat. Cancer Res.. 36. glucocorticoid and androgen receptors in these tumors. The 1886-1893, 1976. present data clearly show the presence of 4 distinct classes of 15. Granberg, J. P., and Ballard. P. L. The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other steroid-binding components in cytosol prepared from DMBA- mammalian tissues. Endocrinology, 100: 1160-1168, 1977. induced mammary tumors. The binding properties are charac 16. HÃ¤hnel,R.. and Twaddle. E. Estrogen receptors in human breast cancer. I. Methodology and characterization of receptors. Steroids. 18: 653-680, teristic of receptors: rapid association rate; high affinity; high 1971. specificity; presence of a fast-moving binding component in 17. Holdaway, I. M., and Friesen, H. G. Hormone binding by human mammary low-ionic-strength sucrose gradient, which is transformed into carcinoma. Cancer Res., 37: 1946-1952, 1977. a slow-moving binding component in high-salt buffer; a prota- 18. Horwitz, K. B., Costlow, M. E.. and McGuire. W. L. MCF-7: a human breast cancer cell line with estrogen, androgen. progesterone and glucocorticoid mine sulfate-precipitable binding complex; and adsorption of receptors. Steroids, 26 785-795, 1975. the labeled complex by HAP. Moreover, these binding proteins 19. Horwitz. K. B., and McGuire, W. L. Progesterone and progesterone recep tors in experimental breast cancer. Cancer Res., 37: 1733-1738, 1977. could not be found in the serum of the same animals (data not 20. Horwitz, K. B., and McGuire, W. L. Estrogen and progesterone, their rela shown). tionship in hormone-dependent breast cancer. In: W. L. McGuire. J P. The second series of experiments clearly demonstrates that Raynaud. and E. E. Baulieu (eds.), Progesterone Receptors in Normal and Neoplastic Tissues, Vol. 4. pp. 103-124. New York: Raven Press. 1977. after combined ovariectomy-adrenalectomy or hypophysec 21. Huggins, C., Briziarelli, G.. and Sutton H.. Jr. Rapid induction of mammary tomy alone, [3H]R5020 binding levels are drastically reduced, carcinoma in the rat and the influence of hormones on the tumors. J. Exp. Med., 109: 25-41. 1959. while glucocorticoid binding remains at a constant level, thus 22. Ip, M. M.. Milholland, R. J.. Kim, V., and Rosen, F. Characterization of indicating that, contrary to some earlier suggestions (13), the androgen receptors in 7,12-dimethylbenz(a)anthracene-induced and trans- binding proteins for progestins and glucocorticoids are differ plantable rat mammary tumors. Cancer Res.. 38: 2879-2885. 1978. 23. Jensen. E. V.. De Sombre, E. R.. and Jungblut. P. W. Estrogen receptors in ent in the cytosol of this experimental mammary tumor. hormone-responsive tissues and tumors In: R. W. Wissler, T. L. Dao. and S. Detailed knowledge of the characteristics of estrogen, pro- Wood. Jr. (eds.). Endogenous Factors Influencing Host-Tumor Balance, pp. gestin, androgen, and glucocorticoid binding in cytosol pre 15-30. Chicago: University of Chicago Press, 1967. 24. Kelly. P. A., Asselin. J., Labrie. F., and Raynaud. J. P. Regulation of hormone pared from DMBA-induced tumors should facilitate studies of receptor levels and growth of DMBA-induced mammary tumors by RU1611 7 the hormonal factors involved in the control of growth and and other steroids in the rat. In: W. L. McGuire. J. P. Raynaud. and E. E. Baulieu (eds.). Progesterone Receptors in Normal and Neoplastic Tissues. development of this tumor. Moreover, it should permit a com Vol. 4, pp. 85-101. New York: Raven Press. 1977. parison with the characteristics of steroid binding in normal 25. Kelly. P. A., Bradley. C., Shiu. R. P. C.. Meites, J.. and Friesen. H. G. mammary gland and perhaps reveal possible differences in the Prolactin binding to rat mammary tumor tissue. Proc. Soc. Exp. Biol. Med., 746:816-819, 1974. hormonal control mechanisms regulating tissue growth in nor 26. Koenders, A. J. M., Geurts-Moespot. A . Zolingen, S. J.. and Benraad, T. J. mal and neoplastic tissue. Progesterone and estradiol receptors in DMBA-induced mammary tumors before and after ovariectomy and after subsequent estradiol administration. In: W. L. McGuire, J. P. Raynaud, and E. E. Baulieu (eds.). Progesterone REFERENCES Receptors in Normal and Neoplastic Tissues. Vol. 4, pp. 71-84. New York: Raven Press. 1977. 1. Asselin, J.. Kelly. P. A., CarÃ³n. M. G., and Labrie. F. Control of hormone 27. Korenman, S. G., and Dukes, B. A. Specific estrogen binding by the receptor levels and growth of 7,12-dimethylbenz(a)anthracene-induced cytoplasm of human breast carcinoma. J. Clin. Endocrinol. Metab.. 30: 639- mammary tumors by estrogens, progesterone and prolactin. Endocrinology. 645. 1970. 101: 666-671, 1977. 28. Kramer, C. Y. Extension of multiple range tests to group means with unequal 2. Asselin. J.. and Labrie. F. Effect of estradiol and prolactin on steroid receptor numbers of replications. Biometrics. 12: 307-310. 1956 levels in 7,12-dimethylbenz(a)anthracene-induced mammary tumors and 29. Leung. B. S , and Sasaki, G. H On the mechanism of prolactin and estrogen uterus in the rat. J. Steroid Biochem.. 9: 1079-1082, 1978. action in 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in 3. Asselin, J., Labrie, F.. Gourdeau, Y., Bonne, C., and Raynaud, J. P. Binding the rat. II. In vivo tumor responses and estrogen receptor Endocrinology, of [3H]methyltrienolone (R1881) in rat prostate and human benign prostatic 97:564-572, 1975. hypertrophy (BPH). Steroids. 28 449-459, 1976. 30. Lowry. O. H.. Rosebrough. N. J., Farr. A. L , and Randall, R. J. Protein 4. Asselin. J., Labrie, F., Kelly, P. A., Philibert. D., and Raynaud. J. P. Specific measurement with the Folin phenol reagent. J Biol. Chem.. /93: 265-275, progesterone receptors in dimethylbenz(a)anthracene (DMBAHnduced 1951. mammary tumors. Steroids, 27: 395-404. 1976. 31. Maass. H., Engel, B.. Trams. G., Nowakowski, H.. and Stolzenbach. G. 5. Asselin, J.. and MÃ©lanÃ§on,R. Characteristics of androgen binding in rat Steroid hormone receptors in human breast cancer and the clinical signifi adrenal tissue using [3H]methyltrienolone (R1881) as tracer Steroids. 30. cance J. Steroid Biochem.. 6 743-749. 1975. 591-604. 1977. 32. Markland. F. S., Jr.. and Hutchens, T. W. Characterization of the progestin 6. Asselin. J., MÃ©lanÃ§on,R.,Gourdeau, Y., Labrie, F., Bonne, C., and Raynaud. receptor from lactating mammary glands of the goat. In: W L. McGuire, J. J. P. Specific binding of [3H]methyltrienolone to both progestin and androgen P. Raynaud. and E. E. Baulieu (eds.), Progesterone Receptors in Normal binding components in human benign prostatic hypertrophy (BPH). J. Ste and Neoplastic Tissues. Vol. 4. pp. 23-38. New York: Raven Press, 1977. roid Biochem., 10: 483-486. 1979. 33. McGuire. W. L , and Julian. J. A. Comparison of macromolecular binding of 7. Blondeau. J. P.. CorpÃ©chot. C.. Le Goascogne. C.. Baulieu. E. E., and estradiol in hormone-dependent and hormone-independent rat mammary Robel, P. Androgen receptors in the rat ventral prostate and their hormonal carcinoma. Cancer Res.. 31: 1440-1445, 1971. control. Vitam. Horm.. 33. 319-345. 1975. 34. Pavlik. E. J.. and Coulson. P. B. Hydroxylapatite 'Batch" assay for estrogen 8. Bonne, C.. and Raynaud, J. P. Methyltrienolone. a specific ligand for cellular receptors: increased sensitivity over present receptor assays. J. Steroid androgen receptors. Steroids. 26. 227-232, 1975. Biochem., 7: 357-368, 1976. 9. Bonne. C.. and Raynaud, J. P. Assay of androgen binding sites by exchange 35. Philibert. D.. and Raynaud. J. P. Progesterone binding in the immature with methyltrienolone (R1881). Steroids, 27. 497-507, 1976. mouse and rat uterus. Steroids. 22 89-98. 1973.

MAY 1980 1621

36. Philibert, D., and Raynaud, J. P. Progesterone binding in the immature rabbit 42. Rochefort, H., and Capony, F. Estradiol dependent decrease of binding and guinea pig uterus Endocrinology, 94: 627-632. 1974. inhibition by anti-estrogens (a possible test of receptor activation). Biochem. 37. Philibert, D., and Raynaud, J. P. Cytoplasmic progestin receptors in mouse Biophys. Res. Commun.. 75. 277-285, 1977. uterus In: W. L. McGuire, J. P. Raynaud, and E. E. Baulleu (eds.). Proges 43. Scatchard. G. The attractions of proteins for small molecules and ions. Ann. terone Receptors in Normal and Neoplastic Tissues, Vol. 4, pp. 227-243. N. Y. Acad. Sci., Si. 660-672, 1949. New York: Raven Press, 1977. 44. Suthers, M. B., Pressley. L. A., and Funder, J. W. Glucocorticoid receptors: 38. PichÃ³n, M. F., and Milgrom. E. Characterization and assay of progesterone evidence for a second non-glucocorticoid binding site. Endocrinology, 99. receptor in human mammary carcinoma. Cancer Res.. 37 464-471, 1977. 260-269, 1976 39. Poortman, J., PreÃ±en,J. A. C., Schwarz, F., and Thijssen, J. H. H. Interaction 45. Terenius, L. Estrogen and progesterone binders in human and rat mammary of 5-androstene-3/Ã¯, 17/i-diol with estradiol and dihydrotestosterone recep carcinoma. Eur. J. Cancer, 9. 291-294, 1973. tors in human myometrial and mammary cancer tissue. J. Clin. Endocrinol. 46. Turkington, R. W. Prolactin receptors in mammary carcinoma cells. Cancer Metab., 40: 373-379, 1975. Res., 34: 758-773, 1974. 40. Raynaud. J. P. R5020, a tag for the progestin receptor. In: W. L. McGuire, 47. Vignon, F., and Rochefort, H. Regulation of estrogen receptors in ovarian- J. P. Raynaud, and E. E. Baulieu (eds.), Progesterone Receptors in Normal dependent rat mammary tumors. I. Effects of castration and prolactin. and Neoplastic Tissues, Vol. 4, pp. 9-21. New York: Raven Press, 1977 Endocrinology, 98 722-729, 1976 41. Raynaud, J. P., Ojassoo, T., DelaRue, J C., Magdelenat, H., Martin, P., and 48. Watson, C., Medina, D.. and Clark. J. H. Estrogen receptor characterization Philibert. D. Estrogen and progestin receptors in human breast cancer. In: in a transplantable mammary tumor. Cancer Res.. 37 3344-3348, 1977. W. L. McGuire, J. P. Raynaud, and E. E. Baulieu (eds.). Progesterone 49. Williams. D., and Gorski, J. Equilibrium binding of estradiol by uterine cell Receptors in Normal and Neoplastic Tissues. Vol. 4, pp. 171-191. New suspensions and whole uteri in vitro. Biochemistry, )3. 5537-5542, 1974. York: Raven Press, 1977

1622 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1980 American Association for Cancer Research. Characteristics of Binding to Estrogen, Androgen, Progestin, and Glucocorticoid Receptors in 7,12-Dimethylbenz( a )anthracene-induced Mammary Tumors and Their Hormonal Control

Jacques Asselin, Réjean Melançon, Gilbert Moachon, et al.

Cancer Res 1980;40:1612-1622.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/40/5/1612

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/40/5/1612. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.