Estrogen-induced progesterone receptor in the Syrian hamster kidney. I. Modulation by antiestrogens and androgens.

0013-7227/78/1036-2119$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 103, No. 6 Printed in U.S.A.

Estrogen-Induced Progesterone Receptor in the Syrian Hamster Kidney. I. Modulation by Antiestrogens and Aindrogens* SARA ANTONIA LI AND JONATHAN J. LI Research and Endocrine Services, Veterans Administration Hospital, 55417; and Departments of Pharmacology and Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455 ABSTRACT. Our previous studies demonstrated a specific progesterone receptor in the estrogen-induced and -dependent hamster renal carcinoma and provided evidence that estrogen treatment induces a 4S progesterone-binding component in renal cytosol of the hamster. This latter finding represented the first physiological change thus far reported during the induction period of estrogen renal tumorigenesis. The present studies indicated that the earliest appreciable increase in specific progesterone binding in kidneys of estrogen-primed hamsters occurred after 3 weeks of hormone treatment. The hormone specificity of the progesterone receptor in renal cytosol of estrogenized hamsters was ascertained by competitive binding assay and sucrose gradient centrifugation. These data indicate that the progesterone receptor is specific, since estrogens, androgens, and most progesterone metabolites did not compete appreciably for this receptor at 100-fold excess, whereas unlabeled progesterone markedly inhibited the binding at corresponding concentrations. Binding equilibrium measurements (Ka = 1.25-1.36 x 109 M~') for the estrogeninduced progesterone receptor in the hamster kidney were similar to those obtained for the hamster uterus and renal carcinoma. Prolonged estrogen treatment did not influence the apparent minimal quantities of specific

progesterone binding in hamster liver, lung, heart, or serum as well as in rat kidney. The amount of specific progesterone binding induced by estrogen in the hamster kidney (40-50 fmol/ml protein) was approximately 30 times greater than untreated levels. On the other hand, the concentration of progesterone receptor in the renal carcinoma (1,050 fmol/mg protein) is at least 520fold higher than unprimed levels and approximately 17-20 times greater than estrogen-primed levels. Data presented herein also show that antiestrogens, such as nafoxidine and enclomiphene, but not MER-25 block the induction of specific progesterone binding by estrogen in the hamster kidney. Furthermore, androgen treatment is as effective as antiestrogens in inhibiting the increase in progesterone binding by estrogen. Similarly, hamsters previously treated with estrogen alone for 3-4 months and then subsequently treated with either nafoxidine, enclomiphene, or androgens with continued estrogen treatment resulted in the suppression of the estrogen-induced progesterone receptor response. These results are consistent with previous reports concerning the inability of estrogen to induce renal carcinoma in the hamster in the presence of antiestrogens or androgens. (Endocrinology 103: 2119, 1978).

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T IS NOW well documented that estrogen would expect to be estrogen responsive. Acpriming enhances specific progesterone re- cordingly, previous studies have demonstrated ceptor concentrations in the mammalian that after relatively brief treatment with esuterus, pituitary, chick oviduct, and dimeth- trogen, the amount of progesterone-binding ylbenz(a) anthracene-induced rat mammary activity in such tissues as rat kidney, liver, carcinoma (1-7). Thus far, the presence of heart, and diaphragm remains unaltered (1, 8, estrogen-dependent progesterone-binding sys- 9). tems have only been described in tissues one The presence of specific estrogen-binding proteins in renal cytosol has now been demonstrated in a number of mammalian species Received February 6, 1978. Address requests for reprints to: Dr. Jonathan J. Li, (10-12). Our studies have indicated a marked Veterans Administration Hospital, 54th Street and 48th concentration of tritiated estradiol in the proxAvenue South, Minneapolis, Minnesota 55417. * This work was supported by Grant CA-22008 from imal tubules of estrogenized hamster kidneys the National Cancer Institute, NIH, DHEW and by the and the presence of a specific 4S binding comGeneral Medical Research Fund, Research Service, V.A. ponent in cytosol of these kidneys as well as Hospital. It was presented in part at the 59th Annual Meeting of The Endocrine Society, Chicago, IL, June in the kidney of untreated castrate animals 8-10, 1977. (11, 13). Therefore, it seemed conceivable to 2119

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us that the hamster kidney could perhaps respond to estrogen treatment by a change in specific progesterone receptor concentration despite essentially negligible levels in untreated animals. Recently, we have reported that prolonged estrogen treatment clearly enhances the amount of specific progesterone receptor in the hamster kidney (14). This dramatic increase in cytosolic progesterone receptor in response to estrogen stimulation is the earliest change thus far reported during the latency period of estrogen carcinogenesis in the hamster kidney (15, 16), and we have utilized it as a marker for estrogen responsiveness at this stage. The present report more fully describes the estrogen-dependent progesterone-binding system in the hamster kidney and the effect of concomitant treatment of a number of antiestrogens and androgens on the concentration of progesterone receptor inducible by estrogen. Materials and Methods Chemicals and buffers [l,2,6,7-3H]Progesterone (103 Ci/mmol), [17amethyl-3H]R5020 (86 Ci/mmol), and nonlabeled R50201 were obtained from New England Nuclear Corp. Nonradioactive progesterone was purchased from Calbiochem and all other unlabeled steroids were provided by Sigma Chemical Co. Enclomiphene citrate, MER-25, and nafoxidine hydrochloride were generously supplied by Dr. Alfred Richardson, Jr., Merrell-National Laboratories (Cincinnati, OH) and by Dr. Gary L. Neil, The Upjohn Co. 1

The following trivial names were used: dexamethasone, 9a-fluoro-ll/?,17a,21-trihydroxy-16a-methylpregna-l,4-diene-3,20-dione; triamcinolone acetonide, 9afluoro-ll/M6a,17a,21-tetrahydroxy-17a,21-trihydroxypregna-4-ene-3,20-dione; diethylstilbestrol (DES), 3,4bis(4'-hydroxyphenyl-)-3-hexane; nafoxidine hydrochloride (U-11.100A), l-[2-(p-(3,4-dihydro-6-methoxy-2phenyl-l-naphthyl)phenoxy)ethyl]pyrrolidinehydrochloride; enclomiphene citrate, l-[p-(-/?-diethyl aminoethoxy)phenyl ] -1,2 - diphenyl - 2 - chloroethylene monocitrate; MER-25, l-(p-2-diethyl aminoethoxyphenyl)-l-phenyl-2p-methoxy phenyl ethanol; R5020, 17a,21-dimethyl-19nor-4,9-pregnadiene-3,20-dione; 5/J-pregnanedione, 5/?pregnan-3,20-dione; 5a-pregnanedione, 5a-pregnan-3,20dione; ll/?-hydroxyprogesterone, 4-pregnen-ll/?-ol-3,20 dione; lla-hydroxyprogesterone, 4-pregnen-lla-ol 3,20 dione; 20/?-hydroxyprogesterone, 4-pregnen-20/?-ol-3-one; 20a-hydroxyprogesterone, 4-pregnen-20a-ol-3-one.

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(Kalamazoo, MI), respectively. Trizma base, Norit A, dextran 80, diethiothreitol, bovine serum albumin, and ovalbumin were obtained from Sigma Chemical Co. Ultrapure sucrose (RNase-free) was supplied by Schwarz-Mann. The following buffers, prepared at room temperature, were used: TED buffer (0.01 M Tris-HCl, 0.0015 M EDTA, and 0.001 M dithiothreitol, pH 7.4) and TEDG buffer (TED buffer and 10% glycerol). All buffers were prepared in glass-distilled water, and dithiothreitol was added to the buffers just before use. Animals and treatment Adult castrated male Syrian golden hamsters (LVG:LAK, outbred strain; Charles River Lakeview Hamster Colony, Newfield, NJ), weighing 80-100 g, and castrated male Sprague-Dawley rats (Charles River Breeding Labs, Wilmington, MA), weighing 180-200 g, were used. All animals were acclimated at least 1-2 weeks before treatment or sacrifice. Pure hormone pellets, prepared by Dr. George M. Krause, Copley Pharmaceutical, Inc. (Boston, MA), contained the following compounds: diethylstilbestrol (DES), 20 mg; dihydrotestosterone (5a-DHT), 20 mg; enclomiphene, 30 mg; MER-25, 30 mg; and nafoxidine hydrochloride, 30 mg. Pellets were implanted in the shoulder region, as described previously (17). Groups of castrated hamsters were subjected to the following regimens before study: 1) no treatment; 2) DES alone; 3) DES and MER-25; 4) DES and enclomphene; 5) DES and nafoxidine; 6) enclomiphene alone; 7) DES and 5a-DHT; and 8) 5a-DHT alone. Castrated male rats received either no treatment or were implanted similarly with DES pellets alone. The mean daily absorption of the DES and 5a-DHT pellets was 115.2 ± 18 and 117.6 ± 21 /xg (SE) daily, respectively. The mean daily absorption for the antiestrogens could not be ascertained, since pellets prepared from these compounds formed liquified encapsulations after implantation. However, preliminary studies indicated that the absorption rate of these antiestrogen pellets, particularly enclomiphene and nafoxidine, was more rapid than the other hormone pellets, and it seemed that the presence of an estrogen pellet enhanced the absorption of the antiestrogen. Therefore, additional antiestrogen pellets were implanted into hamsters every 3-4 weeks. In another series of experiments, groups of 3.0-month-old, DES-treated hamsters were divided into subgroups and implanted with either androgen or antiestrogen pellets. For induction of renal carcinoma, DES pellets were implanted every 3 months in each animal


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to insure the maintenance of estrogen levels. Tumor samples were generally taken from animals that had been treated with DES for 8-11 months. All pellets were removed 24-48 h before sacrifice to clear endogenous hormone.

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DC treatment. In addition, there was no appreciable difference in tritiated progesterone binding in serial dilutions of renal cytosols derived from 4.6month DES-treated hamsters (0.5-16 mg/ml protein concentration) when either 10% or 30% glycerol was used in the homogenization buffer (data not shown). Nevertheless, to determine the amount of Cytosol receptor preparation progesterone binding as a function of protein conAnimals were either decapitated or exsanguin- centration, the affinity constant, and number of ated under ether anesthesia and perfused with 0.15 binding sites in renal cytosols from DES-primed M NaCl in TED buffer until the organs to be excised hamsters and renal carcinoma, 30% glycerol was were blanched. Kidney, liver, heart, lung, and renal routinely present in the homogenization buffer of carcinoma cytosol fractions were obtained as de- these tissues to insure maximum binding under scribed previously (11). Briefly, the chilled tissues conditions of low receptor protein concentrations were washed, minced, and homogenized in appro- and prolonged incubation periods. For Scatchard priate dilutions (1 g/1.5-2.5 ml) of TEDG buffer so analyses (22), the renal carcinoma cytosol was dithat protein concentrations in the various tissue luted to 0.6-1.0 mg/rrjl, and the renal cytosols decytosols would be nearly equivalent. The homoge- rived from DES-treated hamsters were reduced to nates were centrifuged at 100,000 x g for 1 h in a a protein concentration of 7.0-10.0 mg/ml. Aliquots Spinco L2-65B ultracentrifuge, and the cytosols of these cytosols were incubated with varying conwere filtered through a millipore filter (0.45 JUM; centrations (1-10 nM), as previously described (14, Millex). In addition to removing residual lipid and 23). Nonspecific binding was subtracted from these particulate matter, the cellulose ester filter re- data using unlabeled cortisol and progesterone at moved any free progesterone, contained in these 100-fold excess at each concentration. Background samples (18). Serum samples were prepared as re- (cytosol without added radioactivity) was subported elsewhere (11). All subsequent steps were tracted from these data. carried out at 0-3 C, except where noted. Protein concentration of the cytosol fractions Sucrose density gradient analyses was determined by the method of Lowry et al. (19) Cytosols (0.2 ml) were layered on 4.6-ml linear using bovine serum albumin as a standard. 5-20% sucrose gradients prepared in TED buffer, pH 7.4, using a Buchler gradient former. The samTreatment of the cytosols ples applied were centrifuged for 17 h at 39,000 rpm The cytosol fractions (0.5 ml) were incubated in in a Spinco 50.1 rotor at 4 C. Gradient tubes were vitro with 2 nM tritiated progesterone or R5020 for pierced with a 20-gauge needle and nine-drop frac90-120 min on ice with gentle agitation. After the tions were collected in scintillation vials. Details of incubation period, appropriate amounts of each radioactivity sample measurements have been presample were assayed for radioactivity and treated viously reported (14, 23, 24). Samples were counted with dextran charcoal (DC) for 12-15 min by the at 5 C in a Packard Tri-Carb liquid scintillation method described earlier (14). Competition studies spectrometer (model 3375) with a counting efficienwere performed by adding unlabeled compounds cyd of about 43% for tritium. diluted, as previously indicated (20), in excess conSedimentation markers for renal cytosols were centration immediately before the addition of la- bovine serum albumin (4.6S) and ovalbumin (3.7S). beled hormone. Preliminary studies indicated a Sedimentation coefficients were determined by the very gradual linear decrease in the amount of la- method of Martin and Ames (25) using the abovebeled progesterone or R5020 binding after increas- mentioned protein standards and catalase (11.3S), ing periods of DC treatment for at least 2 h in y-globulin (70S), and myoglobin (2.0S). hamster renal cytosols containing 10% glycerol compared to a rapid decline in progesterone binding Results observed in the absence of glycerol (data not shown). This latter finding is similar to that re- Relative progesterone binding in untreated ported previously for rat uterine progesterone re- and DES-primed hamster and rat tissues ceptor (21). Sucrose gradient analyses of hamster tissue cytosols and rat kidney indicated that essenProgesterone binding in tissue cytosols from tially no free hormone remained after 12-15 min untreated and estrogen-primed (3.4 months)


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castrate male hamsters and rats were compared after in vitro incubation of the cytosols with either tritiated progesterone or R5020. Figure 1 summarizes the results of these experiments. Although appreciable progesterone binding was detectable in liver cytosols of untreated and DES-treated hamsters, little of this binding seemed to be specific when incubated with 100-fold excess of nonlabeled cortisol and progesterone, and no appreciable changes in specific binding were found with prior estrogen priming. Similarly, cytosols prepared from hamster heart and lung tissue as CPM

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well as serum exhibited little specific progesterone binding, and the amount of binding was also relatively unresponsive to estrogen treatment. In contrast, there was more than a 30fold increase in specific progesterone binding in renal cytosols of these hamsters treated with DES [Fig. 2; 154 cpm ± 2.0 (sE)/mg protein; n = 5] compared to untreated control levels [5 cpm ±1.0 (sE)/mg protein; n = 10]. No detectable change in progesterone binding was observed when similarly prepared DEStreated rat kidney cytosols were examined (Fig. 1). The small amount of specific progesterone binding in rat serum and liver cytosol was also unaffected by DES treatment (data not shown). Parallel incubations of these cytosols with tritiated R5O2O and corresponding unlabeled compound gave essentially identical

i K RAT

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FIG. 1. Specific progesterone binding in cytosol of untreated (A) and 3.4 month DES-treated (B) hamster tissues and rat kidney. Cytosols were incubated with 2 mM tritiated progesterone alone or in combination with unlabeled cortisol and progesterone at 2 x 10~7 M and then subjected to DC treatment for 12 min. Specific progesterone binding (a) was determined by subtracting the amount bound in the presence of 100-fold excess concentration of nonradioactive cortisol and progesterone (E) from the total binding in the presence of [3H]progesterone (a, ES). K, Kidney; L, liver; H, heart; S, serum. Protein concentration ranged between 18-20 mg/ml for all tissues except the heart (14 mg/ml). Cpm, Counts per min/25 /xl cytosol. Values are expressed as the mean ± SE (hamster tissues, n = 10; rat kidney, n = 4).

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PROTEIN CONCENTRATION (mg/ml)

FIG. 2. Effect of protein concentration on specific progesterone binding in renal cytosol of DES-primed hamsters and pure renal carcinoma. Cytosols from renal carcinoma (•) and kidneys from 2.0- (•) and 4.0- (•) month DES-treated hamsters were diluted in varying amounts in TEDG buffer and then incubated with 2 nM tritiated progesterone alone or in combination with nonlabeled cortisol and progesterone at 100-fold excess concentrations. DC treatment (12 min) was used to remove free hormone. Specific progesterone binding at each protein concentration was determined as indicated in Fig. 1. Each point represents the mean of duplicate determinations containing at least four animals in each group.



results. After weekly sampling for specific progesterone binding in renal cytosol of DEStreated hamsters, the earliest appreciable increase in hormone binding in the hamster kidney was detected 3 weeks after estrogen priming. This increase was approximately 4fold above control levels. Progesterone receptor binding specificity in DES-treated hamster renal cytosol The competition for progesterone-binding sites in renal cytosol of DES-primed hamsters by estrogens, antiestrogens, and androgens after dextran charcoal treatment is essentially nil at 100-fold excess concentrations. Similar results were obtained also at 200-fold excess concentrations of 5a-DHT and enclomiphene. However, both natural and synthetic glucocorticoids as well as aldosterone displaced some of the tritiated progesterone at 100-fold excess. The competition by progesterone and progesterone metabolites was essentially similaj to that estimated for the progesterone receptor in the renal carcinoma and uterus (14). Understandably, progesterone and R5020 were the most effective competitors, while 11/?-, 17a-, and 20-/?-hydroxyprogesterone exhibited appreciable competition for the progesterone binding in DES-primed hamsters (Table 1). Based on our competition experiments, we have estimated that approximately 15-20% of the progesterone binding in the 4S region of the gradient is nonspecific in renal cytosol of estrogenized hamsters. Relative concentration and Scatchard analyses of the progesterone receptor in DEStreated kidney and renal carcinoma cytosols Figure 2 indicates that the amount of specific progesterone binding in renal cytosols derived from 2.0- and 4.0-month DES-treated hamsters is essentially linear with decreasing protein concentrations from 15-1 mg/ml. In contrast, a linear relationship between specifically bound progesterone and protein concentration was not attained in renal tumor cytosols until protein concentrations were reduced to less than 1 mg/ml. These data indicate that the concentration of progesterone receptor in

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TABLE 1. Competitive binding activity of various compounds for [3H]progesterone receptor sites in renal cytosol from DES-primed hamsters Nonradioactive compounds 17/?-Estradiol DES

% Inhibition 9 0 0 4 1 6 33 34 25 35 82 77 27 4 1 47 44 14 47

Nafoxidine hydrochloride Enclomiphene citrate 5a-DHT Testosterone Cortisol Dexamethasone Triamcinolone acetonide Aldosterone Progesterone R5020 5a-Pregnanedione 5/?-Pregnanedione 1 la-Hydroxyprogesterone 1 l/?-Hydroxyprogesterone 17a-Hydroxyprogesterone 20a-Hydroxyprogesterone 20/?-Hydroxyprogesterone Aliquots of renal cytosol (15 mg protein/ml) from 3.0 to 3.5-month DES-treated hamsters were incubated at 0-3 C in a total volume of 0.5 ml with 2 nM tritiated progesterone alone or in combination with competing nonradioactive steroids for 90 min. Nonlabeled compounds were added at 2 x 10"' M (100-fold). Free progesterone was removed by DC adsorption (12 min). [:JH]Progesterone concentration in these cytosols, without competitor, corresponded to 0% inhibition. Values are based on the mean of triplicate determinations in separate experiments containing at least eight animals in each group.

the renal tumor [2600 ± 86 (sE)/mg protein; n = 5] is at least 520 times higher than unprimed levels and approximately 17-20 times greater than DES-treated kidney levels. Calculations made from the same data give progesterone receptor concentration values of 1050, 52, and 36 fmol/mg protein for tumor and renal cytosols of 4.0- and 2.0-month DEStreated hamsters, respectively. These values are in good agreement with our previous estimations. Aliquots of similarly prepared cytosols from kidneys of 2.0- to 4.0-month DESprimed hamsters and renal carcinoma were incubated with appropriate concentrations (1-10 nM) of radioactive progesterone to determine the affinity constant (Ka) and the number of binding sites. Data derived from these Scatchard analyses indicate that the affinity constant for progesterone receptor in DES-treated hamster kidney and renal carcinoma are similar (Ka = 1.25-1.42 X 109 M"1),


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and the number of binding sites are 1.6-2.6 X compared to animals treated with DES alone 10~10 and 3.4 x 10~9 M binding sites/mg pro- (Fig. 4). However, hamsters similarly treated with either nafoxidine or enclomiphene extein, respectively (Fig. 3). hibited essentially no estrogen-induced proEffect of concomitant antiestrogen treatment gesterone receptor response. None of the anon the estrogen-induced progesterone recep- tiestrogens, when administered alone, were capable of eliciting an increase in progesterone tor in the hamster kidney receptor in the hamster kidney. In addition, Groups of castrate male hamsters were im- animals that had been stimulated with estroplanted with pellets containing either MER- gen alone for 3 months, a period sufficient to 25, nafoxidine hydrochloride, or enclomiphene produce maximal progesterone receptor in the citrate and DES for 3 months to examine the kidney, were then treated for 1 month with effect of these antiestrogens on the induction the above antiestrogens in combination with of specifically bound progesterone by estrogen continued DES treatment. Under these conin hamster renal cytosols. Simultaneous treat- ditions, the amount of specifically bound proment with MER-25 and DES did not affect gesterone which had accrued during prior the amount of cytosolic progesterone receptor DES stimulation was reduced to nearly uninduced by estrogen in the hamster kidney treated levels by both nafoxidine and enclomiphene but not by MER-25 treatment. • K 8 = 1.42

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BS= 3.39 x 10~ M O Ka = 136 x 1 0 9 M - ' BS= 2.61 x 1 0 - 1 o M O Ka

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Effect of concomitant androgen treatment on the estrogen-induced progesterone receptor in the hamster kidney Castrated hamsters were also treated with a combination of 5a-DHT and DES pellets for 4.3 months and renal cytosols from these animals were assayed for specific progesterone CPM 600

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FIG. 3. Scatchard plot analysis of specific cytoplasmic progesterone receptor in primary renal carcinoma (•), 2.0- (©), and 4.0- (O) month DES-treated kidney. Incubations were carried out for 22 h at 0-3 C to attain equilibrium and then the samples were treated with DC (12 min). Nonspecific binding was subtracted from these data using unlabeled cortisol and progesterone at 100-fold excess concentrations. Protein concentration was 1.0,7.75, and 5.75 mg/ml for renal tumor and 2.0- and 4.0-month DES-primed kidneys, respectively. Each point represents the mean of triplicate determinations.

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FIG. 4. Effect of antiestrogens on the estrogen-induced progesterone receptor in the hamster kidney. Renal cytosol from untreated control (C), 3.0-month DES-treated, and hamsters treated simultaneously with DES and either MER-25, enclomiphene, or nafoxidine for 3.0 months was incubated alone or in combination with unlabeled cortisol and progesterone at 100-fold excess concentrations. Specific progesterone binding (•) was calculated by subtracting the total amount bound by tritiated progesterone (•, Ea) in the presence of nonlabeled steroids (ED). Protein concentration of the various groups was 17-19 mg/ml. Cpm, Counts per min/25 /U cytosol. Values are expressed as the mean ± SE (n = 3).



binding. Results of these experiments clearly indicate that 5a-DHT is capable of opposing the estrogen induction of progesterone receptor even in the presence of additional DES in one group of animals (Fig. 5). Substitution of a testosterone proprionate pellet for 5a-DHT resulted in a similar inhibition of progesterone receptor induction. Renal cytosols assayed from animals similarly treated with either 5aDHT or testosterone alone did not reveal any augmentation of specific progesterone-binding activity. Sucrose gradient analyses of renal cytosol from combined androgen- and estrogen-primed hamsters indicate that the 4S peak is reduced compared to kidney cytosol of DES-treated animals (Fig. 6A), and competition with 100-fold excess unlabeled cortisol and progesterone indicated no appreciable specific binding in this region of the gradient (Fig. 6B). The latter sucrose gradient profiles also reflect the amount of progesterone binding activity in the untreated hamster kidney and are similar to those obtained from kidneys of either nafoxidine- or enclomiphene-estrogen-treated hamsters. Moreover, both 5aDHT and testosterone in the presence of DES reduced the amount of specific progesterone binding to untreated levels after 1 month of

C

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FIG. 5. Effect of 5a-DHT treatment on the estrogen-induced progesterone receptor in the hamster kidney. Renal cytosol from untreated control (C), 4.3-month DEStreated, 5o-DHT-treated, and 4.3-month DES- and 5aDHT-treated hamsters was incubated with 2 nM tritiated progesterone alone or in combination with nonlabeled cortisol and progesterone at 100-fold excess concentrations. Numbers in parentheses indicate the quantity of hormone pellets implanted. Specific progesterone binding (a) was calculated as previously indicated in Fig. 1. Protein concentration of the various groups ranged between 18-20 mg/ml. Values are expressed as the mean ± SE (n = 3).

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treatment in kidneys of animals that had received estrogen alone for 4.3 months. Discussion We have recently described a progesteronebinding component in renal cytosols of estrogen-primed castrate male hamsters (14). Present studies correlating tissue response to estrogen treatment and enhanced specific progesterone binding, competitive binding, affinity, and sedimentation characteristics of the progesterone receptor in kidneys of estrogenized hamsters indicate it is similar to other progesterone-binding systems previously described (1, 3, 9, 21, 26). The sedimentation of the progesterone receptor in estrogen-primed hamster kidneys as a 4S component probably reflects in part a low binding site concentration compared to the renal carcinoma. In support of this contention, studies in our laboratory indicate that serial dilution of the 7S progesterone receptor in the renal carcinoma resulted in considerable change in sedimentation coefficient (7S to 4-5S; Li, J. J., S. L. Yun, and S. A. Li, unpublished observations). Consistent with this notion are reports indicating a change in concentration of the uterine cytosol progesterone receptor during the estrus cycle, which is reflected as an alteration in sedimentation coefficient value [a 7S binder in proestrus and a 4S binder in diestrus (21, 26)]. In addition, a considerable shift in sedimentation coefficient value has been shown when calf uterine cytosol estrogen receptor was diluted 30-fold (27). Nevertheless, the relationship between receptor sedimentation value and protein concentration remains unclear since other factors are also probably involved. Experiments reported herein modulating the estrogen-induced progesterone receptor concentration with certain antiestrogens and androgens provide a means of monitoring the effect of at least some of the hormonal influences on the progression of estrogen-induced carcinogenesis in the hamster kidney. In this regard, we have previously reported that antiestrogens, such as nafoxidine and enclomiphene, which compete effectively for the kid-


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2126 FIG. 6. Sucrose gradient centrifugation of renal cytosols from (A) 4.3-month DES-primed hamsters and (B) 4.3-month DES and 5aDHT-treated hamsters. Aliquots of 0.2 ml were layered on 5-20% sucrose gradients from cytosols of DES-treated kidney and [ ^ p r o gesterone (•), DES-treated kidney and nonlabeled cortisol and progesterone and [3H]progesterone (O), DES and 5a-DHTtreated kidney and [3H]progesterone (•), and DES and 5a-DHTtreated kidney and nonlabeled cortisol and progesterone and [3H]progesterone (•). Protein concentration was 20 mg/ml for all samples. Bovine serum albumin (BSA) and ovalbumin (OA) served as sedimentation coefficient standards.

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ney 4S estrogen receptor in unprimed and estrogen-primed hamsters, completely block renal tumorigenesis when administered together with estrogen, whereas MER-25, which exhibits no appreciably competition for the renal estrogen receptor, does not block this transformation in the hamster kidney under similar conditions (28). Accordingly, it is not surprising that both nafoxidine and enclomiphene, but not MER-25, effectively suppress the induction of progesterone receptor by estrogen in the hamster kidney. This effect would be consistent with the ability of these antiestrogens to block tumor induction. Interestingly, Kirkman (29) indicated that androgens, when administered together with estrogen, are capable of inhibiting renal tumorigenesis. In this regard, our studies demonstrate that simultaneous treatment with androgen and estrogen results in the suppression of the estrogen-induced progesterone receptor response and this treatment may be a more affective inhibitor than antiestrogen. It is clear that the inhibitory actions of androgen and antiestrogen on the estrogen-induced progesterone receptor response are through different nechanisms, since androgens do not compete ippreciably for the estrogen receptor in the lamster kidney even at high concentrations.

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Preliminary results in our laboratory indicate that the ability of estrogen to induce progesterone receptor in the kidney was not appreciably impaired in hamsters that had been exposed to combined progesterone and estrogen for 4 months compared to animals receiving estrogen alone for the same period. Since it is known that progesterone is also capable of preventing estrogen-induced renal tumorigenesis in the hamster (29), our findings provide at least an initial mechanism for this inhibition to occur in the absence of any specific progesterone binding in kidneys of untreated hamsters. Our data indicate that progesterone is able to bind to markedly increased quantities of its receptor as a result of estrogen induction even in the presence of progesterone and, perhaps as a consequence of this increased hormone-receptor interaction, produce its inhibitory effects on renal tumorigenesis. It would be of interest if similar modulations of the estrogen-primed progesterone receptor in other systems could be effected by androgen and antiestrogen treatment, as in the hamster kidney. Such studies would be particularly interesting if tumor systems were used which exhibit enhanced progesterone binding as a result of estrogen treatment. Modulation of



this estrogen response could aid in determining the effect of various hormonal agents on tumor induction and regression. Acknowledgment We are grateful for the excellent technical assistance of Mr. Terry L. Cuthbertson, Research Service, V.A. Hospital, in these studies.

References 1. Milgrom, E., and E. E. Baulieu, Progesterone in uterus and plasma. I. Binding in rat uterus 105,000 g supernatant, Endocrinology 87: 276,1970. 2. Feil, P. D., S. R. Glasser, D. O. Toft, and B. W. O'Malley, Progesterone binding in the mouse and rat uterus, Endocrinology 91: 738,1972. 3. Reel, J. R., and Y. Shih, Oestrogen-inducible uterine progesterone receptors. Characteristics in the ovariectomized and immature and adult hamster, Ada Endocrinol 80: 344, 1975. 4. Rao, B. R., W. G. Wiest, and W. M. Allen, Progesterone "receptor" in rabbit uterus. I. Characterization and estradiol-17/? augmentation, Endocrinology 92: 1229, 1973. 5. Leavitt, W. W., J. J. Chen, and T. C. Allen, Regulation of progesterone receptor formation by estrogen action, Ann NYAcad Sci 286: 210, 1977. 6. Sherman, M. R., P. L. Corvol, and B. W. O'Malley, Progesterone-binding components of chick oviduct. I. Preliminary characterization of cytoplasmic components, JBiol Chem 245: 6085, 1970. 7. Koenders, A. J. M., A. Geurts-Moespot, S. J. Zolingen, and Th. J. Benraad, Progesterone and estradiol receptors in DMBA-induced mammary tumors before and after ovariectomy and after subsequent estradiol administration, In McGuire, W. L., J. P. Raynaud, and E.-E. Baulieu (eds.), Progesterone Receptors in Normal and Neoplastic Tissues, Raven Press, New York, 1977, p. 71. 8. Feil, P. D., and C. W. Bardin, Cytoplasmic and nuclear progesterone receptors in the guinea pig uterus, Endocrinology 97: 1398, 1975. 9. Falk, R. J., and C. W. Bardin, Uptake of tritiated progesterone by the uterus of the ovariectomized guinea pig, Endocrinology 86: 1059, 1970. 10. King, R. J. B., Fixation of steroids to receptors, Arch Anat Microsc Morphol Exp (Suppl 3, 4) 56: 570, 1967. 11. Li, J. J., D. J. Talley, S. A. Li, and C. A. Villee, An estrogen binding protein in the renal cytosol of the intact, castrated, and estrogenized golden hamsters, Endocrinology 95: 1106, 1974. 12. Bullock, L. P., and C. W. Bardin, The presence of estrogen receptor in kidneys from normal and androgen-insensitive tfm/y mice, Endocrinology 97: 1106, 1975.

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Structural requirements for the in vitro transformation of Syrian hamster embryo cells by stilbene estrogens and triphenylethylene-type antiestrogens.

Significance of the progesterone receptor in the estrogen-induced and -dependent renal tumor of the Syrian golden hamster.

Mechanism of action of antiestrogens and androgens on the estrogen receptor.

Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors.

Metabolism of androgens by the baboon kidney.

Early periodontal disease in the Syrian hamster.

Lack of modulation effects of praziquantel on DMN-induced lesion development in the Syrian hamster liver.

Characterization of a specific progesterone receptor in decidualized hamster uterus.

Immunohistochemical localization of antioxidant enzymes in adult Syrian hamster tissues and during kidney development.

9S binding protein for androgens and progesterone.

Streptozotocin and renal amyloidosis in the Syrian hamster.

Serum gonadotropins and follicular development in the Syrian hamster.

Female-biased anorexia and anxiety in the Syrian hamster.

Morphological transformation of Syrian hamster embryo cells by mezerein.

The production of progesterone, androgens and oestrogens by human granulosa cells in vitro and in vivo.

Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium.

Polarizing activity in the developing limb of the Syrian hamster.

The intrarenal distribution of 125I-albumin in the Syrian hamster.

Vascularization induced in the cheek pouch of the Syrian hamster by tumor and nontumor substances.

The cholesterol-raising effect of coffee in the Syrian hamster.

Modulation of neointimal lesion formation by endogenous androgens is independent of vascular androgen receptor.

Acute cadmium chloride-induced renal toxicity in the Syrian hamster.

Morphine withdrawal induced behavior in the Syrian hamster (Mesocricetus auratus).

Amyloidosis and female protein in the Syrian hamster. Concurrent regulation by sex hormones.