Steroid metabolism and binding activity in a murine renal tumor cell line

J. steroid Biochem.

Vol. 21, NO. 5, pp. 505-511, 1984 Printed in Great Britain. All rights reserved

STEROID

1X22-4731/84 163.00 + 0.00 Copyright 0 1984 Pergamon Press Ltd

METABOLISM AND BINDING ACTIVITY MURINE RENAL TUMOR CELL LINE

IN A

SHEILAM. JUDGE*,MARTHA M. PHILLIPSand SHUIXJNG LIAO~ The Ben May Laboratory for Cancer Research and The Department of Bi~h~ist~, The University of Chicago, 950 E. 59th Street, Chicago, IL 60637, U.S.A. (Received 19 December

1983)

Summary-The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextrancoated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific Sadihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, RI881 and GRG2058 all completely inhibited rH]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-liter assay, with an estimated II, for methyIt~enolone (Rl881) of 5 nM at 0°C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a xl, of 6nM for Dx and #nM for corticosterone at 0°C. Glucocorticoid receptor levels were estimated to be 182 fmoi/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and (‘H]DHT by RAG cells was examined I,4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.

INTRODUCTION Advanced renal carcinoma is characterized by high mortality rates and apparent refractoriness to available chemothera~utic and radiation treatments [I]. Any treatment protocol, including hormonal therapy, that would benefit even a small percentage of patients would be valuable. Recent studies suggest that only a small percentage of renal neoplasms respond to hormonal manipulation [2]. However, such studies may underestimate the efficacy of hormonal therapy at early stages of the disease when the tumor is more likely to be hormone-dependent. Screening tumors at early stages of the disease for hormonal sensitivity would ensure that those patients most likely to benefit

from hormonal therapy would receive such treatment. Steroid receptor concentration in tumor tissue is the conventional criterion for determining the likelihood of a response to hormonal therapy and has been used as a guide in the treatment of breast [3,4] and prostatic cancers [5,6]. However, the application of similar stratagems to kidney cancer has met with little success. The current use of estrogen and progesterone receptor levels as prognostic indices for kidney cancer are based upon observations made on the diethylstilbestrol-induced renal tumor in Syrian hamster. These tumors are dependent upon estrogens for growth and are inhibited by medroxyprogesterone acetate /7]. Receptors for estrogens and progestins have been detected in these tumors [8,9], which led to the testing of human renal *Present address: Division of Urology, Department of Surgery, Unive~ity of Chicago, Chicago, IL 60637, tumors for similar properties. The Syrian hamster U.S.A. renal tumor model, however, appears to correlate tTo whom correspondence should be addressed. poorly with clinical experience with human renal The following trivial names and abbreviations have been tumors, which have been found to contain only Iow used: T, testosterone; DHT, Sa-dihydrotestosterlevels of estrogen and progesterone receptors or lack one (17fi-hydroxy-SK-androstan-3-one); cyproterone them altogether [9, 10, 111.Furthermore, there is little acetate, 17a-hydroxy-6-chloro-l,2K-methylene-4,6-pregnadiene-3,20-dione 17a-acetate; androstene dione, evidence to suggest that estrogens or progestins play 4-androstene-3,17-dione; androstanediol 3K,17/% an important role in the function or growth of dihydroxy-Sa-androstane; androstanedione, SK-androstane-3,1 ‘I-dione; Dx, dexamethasone (9-fluoro- 11/3,17a, normal renal tissue. Most observed effects of such progestins as medroxyprogesterone acetate on kidney 2 1-trihydroxy- 16~ -methyl-I ,4-pregnadiene-3,20-dione); medroxyprogesterone acetate, (f7a-acetoxy-tia-methylcell function appear to be mediated by the androgen ~pm~ene-3,2~~one); Ri 88 1, methyltrienoione (I 78 - receptor [12]. h~dr~xy-l7a~ethyI~,9,1 I-estratrien&ne); OR0 2Oj8, In the present study we use a cloned line of murine (l~-ethyl-2l-hydroxy-l9-nor-pm~-4-en-3,2~ione); renal tumor cells (RAG), that was developed as an DMEM, Dulbecco’s minimal essential medium; FBS, fetal bovine serum. 8-azaguanine resistant mutant of the Renal 2a SO5

SHEKA

506

M.

line f 131,as an in vitro model for renal neoplasia. The parental Renal 2a line was derived from a renal adenocarcinoma that developed spontaneously in a male Balb/cf/Cd mouse [14]. The RAG cell line has been characterized with respect to karyotype, morphology, growth in soft agar and nude mice, and is available as a registered cell line through the American Type Culture Collection. In the work reported here, we found that RAG cells possess androgen and glucocorticoid receptors whereas we were unable to detect receptors for estrogen and progestins. The metabolism of androgens differed markedly from the patterns of androgen metabolism reported for normal mouse kidney in viva. MATERIALS AND METHODS

Tissue culture medium and fetal bovine serum were obtained from Grand Island Biological Co. (Grand Island, NY). [6,7-3H]ORG 2058 (54 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). 17fi[6,7-‘Hlestradiol(52 Ci/mmol), [ 1,2,4,5,6,7, 17-3H] DHT ( 179 Ci/mmol), [ 1,2,6,7-‘HItestosterone (102~i/mmol), [17a-me~y13~R1881 ~87Ci~mmol), and [6,7-3H]dexamethasone (38 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Hydroxylapatite (Bio-Gel HT) was purchased from Bio-Rad Laboratories (Richmond, CA). Silica gel Red&plates (250 microns) were obtained from Fischer (Itasca, IL). Cell culture RAG cells at passage number 263 were obtained from the American Type Culture Collection (Rockville, MD). The cells were maintained in Eagle’s minimal essential medium with Hank’s salts supplemented with 10% fetal bovine serum, 2 x amino acids and vitamins, in closed monolayer cultures at 37°C. Confluent cultures of RAG cells were subcultured by treating the cells briefly with 0.25% trypsin to remove the cells from the culture flask, followed by a 1:4 dilution of the cells in the fresh culture medium and seeding into new flasks. A subline, designated RAG-2, was obtained in our laboratory that exhibits modest differences in its karyotype from the parent line (S. Judge, unpublished data). RAG-2 cells were maintained as monolayer cultures at 37°C in Dulbecco’s modified minimal essential medium supplements with 10% fetal bovine serum. RAG-2 cell cultures were passaged every 4 days by a brief treatment with 0.25% trypsin to remove the cells from the culture flask followed by a 1:6 or 1: 10 dilution with fresh culture medium and seeding into new flasks. Preparation of cell cytosof

Confluent cultures to be used for receptor assay were exposed to medium containing 5% charcoalstripped fetal bovine serum [15] for 18 h before cell harvest to reduce the level of endogeneous androgens.

JUDGEet al.

The cells were harvested by repeated washing of the cultures with ice-cold calcium- and magnesium-free phosphate buffered saline (4 mM KCl, 137 mM NaCl and 11.1 mM glucose in 0.608 mM Na,HPG& 147 mM KH2P0, buffer) followed by treatment with 0.15 M EDTA in phosphate buffered saline. Cell suspensions were centrifuged at 8OOg for 3-4min at 0°C. The cell pellets were washed saline and once with phosphate-buffered twice with homogenization buffer (TED,G,,Mo,,: 1OmM Tris-HCl and 1.5 mM EDTA, pH 7.4 containing 1 mM dithiothreitol, 10% glycerol and 10 mM sodium molybdate). The cells were resuspended in 5 ml TED2G10Mo,, and then homogenized by 15 s bursts 5 times at a setting of 6 on a Brinkman Polytron. The extent of cell disruption (usually 9&95x) achieved by this procedure was determined by trypan-blue dye exclusion by the cells under phase-contrast. The homogenates were centrifuged at 125,OOOgfor 30min at 0°C and the resulting supernatant fractions were used as cytosol for all subsequent receptor assays. The protein content of the cytosols was determined by the method of Bradford[ 161. Hydroxylapatite-filter assay

Saturation analyses and competition studies were carried out using a hydroxylapatite-filter assay [ 171to measure the binding of [‘HIsteroid to receptor. For these assays 0.2 ml cytosol ~containing 3-6 mg protein) was incubated with rH]steroid plus or minus a IOO-fold molar excess of the appropriate unlabeled steroid in a final volume of 0.5 ml in TED,G,,Mo,, for 2 h at 0°C. Then 0.5 ml of a 60% (v/v) slurry of hydroxylapatite in 10 mM sodium phosphate20mM Tris-HCl buffer, pH 7.2 was added to each sample. The sampies were vortexed and then incubated for 20 min at 0°C. Then 5 ml of wash buffer, consisting of 0.1% Triton X-100 and 0.1 M KC1 in 50 mM Tris-50 mM sodium phosphate buffer, pH 7.2, was added to each sample and rapidly mixed. The contents of each tube was poured over a Whatman GF-C filter in a Millipore filtration unit under constant vacuum. The tubes were washed once with 5 ml of wash buffer and their contents were transferred to the filters. The filters were then washed five times with 5 ml of wash buffer. The filters were then removed into vials containing 10 ml scintillation fiuid and counted in a Packard scintillation counter. Specific binding activity was determined by subtracting the radioactivity in the samples that were incubated with [3H]steroid plus excess unlabeled steroid from the radioactivity obtained with corresponding samples that were incubated with [3H]steroid in the absence of unlabeled steroid. Dextran-coated receptor

charcoal

assay for

glucocorticoid

The glucocorticoid receptor activity of RAG-2 cell cytosol was examined by dextran-coated charcoal

507

Steroid receptors in renal tumor ceils assay (DCC assay). Cytosol (0.2 ml) was incubated with rH]Dx with and without the appropriate unlabeled steroid in a final volume of 0.25ml of TED,G,,Mo,, buffer for 2h at 4°C. Then 0.25 ml of DCC suspension (0.1% dextran T-70, 1% ‘acidwashed Norit A in 0.01 M Tris-HCl, pH 8.0) was added to each sample with vortexing. The samples were incubated with DCC for 30min at 4°C. DCC was removed by centrifugation at 5000 rpm for 20 min at 4°C in a Sorvall HS-4 rotor and 0.35 ml of the supernatant was removed for counting. Specific binding was calculated by subtracting the radioactivity dpm bound in the presence of IOO-fold molar excess of unlabeled steroid from that bound in the absence of unlabeled steroid. Sucrose gradient analysis of steroid receptor content Cytosols (0.2 ml) were incubated with [3H]steroid with or without unlabeled steroid in a final volume of 0.25 ml in TEDzG,,,, pH 7.4 for 2 h at 0°C. The samples were then transferred onto pellets of DCC (prepared by centrifuging 1 ml of a suspension of 0.25% Norit A, 0.0025% dextran T-70 in 0.01 M Tri-HCl, pH 8.0 at 2000g for 10 min), vortexed, and incubated for 15 min at 0°C. The samples were centrifuged at 2000g for 10min to pellet the DCC and 0.2 ml aliquots of the su~~atan~ were transferred onto linear 5-20x sucrose gradients that were made up in TEDrG,o, pH 7.4 (some gradients also contained 0.6 M KC1 or 10 mM sodium molybdate). Gradients were centrifuged at 315,000g for 20 h at 0°C in a Beckman SW-60 rotor and then the gradients were fractionated from the bottom into 0.2ml fractions and counted. Androgen metabolism studies Confluent ceil cultures were incubated for various periods of time with either 0.1 PM [3H]testosterone (102 Ci/mmol) or [-‘H]DHT (179 Cifmmol) in serumfree Dulbecco’s minimal essential medium. The medium was then collected and stored at -20°C until extraction. The cells were harvested by a brief treatment with 0.25% trypsin and centrifuged at 800g for 4 min, resuspended in 3 ml of cold 1.5 mM EDTA in 10 mM Tris-HCl, pH 7.4 and homogenized as described above. The homogenates were centrifuged at 600g for 20 min at 4°C. The supernatants were referred to as cytosol fractions and the pellet as nuclear-microsomal fraction (N-M). The pellets were resuspended in Tris-EDTA buffer and both cytosol and N-M fractions were extracted with 2 vol of methylene chloride. The methylene chloride was removed by flushing the extracts with N2, then the residues were redissolved in 1 ml of methylene chloride and 10,000 cpm of [4-14C]testosterone were added to each sample. The samples were appiied to silica gel G-coated thin layer plates and the plates were developed in chloroform&hyl acetate-methanol (85: 15:3, v/v). A mixture of standards (testosterone, DHT, androstanediol, an-

drostanedione and androsten~ione) was cochromatographed with the samples. Another mixture of standards and individual standards were applied to a second TLC plate that was developed simultaneously with the sample plate. The chromatograms that contained the ‘H-labeled samples were divided into 0.5 cm sections for each sample lane and the silica gel in these sections was scraped directly into scintillation vials, Scintillation fluid (10 ml) was added to each vial and the samples were counted on a double-label program for 14C and 3H. The Rf value for each peak of labeled material was compared to the internal that of the standards and [‘4C]testosterone standard. The relative proportion of each peak of [3H]steroid was calculated by dividing the dpm in that peak by the sum of the dpm in all of the peaks obtained for that sample. RESULTS

To determine if murine renal tumor cells in continuous culture contain receptors for steroid hormones cytosol preparations (7-10 mg protein) were incubated with either [‘H]R1881, [3H]ORG 2058, 17/?-[3H]estradiol or f’H]Dx for 2 h at 0°C with or without a IOO-fold molar excess of the appropriate unlabeled steroid. Fo~lo~ng a brief treatment with dextran-coated charcoal to remove free steroid the samples were layered over linear 5-20x sucrose gradients and centrifuged. Analysis of the gradient fractions post centrifugation showed that there was little specific binding activity for either [‘H]ORG 2058 or 17J?j3H]estradiol, whereas a distinct peak of specific binding activity could be detected in the 8 S region of the gradients containing samples that were incubated with [3H]R1881 or [3H]Dx (Fig. 1). All of the samples that were analyzed in this fashion contained a peak of nonspecific binding activity in the 4s region of the gradient that co-sedimented with the bovine [14C]albumin standard. SDS-polya~y~amide gel analysis [I81 of RAG cytosol proteins revealed a band that co-migrated with albumin standards. Further examination of androgen binding activity on sucrose gradients showed that specific [3H]DHT binding activity sedimented as a 4s peak in gradients that contained 0.6 M KCl, whereas in the absence of salt DHT-receptor sedimented in the 8s region of the gradient. The inclusion of Na,MoO, in the gradient buffer resulted in the appearance of a 10s form of the receptor (not shown). The specificity of f3H]DHT binding activity was tested by incubation with a lOO-fold molar excess of either DHT, R1881, diethylstilbestrol, RS020 or Dx. DHT, RI881 and R5020 all completely inhibited E3H]DHT binding whereas DES and Dx were ineffective. Saturation analyses, in which various concentrations of [3H]steroid were incubated with cytosol with or without a lOO-fold excess of unlabeled steroid, were performed by hydroxylapatite-filter assay. As shown in Fig. 2 these studies corroborated the

SHEEAM.

508

I A

4000

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et al.

E

4

3000

~

Fractton

No

Fig. f Sucrose gradient analysis of the receptor content of RAG cytosoi. RAG cell cytosol (8 mg protein/ml in TED2G,,Mo,,, pH 7.5) was incubated with (A) 4OnM 17/Ij3H]estradiol with or without 4 p M 17/3estradiol, (B) 20 nM [3H]ORG 2058 with and without 2 p M cold ORG 2058, (C) 50 nM Dx with or without 5 @I cold Dx, or (D) 20nM [‘H]R1881 with or without 2pM R1881, for 2 h at 4”C, then treated with DCC to remove free steroid and layered on linear 5-2PA sucrose gradients. Gradients were centrifuged at 150,000g for 20 h at 4°C then fractionated from bottom to top. Symbols: with (A) or without (Of additional nonradioactive steroids,

results obtained by sucrose gradient analysis of steroid binding activity in RAG-2 cytosol. High affinity, specific binding activity was detected only in those samples that were incubated with either t3HjDx or [W]R 188 1, whereas significant [3H&stradiol and

[‘H]ORG 2058 binding activity appeared to be absent in RAG-2 cytosol.

Scatchard analysis of the data from the saturation experiments provided an estimate of the binding affinity for Dx at 4°C as 2.4 nM. The concentration of Dx bin~ng sites in RAG-2 cytosol obtained by this method was approx. 5Ofmol/mg cytosol protein. Comparison of the receptor levels measured by this method to those obtained by sucrose gradient anal&

A

B/F .20

.

16 .I2 08

4 2.0 i.5 Bound (10”~ hi)

04 ‘I‘:; 0.5 Bound ( IO% M 1

1.0

Fig. 2. Saturation analysis of 13H]Dx and j3H]R1881 binding by hydroxylapatite-alter assay. (A) Various concentrations of [)H]Dx were incubated with RAG-2 cytosol(3.4 mg protein/ml) with or without 100-fold molar excess Dx in TED,G,,Mo,e, pH 7.5 for 2 h at 4°C. (B) Various concentrations of [rHlR1881 were incubated with RAG-2 cytosol (5.56mg protein/ml) with or without NO-fold molar excess R1881 in TED,G,,Mo,,, pH 7.5 for 2 h at 4°C. Symbols: with (A) or without (0) excess nonradioactive steroids; specific binding (0).

so9

Steroid receptors in renal tumor cells Table

1. Specificity of [‘HfR1881 binding sites in RAG-2 cells*

Table 2. Metabolism of [‘Hjtestosterone by RAG-2 cell cultures* % Metabolite recovered? 4h lh

Relative binding affinity (RBAft

Competitor

1.0 0.4 0.3 0.35 0.23 0.10 0.038 0.00005

R1881 Testosterone Sa-Dihydrotestosterone Cyproterone acetate Medroxyprogesterone acetate 17/?-Estradiol Progesterone 0RQ 20%

Standard --Androstanediol Testosterone Sa-Dihydrotestosterone Androstenedione Androstanedione

*Cytosol prepared from RAG-2 calls was incubated with 2nM [‘H]R1881in the absence or presence of various concentrations of unlabeled competitor at molar ratios of I : 1, 1: IO, I: 100 and 1: 1000 for 2 h at 4°C. Bound [3H]R1881 was determined by the hydroxylapatite-filter assay. Specific [3H]R1881 binding was piotted vs the molar concentration of the competing steroid to produce competition curves for each steroid. tTo determine the RBA of each steroid for the R1881-receptor the concentration of R1881 that was required to reduce f3H]R1881 binding by 50% was estimated from the competition curve and then divided by the concentration of cold competitor required to produce 50% inhibition of (‘H]R1881 binding.

ysis (87 fmol/mg cytosol protein) showed that the hydroxylapatite-filter assay tends to underestimate Dx binding activity whereas R1881 binding activity was comparable to that obtained by sucrose gradient methods (N 10 fmol/mg cytosol protein). Therefore these experiments were repeated using the DCC method for separation of bound and free [3H]Dx. By this method ~uc~orticoid receptor levels were estimated to be 182 fmol/mg cytosol protein and the X, for Dx was 6 nM at 4°C. The affinity of corticosterone for [3H]Dx binding sites was 44 nM. Examination of the steroid specificity of the Dx receptor in RAG-2 cytosol showed similarities between this receptor and the type II glucocorticoid receptor in the rat kidney [19] in that the Dx receptor exhibited a greater affinity for Dx than deoxycorticosterone, relative binding affinity (RBA = 0.92) and deoxycorticosterone was a more effective competitor than corticosterone (RBA = 0.207) for [)H]Dx binding sites. Competition for [3H]Dx binding sites was measured by DCC assay using 10 nM 13H]Dx and various concentrations (10 nM-10 FM) of unlabeled competitor. Analysis of [jH]R1881 binding activity in RAG and RAG-2 cytosol showed that androgen binding activity was very similar in the two cell populations despite differences in the culture media that the cells

Cytosol

N-M

0.22 ND 22.1 42.7 32.1

ND ND 29.0 36.4 34.6

ND 57.9 27.2 13.5

N-M 1.5 ND 71.8 7.1 1.7

were grown in. The K,, for R1881 binding at 4°C was estimated to be 5 nM in RAG-2 cells and 6 nM in RAG cell cytosoi preparations. The level of [3H]R188 1 binding activity in these cells ranged from 9-17 fmol/mg cytosol protein. The relative binding affinity of the [3H]R1881-receptor complex for various steroids was tested by competition studies using the hydroxylapatite-filter assay (Table 1). These steroids can be arranged in order of relative binding affinity as: RI881 > testosterone > DHT > cyproterone acetate > m~roxyprogesterone acetate > estradio1 > progesterone>>ORG 2058. Both of the synthetic glucocorticoids, Dx and triamcinolone acetonide, were completely ineffective as competitors for t3HJR1881 binding sites. The ability of medroxyprogesterone acetate to compete for androgen receptor sites in normal mouse kidney has been previously reported by Bardin and coworkers[20] and it appears likely that the biological activity of medroxyprogesterone acetate in that system is mediated by the androgen receptor [ 121.It is interesting to note that the anti-androgen cyproterone acetate was a very potent competitor for RI881 binding sites in RAG-2 cytosol. Androgen receptor interaction in target tissues is frequently modulated by androgen metabolism in situ [21]; for example, DHT is rapidly converted to androstanediol in murine kidney in vivo so that little DHT is recovered from kidney nuclei after incubation with [3H]DHT [22]. Androgen metabolism in RAG and RAG-2 renal adrenocarcinoma cells apby RAG-2 cell cultures*

% Metabolite recovered? lh -.....-. Androstanedioi Testosterone 5a-Dihydrotestosterone Androstenedione Androstanedione Polar metabolites

1.95

*Replicate confluent RAG-2 cell cultures that were grown in DMEM-10% FBS were incubated with lo-’ M [‘HItestosterone (102 Ci/mmol) in serum-free DMEM at 37°C for 1 or 4 h. tMethylene chloride extracts of cytosol and nuclear-microsomal fractions (N-M) were analyzed by thin-layer chromatography. Results are expressed as a percentage of the total radioactivity recovered. ND: not detected.

Table 3. Metabolism of Sa-[‘Hldihydrotestosterone

Standard

Cytosol

4h

6h

Cytosol

N-M

Cytosoi

N-M

Cytosol

N-M

6.3 ND 68.8 21.6 ND 3.2

ND ND 72.6 23.7 ND ND

4.46

ND ND 86.2 13.0 0.7 ND

4.1 ND 87.0 6.6 ND 1.7

3.9 ND 87.6 7.2 1.7 ND

& 11.9 ND ND

*Replicate confluent RAG-2 cell cultures that were grown in DMEM-10% FBS were incubated with IO-’ M 5aj’Hldihydrotestosterone (179ci/mmol) for I, 4 or 6 h at 37°C. tMethylene chloride extracts of cytosol and nuclear-microsomal (N-M) cell fractions were analyzed by thin-layer chromatography. Results are expressed as a percentage of total radioactivity recovered. ND = not detected.

510

&IFZA

M.

peared to differ markedly from that of normal murine kidney. As shown in Table 2 the primary intracellular product of [3HJtestosterone metabolism in the tumor cells co-migrated with the DHT standard on TLC. This was especially apparent in the nuclearmicrosomal fraction prepared from RAG-2 cells that were incubated with lo-’ M [3H]testosterone for 1 or 4 h. Virtually no labeled testosterone was recovered from the cytosol or nuclear-microsomal fraction after a 1 h incubation. The other major by-products of testosterone metabolism, androstanedione and androstenedione, were recovered from the cytosol and media. When IO-‘M [3H]DHT was incubated with RAG-2 cells for 1,4 or 6 h, there was little conversion of DHT to androstane~ol or androstanedione (Table 3). These results contrast markedly with those obtained by incubating cytosol prepared from normal mouse kidney with labeled DHT; in those studies 80% of [3H]DHT was reduced to androstanediol within 1 h of incubation[23].

DISCUSSION

Examination of the RAG mouse renal adenocarcinoma cell line and its subline RAG-2 revealed severai significant differences between these cancer cells and normal rodent kidney cells. In terms of receptor content we found that unlike normal mouse kidney [24] these cells lack specific estrogen receptors. These results also differ from observations on estrogen-induced renal adenocarcinomas in Syrian hamsters which have been reported to possess both and estrogen-induced progesterone estrogen receptors [9, 10,251. The absence of these receptors in RAG cells may reflect the clonal origins of this line since it is possible that these receptors are present in only a subpopuIation of kidney cells. In this respect it is interesting to note that only a small fraction of human renal tumors examined thus far contain significant estrogen and progesterone binding activity[26]. Indeed, the significance of finding ER and PgR in normal tissue may be questioned, since a physiological function of these steroids in renal cell activity has not yet been observed. The [3H]Dx binding activity that was observed in RAG-2 cells may be comparable to the type II binder described in normal rat kidney [19]. An approximate & of 6 nM for Dx binding at 0°C in RAG-2 cells appears to be within the range reported for [‘H]Dx receptor in rat kidney: 5 nM at 25°C and 10 nM at 0°C [19]. The steroid specificity exhibited by the Dx receptor was also similar to that of the glucocorticoid receptor in rat kidney [ 191. On the basis of its affinity for Dx and low affinity for cortisol the Dx binding activity of the RAG-2 cells appears to be distinct from both the aldosterone (type I) and corticosterone (type III) receptors found in rat kidney. The aldosterone and corticosterone binding capacity of the RAG-2 cells have not been examined. It should be

JUDGE ef al.

noted that the level of Dx binding activity observed in RAG-2 cytosol (182 fmol~mg cytosol protein) is within the range reported for normal rat kidney; glucocorticoid binding activity in normal mouse kidney has not been examined. A comparison of the binding characteristics of androgen receptors in RAG and RAG-2 cells to those of normal murine kidney receptor revealed several differences. First, the affinity of RAG cell androgen receptor for R1881 (&5-6nM) is lower than that described for normal mouse kidney (0.75 nM) under similar conditions [27]. Secondly, androgen receptor levels in the renal tumor cells are approximately half those of normal mouse kidney. The lower level of androgen receptors in RAG-2 cells may reflect either a deficiency in cell culture conditions or transformation-associated alterations in kidney cell phenotype. An examination of androgen metabolism by RAG cells presents another source of conflict with data collected on mouse kidney in that the primary intracellular androgen in RAG and RAG-2 cells appears to be DHT. These findings contrast with those obtained with normal murine kidney in which testosterone, rather than DHT, is the primary intracellular androgen 1211.Since both the RAG cell line and the mice used in studies by others are of ~~~bl~ origin it seems unlikely that these discrepancies arise from differences in the strain in the donor tissue. The apparent changes in the pattern of kidney cell response to androgens may be more readily attributed to the clonal nature of the cell line vs whole kidney or to alterations associated with neoplasia. One implication of these studies is that the information acquired by studying the mechanism of androgen action in normal kidney cells should not be extrapolated in toto to renal tumor cells. The success of hormonal therapy for renal carcinoma depends upon the development of expe~mental models that will reflect the differences between normal and neoplastic renal tissue. Our studies suggest that the androgen receptor content of renal tumor tissue may be a useful indicator for hormonal therapy. There are few reports of measurements of androgen receptors in tumor tissue and extensive clinical evaluation of androgen receptor as a marker for hormone-sensitive renal cancer has not yet been undertaken. A potential role for Sa-reductase inhibitors [28,29] in the treatment of hormone-sensitive renal tumors should be examined since it is apparent from these studies on the RAG cells that some renal tumor cells can convert testosterone to dihydrotestosterone. Androgen metabolism in normal and neoplastic renal tissue should be examined to determine if similar patterns exist in human renal cells. Acknowledgements-This work was supported by an NIH postdoctoral traineeship CA09183 to SMJ and NIH re-

search grant HD 7103 and American Cancer Society grant BC-151.

Steroid receptors in renal tumor cells REFERENCES

1. McDonald M. W.: Current therapy for renal cell carcinoma. J. Ural. 127 (1982) 211-217. 2. Hrushesky W. J. and Murphy G. P.: Current status of the therapy of advanced renal carcinoma. J. Surg. Oncol. 9 (1977) 277-288. 3. DeSombre E. R., Greene G. L. and Jensen E. V.: Estrogen receptors and the hormone dependence of breast cancer. In Breast Cancer: New Concepts in Etiology and Control (Edited by M. J. Brennan, C. M. McGrath and M. A. Rich). Academic Press, New York (1980) pp. 69-88. 4. McGuire W. L.: Steroid receptors in human breast cancer. Cancer Res. 38 (1978) 4289. 5. Concolino G., Marocchi A., Margiotta G., Conti C., DiSilverio F., Tenaglia R., Ferraro F. and Bra& U.: Steroid receptors and hormone responsiveness of human prostatic carcinoma. The Prostate 3 (1982) 475-482.

6. Ekman P., Dahlberg E., Gustafsson J. A., Hogberg B., Pousette A. and Snochowski M.: Present and future clinical value of steroid receptor assays in human prostatic carcinoma. Cancer Horm. 14 (1980) 361-370. I. Kirkman H.: Estrogen-induced tumor of the kidney in the Syrian hamster. J. natn. Cancer Inst. Monogr. 1 (1959) l-57. 8. Steggles A. W. and King R. J. B.: Oestrogen receptors in hamster tumours. Eur. J. Cancer 8 (1972) 323-334. 9. Li S. A., Li J. J. and Villee C. A.: Significance of the progesterone receptor in the estrogen-induced and dependent renal tumor of the Syrian golden hamster. Ann N. Y. Acad. Sci 286 (1977) 36937%

10. Li J. J.. Tallev D. J.. Li S. A. and Villee C. A.: An oestrogdn binding protein in the renal cytosol of intact, castrated and estrogenized golden hamsters. Endocrinology 95 (1974) ii341141. 11. Concolino G.. Marocchi A.. Conti C.. Tenaalia R.. DiSilverio F. ‘and Bracci U:: Human renal &ll car: cinema as a hormone-dependent tumor. Cuncer Res. 38

(I 978) 434&4344. 12. Gupta C., Bullock L. P. and Bardin C. W.: Further studies on the androgenic, antiandrogenic and synandrogenic actions of progestins. Endocrinology 102 (1978) 736-744. 13. Klebe R. J., Chen T. R. and Ruddle F. H.: Controlled production of proliferating somatic cell hybrids. J. cell Biol. 45 (1970) 74-82.

14. Felluga B., Claude A. and Mrena E.: Electron microscope observations on virus particles associated with a transplantable renal adenocarcinoma in Balb/cf/Cd mice. J. natn. Cancer Inst. 43 (1969) 319-333. 15. Horwitz K. B., Costlow M. E. and McGuire M. L.: MCF-7: a human breast cancer cell line with estrogen, androgen, progesterone and glucocorticoid receptors. Steroids 26 (1975) 785-195.

511

16. Bradford M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biothem. 72 (1976) 248-254. 17. Liao S., Witte D., Schilling K. and Chang C.: The use of a hydroxylapatite filter steroid receptor assay method in the study of the modulation of androgen receptor interaction. J. steroid Biochem. 20 (1984) 1l-17. 18. Laemmeli U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685.

19. Funder J. W., Feldman D. and Edelman I. S.: Glucocorticoid receptors in rat kidney: the binding of tritiated-dexamethasone. Endocrinology 92 (1973) 1005-1013.

20. Brown R., Bullock L. and Bardin C. W.: In vitro and in uioo binding of progestins to the androgen receptor of mouse kidney: correlation with biological activity. Endocrinology lbs (1979) 1281-1287. 21. Bardin C. W. and Catterall J. F.: Testosterone: a major determinant of extragenital sexual dimorphism. Science 211 (1981) 1285-1294. 22. Bullock L. P., Bardin C. W. and Ohno S.: The androgen insensitive mouse: absence of intranuclear androgen retention in the kidney. Biochem. biophys. Res. Commun. 44 (1971) 1537-1543.

23. Bullock L. P. and Bardin C. W.: Androgen receptors in mouse kidney: a study of male, female and androgeninsensitive (tfm/y) mice. Endocrinology 94 (1974) 746-756.

24. Bullock L. P. and Bardin C. W.: The presence of estrogen receptor in kidneys from normal and androgen-insensitive tfm/y mice. Endocrinology 97 (1975) 10061111. 25 Li S. A. and Li J. J.: Estrogen-induced progesterone ’ receptor in the Syrian hamster kidney. I. Modulation by antiestrogens and androgens. Endocrinology 103 (1978) 2119-2128.

26, DiFronzo G., Ronchi E., Bertuzzi A., Vezzoni P. and Pizzacaro G.: Estrogen receptors in renal carcinoma. Eur. J. Ural. 6 (1980) 307-311. 27 Wright W. W., Chan K. C. and Bardin C. W.: Charac’ terization of the stabilizing effect of sodium molybdate on the androgen receptor in mouse kidney. Endocrinology 108 (1981) 2210-2216. 28 Liang T. and Weiss C. E.: Inhibition of Sa-reductase, receptor binding, and nuclear uptake of androgens in the prostate by a 4-methyl-2-aza steroid. J. biol. Chem. 256 (1981) 7998-8005. 29. Brooks J. R., Baptista E. M., Berman C., Ham E. A., Hichens M., Johnston D. B. R., Primka R. L., Rasmusson G. H.. Revnolds G. F.. Schmitt S. M. and Arth G. E.: Response of rat ventral prostate to a new and novel Sa-reductase inhibitor. Endocrinology 109 (1981) 83&836.