Characterization and Measurement of the Androgen Receptor in Human Benign Prostatic Hyperplasia and Prostatic Carcinoma MAREK SNOCHOWSKI,* AKE POUSETTE,* PETER EKMAN,| DOMINIQUE BRESSION,* LENNART ANDERSSON,| BERTIL HOGBERG,f AND JAN-AKE GUSTAFSSON * Department of Chemistry and \Department of Pharmacology, Karolinska Institutet, tDepartment of Urology, Karolinska Sjukhuset, S-104 01 Stockholm 60, Sweden ABSTRACT. [6,7-3H]Methyltrienolone (R 1881) has been used as ligand to measure and characterize the androgen receptor in human benign prostatic hyperplasia and prostatic carcinoma. [3H]R 1881 was bound with high affinity and low capacity to cytosol from six out of nine specimens of transvesically enucleated benign prostatic hyperplasia and from two specimens of prostate carcinoma obtained by Veenema biopsy or transvesical enucleation. The dissociation constant of the [3H]R 1881receptor complex was in the range of 0.3-1.8 x 10~9M and the number of binding sites 9-26 fmoles/mg protein. [3H]R 1881 was displaced from its binding sites on the receptor by 5a-dihydrotestosterone and testosterone; much less efficient competitors were LS 1727, cyproterone acetate, 17/3-estradiol, R 5020, 5a-androstane-3a,17/3-diol and progesterone, whereas 4-androstene-3,17-dione, cyproterone, estramustine and cortisol did not
T
HE PROSTATE gland is completely dependent upon androgens both for normal growth and function. The mechanism by which androgens exert their effects on prostatic tissue has been intensely studied during recent years. In the prostatic cell, testosterone is metabolized to 5a-dihydrotestosterone1 (DHT) that is bound to a re-
Received January 19, 1977. Supported by grants from LEO Research Foundation, Riksforeningen mot Cancer, Loo and Hans Ostermans Stiftelse, and Svenska Lakaresallskapet. 1 The following abbreviations and trivial names are used: methyltrienolone (R 1881) = 17/3-hydroxy-17amethylestra-4,9,ll-trien-3-one; 5a-dihydrotestosterone (DHT) = 17/3-hydroxy-5a-androstan-3-one; cyproterone = 6-chloro-17o:-hydroxy-l,2a-methylenepregna-4,6-diene-3,20-dione; cyproterone acetate = cyproterone 17-acetate; R 5020 = I7a,21-dimethyl19-nor-pregna-4,9-diene-3,20-dione; G-302 = 2formyl-17a-methylandrosta-l,4-diene-ll/3,17/3-diol-3-
and
compete. The receptor was stable at 0 C but was degraded rapidly (t,/2 = 14 min) at 37 C. The rate of dissociation of the [3H]R 1881-receptor complex increased at higher temperatures (t]/2 = 810 and 50 min at 0 C and 37 C, respectively). The [3H]R 1881receptor complex had an isoelectric point of about pH 5. 5a-[3H]Dihydrotestosterone was found to be an unsuitable ligand in assays of the androgen receptor in benign prostatic hyperplasia since this steroid also binds to testosterone-binding globulin that probably contaminates all prostate cytosol preparations. Furthermore, 5a-[3H]dihydrotestosterone was rapidly metabolized in prostate cytosol, even at 0 C, whereas [3H]R 1881 was not. Electroresected specimens did not contain detectable levels of androgen receptor, probably due to heat denaturation. (J Clin Endocrinol Metab 45: 920, 1977)
ceptor protein and the steroid-receptor complex is translocated into the cell nucleus where it probably affects transcription of DNA to messenger-RNA (1). Most of these studies have been carried out on experimental animals and relatively little information is available concerning protein binding of steroid hormones in human prostate. Evidence for the existence of a specific androgen-binding protein in human benign prostatic hyperplasia has been presented by Rosen et al. (2) using glycerol gradient centrifugation, and by Geller et al. (3) and Attramadal et al. (4) using gel filtration. All these studies on the androgen receptor in human prostate have been carried out with one; estramustine (LS 275) = 17/3-estradiol-3-N-bis(2chloroethyl)carbamate; LS 1727 = 19-nortestosterone 17-N-/3-chloroethyl-N-nitroso-carbamate.
920
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR IN HUMAN PROSTATE [ 3 H]DHT that unfortunately has a high affinity for testosterone-binding globulin (TeBG) in plasma. This creates a potential source of error during receptor quantitation that is difficult to eliminate completely. In fact, Mobbs et al. have concluded that the steroid specificity of the binding occurring in human prostate tissue appeared closer to that of TeBG than to that of the androgen receptor in rat prostate (5). Bonne and Raynaud recently reported that the synthetic androgen, methyltrienolone (R 1881), binds specifically to the androgen receptor in rat prostate cytosol but not to plasma proteins such as sex hormonebinding globulin (6,7). This important finding has encouraged us to use [3H]R 1881 as a ligand in studies on characteristics of the androgen receptor in human prostate and in measurements of receptor levels in specimens of benign prostatic hyperplasia and prostatic carcinoma. Materials and Methods Steroids [l,2,6,7- 3 H]Testosterone (specific radioactivity 84.7 Ci/mmol) and [1,2-3H]DHT (specific radioactivity 40.3 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, England. [6,7-3H] Methyltrienolone (R 1881, specific radioactivity 52.8 Ci/mmol) and unlabelled methyltrienolone as well as 17a,21-dimethyl-19nor-pregna-4,9-diene-3,20-dione (R 5020) was kindly supplied by Dr. J.-P. Raynaud, RousselUCLAF, Paris, France. The purity of the labelled compounds (>99%) was checked each month by thin-layer chromatography. Cyproterone acetate was generously supplied by Dr. F. Neumann, Shering Co., Berlin, Germany. Estramustine, 17/8 -estradiol -3 -N -bis(2 -chloroethyl)carbamate (LS 275) and 19-nortestosterone 17-N-/8-chloroethyl-N-nitrosocarbamate (LS 1727) were synthesized at LEO Research Laboratories, Helsingborg, Sweden. The alkylating agent estramustine is the dephosphorylated metabolite of Estracyt®, a drug used in treatment of prostatic carcinoma (8,9). LS 1727 is a steroidal alkylating agent with androgenic activity. G-302 (2-Formyl-17amethylandrosta-l,4-diene-llj3,17/3-diol-3-one)
921
that was synthesized at LPB Istituto Pharmaceutico S.p.a., is an anabolic steroid without or with weak androgenic activity. Testosterone, 4androstene-3,17-dione, 5a-dihydrotestosterone, 5a-androstane-3a,17/3-diol, 17/3-estradiol, progesterone and cortisol were gifts from Dr. J. Babcock, Upjohn Co., Kalamazoo, Michigan. Tissue specimens In 7 cases prostate tissue was obtained by transurethral electro-resection. From another 16 patients prostate tissue was obtained after transvesical enucleation. Parts of the clinical specimens were in all cases taken for histological examination. In two cases unexpected diagnoses of cancer were made. Several cancer specimens were obtained by punch biopsy according to Veenema (10). Each biopsy punch was divided in two halves, one of which was taken for histological examination. Unfortunately, all but one of the biopsy specimens appeared to be more or less contaminated with surrounding tissue (benign prostatic hyperplasia, fat, muscle) and therefore they had to be excluded from the study. Immediately after operation the tissue specimens were cooled on ice and were frozen to - 7 0 C within 3 h. Venous blood was also taken from patients and pooled for analysis of high-affinity, lowcapacity steroid binding. Preparation of cytosol Human prostate cytosol (ratio 1:3) was prepared from frozen material using a 50 mM Tris-HCl buffer containing 1 mM EDTA, 0.1 mM dithioerythreitol (DTE) and 10% (vol/vol) glycerol. For homogenization an Ultra-Turrax homogenizer (Janke and Kunkel, KF, Staufen i.Br., Germany) was used; the sample was homogenized for 5 sec under cooling and was then kept in an ice bath for 30 sec. This procedure was repeated three times. The homogenate was centrifuged at 105,000 x g for 60 min, and the supernatant was carefully removed. Aliquots for protein analysis were stored at - 2 0 C. For characterization of the androgen receptor a pool of cytosol was prepared from six specimens of transvesically enucleated benign prostatic hyperplasia and stored in aliquots at - 2 0 C. No loss of receptor binding activity was observed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
922
SNOCHOWSKI ET AL.
after storage for three months. Aliquots of the pool were thawed on ice prior to receptor analysis. Protein was measured according to Lowry et al. (11). Charcoal adsorption assay
JCE & M • 1977 Vol 45 • No 5
form/ethyl acetate, using the solvent ratios (vol/ vol) 4/1 and 1/1 for the [ 3 H]DHT and [3H]R 1881 metabolites, respectively. [3H]Labelled DHT (Rf = 0.67), 5a-androstane-3a,17/3-diol (Rf = 0.49) and 5a-androstane-3,17-dione (Rf = 0.76) were used as reference standards. Radioactivity was localized using a Berthold thin-layer scanner, Modell II (Berthold, Wildbad, Germany) and measured by scintillation counting after extraction.
Dry, washed charcoal (0.5%, wt/vol) was suspended in a solution containing 0.05% (wt/vol) dextran, 0.1% (wt/vol) gelatin, 1 mM EDTA, 0.1 mM DTE, 50 mM Tris, and 10% (wt/vol) glycerol. After binding of labelled androgen to the receptor under different conditions (see Rate of association below), free steroids were removed by treatment The rate of association between receptor and with dextran-coated charcoal (DCC). DCC sus- [3H]R 1881 was studied at three temperatures pension, 0.5 ml, was added per 0.1 ml of cyto- (0 C, 15 C, 23 C) by measuring the amount of sol and after incubation at 0 C for 20 min the androgen-receptor complex formed at various tubes were centrifuged at 6,700 X g for 15 min. times after incubation of aliquots of cytosol with An aliquot, 0.5 ml, of the supernatant was 5 x 10~9M [3H]R 1881. Specific binding was caltaken for determination of radioactivity using 7 culated as described above. No correction for ml of Instagel® (Packard Instruments Co., Inc., degradation of receptor was made. Warrenville, Downess Grove, 111.) as scintillation liquid. The vials were counted for 10 min in Rates of degradation and dissociation an Intertechnique SL 30 liquid scintillation specCytosol was preincubated over night at 0 C trometer. Determination of the total amount of 9 3 radioactivity incubated was performed by count- with 5 x 10~ M [ H]R 1881 with or without a 100-fold excess of unlabelled R 1881 and divided ing the radioactivity in 0.5 ml aliquots of samples treated with buffer instead of DCC-suspension. into two parts. One sample was used to study Binding data were analyzed according to Scat- rate of receptor degradation. The other sample chard (12) and dissociation constants (KJ and was treated for 5 min at 0 C with 2% (wt/vol) number of binding sites (Bmax) were calculated of dry, washed charcoal, centrifuged for 5 min using a Wang programmable electronic calcula- at 12,000 x g and filtered through glass fiber tor (Model 720). All presented data on specific- paper to remove all free steroids (no loss of rebinding were obtained following subtraction of ceptor binding activity was observed). This non-specific binding from total binding. Non- sample was used to study the rate of steroid-respecific binding was calculated from samples ceptor dissociation following addition of 10"6M containing a 100-fold excess of unlabelled steroid unlabelled R 1881 to prevent reassociation of in addition to labelled ligand (13). Non-specific receptor with radioactive ligand. Specific binding binding varied from 1 to 3% of total binding. (high-affinity, low-capacity) was calculated as the difference in bound radioactivity (measured 3 3 by the use of dextran-coated charcoal treatment) Metabolism of [ H]R 1881 and [ H]DHT between samples preincubated without and Samples containing 0.2 ml of cytosol and with a 100-fold excess of unlabelled R 1881 10"8M [ 3 H]DHT or [3H]R 1881 were incubated (c/., above). at 0 C, 15 C or 20 C for 20 min, 1 h, 4 h or 16 h. Rates of degradation and dissociation of the Incubations were terminated by addition of 0.2 [3H]R 1881-receptor complex were studied at 0, ml of ethyl acetate (saturated with H2O) con- 15, 23 and 37 C for various periods up to 20 h. taining 3 fx,g of DHT, 5a-androstane-3/3,17/3- Rate of dissociation was determined after cordiol and 5a-androstane-3,17-dione. Radio- rection for degradation of receptor. activity was extracted with 2 x 1 ml of ethyl acetate and the extract evaporated under nitro- Ligand specificity gen. Extracts were applied on precoated silica The specificity of the androgen receptor was gel plates (250 /x, Merck, Darmstadt, Germany) that were developed in the solvent system chloro- investigated by incubating aliquots of cytosol
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
923
ANDROGEN RECEPTOR IN HUMAN PROSTATE with 5 X 10~9M [3H]R 1881 and varying concentrations of non-radioactive compounds (0.2 to 500 9
x 10- M) for 18 h at 0 C.
cpm A 3 [ H]R1881 400 PLASMA
[ 3 H]DHT PLASMA
Isoelectric focusing Two 10 ml glass columns were connected by a plastic tube. The lower parts of the two columns and the connecting tube were filled with 70% (w/v) sucrose-2% (v/v) ethylenediamine (lower electrode buffer). One column was used for balance and was filled with 35% sucrose-0.5% ethylenediamine. In the other column, 0.5 ml of 65% sucrose-3% (v/v) Ampholine® (LKB-Produkter, Stockholm, Sweden) was layered on top of the 70% sucrose solution to protect the sample that was distributed in a discontinuous 60-10% sucrose, 4 — 1% Ampholine gradient with a total volume of 6 ml. On top of the sucrose gradient, 0.5 ml of 7% sucrose-0.5% Ampholine solution was applied in order to protect the sample from the upper electrode buffer that consisted of 1% (vol/vol) H2SO4. Focusing was performed for 15-20 h at increasing voltage up to 1200 V. After focusing, the columns were fractionated into counting vials. pH was determined using a surface electrode (type 403-30, Ingold, Zurich, Switzerland) and radioactivity measurements were performed as described above. Ferritin (pi = 5.0) and hemoglobin (pi = 7.2 and 7.6) were used as standards.
Results High-affinity binding of [3H]R 1881, [3H]DHT and [3H]testosterone to human prostate cytosol Binding of [3H]R 1881, [ 3 H]DHT and [3H] testosterone to human prostate cytosol analyzed according to Scatchard yielded the TABLE 1. Kj and Bmax values calculated from Scatchard analyses of binding data from incubations of human prostate cytosol with [3H]R 1881, [3H]DHT and [3H] testosterone max
Steroid
K,, ± SD (IIM)
(95% confidence limit) (fmoles/mg protein)
R 1881 DHT Testosterone
1.48 ± 0.11 2.60 ± 0.08 8.65 ± 1.59
29.2 (28.3-30.1) 201(199-203) 45.3 (44.4-46.2)
200
cpm 400
FIG. 1. Isoelectric focusing of: a) [3H]R 1881 incubated with human plasma; b) [3H]DHT incubated with human plasma; c) [3H]R 1881 incubated with human prostate cytosol; d) [3H]DTH incubated with human prostate cytosol.
IQj and B max values shown in Table 1. DHT has considerably more binding sites than R 1881 and testosterone. Since R 1881 did not show any high-affinity, low-capacity binding to serum proteins (cf, below) this ligand does not appear to bind to TeBG. Therefore, the large amount of DHTbinding sites in prostate cytosol is probably explained by the presence of contaminating TeBG. The relatively small amount of testosterone-binding sites in prostate cytosol is in agreement with previous reports on the higher affinity of DHT than testosterone to TeBG (for ref., see 14). Characterization of high-affinity [3H]R 3 1881- and [ H]DHT-binding protein in human prostate cytosol by isoelectric focusing Plasma obtained from the third trimester of pregnancy was diluted 40 times and incubated with [3H]R 1881 and [ 3 H]DHT as described above for prostate cytosol. After treatment with DCC, the labelled steroidprotein complexes were desalted by
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
SNOCHOWSKI ET AL.
924
chromatography on Sephadex G-50. [3H]R 1881- and [ 3H]DHT-protein complexes from prostate cytosol were treated in the same way and all samples were analyzed by isoelectric focusing employing the pH range of 4-6. The [3H]R 1881- and [3H]DHT-protein complexes from prostate cytosol were found to focus at the same pH (pi 5.0-5.2) (Fig. 1). However, [ 3 H]DHT also bound to a plasma protein (conceivably TeBG) and this complex focused at the same pH as the cytosol complex. [3H]R 1881 did not bind to any plasma protein, confirming previous data reported by Bonne and Raynaud (7). Furthermore, the [3H]DHT-cytosol protein complex gave a more complicated electrofocusing pattern than the homogeneous peak formed by the [3H]R 1881- receptor complex (Fig. 1) indicating that the latter complex may be more stable during isoelectric focusing than the DHT-receptor complex. These results point out some of the great advantages—no binding to TeBG or other plasma proteins, high stability of steroid-receptor complex—in utilizing [3H]R 1881 rather than [ 3 H]DHT as ligand in studies on the androgen receptor in human prostate cytosol. Metabolism of [3H]R 1881 and [3H]DHT in human prostate cytosol Figure 2 shows another great advantage in using [3H]R 1881 rather than [ 3 H]DHT in studies on the androgen receptor in
JCE & M • 1977 Vol 45 • No 5
human prostate cytosol. Although [ 3 H]DHT was rapidly metabolized even at 0 C, [3H]R 1881 was not significantly metabolized even at 23 C. Specificity of[3H]R 1881 binding to human prostate cytosol The competition data are summarized in Fig. 3 and Table 2. The most efficient competitors to R 1881 were DHT and testosterone. Competition by LS 1727, cyproterone acetate, 17/3-estradiol, R 5020, 5aandrostane-3a,17/3-diol and progesterone was also observed but to a smaller extent. Androstenedione, cyproterone, cortisol, estramustine or G-302 did not compete significantly with R 1881 for binding sites on the receptor. These results indicate that R 1881 binds to an androgen receptor in human prostate cytosol with high specificity for androgens. Recent reports that R 1881 also shows high affinity to the progesterone receptor (15,16) do not influence this interpretation since progestins like cyproterone acetate, R 5020 and progesterone were only weak competitors to R 1881. Rates of degradation and dissociation of the [3H]R 1881-receptor complex in human prostate cytosol Rates of degradation and dissociation of the [3H]R 1881-receptor complex were determined as described in Materials and Methods and the results are shown in Figs.
A •DHT20-C • DHT 15 C • DHT 0 C
FIG. 2. Metabolism of [3H]R 1881 and [3H]DHT at different temperatures. Metabolites were measured by thin-layer chromatography as described in Materials and Methods. Per cent conversion of the labelled substrates represent total metabolism.
1 A 16 INCUBATION TIME (HOURS)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
925
ANDROGEN RECEPTOR IN HUMAN PROSTATE
100
G3O2
gfe 50
ANOROSTANEDIOL ESTRADIOL TESTOSTERONE LS 1727 R1881 n-CYPAC. DHT 2-10"1'
10"
10"8
CONCENTRATION (M) OF ADDED COMPETITOR
FIG. 3a. Competition of DHT, testosterone, LS 1727, cyproterone acetate, 17/3-estradiol (estradiol) and 5aandrostane-3a,17/3-diol (Fig. 3a) and of R 5020, progesterone, androstenedione, cyproterone, estramustine and cortisol (Fig. 3b) for high-affinity, low-capacity binding of [3H]R 1881 to human prostate cytosol. Samples were incubated with 5 x 10~9M [3H]R 1881 and varying concentrations of non-radioactive steroids (0.2-500 x 10" 9 M) at 0 C for 18 h. Free and bound steroid were separated using treatment with DCC. Relative binding affinities of the different competitors calculated on the basis of these results are summarized in Table 2.
100-
CORTISOL
ESTRAMUSTINE CYPROTERONE ANDROSTENEDIONE
00 u.
crO
50 PROGESTERONE R5020
R1881 2-K)r10
10"°
10"'
CONCENTRATION (M) OF ADDED COMPETITOR
FIG. 3b. See legend to Fig. 3a.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
926
SNOCHOWSKI ET AL.
TABLE 2. Relative binding affinity of various steroids to the androgen receptor in human prostate cytosol
JCE & M • 1977 Vol 45 • No 5
0 C the complex dissociated with a halflife of 13.5 h.
Relative binding affinity
Steroid R1881
Rate of association of [3H]R 1881 to the androgen receptor in human prostate cytosol
1
0.48 0.082 0.035 0.025
T
HE PROSTATE gland is completely dependent upon androgens both for normal growth and function. The mechanism by which androgens exert their effects on prostatic tissue has been intensely studied during recent years. In the prostatic cell, testosterone is metabolized to 5a-dihydrotestosterone1 (DHT) that is bound to a re-
Received January 19, 1977. Supported by grants from LEO Research Foundation, Riksforeningen mot Cancer, Loo and Hans Ostermans Stiftelse, and Svenska Lakaresallskapet. 1 The following abbreviations and trivial names are used: methyltrienolone (R 1881) = 17/3-hydroxy-17amethylestra-4,9,ll-trien-3-one; 5a-dihydrotestosterone (DHT) = 17/3-hydroxy-5a-androstan-3-one; cyproterone = 6-chloro-17o:-hydroxy-l,2a-methylenepregna-4,6-diene-3,20-dione; cyproterone acetate = cyproterone 17-acetate; R 5020 = I7a,21-dimethyl19-nor-pregna-4,9-diene-3,20-dione; G-302 = 2formyl-17a-methylandrosta-l,4-diene-ll/3,17/3-diol-3-
and
compete. The receptor was stable at 0 C but was degraded rapidly (t,/2 = 14 min) at 37 C. The rate of dissociation of the [3H]R 1881-receptor complex increased at higher temperatures (t]/2 = 810 and 50 min at 0 C and 37 C, respectively). The [3H]R 1881receptor complex had an isoelectric point of about pH 5. 5a-[3H]Dihydrotestosterone was found to be an unsuitable ligand in assays of the androgen receptor in benign prostatic hyperplasia since this steroid also binds to testosterone-binding globulin that probably contaminates all prostate cytosol preparations. Furthermore, 5a-[3H]dihydrotestosterone was rapidly metabolized in prostate cytosol, even at 0 C, whereas [3H]R 1881 was not. Electroresected specimens did not contain detectable levels of androgen receptor, probably due to heat denaturation. (J Clin Endocrinol Metab 45: 920, 1977)
ceptor protein and the steroid-receptor complex is translocated into the cell nucleus where it probably affects transcription of DNA to messenger-RNA (1). Most of these studies have been carried out on experimental animals and relatively little information is available concerning protein binding of steroid hormones in human prostate. Evidence for the existence of a specific androgen-binding protein in human benign prostatic hyperplasia has been presented by Rosen et al. (2) using glycerol gradient centrifugation, and by Geller et al. (3) and Attramadal et al. (4) using gel filtration. All these studies on the androgen receptor in human prostate have been carried out with one; estramustine (LS 275) = 17/3-estradiol-3-N-bis(2chloroethyl)carbamate; LS 1727 = 19-nortestosterone 17-N-/3-chloroethyl-N-nitroso-carbamate.
920
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
ANDROGEN RECEPTOR IN HUMAN PROSTATE [ 3 H]DHT that unfortunately has a high affinity for testosterone-binding globulin (TeBG) in plasma. This creates a potential source of error during receptor quantitation that is difficult to eliminate completely. In fact, Mobbs et al. have concluded that the steroid specificity of the binding occurring in human prostate tissue appeared closer to that of TeBG than to that of the androgen receptor in rat prostate (5). Bonne and Raynaud recently reported that the synthetic androgen, methyltrienolone (R 1881), binds specifically to the androgen receptor in rat prostate cytosol but not to plasma proteins such as sex hormonebinding globulin (6,7). This important finding has encouraged us to use [3H]R 1881 as a ligand in studies on characteristics of the androgen receptor in human prostate and in measurements of receptor levels in specimens of benign prostatic hyperplasia and prostatic carcinoma. Materials and Methods Steroids [l,2,6,7- 3 H]Testosterone (specific radioactivity 84.7 Ci/mmol) and [1,2-3H]DHT (specific radioactivity 40.3 Ci/mmol) were purchased from the Radiochemical Centre, Amersham, England. [6,7-3H] Methyltrienolone (R 1881, specific radioactivity 52.8 Ci/mmol) and unlabelled methyltrienolone as well as 17a,21-dimethyl-19nor-pregna-4,9-diene-3,20-dione (R 5020) was kindly supplied by Dr. J.-P. Raynaud, RousselUCLAF, Paris, France. The purity of the labelled compounds (>99%) was checked each month by thin-layer chromatography. Cyproterone acetate was generously supplied by Dr. F. Neumann, Shering Co., Berlin, Germany. Estramustine, 17/8 -estradiol -3 -N -bis(2 -chloroethyl)carbamate (LS 275) and 19-nortestosterone 17-N-/8-chloroethyl-N-nitrosocarbamate (LS 1727) were synthesized at LEO Research Laboratories, Helsingborg, Sweden. The alkylating agent estramustine is the dephosphorylated metabolite of Estracyt®, a drug used in treatment of prostatic carcinoma (8,9). LS 1727 is a steroidal alkylating agent with androgenic activity. G-302 (2-Formyl-17amethylandrosta-l,4-diene-llj3,17/3-diol-3-one)
921
that was synthesized at LPB Istituto Pharmaceutico S.p.a., is an anabolic steroid without or with weak androgenic activity. Testosterone, 4androstene-3,17-dione, 5a-dihydrotestosterone, 5a-androstane-3a,17/3-diol, 17/3-estradiol, progesterone and cortisol were gifts from Dr. J. Babcock, Upjohn Co., Kalamazoo, Michigan. Tissue specimens In 7 cases prostate tissue was obtained by transurethral electro-resection. From another 16 patients prostate tissue was obtained after transvesical enucleation. Parts of the clinical specimens were in all cases taken for histological examination. In two cases unexpected diagnoses of cancer were made. Several cancer specimens were obtained by punch biopsy according to Veenema (10). Each biopsy punch was divided in two halves, one of which was taken for histological examination. Unfortunately, all but one of the biopsy specimens appeared to be more or less contaminated with surrounding tissue (benign prostatic hyperplasia, fat, muscle) and therefore they had to be excluded from the study. Immediately after operation the tissue specimens were cooled on ice and were frozen to - 7 0 C within 3 h. Venous blood was also taken from patients and pooled for analysis of high-affinity, lowcapacity steroid binding. Preparation of cytosol Human prostate cytosol (ratio 1:3) was prepared from frozen material using a 50 mM Tris-HCl buffer containing 1 mM EDTA, 0.1 mM dithioerythreitol (DTE) and 10% (vol/vol) glycerol. For homogenization an Ultra-Turrax homogenizer (Janke and Kunkel, KF, Staufen i.Br., Germany) was used; the sample was homogenized for 5 sec under cooling and was then kept in an ice bath for 30 sec. This procedure was repeated three times. The homogenate was centrifuged at 105,000 x g for 60 min, and the supernatant was carefully removed. Aliquots for protein analysis were stored at - 2 0 C. For characterization of the androgen receptor a pool of cytosol was prepared from six specimens of transvesically enucleated benign prostatic hyperplasia and stored in aliquots at - 2 0 C. No loss of receptor binding activity was observed
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
922
SNOCHOWSKI ET AL.
after storage for three months. Aliquots of the pool were thawed on ice prior to receptor analysis. Protein was measured according to Lowry et al. (11). Charcoal adsorption assay
JCE & M • 1977 Vol 45 • No 5
form/ethyl acetate, using the solvent ratios (vol/ vol) 4/1 and 1/1 for the [ 3 H]DHT and [3H]R 1881 metabolites, respectively. [3H]Labelled DHT (Rf = 0.67), 5a-androstane-3a,17/3-diol (Rf = 0.49) and 5a-androstane-3,17-dione (Rf = 0.76) were used as reference standards. Radioactivity was localized using a Berthold thin-layer scanner, Modell II (Berthold, Wildbad, Germany) and measured by scintillation counting after extraction.
Dry, washed charcoal (0.5%, wt/vol) was suspended in a solution containing 0.05% (wt/vol) dextran, 0.1% (wt/vol) gelatin, 1 mM EDTA, 0.1 mM DTE, 50 mM Tris, and 10% (wt/vol) glycerol. After binding of labelled androgen to the receptor under different conditions (see Rate of association below), free steroids were removed by treatment The rate of association between receptor and with dextran-coated charcoal (DCC). DCC sus- [3H]R 1881 was studied at three temperatures pension, 0.5 ml, was added per 0.1 ml of cyto- (0 C, 15 C, 23 C) by measuring the amount of sol and after incubation at 0 C for 20 min the androgen-receptor complex formed at various tubes were centrifuged at 6,700 X g for 15 min. times after incubation of aliquots of cytosol with An aliquot, 0.5 ml, of the supernatant was 5 x 10~9M [3H]R 1881. Specific binding was caltaken for determination of radioactivity using 7 culated as described above. No correction for ml of Instagel® (Packard Instruments Co., Inc., degradation of receptor was made. Warrenville, Downess Grove, 111.) as scintillation liquid. The vials were counted for 10 min in Rates of degradation and dissociation an Intertechnique SL 30 liquid scintillation specCytosol was preincubated over night at 0 C trometer. Determination of the total amount of 9 3 radioactivity incubated was performed by count- with 5 x 10~ M [ H]R 1881 with or without a 100-fold excess of unlabelled R 1881 and divided ing the radioactivity in 0.5 ml aliquots of samples treated with buffer instead of DCC-suspension. into two parts. One sample was used to study Binding data were analyzed according to Scat- rate of receptor degradation. The other sample chard (12) and dissociation constants (KJ and was treated for 5 min at 0 C with 2% (wt/vol) number of binding sites (Bmax) were calculated of dry, washed charcoal, centrifuged for 5 min using a Wang programmable electronic calcula- at 12,000 x g and filtered through glass fiber tor (Model 720). All presented data on specific- paper to remove all free steroids (no loss of rebinding were obtained following subtraction of ceptor binding activity was observed). This non-specific binding from total binding. Non- sample was used to study the rate of steroid-respecific binding was calculated from samples ceptor dissociation following addition of 10"6M containing a 100-fold excess of unlabelled steroid unlabelled R 1881 to prevent reassociation of in addition to labelled ligand (13). Non-specific receptor with radioactive ligand. Specific binding binding varied from 1 to 3% of total binding. (high-affinity, low-capacity) was calculated as the difference in bound radioactivity (measured 3 3 by the use of dextran-coated charcoal treatment) Metabolism of [ H]R 1881 and [ H]DHT between samples preincubated without and Samples containing 0.2 ml of cytosol and with a 100-fold excess of unlabelled R 1881 10"8M [ 3 H]DHT or [3H]R 1881 were incubated (c/., above). at 0 C, 15 C or 20 C for 20 min, 1 h, 4 h or 16 h. Rates of degradation and dissociation of the Incubations were terminated by addition of 0.2 [3H]R 1881-receptor complex were studied at 0, ml of ethyl acetate (saturated with H2O) con- 15, 23 and 37 C for various periods up to 20 h. taining 3 fx,g of DHT, 5a-androstane-3/3,17/3- Rate of dissociation was determined after cordiol and 5a-androstane-3,17-dione. Radio- rection for degradation of receptor. activity was extracted with 2 x 1 ml of ethyl acetate and the extract evaporated under nitro- Ligand specificity gen. Extracts were applied on precoated silica The specificity of the androgen receptor was gel plates (250 /x, Merck, Darmstadt, Germany) that were developed in the solvent system chloro- investigated by incubating aliquots of cytosol
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
923
ANDROGEN RECEPTOR IN HUMAN PROSTATE with 5 X 10~9M [3H]R 1881 and varying concentrations of non-radioactive compounds (0.2 to 500 9
x 10- M) for 18 h at 0 C.
cpm A 3 [ H]R1881 400 PLASMA
[ 3 H]DHT PLASMA
Isoelectric focusing Two 10 ml glass columns were connected by a plastic tube. The lower parts of the two columns and the connecting tube were filled with 70% (w/v) sucrose-2% (v/v) ethylenediamine (lower electrode buffer). One column was used for balance and was filled with 35% sucrose-0.5% ethylenediamine. In the other column, 0.5 ml of 65% sucrose-3% (v/v) Ampholine® (LKB-Produkter, Stockholm, Sweden) was layered on top of the 70% sucrose solution to protect the sample that was distributed in a discontinuous 60-10% sucrose, 4 — 1% Ampholine gradient with a total volume of 6 ml. On top of the sucrose gradient, 0.5 ml of 7% sucrose-0.5% Ampholine solution was applied in order to protect the sample from the upper electrode buffer that consisted of 1% (vol/vol) H2SO4. Focusing was performed for 15-20 h at increasing voltage up to 1200 V. After focusing, the columns were fractionated into counting vials. pH was determined using a surface electrode (type 403-30, Ingold, Zurich, Switzerland) and radioactivity measurements were performed as described above. Ferritin (pi = 5.0) and hemoglobin (pi = 7.2 and 7.6) were used as standards.
Results High-affinity binding of [3H]R 1881, [3H]DHT and [3H]testosterone to human prostate cytosol Binding of [3H]R 1881, [ 3 H]DHT and [3H] testosterone to human prostate cytosol analyzed according to Scatchard yielded the TABLE 1. Kj and Bmax values calculated from Scatchard analyses of binding data from incubations of human prostate cytosol with [3H]R 1881, [3H]DHT and [3H] testosterone max
Steroid
K,, ± SD (IIM)
(95% confidence limit) (fmoles/mg protein)
R 1881 DHT Testosterone
1.48 ± 0.11 2.60 ± 0.08 8.65 ± 1.59
29.2 (28.3-30.1) 201(199-203) 45.3 (44.4-46.2)
200
cpm 400
FIG. 1. Isoelectric focusing of: a) [3H]R 1881 incubated with human plasma; b) [3H]DHT incubated with human plasma; c) [3H]R 1881 incubated with human prostate cytosol; d) [3H]DTH incubated with human prostate cytosol.
IQj and B max values shown in Table 1. DHT has considerably more binding sites than R 1881 and testosterone. Since R 1881 did not show any high-affinity, low-capacity binding to serum proteins (cf, below) this ligand does not appear to bind to TeBG. Therefore, the large amount of DHTbinding sites in prostate cytosol is probably explained by the presence of contaminating TeBG. The relatively small amount of testosterone-binding sites in prostate cytosol is in agreement with previous reports on the higher affinity of DHT than testosterone to TeBG (for ref., see 14). Characterization of high-affinity [3H]R 3 1881- and [ H]DHT-binding protein in human prostate cytosol by isoelectric focusing Plasma obtained from the third trimester of pregnancy was diluted 40 times and incubated with [3H]R 1881 and [ 3 H]DHT as described above for prostate cytosol. After treatment with DCC, the labelled steroidprotein complexes were desalted by
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
SNOCHOWSKI ET AL.
924
chromatography on Sephadex G-50. [3H]R 1881- and [ 3H]DHT-protein complexes from prostate cytosol were treated in the same way and all samples were analyzed by isoelectric focusing employing the pH range of 4-6. The [3H]R 1881- and [3H]DHT-protein complexes from prostate cytosol were found to focus at the same pH (pi 5.0-5.2) (Fig. 1). However, [ 3 H]DHT also bound to a plasma protein (conceivably TeBG) and this complex focused at the same pH as the cytosol complex. [3H]R 1881 did not bind to any plasma protein, confirming previous data reported by Bonne and Raynaud (7). Furthermore, the [3H]DHT-cytosol protein complex gave a more complicated electrofocusing pattern than the homogeneous peak formed by the [3H]R 1881- receptor complex (Fig. 1) indicating that the latter complex may be more stable during isoelectric focusing than the DHT-receptor complex. These results point out some of the great advantages—no binding to TeBG or other plasma proteins, high stability of steroid-receptor complex—in utilizing [3H]R 1881 rather than [ 3 H]DHT as ligand in studies on the androgen receptor in human prostate cytosol. Metabolism of [3H]R 1881 and [3H]DHT in human prostate cytosol Figure 2 shows another great advantage in using [3H]R 1881 rather than [ 3 H]DHT in studies on the androgen receptor in
JCE & M • 1977 Vol 45 • No 5
human prostate cytosol. Although [ 3 H]DHT was rapidly metabolized even at 0 C, [3H]R 1881 was not significantly metabolized even at 23 C. Specificity of[3H]R 1881 binding to human prostate cytosol The competition data are summarized in Fig. 3 and Table 2. The most efficient competitors to R 1881 were DHT and testosterone. Competition by LS 1727, cyproterone acetate, 17/3-estradiol, R 5020, 5aandrostane-3a,17/3-diol and progesterone was also observed but to a smaller extent. Androstenedione, cyproterone, cortisol, estramustine or G-302 did not compete significantly with R 1881 for binding sites on the receptor. These results indicate that R 1881 binds to an androgen receptor in human prostate cytosol with high specificity for androgens. Recent reports that R 1881 also shows high affinity to the progesterone receptor (15,16) do not influence this interpretation since progestins like cyproterone acetate, R 5020 and progesterone were only weak competitors to R 1881. Rates of degradation and dissociation of the [3H]R 1881-receptor complex in human prostate cytosol Rates of degradation and dissociation of the [3H]R 1881-receptor complex were determined as described in Materials and Methods and the results are shown in Figs.
A •DHT20-C • DHT 15 C • DHT 0 C
FIG. 2. Metabolism of [3H]R 1881 and [3H]DHT at different temperatures. Metabolites were measured by thin-layer chromatography as described in Materials and Methods. Per cent conversion of the labelled substrates represent total metabolism.
1 A 16 INCUBATION TIME (HOURS)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
925
ANDROGEN RECEPTOR IN HUMAN PROSTATE
100
G3O2
gfe 50
ANOROSTANEDIOL ESTRADIOL TESTOSTERONE LS 1727 R1881 n-CYPAC. DHT 2-10"1'
10"
10"8
CONCENTRATION (M) OF ADDED COMPETITOR
FIG. 3a. Competition of DHT, testosterone, LS 1727, cyproterone acetate, 17/3-estradiol (estradiol) and 5aandrostane-3a,17/3-diol (Fig. 3a) and of R 5020, progesterone, androstenedione, cyproterone, estramustine and cortisol (Fig. 3b) for high-affinity, low-capacity binding of [3H]R 1881 to human prostate cytosol. Samples were incubated with 5 x 10~9M [3H]R 1881 and varying concentrations of non-radioactive steroids (0.2-500 x 10" 9 M) at 0 C for 18 h. Free and bound steroid were separated using treatment with DCC. Relative binding affinities of the different competitors calculated on the basis of these results are summarized in Table 2.
100-
CORTISOL
ESTRAMUSTINE CYPROTERONE ANDROSTENEDIONE
00 u.
crO
50 PROGESTERONE R5020
R1881 2-K)r10
10"°
10"'
CONCENTRATION (M) OF ADDED COMPETITOR
FIG. 3b. See legend to Fig. 3a.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 21:02 For personal use only. No other uses without permission. . All rights reserved.
926
SNOCHOWSKI ET AL.
TABLE 2. Relative binding affinity of various steroids to the androgen receptor in human prostate cytosol
JCE & M • 1977 Vol 45 • No 5
0 C the complex dissociated with a halflife of 13.5 h.
Relative binding affinity
Steroid R1881
Rate of association of [3H]R 1881 to the androgen receptor in human prostate cytosol
1
0.48 0.082 0.035 0.025
