Concentration and cellular distribution of androgen receptor in human prostatic neoplasia: Can estrogen treatment increase androgen receptor content?

J. steroid Biochem. Vol. 19. No. 3, pp. 1279-1290, Printed in Great Britain. All rights reserved

1983 Copyright

0022.4731/83 $3.00 + 0.00 Q 1983 Pergamon Press Ltd

CONCENTRATION AND CELLULAR DISTRIBUTION OF ANDROGEN RECEPTOR IN HUMAN PROSTATIC NEOPLASIA: CAN ESTROGEN TREATMENT INCREASE ANDROGEN RECEPTOR CONTENT? B. G.

MOBBS,

I. E. JOHNSON,

Department

of Surgery,

J. G. CONNOLLY University

and J. THOMPSON

of Toronto,

Canada

Summary-The concentration of androgen receptor in cytosol (free and total sites) and nuclear fractions from benign (28 specimens) and malignant prostatic tissue from treated (16 specimens) and untreated patients (10 specimens) were assayed using [3H]methyltrienolone (‘H R-1881) as ligand under conditions which stabilize AR and prevent binding of 3H R-1881 to progesterone receptor. It was found that optimum results were obtained when sodium molybdate (10 mM) was added after separation of the nuclear pellet rather than during tissue homogenization; when cytosol and nuclear exchange assays were carried out at 15°C rather than at 0°C; and when hydroxylapatite was used to separate free and bound steroid in the nuclear assay. Although AR values were variable in both BPH and carcinoma tissue, certain patterns of concentration, occupancy, and cellular distribution were observed in different patient groups. In BPH and untreated carcinoma tissue, the mean occupancy of cytosol AR by endogenous androgens was high, but the mean nuclear AR concentration was higher in BPH than in carcinoma tissue. Androgen receptor concentrations in tissue from orchiectomized patients were consistent with the effects of androgen deprivation: total cell AR was depleted, and a higher proportion was present as free cytosol AR. However, in tissue from most patients who had been treated with diethylstilbestrol (DES) on a long-term basis, total cell AR values were high. Although most of the AR was present as free cytosol AR, in three of four patients who had been treated with both orchiectomy and DES, the concentrations of bound cytosol AR and nuclear AR were similar to those in untreated patients.

INTRODUCTION A large cinoma usually estrogens

percentage respond brought and/or

of

patients

initially about

to

with androgen

by administration

orchiectomy.

Such

prostatic

car-

deprivation, of synthetic treatment

may

for long periods of time, but eventually hormonal control is lost and the disease progresses. Work with animal tumour models has been suggested that progression is due to the growth of the hormone-insensitive cells in the tumour cell population over-riding the effect of the endocrine manipulation on the hormone-sensitive cells [ 1,2]. Recent data on the androgen receptor (AR)* content of prostatic carcinoma tissue from some patients treated with estrogens for long periods now lead us to speculate that in these patients another mechanism may also be operating to induce apparent relapse from hormonal control. control

tumour

growth

*Abbreviations: AR, Androgen receptor; BPH, benign prostatic hypertrophy; DCC, dextran-coated charcoal; DES, diethylstilbestrol, I-a’-diethylstilbenediol; 5a-DHT. Sa-dihydrotesterone, 17P-hydroxy-Sa-androstan-3-one; HAP, hvdroxvlanatite; MOO:. sodium molvbdate. Na,MoO;.2H,b; k-1881, methyltrienolone; SHBG, sex hormone binding globulin; SN, supematant; SDD, single saturating dose; TA, triamcinolone acetonide; TURP, transurethral prostatectomy. Address for reprint requests: Dr B. G. Mobbs, Room 7336, Medical Sciences Building, University of Toronto, Toronto, Canada MSS lA8. 1279

Using a protamine sulphate precipitation assay, we previously observed very high concentrations of cytosol AR in some prostatic carcinomas after long-term estrogen treatment [3]. Since these patients were undergoing surgery for the relief of symptoms which had developed during endocrine treatment, their tumours were apparently insensitive to hormonal manipulation at the time the tissue specimens were taken for AR assay, and thus the presence of elevated AR values was unexpected. We have now extended this investigation to include assay of salt-extracted nuclear AR in a further series of patients, so as to obtain a more complete picture of the concentration and cellular distribution of AR in human benign and malignant prostatic tissue, and of the differences in AR concentration, distribution and occupancy by endogenous androgen between treated and untreated groups of patients. In this series, we have used methyltrienolene (R188 1) as ligand in free site and exchange assays in the cytosol under conditions which prevent binding to progesterone receptor. This method was less time consuming than the protamine sulphate precipitation assay, and gave equivalent results provided sodium molybdate was included in the cytosol assay buffer to conserve receptor [4,5]. [3H]R-1881 was also used as ligand in an exchange assay on KC1 extracts of the nuclear pellet. Optimum incubation conditions with respect to ligand concentration, time, temperature and cytosol protein concentration were established

largely on benign prostatic hypertrophy (BPH) tissue obtained at open operation. These conditions were used in single saturating dose (SSD) assays to examine the quantity and cdfutar distr~butio?~ of AR in untreated and treated prostatic neoplasia. During the course of the investigation we also evaluated (i) the efl‘ect of avoiding extraction of nuclear AR with moiybdate by adding the latter to the supernatant fraction after separation of the n&ear pellet, (ii) separation of free and bound ligand in the nuclear extract by dextran-coated charcoal (DCC) and hydroxylapatite (HAP). and (iii) the effect of excision of prostatic tissue by transurethral prostatectomy (TURP) as compared with open excision.

~H]Meth~itrienolone (R-1881) (SA X7.0 Ci/mmol) and radioinert methyitrienolone were obtained from New England Nuclear Corp. On arrival [3H]R-1881 was diluted to 50hCi/mi in redistilled benzeneethanol (9: I. v&f and stored at “C. UntabeIled steroids, DNA standard (sodium salt from salmon testes), and BSA were obtained from Sigma Chemical Company (St. Louis, MO); Dextran T70 from Pharmacia (Montreal); charcoal (Norit A) from Matheson, Coleman and Bell: hydroxyiapatite (DNA Grade) from Bio-Rad Laboratories (Richmond, CA). The scintiitators used were either 5 g 2,5-diphenyloltazole (PPO) and 0. I g p-h& [2-( %phenyf-oxazofyl)].-benzene (POPOP) (New England Nuclear), per I. of toiuene; or PCS (Amersham): toluene (2: I, v/v).

The pH of aIi buffers was 7,4. TEM: tOmM Tris, 0.5 mM EDT& 0.5 mM ~-mercaptoeth~nol; TEM: IOmM MOO;: TEM + IOmM NaZMo0,.2Hz0; TEM: 110 mM MOO,‘: TEM + 1 IO mM Na,MoO,” 2Hz0; STMg: IOmM Tris, 0.25 M sucrose, 3 mM MgCi,; TEMK: 0.5 M KC1 in TEM; TK,,: IO mM Tris. 5OmM KCl; DCC: 0.57; charcoal, 0.05% dextran T70 in TEM for nuclear receptor assay: or 0.33?<,charcoal, 0.033% dextran 7-70 in TEM 10 mM MOO; for cytosol receptor assay; HAP slurry: hydroxyiapatite (HAP) was washed with TK,, buffer until pH of SN was 7.4. Final ratio of HAP: buffer was 0.7: I; Tris--Tween wash: f 0 mM Tris-HCi, 1:‘;; Tween X0
Immediately after removal, the tissue was placed in a glass vial refrigerated on ice until it was transferred in ice to the laboratory (usually within half an hour). All tissue handii~~ and assay procedures were carried out at %4 C with precooled equipment, gtassware and buffer solutions unless otherwise stated. Tissue from transurethral resections was carefully selected: chips that were badly damaged and/or less than 2 mm thick were discarded. The remainder were trimmed of damaged portions and the surfaces were scraped with

a scalpel. Representative portions ol‘ all tissues were fixed for histological examination and the rest of the tissue was rinsed in hoinogen~z~~tion bufrer. Excess buffer was removed with Kimwipes. The tissue was then wrapped in aluminum foil and snap frcLe:enin liquid nitrogen, where it was stored for not more than 3 weeks before assay.

Frozen tissue was pulverized in a Thermovac pulverizer {Thermovac Industries Corp. Copiague, N.Y.), cooled with liquid nitrogen and then homogenized (150 mg tissue/ml buffer) in either TEM or TEM lOmM,MoO; buffer using a Polytron P-IO homogenizer (Brinkman Instruments Inc.), for two bursts of IS--20 s, (setting 31, with a 30 s cooling interval. The homoge~te was centrifuged at 3000~ for IO min to yield a crude nuclear pellet used for nuclear receptor assay, and a crude supernatant which was immediately adjusted to 10 mM molybdate by the addition of I /lOth vol of TEM I 10 mM- MOOT if the homogen~~at~on was carried out in TEM buffer. DNA estimation was carried out by the method of Dische&], either on an aliquot of the homogenate, or on the washed nuclear pellet after KC1 extraction. Preliminary experiments established that virtually no loss of DNA occurred during processing of the crude nuctear pellet. The crude SN was then centrifuged at t45,001fg for i h to separate the cytosol. An aliquot of cytosoi was taken for protein assay 171.

The techniques used derived largely from the work of Bonne and RaynaudfX]. Zava Edni@], Gaubert rr a!.[51 and Garola and McGuire[lO~.

Both free (i.e. unoccupied by endogenous androgen) and totat cytosol AR sites were assayed using f%I]R-I88 I as ligand, using excess radioinert R- I88 f to correct for non-specific binding. Duplicate 1.0 mX aliquots were incubated with [XH]R-188i & lOO-fold excess R- 1881 in the presence of SOO-fold excess triamcinolone acetonide to prevent binding to progesterone receptor [9]. Preliminary experiments confumed the conclusion of Shain PI ui. that progesterone receptor does sot interfere in AR assays at I5 ‘C [Ii]. Incubation was for 2 h at 0 C for the free site assay and 2 h at 0°C followed by 16 h at 15°C for the exchange assay for total sites. Unless otherwise stated, incubated cytosol protein concentrations were [email protected] mg/mt. After incubation, 0.25 ml replicates were transferred to glass tubes and free steroid was removed by the addition of 0.05 mi of DCC suspension (final concentration 0.257;; charcoal, 0.025”,, dextran) for 15 min at 0°C. The DCC was removed by two 10 min spins at 30006 and 0.4 ml of the SN counted in PCS--toluene (2: 1) sointillator. Highaffinity binding was calculated by subtracting Iow-

AR in prostatic neoplasia: effect of treatment

affinity binding (in the presence of excess unlabelled R-1881) from total binding (in the absence of excess unlabelled R- 188 1). Free cytosol AR saturation analysis was carried out on cytosol derived from carcinoma tissue with a high concentration of free sites, using O.&lOnM [3H]R-1881. Saturation of high-affinity binding occurred at a ligand concentration between 5 and 10 nM, with low-affinity binding comprising 40% of the total binding at 10 nM ligand concentration. The & determined by Scatchard analysis 1121 was 1.8 x lo-‘* M. We have previously established that in rat ventral prostate preparations assayed under these conditions, a small amount of exchange (approx 12% of occupied sites) also takes place. It is not possible to verify this in human tissue, but it is possible that free site concentrations are slightly overestimated by this assay. Saturation analysis for total cytosol AR by exchange was carried out by BPH tissue in which most cytosol AR is already bound to endogenous DHT. Cytosol was incubated for 2 h at 0°C followed by 16 h at 15°C with concentrations of [3H]R-1881 ranging from 2.5-45 nM & loo-fold excess radioinert R- 1881. Maximum high-afl?nity binding occurred between 20 and 25 nM, with a Kc,of 2 x lOmyM. The proportion of low-affinity binding ranged from 22% of the total binding at 2.5 nM to 77% at 45 nM [3H]R-1881. When the SSD exchange assay was carried out with 25 nM [‘HJR-1881 on cytosol diluted to give a range of protein concentrations from 0.44.0 mg protein/ml, it was found that high-affinity binding values dropped off sharply at protein concentrations below 2.0 mg/ml. Replication was also very poor below 1 mg/ml. Owing to the high proportion of low-affinity binding in specimens with low receptor activity, accuracy of the assay in such specimens is

decreased, and the sensitivity achieved is not very high. We do not feel that cytosol assays on less than 300 mg of tissue are reliable unless the specimens have high receptor activity, and that in a specimen of this size, a total high-affinity binding value for the cytosol of less than 25 fm/mg cytosol protein should be considered below the limit of sensitivity. The effect of the incubation temperature on the cytosol exchange assay was examined by incubating aliquots of the same BPH cytosol with 25 nM [3H]R-1881 + loo-fold excess radioinert R-1881 for either 2 h at 0°C followed by 16 h at 1YC, or for 24 h at 0°C. The latter incubation was carried out in the

1281

presence of 5 ~1M TA. Incubation in the cold resulted in high-affinity binding values only approx 25% of those achieved at 15°C on the same cytosols. There was no significant difference between the low-a~njty binding values at 15°C and O”C, and the precision of the assay was similar at both temperatures (Table 1). Cytosol receptor may have been stabilized by filling empty sites at 0°C before raising the temperature for exchange, as well as by the presence of molybdate. Cytosol binding was expressed as fm/mg cytosol protein and as fm/mg DNA in the crude nuclear pellet so as to compensate for differences in cellularity between specimens, and also to permit comparison between amounts of high-affinity binding in the cytosol and nuclear fractions. Preparation ofnuclear extract. Immediately after removal of the crude SN from the 3000g spin of the tissue homogenate, the pellet was washed three times by resuspension in STMg buffer (approx 10 vol) and centrifugation at 800g (3 x 5 min). The crude nuclear pellet was then resuspended in TEMK buffer and extracted for 45 min at O”C, vortexing every 10 min. The pellet was separated from the extract by a 10 min spin at 25,000g and extracted for 15 min with additional TEMK (for a total of 1 ml/200 mg tissue equivalent). After centrifugation (25,OOOg, 10 min), the pellet was frozen for DNA assay. The extracts were combined and clarified by a 30min spin at 140,OOOg.Typically the nuclear extract from 200 mg tissue contained 2-3 mg protein and represented 0.5-0.8 mg DNA/ml. Aliquots (1 .Oml) of the nuclear extract were diluted with TEM buffer containing 13H]R-188 1 & 1OO-fold excess R- 188 1 to give the desired ligand concentration. For saturation analysis, the ligand range was 4-45 nM. In separate experiments carried out with 25 nM ligand, the incubation time was varied from O-29 h, temperature from 0-25°C and nuclear extract protein concentration from 0.25-2.0 mg/ml. incubated extracts were treated with HAP or DCC to separate free and bound steroid. Hydroxylapatite separation. The incubated samples were cooled on ice and replicate aliquots, containing 0.5-0.75 mg protein, were added to 0.25 ml HAP slurry in polystyrene tubes. AR was allowed to adsorb to the HAP for 30 min at 0°C with vortexing every 10 min. Tris-Tween (2 ml) was added to each

Table 1. Effect of incubation temperature on cytosol exchange assay High-affinity binding (fmjmg protein)*

Low-affinity binding (fm/mg protein)*

Exchange conditions

A

B

A

B

O’-24 h CJ-2 hr then 117-16 h

28 i_ 7 72 -+ 3

42 * 5 138 + - 10

184+_5 166+2 -

163+3 168f2 _

*Mean k SD calculated from 8 replicates per assay (4+, 4 - radioinert R-1881); A, B: two specimens of BPH; cytosol was incubated with 25nM [3H]R-1881 f2.5j~M R-1881; free and bound steroid were

separated with DCC.

tube which was then vortexed and centrifuged at 3000~ for 2 min. The SN was aspirated and the pellet was washed three times with 2ml Tris-Tween to remove all free steroid (vortex+ spinning 2 min at 3OOOa and aspirating the SN each time). Radioactive steroid was extracted from each HAP pellet by vortexing with 2ml ethanol and leaving at room temperature overnight. After the HAP was repelleted (3OOOg for 5min), the supernatants were decanted into scintillation vials. The HAP pellets were washed with an additional 0.5ml ethanol and respun. The supernatants were combined and toluene scintillator was added for counting. DC’c’.repurution. After exchange incubation. 0.5 ml replicate aliquots of the nuclear extract were mixed with OS ml DCC suspension (in TEM buffer), bringing the concentration of KC1 to 0.15 M. The tubes were vortexed and kept on ice for I5 min then centrifuged at 3000~ for IO min. The supernatants were decanted and respun (3000~ for IO min) to remove all charcoal. Aliquots (0.5ml) of the final supernatants were counted with PCSWoluene scintillator. High-affinity binding in the nuclear assay was expressed as fm per mg DNA in the washed nuclear pellet. Experiments were done to compare the sensitivity and precision of the nuclear assays using the two separation methods. Incubated BPH nuclear extract was adjusted to a range of concentrations from 0.254.0 mg nuclear protein/ml and separation of free and bound steroid was carried out by both methods on replicate aliquots at each dilution. In addition, nuclear extracts from seven BPH specimens were assayed using both separation methods at the protein concentration normally used for the SSD assays (2-3 mg/ml). The results confirmed the observation of Trachtenberg ef ul. that HAP separation in the nuclear extract is superior to separation with DCC [4]. Intra-assay variation was lower with HAP and the assay was very sensitive, being linear down to 0.5 mg protein~ml nuclear extract. In the series of specimens assayed by both separation methods, the mean values for each method were not significantly different, but precision with HAP separation was much higher, due to better replication resulting from much lower non-specific binding values. The contributjon of non-specific to total binding was 37’114 SD 7”,, after HAP separation compared with 81”); _t SD IO”: after DCC separation in the same extracts. Using HAP separation it is possible to assay nuclear AR in diluted nuclear extracts from pellets containing as little as 2OOpg of DNA. Using a ligand concentration of 25 nM for the nuclear exchange assay at 15 C, high-affinity binding values increased from 2 to 20 h, and then reached a plateau. Incubation with 25 nM [‘H]R-1881 for 20 h at temperatures from 0 -30°C resulted in maximum specific binding between I5 and 23’ C with a rapid drop at temperatures above this. Specific binding was

SO”,, higher at IS than at 0 C. and the amount ot non-specific binding was not significantly diKerent at these temperatures. Incubation for saturatiol~ analysis was carried out at 15 ‘C for 20 h with 4-45 nM [jH]R- I88 I. Scatchard analysis showed that specific binding had not completely levelled off at 45 nM, and Rosenthal analysis of the Scatchard plot [ 131 revealed that in addition to the main high-affinity component (K,, -. 2 x’ IO-” M), a second component with an affinity approx 100-fold lower was present. which was not saturated under the conditions used (Fig. I). At the ligand concentrations used for the SSD assays (see below). this component comprised less than 20”;, of the total specific binding. Thus the SSD assays may slightly overestimate the nuclear high-a~nity AR content. The significance of the lower-affinity binding component observed on Scatchard analysis of nuclear binding needs further investigation: possibly it represents a type II receptor component such as that reported for estrogen receptor by Markevich cl rr/.jl4]. Biphasic nuclear androgen binding has also been observed by Rennie ct ttl. in an androgen sensitive rat prostatic adenocarcinoma [l5]. Singlt~ -.sutura ring dose a.s.r~~~s

The assay of free and total cytosol and nuclear androgen receptor in the majority of the tissue speci-

NUCLEAR

EXCHANGE

L,,-L___i_i_..

0

ASSAY

(HUMAN

30

BPH)

40

Fig. I. Binding of [“H]R- 188 I to 0.6 M KCI nuclear extract from human BPH. The extract was incubated with [3H]R-1881 +- IOO-fold excess radioinert R-1881 for 20 h at 1.5’C. Free and bound steroid were separated with HAP. Motybdate was omitted from the homogenization buffer. The Scatchard plot was anatyzed by the graphic method of Rosenthalf 1.71.

AR in prostatic neoplasia: effect of treatment

mens was carried out using a single ligand concentration for each assay. For free cytosol sites, [3H]R- 1881 was used at 10 nM in the presence of 5 pM TA, with and without 1 FM unlabell~ R-1881 to permit correction for low-affinity binding. Incubation was for 2 h at @4”C. For the exchange assay for total cytosol sites, incubation was carried out with 25 nM [‘H]R-1881 with and without 2.5pM unlabelled R-188 1 for 2 h at 0°C followed by 16 h at 15°C. Nuclear extract was incubated for 20 h at 15°C with 25 nM rH]R-1881 with and without 2.5 pM unlabelled R-1881. Free and bound steroid were separated with DCC for the cytosol assays and with either DCC or HAP for the nuclear assays. i@ect nf m@bdate on cytosol and nuclear assays. A recent report indicated that the increased cytosol AR values obtained after homogenization in the presence of 25 mM molybdate were due in part to AR extraction from the nucleus [14]. We examined this possibility under the conditions of our assays, using 10 mM molybdate. Frozen BPH tissue was pulverized, mixed and divided in two approx equal portions. One portion was homogenized in TEN buffer and the crude SN obtained after centrifugation was immediately adjusted to 1OmM molybdate by the addition of TEM. 110 mM .MoO;. The other portion was homogenized in TEM . 10 mM MOO;, Free and total cytosol AR and nuclear AR were assayed by single saturating dose (SSD) assays. In all three specimens, mean cytosol AR values were slightly higher when molybdate was included at the time of homogenization, but the differences were not significant (Table 2). In the nuclear assay the omission of sodium molybdate from the homogenization step appeared to have the greatest effect on specimens with high nuclear AR content (Table 2). In two BPH

I283

specimens with nuclear AR values >400 fm/mg DNA, the mean values were significantly higher (P < 0.05) when molybdate was omitted from the homogenization buffer: however, in another specimen with a nuclear AR value of approx lOOfm/mg DNA, the presence or absence of molybdate at the time of homogenization made no significant difference. We have concluded that it is preferable to add molybdate to a crude supernatant fraction immediately after separation of the nuclear pellet by a short, low-speed centrifugation. Ligand specificity

Aliquots of BPH cytosol and nuclear extract were incubated with 25 nM [‘H]R-1881 in the presence of a series of competitors at concentrations ranging from 25 nM-1.25 FM under exchange conditions. Free steroid was removed from the cytosol with DCC and from the nuclear extract with HAP. The relative binding affinity (RBA) of each competitor was calculated by dividing the concentration of R-1881 necessary to reduce high-affinity binding by 50% by the concentration of competitor necessary to cause the same reduction. In both nucleus and cytosol, the steroid specificity was typical of an androgen receptor (Table 3). Patients and treatment

The data from the SSD assays, on which the results to be presented are based, were derived from specimens of benign hypertrophic or carcinomatous prostatic tissue from 64 patients. Fourteen of the 39 BPH specimens were obtained at open operation and the remaining 25 by transurethral resection (TLJRP). All but two of the 26 carcinoma specimens were of the primary tumour obtained by TURP; the remaining

Table 2. Effect of omission of MOO; from homogenization buffer on cytosol and nuclear AR concentrations Cytosol AR (fm~mg DNA)* Total Free

Nuclear AR (frn~rn~ DNA)*

MOO; included in homogenization buffer and throughout assay Specimen C Specimen D Specimen E

243 + 106 878 + 240 708 + 58 127 + 83 355 +_161 91 + 26

503 + 24” 437 + 18h 108k8

MOO; added to crude SN after separation of nuclear pellet Specimen C Specimen D Specimen E

173f70 105 + 16 94+ 55

727 + 22” 534 & 25b 105k5

792&113 684 + 140 279 f 138

a vs a, b vs b: P < 0.05. *Mean & SD calculated from 6 or 8 replicates (half+, half- excess radioinert R- 188I). C, D. E: three specimens of BPH. Cytosol was incubated with 10 nM [‘H]R-1881 & 1FM R-1881 in the presence of 5gM TA for 2 h at O”C, for the free site assay. For total site exchange assays, cytosol was incubated with 25 nM [3H]R-1881 + 2.5 PM R-1881 for 2 h at 0°C. followed by 16 h at lS”C, and the 0.6 M KCI nuclear extract was incubated with 25 nM [3H]R-1881 for 20 h at 15°C. Bound and free steroid were separated with DCC in the cytosol assays, and HAP in the nuclear assays.

I284

B. G. Motres

Table 3. Steroid

Competitor

specificity

in cytosol assays

and nuclear

exchange

Relative binding affinity Nuclear Cytosol (exchange assay) (exchange assay)

R-1881 DHT Cyproterone acetate Estradiol Cortisol Progesterone DES

1.o

I .o 0.3

0.4

0.06

0.02

<0.02
0.01 < 0.003 0.007 N.D.

RBA: [R-1881]/[Competitor] necessary to reduce affinity binding of [3H]R-1881 by 502,. N.D.: not mined. Cytosoi and n&ear extract w&e incubated 25 nM r3H1R- 188 I under exchange conditions with petitor; ai concentrations in thi range 25 nM-10 Free and bound steroid were separated with DCC cytosol assay and HAP in the nuclear assay.

highdeterwith comPM.

in the

two were biopsies of carcinoma metastatic to lymph nodes. Carcinoma specimens from which the data were derived contained at least 707: malignant tissue. Of the carcinoma patients, IO had had no hormonal treatment prior to surgery, eight had been orchiectomized for periods ranging from 4 days to 6 years, four had been treated with diethylstilbestrol (DES) for periods ranging from 3 days to 8 years, and four had undergone both orchiectomy and DES treatment for periods ranging from 2 to 7 years. Assessment

of‘ endocrine

stntus

In order to establish the extent of the effect of hormone manipulation, 10 ml nonheparinized venous blood was taken from all carcinoma patients and all except five BPH patients at time of surgery. The serum was spun off and stored at - 17°C until assay of the serum testosterone concentration and the high affinity [‘H]DHT-binding capacity of the serum, which is considered equivalent to the SHBG concentration. Total serum testosterone concentration was assayed in the Department of Clinical Chemistry, Mt Sinai Hospital, Toronto. For the measurement of [3H]DHT binding capacity, serum was diluted I : 100 or I :200-500 (v/v) (in estrogen-treated patients) with IO mM Tris-HCl buffer (pH 7.4) containing I.5 mM EDTA and glycerol IO’:<;v/v (TEG buffer). 0.2 Milliliter aliquots were incubated with an equal volume of 2 nM [3H]DHT f 200 nM cold DHT in TEG buffer for 2 h at O’C. 0.5 Milliliter of TEG containing a suspension of 0.5% charcoal and 0.050/ dextran was added to each aliquot. The tubes were vortexed, allowed to stand in an ice bath for 15 min and centrifuged at 3000 g for IO min. The supernatant was removed and recentrifuged to remove the charcoal completely, and 0.5 ml aliquots of the supernatant were counted in 10 ml PCSWoluene scintillator. Tissue ,fbr SSD ussuys

Control

tissue (i.e. not from androgen

target

or-

<‘/ (I/.

gans) was obtained from two patients: the specimens were pyramidalis muscle from a patient with BPH. and pelvic lymph nodes with no malignant involvement removed from a previously untreated patient with prostatic carcinoma. The proportion of malignant tissue present in the carcinoma specimens and the degree of dilt‘erentiation was estimated both in the tissue fixed in our laboratory and the diagnostic specimen obtained by the pathology department directly from the operating room. The evaluation of the degree of differentiation of each specimen was based on the gland&n differentiation and the gland growth pattern in relation to stroma. There was generally good agreement between both specimens, indicating that the specimen sent for assay was representative of the tumour as a whole. The proportion of tissue that was malignant varied very widely between tumours but, as already mentioned. all the data from carcinoma specimens presented here are from tissue with a malignant component of at least 70”,,. These specimens were all poorly and/or moderately well differentiated. Two were anaplastic. RESULTS Serum

testosterone

concmtrution

mid

[‘H]DHT-

binding cupucitv

The total serum testosterone concentration and serum [‘HIDHT-binding capacity (SHBG) was assayed for all patients except three with BPH. The results are presented in Table 4. No significant differences were observed in values obtained from serum from untreated carcinoma and BPH patients. The results in the treated carcinoma patients were consistent with their treatment: all had subnormal testosterone levels; in the orchiectomized and tongterm DES-treated groups, serum testosterone was < 80 ng/dl. One patient who had been treated with DES for only 3 days had a serum testosterone level of 105 ng/dl. The [‘HIDHT-binding capacity in the orchiectomized patients was similar to that in untreated patients but, as expected. was raised in all the Table 4. Serum lestosterone m patients

yielding

Diagnosis and treatment (No. of patients) BPH (34) Carcinoma Untreated (I 0) Orchiectomy <2m (2) >6m (6) DES < I wk (2) >5 yr (2) Orchiectomy + DES >2yr (4) -. _

‘Mean + su

and [‘H]DHT prostatic specimens Serum testosterone (ng:dl)*

binding capacit! for AR assays Serum [‘H]DHT binding capacity (/lg,dl)*

520 * 215

0.72 & 0.42

I

0.7x * 0.32

437 i_ 20 37. 43*

26 IX

1.26 0.79-i

0.22 0.62

70. ‘7.

105 43

5.94. 2.64.

1.01 5.70

41 *29

3.65 & 1.95

*Mean + SD. tNumbers in parentheses:

MOO; added to crude SN Control: Uninvolved node (1) BPH--open (7) -TURP (10) Carcinoma-open (I) -TURP (4) -TURP (2) -TURP (1)

number

treatment

in benign

of specimens

assayed.

N.D.:

None None None None None Orchiex: long term DES: short-term

None None None None None Orchiex: short term Orchiex: long term DES: short term DES: long term Orchiex + DES: long term

Patient

5. AR concentrations

MOO; in homogenization buffer Control: Pyramidalis muscle (I)? BPH-open (7) -TURP (14) Carcinoma-pen (I) -TURP (4) -TURP (2) -TURP (4) -TURP (1) -TURP (2) -TURP (4)

Tissue

Table

not determined.

16 91 +28 63 + 28 217 71*41 152, 87 58

N.D. 98 k 52 90*51 425 101 * 71 41, 25 60 k 25 29 711, 251 1032 f 504

51 725 k 189 329 * 124 669 198 & 155 465, 482 226

221 770 + 526 407 +217 1157 396 k 285 207, 72 240 k 122 253 1582, 1220 3740 k 1574

36 627 k 189 278 If: 133 120 138 + 101 3, 263 148

125 651 f 493 443 + 229 333 351 * 281 11, 51 114* 118 141 137, 20 510 + 395

DNA* Bound

patients

20 557 + 157”,’ 271 T 129”,d 259 148 k 80“ 41, 49 24

0 347 + 161’ 241 f 145 215 142 + 41’ 0, 84 29 k 41’ 177 42, 15 136k 111

Salt-extractable nuclear AR (fm/mg DNA)*

and treated

71 1281 k 325b 599 * 199b 928 476 + 267 512. 531 250

221 1117+539 69Ok217 1372 534 + 302’ 207, 166 269 + 88’ 430 1624, 1235 3876 + 1602

Cytosol and salt-extractable nuclear AR (fm/mg DNA)*

. -.

- ,^ . . - _.. _”

_,.

- -

-

.. .^ - _..

a vs a, b vs b: P < 0.001; c vs c: P i 0.01; d vs d, e vs e, f vs f: P < 0.05.

11 79 k 26 54 * 30 39 40 f 34 1, 47 39

N.D. 83+51 81 +48 122 90&71 2, 18 29 & 25 13 62, 4 160 f 109

fm/mg Total

AR

tissue from untreated

Cytosol

prostatic

fm/mg cytosol protein* Bound Total

and malignant

_.. -

-

Cytosol protein yields from BPH and carcinoma tissue were very similar: 21.5 $ 4.g mggm of BPH tissue, and 22.2 r4:4.8 mg:gm of carcinoma tissue. The DNA concentration was signi~c~~ntiy grcatcr (P < 0.001) in the carcinoma tissue (6.0 * 2.0 mg DNA/gm of tissue) than in the WI-1 tissue (3.X J 0.9 mg DNAigm of tissue), reflecting the greater cellularity and/or ancuploidy [I61 of the carcinoma tissue.

These data are presented in Table 4 and Figs 7---3. The two control tissues contained < 250 fm:mg DNA total cellular receptor, and negligible amounts were present in the nuclear fraction (420 fmimg DNA). Benign

prastutit~

Itypwttwpltj~.

Total

celi,

c qeOSD! AR occupied a,,cell AR

I”

““Cl*“*

85 + 7

81177

18

51 tt7

3-I

a -

‘%+I1

28

33116

28

5

Fig. 2. AR conccntratians (mean? SD) in tissue from untreated patients. Homogenization was carried out either in the presence (upper panel) or absence (lower panel) of IOmM sodium molybdate. In the latter, the supernatant was adjusted to IO mM molybdate after separation of the nuclear peilet. Cytosol was incubated with 10 nM [ZH]R-1881 + 1pM mdioinert R-1881 in the presence of 5 LIM TA for 2 h at DC (free site assay), and with 25 nM [7H]R-IRXI f2.5icM R-1881 for 2 h at 0°C followed by I6 h at 15 C (total site assay). Nuclear extract was incubated with 2.5nM [‘HJR-18X1 &2.5pM R-1881 for 20h at 15°C. Data in the upper panel are from different specimens from those in the lower panel. Control tissues were ~~rarn~d~i~ muscic (upper panel) and a lymph node with no malignant involvement (lower panel).

TREATED:

LOW

SERUM

T

HOrn”c?

Fig. 3. AR concentrations in carcinoma those in Fig. 4. Left panel: homogenized

cytosol

nuclear receptor concentrations were variabte in BPH tissue, whether this was obtained at open operation or by TURP, and whether MoO, was present or absent from the homogenization buffer. The mean concentration of both cytosol and nuclear reccptot was higher in the “open” than in the “TURP” specimens, hut this difference was significant only for total cell and nuclear receptor in the specimens assayed in the absence of MOO; in the homogenization buffer (P < O.OOl). A barely significant difference was also observed between mean nuclear AR concentration in open specimens assayed with and without MOO,” in the hoIno~er~iz~lt~on hutfer (0.05 > P > 0.02), but this difference was not observed in the “TURP“ specimens, In al] the BPH groups, the mean proportion ofoytosol sites occupied

and

MOO:

tissue from treated patients. Assay conditions were identical with in the presence of MOO;: right panel: Moo; omitted from the homoge~i~t~on buffer.

1287

AR in prostatic neoplasia: effect of treatment 0

Free

m

Occupied Nuclear

AR

BPH

Fig. 4. Concentration, occupancy and distribution of AR in BPH and prostatic carcinoma tissue. The areas of the circles are proportional to the mean AR concentration in the tissue of each patient group, and the areas of the segments are proportional to the mean concentrations of free cytosol, occupied cytosol and nuclear AR. This chart is derived from the results obtained from specimens homogenized in the presence of molybdate and assayed by the SSD method (see Table 5). by endogenous androgens was >80%. The cellular distribution of receptor (i.e. between cytosol and nucleus) was almost identical in the “open” and “TURP” specimens whether or not they had been homogenized in the presence of molybdate, but the mean proportion of total cell AR which was present in the nucleus was slightly higher in both types of specimen when molybdate was omitted from the homogenization buffer (43.5 + 5% in the “open” and 46 f 11% in the “TURP” specimens) than when homogenization took place in the presence of molybdate (35 k 17% in the “open” specimens and 37 & 20% in the “TURP” specimens). However, this difference was not significant (P > 0.1). Untreated prostatic carcinoma. When the results from the untreated carcinoma “TURP” specimens were compared with those from the BPH “TURP” specimens, no significant differences were observed in total cell, cytosol, or nuclear AR content when molybdate was present in the homogenization buffer. However, when molybdate was omitted from the homogenization, the mean nuclear receptor concentration and the mean proportion of cytosol receptor occupied by endogenous androgen was significantly lower in the carcinoma specimens than in the BPH specimens (nuclear AR concentration in carcinoma 148 k 80 fm/mg DNA vs 271 k 129 fm/mg DNA in BPH, P < 0.05; 51 f 17% cytosol sites occupied in carcinoma vs 8 1 f 17% cytosol sites occupied in BPH, P < 0.02). The data from the two carcinoma specimens obtained by open biopsy (both were lymph nodes virtually replaced by tumour) showed a different pattern: both had a receptor content which was considerably higher than the mean for the carcinoma “TURP” specimens, but in both cases over 80% of the total cytosol receptor content was assayable under free site assay conditions, the occupied cytosol receptor concentration being comparable with that in the other untreated carcinoma sDecimens. 1

Nuclear AR concentrations were marginally higher than those in the carcinoma “TURP” specimens. Treated carcinoma specimens. Carcinoma specimens from orchiectomized patients had a significantly lower mean AR receptor concentration than specimens from untreated patients when homogenized in the presence of molybdate (P < 0.05), mainly due to the depletion of nuclear AR content (35 f 48 fm/mg DNA in orchiectomized patients vs 142 f 41 fm/mg DNA in untreated patients, P < 0.01). The proportion of cytosol AR occupied by endogenous androgen was also significantly lower (42 f 33% vs 85 f 16% in untreated patients, P < 0.05). Only two specimens from orchiectomized patients were homogenized in the absence of molybdate. In these, the total AR content was not depleted compared to that in untreated patients, but a similar distribution of receptor between the cytosol and nucleus to that in the other orchiectomized patients was observed. A mean of 72% of the total tissue AR was assayable in the cytosol under free site conditions. This pattern of depleted nuclear receptor and a high proportion of free cytosol receptor is consistent with androgen deprivation and was observed in all the orchiectomized patients, irrespective of the time which had elapsed since orchiectomy. Two patients had undergone DES treatment for < 1 week, and total cell AR concentrations were not significantly lower than in untreated patients. In one, whose serum testosterone was higher than in the other treated patients, nuclear AR was not depleted. In both the proportion of cytosol sites assayable only under exchange conditions was approx 60%. Thus, in these patients, treatment had not yet resulted in sufficient androgen deprivation to complete the expected changes in receptor distribution. In the two patients who had undergone long-term DES treatment, nuclear receptor concentrations were similar to those in orchiectomized patients. However, cytosol AR concentrations were significantly higher than those in untreated carcinoma patients. Most of this cytosol AR was assayable under free site conditions, but in one the occupied cytosol AR concentrations was within the range of that in untreated patients. Even higher concentrations of free cytosol AR were observed in all four patients who had been orchiectomized and received DES for more than 2 years. In those cases, although only a small proportion (13 + 5%) of cytosol AR appeared to be bound, the absolute concentrations were as high or higher than in untreated patients, and in three of the four patients nuclear AR values were also as high as those in untreated patients. The results of the SSD assays in untreated and treated groups of patients are summarized in Fig. 4. DISCUSSION

The number of patients in each treatment group was small, but certain patterns of AR distribution

I xx

H.
within the cell, and of cytosol AR occupancy bq endogenous androgens. appeared to be characteristic of dill‘erent treatment groups. and the results confirm our previous investigations on cytosol AR 131. Wide variations in both cytosol and nuclear AR concentrations were observed in benign and malignant tissue from untreated patients. When homogenized in the presence of MOO,. the mean values for cytosol and nuclear AR concentration. the proportion of cytosol AR occupied by endogenous androgens, and the proportion of total AR which was present in the nucleus, were not significantly different between benign and malignant tissue or between benign tissue obtained by “open” prostatectomy and by “TURP”. Others have also observed variable cytosol and nuclear concentrations. with no signiticant differences between benign and malignant tissue, using assays from which molybdate was omitted throughout the assays [7- 191: however, reported cytosol AR concentrations tend to be rather low compared to our results when exchange was carried out at 4 C [ 191. Addition of molybdate to the crude supernatant after separation of the nuclear pellet decreased the variability of the results and revealed significantly higher total cell and nuclear AR concentrations in open BPH than in “TURP” BPH specimens. However, the pattern of AR distribution in the BPH tissue was similar in the “open” and “TURP” specimens. Nuclear AR concentrations were higher in the “open” BPH specimens homogenized in the absence of molybdate than in similar specimens homogenized in the presence of molybdate. A significant difference was also observed between the salt-extractable nuclear AR from BPH (TURP) and carcinoma (TURP) specimens when molybdate was added after separation of the nuclear pellet. The two carcinoma specimens obtained at “open” biopsy differed from those obtained by “TURP” in that they had higher total AR concentrations most of which was in the cytosol and assayable under free site conditions. Since these were both lymph nodes virtually replaced by malignant tissue, these characteristics may be related to the stage of the disease rather than the method of excision. It is of interest that others have also reported high cytosol binding in some specimens of poorly differentiated carcinoma [ 18.201. In our specimens, bound cytosol AR and nuclear AR concentrations were similar to those observed in the untreated carcinoma specimens obtained by “TURP”. The trend towards a lower proportion of occupied cytosol sites compared with BPH tissue was also observed in the carcinoma specimens excised from the primary tumours by “TLJRP”, but the mean proportion of occupied cytosol sites was significantly lower than that in BPH only in specimens homogenized in the absence of molybdatc. To date only three treated carcinomas have been assayed after homogenization in the absence of molybdate, so discussion of the data from treated pa-

Cents will be hmlted to tumours homogenired 111 ~hc presence of molybdate. In most respects the result\ from tumours from treated patients were consistent with androgen deprivation, i.e. a shift in AR dictribution from nucleus to cytosol was observed and ;I higher proportion of cytosol sites was present as free AR. In orchiectomized patients a depletion in total cell AR concentration was also observed. The\e effects of treatment confirm earlier observations [3, 17. 191 but owing to the large variations betwjccn tumours, significant dilrerences have not previously been reported between data from treated a~nd LIIItreated patients. The results from patients who had undergone long-term DES treatment suggest that AR content in these patients’ tumours may have been modulated by influences in addition to androgen deprivation. Since these patients had been admitted for “TURP” for urinary tract obstruction. it must be assumed that their disease was not hormone-sensitive at the time of operation. Therefore, the possibility must be considered that high unoccupied cytosol AR concentrations may not be entirely due to treatment, but may accompany progression of the disease from a hormone sensitive to an autonomous state, which could occur in the absence of treatment. This possibility is supported by occasional observations of high free cytosol AR concentrations in some untreated carcinomas. usually metastatic and:or poorly differentiated [3, IX, 201. It has been shown that prostatic carcinoma contains less Sr-reductase activity [20. 311 and lower mean DHT concentrations [22.23] than BPH. and that autonomous carcinoma tissue contains less DHT than hormone-sensitive tissue [23, 241. If present, cytosol AR might, therefore. be expected to be unbound or to be bound to androgens with relatively low affinity for AR which might well be replaced by the labelled ligand under “free” site conditions. Diethylstilbestrol treatment would augment this tendency by reducing circulating serum testosterone levels, and possibly also by inhibiting Sa-reductase activity, although evidence on this point is conflicting [25. 261. However. freeing of existing AR sites alone is unlikely to account for the extremely elevated values observed in the estrogentreated patients in this present series. Similar clevations were not observed in tumours from patients in this series treated by orchiectomy alone. It is of interest that estrogen treatment has been observed to increase free and/or total cytosol AR in a number of experimental systems [27 301 and it is tempting to speculate that AR synthesis might actually be iI: duced in men treated with estrogens for prostatic carcinoma. From the point of view of patient management. this would be important only if translocation of AR into the nucleus occurred and was followed by tumour activation. A report by Fowler and Whitmore[31] that exacerbation of symptoms occurred when patients who had relapsed from hormonal control were treated with testosterone suggests

AR in prostatic neoplasia: effect of treatment

that functional AR was still present in the tumours of those patients. In our series, the tumours of three of the four patients admitted for “TURP” after both orchiectomy and estrogen treatment appeared to contain occupied cytosol and nuclear AR in similar concentrations to those in untreated patients. Since circulating serum testosterone levels were very low, this raises the question of the nature of the endogenous ligand. There have been a number of reports indicating that adrenal activity may be stimulated by estrogen treatment. A small secondary rise in serum testosterone some months after suppression by estrogen treatment was observed in one study; this effect could be reversed by adrenal suppression [32]. Conversely, in another investigation, stimulation of the adrenal by synthetic corticotrophin in orchiectomized and/or estrogen-treated patients resulted in a rise of serum testosterone and androstenedione [33]. Increases in serum adrenal androgen levels have also been demonstrated in oophorectomized and post-menopausal women after treatment with conjugated estrogens [34,35]. In view of the raised levels of SHBG in estrogen-treated prostatic carcinoma patients, it seems unlikely that the levels of unbound testosterone likely to be present (even if a secondary rise occurs) would be sufficient to result in translocation of AR into the nucleus of the carcinoma cell. However, it is possible that adrenal androgens, which have lower affinity for SHBG, could be metabolized in the prostatic tissue to more potent androgens [36], which might cause reactivation of the growth of hormonesensitive tumours. This possibility is supported by the observation by Geller et ul. that the prostatic tissue of two patients who had relapsed after hormonal treatment contained significant concentrations of SIX-DHT[23]. Another possibility is that the specimens contained unoccupied AR “resident” in the nuclei, similar to the “resident” nuclear estrogen receptor reported by several investigators to be present in some breast carcinomas [37,38]. Since our nuclear extract incubations were carried out at 15°C any unoccupied AR would have been included in the total salt-extractable AR assayed. Separate incubation at 0 and 15°C would discriminate between unoccupied and occupied nuclear AR. Finally, the possibility cannot be dismissed that the nuclear AR observed in these three specimens is artifactual. Although the concentrations observed were significant, they represent a small proportion of the total cell AR. The nuclear assay is carried out on a crude washed nuclear pellet, and a small amount of cytoplasmic contamination would result in the “nuclear” AR values observed in this group of patients. Nevertheless, there is no doubt of the presence of elevated concentrations of cytosol AR in this group of patients. Only further investigation will allow evaluation of the significance of this receptor and determination of its role in the progression and/or reactivation of prostatic carcinoma. \”

IY/3-

-0

1289

Acknowledgements-This investigation was supported by grant No. 251 from the Ontario Cancer Treatment and Research Foundation, and grant No. CA-27412 from the National Institutes of Health (U.S.A.). The majority of the tissue and blood specimens were obtained by one of the authors (J.G.C.), and we also gratefully acknowledge the cooperation of Drs M. Barkin, R. Comisarow, P. CrassWeller, M. Jewett, A. Keresteci, J. Rankin, G. Ranking, J. Russell and N. Struthers in providing prostate and blood specimens from their patients undergoing urologic surgery. The collaboration of Dr S. Urbanski in evaluating the histopathology, and of Dr M. da Costa, for the testosterone radioimmunoassays, is also greatly appreciated.

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27. Tokarr R. R., Harrtson R. W. and Scaver S. S.. I’hc mechanism of androgen and estrogen synergism tn the chick oviduct. ./. h&l. C‘lrrrn. 254 ( 1979) Y17X Y I X4. 2X. Moore R. J.. Gazak J. M. and Wtlson J. 0.: Regulation of cytoplasmic dihydrotestoerone binding in dog pro\tate by 17/I-estradiol. J. r,/in. /nrc.\r. 63 (1979) 351 357. 29. Trachtenberg J.. Hicks L. L. and Walsh P. c‘.: Androgenand estrogen-receptor content in spontaneous and experimentally induced canine prostatic hyperplasia. J. c/in. Jnw.v/. 65 (1980) 1051 1059. 30. Bouton M. M., Pornin C. and Grandadam J. A.: Estrogen regulation of rat prostate androgen receptor. .I. steroid Biochem. 15 ( I98 I) 4033408. 31. Fowler J. E. and Whitmore W. F.: The response of metastatic adenocarcinoma of the prostate to exogenous testosterone. J. l:ro/. 126 (1981) 372375. 32. Robinson M. R. G. and Thomas B. S.: Effect ot hormonal therapy on plasma testosterone levels in prostatic carcinoma. Br. Med J. 4 (1971) 391-394. 33. Cowley T. H.. Brownsey B. G.. Harper M. E., Peeling W. B. and Griffiths K.: The effect of ACTH on plasma testosterone and androstenedionc concentrations in patients with prostatic carcinoma rlc/rr emlou 81 (1976) 310-320. 34. Abraham G. E. and Maroulis G. B.: Effect of exogenous estrogen on serum pregnenolone. cortisol and androgens in postmenopausal women. Ohsrer. G~~nrcol. 45 (1975) 271 -274. 35. Lobo R. A.. Goebelsmann IJ.. Brenncr P. F. and Mishell D. R.: The effects of estrogen on adrenal androgens in oophorectomiaed women. ilm. .J. Ohster. Gywo/. 142 ( 1982) 47 I -47X. 36. Harper M. E.. Pike A., Peeling W. B. and Grifhths K.: Steroids of adrenal origin metabolized by human prostatic tissue both in ciw and in vitro. J. Endocr. 60 (1974) 117-125. 37. Geier A.. Ginsburg R., Stauber M. and Lunenfeld B.: Unoccupied binding sites for oestradiol in nuclei from human breast carcinomatous tissue. J. Endocr. 80 (1979) 28 lL2XX. 38. Thorsen T.: Occupied and unoccupied nuclear oestrddiol receptor in human breast tumours: relation to oestradiol and progesterone cytosol receptors. J. .stcwid Biochem. 10 ( 1979) 66 I -66X.