0013-7227/91/1282-0872$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
Epithelial- Stromal Interactions in the Regulation of Rat Ventral Prostate Function: Identification and Characterization of Pathways for Androgen Metabolism in Isolated Cell Types* JOHN ORLOWSKIf AND ALBERT F. CLARK Departments of Biochemistry and Pathology, Queen's University, and Kingston General Hospital, Kingston, Ontario, Canada K7L 3N6
ABSTRACT. Androgen metabolism plays a significant role in the androgen regulation of prostate cell function. In this report the various pathways for androgen metabolism in primary cultures of rat ventral prostate epithelial and stromal cells were identified and characterized by in vitro whole cell assays, using HPLC. Confluent cultures of both cell types were incubated with supraphysiological concentrations (50 nM) of tritiated androgens (testosterone, 5a-dihydrotestosterone, 5a-androstane-3a(and 3/3),17/3-diols, and A4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5a-reductase activity, the oxidative reactions of 3a-, 3)3-, and 17/3-hydroxysteroid oxidoreductases, and the reductive reaction of 3/3-hydroxysteroid ox-
idoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3a- and 17/3-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6a/7a-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5adihydrotestosterone directly to a new unidentified polar androgen metabolite (H05a-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5a-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites. {Endocrinology 128: 872884,1991)
I
ment, 5a-DHT receptors are present exclusively in the stroma (7, 8). Cunha and associates (9) have concluded that epithelial responses (development, growth, and cytodifferentiation) to androgen are mediated via trophic factors emanating from the androgen-sensitive stroma. Postnatally, however, 5a-DHT receptors are found at higher levels in the epithelium, suggesting a role for the stroma in the development of epithelial androgen receptor activity (10). In the adult gland, regulation of differentiated prostate function appears to occur predominantly in the epithelial cells, presumably directly mediated by androgen receptors. The role of the stroma in the adult prostate is less well defined. One central aspect of the epithelial-stromal cellular interaction that is poorly understood is the metabolic regulation of intracellular androgen concentrations, which is important to considerations of biological activity in the prostate as well as other male reproductive tract organs. The androgen responsiveness of epithelial and stromal cells during cellular replication and differentiation may be modulated by the overall rates of formation and removal of 5a-DHT. Prostatic 5a-DHT concentrations are regulated by a complex series of steroid metabolic pathways (see Fig. 1). Some experimental evidence has been presented which suggests that alterations in
N THE reproductive tract of a number of species, it has been established that sex steroids are potent systemic regulators of the development, growth, and cytodifferentiation of the accessory sex organs (1-3). In addition to hormonal influences, there has been considerable evidence from in vivo and in vitro morphological tissue recombinant experiments demonstrating an important role for epithelial-stromal cellular interactions in the embryonic development of the prostate and other organs (4-6). With regard to the fetal rodent prostate (urogenital sinus), these studies have indicated that the urogenital stroma is essential for the development and function of the urogenital epithelium. The urogenital stroma requires the presence of 5a-dihydrotestosterone (5a-DHT), the principal metabolite of circulating testosterone (T) and biologically active intracellular androgen, for this inductive effect. During this stage of developReceived August 16,1990. Address all correspondence and requests for reprints to: Dr. Albert F. Clark, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6. * This work was supported in part by grants from the Medical Research Council of Canada (MT-2338 and MA-10212). t Current address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267.
872 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:41 For personal use only. No other uses without permission. . All rights reserved.
•
ANDROGEN METABOLISM IN PROSTATE CELLS
873
FIG. 1. Overview of principal pathways for androgen metabolism in the prostate. Enzyme abbreviations are defined in Footnote 1.
HO*
OH 6«-Atriol
androgen metabolism may be involved in the pathogenesis of human and canine benign prostatic hyperplasia (BPH) (11, 12). However, it is not known whether these changes are causal or secondary to the disease process. In humans, these metabolic changes appear to be occurring predominantly in the stromal cells (13,14). Interestingly, BPH occurs rarely in the rat, and the disease does not resemble the human condition. This makes the rat a useful model for comparison to the human and canine in terms of its ability to regulate androgen action in the prostate. At present, there is considerable controversy concerning the cellular distribution of enzymes involved in androgen metabolism, particularly 5a-reductase activity (15,16). Also, the cellular locations of the other important enzymes involved in regulating 5a-DHT concentrations have not been characterized fully. With the recent development (17) of a HPLC system to measure 5a-reduced androgens, these studies are now more feasible. To study intra- and intercellular androgen-mediated actions in postnatal prostate development, primary cultures of rat ventral prostate epithelial and stromal cells from immature animals have been developed (18). In the present study time-course experiments of androgen metabolism by primary cultures of both cell types has allowed for a more complete analysis of the various steroid metabolic pathways. Materials and Methods Materials Animals. Experiments were performed using immature male Sprague-Dawley rats (21-22 days old) obtained from Charles
M .
OH 6or-Adiol-l7-one
7a-Adiol-l7-one
River Laboratories Ltd. (Montreal, Quebec, Canada). All animals are housed in a controlled environment [room temperature, 21 ± 2 C; normal atmospheric pressure and humidity, and a regulated lighting cycle (14 h of light, 10 h of darkness)]. Tap water and Purina rat chow (Ralston-Purina, St. Louis, MO) were available ad libitum. Steroids. Radioinert steroids were obtained from Steraloids (Wilton, NH) and Sigma Chemical Co. (St. Louis, MO). [1,2'HJA 1 (46.1 Ci/mmol), [4-14C]A (57.8 mCi/mmol), [1,2- 3 H]T (59.0 Ci/mmol), [4-14C]T (57.5 mCi/mmol), [l,2- 3 H]5a-DHT (50.6 Ci/mmol), [4- 14 C]5a-DHT (57.5 mCi/mmol), and [1,23 H]3a-Adiol (30.1 Ci/mmol) were obtained from New England Nuclear Corp. (Lachine, Quebec, Canada) and purified before use by paper chromatography, as described by Bush (19). [414 C]5a-A was prepared by chromic acid oxidation of [4- 14 C]5aDHT, as previously described (20). [4-14C]3a-Adiol and [4-14C] An were prepared in our laboratory by reduction of [4-14C]5aD H T and [4-14C]5a-A, respectively, using NADPH and a 50% 1 The following abbreviations are used: 3a-Adiol, 3a,17/3-dihydroxy5a-androstane; 3/3-Adiol, 3/3,17/3-dihydroxy-5a-androstane; A, androstenedione; 5a-A or 5a-androstanedione, 5a-androstane-3,17-dione; An, androsterone; IsoAn or isoandrosterone, 3/3-hydroxy-5a-androstan17-one; 6a-Atriol, 3j8,6a,17/3-trihydroxy-5a-androstane; 7a-Atriol, 3;8,7a,17/3-trihydroxy-5a-androstane; 6a-Adiol-17-one, 3/3,6a-dihydroxy-5a-androstan-17-one; 7a-Adiol-17-one, 3/?,7a-dihydroxy-5a-androstane-17-one; 5a-d 9 O 2 , refers to 5a-DHT, 3a-Adiol, 3/3-Adiol, 5aA, An, and IsoAn; 5a-Ci9O3, refers to 6a-Atriol, 7a-Atriol, 6a-Adiol17-one, and 7a-Adiol-17-one; 17-oxo-Ci9O2, refers to A, 5a-A, An, and IsoAn; 17/3-OH-Ci9O2, refers to T, 5a-DHT, 3a-Adiol, and 3/3-Adiol; 5a-R, A4-3-ketosteroid-5a-reductase (EC 1.3.1.4); 3a-HSOR, 3a-hydroxysteroid oxidoreductase (EC 1.1.1.50), 3/3-HSOR, 30-hydroxysteroid oxidoreductase (EC 1.1.1.51); 170-HSOR, 170-hydroxysteroid oxidoreductase (EC 1.1.1.63); subscripts Ox or red after HSOR indicate enzyme activities in oxidative or reductive directions, respectively. 6aH, 6a-hydroxylase; 7a-H, 7a-hydroxylase; 6a/7a-H, mixture of 6a-H and 7a-H activities; HBSS, HEPES-sodium hydroxide-buffered saline solution; Bes, iV,iV-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid.
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874
ANDROGEN METABOLISM IN PROSTATE CELLS
rat prostate or lung homogenate in 0.1 M Tris-HCl buffer (pH 7.4) as the source of 3a-HSORred activity. [1,2-3H]3(8-Adiol, [414 C]3/3-Adiol, and [4-14C]IsoAn were prepared from [l,2-3H]5aDHT, [4-14C]5a-DHT, and [4-14C]5a-A, respectively, by reduction with NaBH4, as previously described (20). [4-14C]6a-Adiol, [4-14C]7a-Atriol, [4-14C]6a-Atriol-17-one, and [4-14C]7a-Adiol17-one were prepared in our laboratory by hydroxylation of [414 C]3/3-Adiol using rat ventral prostate epithelial cell cultures as the source of the 6a/7a-H activity. The hydroxylated metabolites of [4-14C]3j8-Adiol were isolated and identified by their capacity factors on HPLC (21). Authentic reference steroids were not available commercially for confirmation of their identification. All radioactive steroids were at least 97% pure, as determined by TLC and paper chromatography (20). 14C-Labeled steroids for monitoring recovery and assessing purity were prepared in methanol and contained approximately 5000 dpm of the appropriate 14C-labeled steroid and 25 ng radioinert steroid/100 pi. Chemicals. Sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate monohydrate, sodium citrate, citric acid, and anhydrous dextrose were of reagent grade and obtained from Canadian Laboratory Supplies (Toronto, Ontario, Canada). Benzene, chloroform, hexane, heptane, and methylene chloride were obtained from Fisher Scientific Co. (Ville St. Pierre, Quebec, Canada). HPLC grade acetonitile and methanol were purchased from Fisher Scientific Co. (Pittsburgh, PA). Hoescht 33258 stain was purchased from Calbiochem-Behring (La Jolla, CA). Collagenase type IV (140 U/mg), beef pancreatic DNase-I, HEPES, Bes, Trizma base (Tris), phenol red, insulin, transferrin, retinoic acid, dexamethasone, and highly polymerized calf thymus DNA were obtained from Sigma Chemical Co. Percoll was purchased from Pharmacia Fine Chemicals (Montreal, Quebec, Canada). Fetal bovine serum, chicken serum, F-12 medium (powder) with sodium bicarbonate (F12), Dulbecco's Modified Eagle's Medium (DME; powder) with sodium bicarbonate, and trypan blue exclusion dye were purchased from Flow Laboratories Ltd. (Mississauga, Ontario, Canada). Lyophilyzed penicillin (10,000 U/ml), streptomycin (10,000 Mg/ml), fungizone (250 Mg/ml), trypsin (1:250; Difco certified), and 0.05% trypsin-0.02% EDTA solution were obtained from Gibco (New York, NY). Methods Establishment of primary cultures of prostate epithelial and stromal cells. Primary cell cultures were established by enzymatic dissociation of the ventral prostate lobes from immature rats (21-22 days old), as described previously (22). The epithelial and stromal cells were separated by isopycnic centrifugation (22) and selective attachment of fibroblast-type cells to plastic culture flask surfaces (22, 23). Both cell types were maintained in F12-DME (1:1) culture medium supplemented with 10% fetal bovine serum, HEPES (20 mM), NaHCO3 (10 mM), insulin (5 Mg/ml), transferrin (5 /ig/ml), T (50 nM), dexamethasone (50 nM), penicillin (100 U/ ml), streptomycin (100 /xg/ml), and fungisone (1 ng/n\\) at 37 C in a gas phase of 98%:2% (vol/vol) air to carbon dixoide ratio. The culture medium was changed every 48 h. Cultures of both cell types were monitored daily using a Leitz
Endo • 1991 Vol 128 • No 2
(Wetzlar, Rockleigh, NJ) inverted phase contrast light microscope. Representative cultures were fixed in 100% methanol for 5 min and stained with 2% Geisma stain for 5-30 min. Cell culture conditions for quantitation of androgen metabolism. Primary monolayer cultures of prostate epithelial and stromal cells were cultured to confluency in 10-cm2 culture dishes. The culture medium was removed and replaced with serum-free and steroid-free culture medium for 24 h. After this time, the medium was replaced with fresh serum and steroid-free culture medium to which 50 nM 3H-labeled androgens (T, 5«-DHT, 3a-Adiol, 3/3-Adiol, and A; 0.1-0.2 /iCi/lOO pmol steroid-2 ml culture medium) in an ethanol medium were added; the final ethanol concentration of the medium (95%) of rat ventral prostate epithelial and stromal cells were obtained. Epithelial cells in culture reached confluency in approximately 7 days. As previously described (18, 22), cells examined by light microscopy were polygonal in shape and closely associated with each other, which is characteristic of this cell type in culture.
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ANDROGEN METABOLISM IN PROSTATE CELLS The stromal cells were also confluent after approximately 7 days. They were elongated, spindle-shaped, and loosely associated with each other. This is characteristic of fibroblast-type cells in culture. In the stromal cell cultures, confluency generally was attained at a lower cell population density than epithelial cells. The appearance of both cell types was not altered when they were grown in the absence or presence of androgen. The functional integrity of the epithelial cells was further demonstrated by their capability to express secretory acid phosphatase activity, a well characterized androgenregulated secretory protein of the rat ventral prostate (25). Androgen metabolism by epithelial and stromal cell cultures To determine the presence of steroid-metabolizing enzymes in the two cell types, 3H-labeled androgens (T, 5a-DHT, 3a-Adiol, 3/3-Adiol, and A) at supraphysiological concentrations (50 nM; 0.1-0.2 juCi/lOOpmol steroid2 ml) were incubated for 3-24 h with confluent monolayers. The radioactive androgen metabolite profiles described below have been obtained by HPLC of androgens extracted from serum-free culture medium plus cells. Extraction recoveries of unchanged radioactive substrate from 24-h control incubations in the absence of cells were 90-95%. In the presence of cells, recovered radioactive substrate and metabolites accounted for 85-95% of the added radioactivity for both cell types. 14C-Labeled androgen standards were added to the organic solvent containing medium plus cells just before steroid extraction to monitor losses. This allowed for correction of recovered radioactivity to 98-102% of the starting levels. Undefined aqueous radioactive metabolites not extracted from the culture medium by the organic solvent with the various androgen substrates accounted for 1-2% of the total radioactivity and remained constant throughout the 24-h incubation period, with the exception of cultures using 3/?-Adiol as substrate. For the latter substrate, the undefined aqueous radioactive metabolites increased gradually with incubation time from approximately 2.8% to 3.5% in epithelial and 5.0% to 10.0% of the total radioactivity in stromal cell cultures. The aqueous radioactive metabolites were not analyzed further.
875
period, 5a-A was the major metabolite, accounting for 55.1% of the total radioactive androgens. Increases were observed in the amounts of 5a-Ci9O3 polar metabolites (12.3%) and IsoAn (7.1%). 3/3-Adiol (2.3%), 3a-Adiol (2.1%), and An (0.7%) were present in smaller amounts. These results indicated that epithelial cells have high 5a-R and 17/3HSOROX activities. In contrast, stromal cells revealed a less complex metabolic pattern (Fig. 2B). Very little metabolism of T (21.0%) to 5«-reduced products occurred during the 24h incubation period. Stromal cells appeared to have lower 5a-R activity than epithelial cells. Stromal cells also produced small amounts of 3a-Adiol as well as an unidentified androgen metabolite (H05a-DHT; 6.2%) that eluted in the polar region of the HPLC profile. This was the only polar metabolite found. Based on the capacity factor (K) of H05a-DHT on HPLC, this metabolite did not comigrate with any of the other polar androgen metabolites previously reported for the prostate (21). Also, 17-oxo-Ci9O2 metabolites were not detected, suggesting that 17/?-HSORox activity in the stromal cells was negligible. Localization of 3a-HS0RTe
Vol. 128, No. 2 Printed in U.S.A.
Epithelial- Stromal Interactions in the Regulation of Rat Ventral Prostate Function: Identification and Characterization of Pathways for Androgen Metabolism in Isolated Cell Types* JOHN ORLOWSKIf AND ALBERT F. CLARK Departments of Biochemistry and Pathology, Queen's University, and Kingston General Hospital, Kingston, Ontario, Canada K7L 3N6
ABSTRACT. Androgen metabolism plays a significant role in the androgen regulation of prostate cell function. In this report the various pathways for androgen metabolism in primary cultures of rat ventral prostate epithelial and stromal cells were identified and characterized by in vitro whole cell assays, using HPLC. Confluent cultures of both cell types were incubated with supraphysiological concentrations (50 nM) of tritiated androgens (testosterone, 5a-dihydrotestosterone, 5a-androstane-3a(and 3/3),17/3-diols, and A4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5a-reductase activity, the oxidative reactions of 3a-, 3)3-, and 17/3-hydroxysteroid oxidoreductases, and the reductive reaction of 3/3-hydroxysteroid ox-
idoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3a- and 17/3-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6a/7a-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5adihydrotestosterone directly to a new unidentified polar androgen metabolite (H05a-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5a-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites. {Endocrinology 128: 872884,1991)
I
ment, 5a-DHT receptors are present exclusively in the stroma (7, 8). Cunha and associates (9) have concluded that epithelial responses (development, growth, and cytodifferentiation) to androgen are mediated via trophic factors emanating from the androgen-sensitive stroma. Postnatally, however, 5a-DHT receptors are found at higher levels in the epithelium, suggesting a role for the stroma in the development of epithelial androgen receptor activity (10). In the adult gland, regulation of differentiated prostate function appears to occur predominantly in the epithelial cells, presumably directly mediated by androgen receptors. The role of the stroma in the adult prostate is less well defined. One central aspect of the epithelial-stromal cellular interaction that is poorly understood is the metabolic regulation of intracellular androgen concentrations, which is important to considerations of biological activity in the prostate as well as other male reproductive tract organs. The androgen responsiveness of epithelial and stromal cells during cellular replication and differentiation may be modulated by the overall rates of formation and removal of 5a-DHT. Prostatic 5a-DHT concentrations are regulated by a complex series of steroid metabolic pathways (see Fig. 1). Some experimental evidence has been presented which suggests that alterations in
N THE reproductive tract of a number of species, it has been established that sex steroids are potent systemic regulators of the development, growth, and cytodifferentiation of the accessory sex organs (1-3). In addition to hormonal influences, there has been considerable evidence from in vivo and in vitro morphological tissue recombinant experiments demonstrating an important role for epithelial-stromal cellular interactions in the embryonic development of the prostate and other organs (4-6). With regard to the fetal rodent prostate (urogenital sinus), these studies have indicated that the urogenital stroma is essential for the development and function of the urogenital epithelium. The urogenital stroma requires the presence of 5a-dihydrotestosterone (5a-DHT), the principal metabolite of circulating testosterone (T) and biologically active intracellular androgen, for this inductive effect. During this stage of developReceived August 16,1990. Address all correspondence and requests for reprints to: Dr. Albert F. Clark, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6. * This work was supported in part by grants from the Medical Research Council of Canada (MT-2338 and MA-10212). t Current address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267.
872 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 19:41 For personal use only. No other uses without permission. . All rights reserved.
•
ANDROGEN METABOLISM IN PROSTATE CELLS
873
FIG. 1. Overview of principal pathways for androgen metabolism in the prostate. Enzyme abbreviations are defined in Footnote 1.
HO*
OH 6«-Atriol
androgen metabolism may be involved in the pathogenesis of human and canine benign prostatic hyperplasia (BPH) (11, 12). However, it is not known whether these changes are causal or secondary to the disease process. In humans, these metabolic changes appear to be occurring predominantly in the stromal cells (13,14). Interestingly, BPH occurs rarely in the rat, and the disease does not resemble the human condition. This makes the rat a useful model for comparison to the human and canine in terms of its ability to regulate androgen action in the prostate. At present, there is considerable controversy concerning the cellular distribution of enzymes involved in androgen metabolism, particularly 5a-reductase activity (15,16). Also, the cellular locations of the other important enzymes involved in regulating 5a-DHT concentrations have not been characterized fully. With the recent development (17) of a HPLC system to measure 5a-reduced androgens, these studies are now more feasible. To study intra- and intercellular androgen-mediated actions in postnatal prostate development, primary cultures of rat ventral prostate epithelial and stromal cells from immature animals have been developed (18). In the present study time-course experiments of androgen metabolism by primary cultures of both cell types has allowed for a more complete analysis of the various steroid metabolic pathways. Materials and Methods Materials Animals. Experiments were performed using immature male Sprague-Dawley rats (21-22 days old) obtained from Charles
M .
OH 6or-Adiol-l7-one
7a-Adiol-l7-one
River Laboratories Ltd. (Montreal, Quebec, Canada). All animals are housed in a controlled environment [room temperature, 21 ± 2 C; normal atmospheric pressure and humidity, and a regulated lighting cycle (14 h of light, 10 h of darkness)]. Tap water and Purina rat chow (Ralston-Purina, St. Louis, MO) were available ad libitum. Steroids. Radioinert steroids were obtained from Steraloids (Wilton, NH) and Sigma Chemical Co. (St. Louis, MO). [1,2'HJA 1 (46.1 Ci/mmol), [4-14C]A (57.8 mCi/mmol), [1,2- 3 H]T (59.0 Ci/mmol), [4-14C]T (57.5 mCi/mmol), [l,2- 3 H]5a-DHT (50.6 Ci/mmol), [4- 14 C]5a-DHT (57.5 mCi/mmol), and [1,23 H]3a-Adiol (30.1 Ci/mmol) were obtained from New England Nuclear Corp. (Lachine, Quebec, Canada) and purified before use by paper chromatography, as described by Bush (19). [414 C]5a-A was prepared by chromic acid oxidation of [4- 14 C]5aDHT, as previously described (20). [4-14C]3a-Adiol and [4-14C] An were prepared in our laboratory by reduction of [4-14C]5aD H T and [4-14C]5a-A, respectively, using NADPH and a 50% 1 The following abbreviations are used: 3a-Adiol, 3a,17/3-dihydroxy5a-androstane; 3/3-Adiol, 3/3,17/3-dihydroxy-5a-androstane; A, androstenedione; 5a-A or 5a-androstanedione, 5a-androstane-3,17-dione; An, androsterone; IsoAn or isoandrosterone, 3/3-hydroxy-5a-androstan17-one; 6a-Atriol, 3j8,6a,17/3-trihydroxy-5a-androstane; 7a-Atriol, 3;8,7a,17/3-trihydroxy-5a-androstane; 6a-Adiol-17-one, 3/3,6a-dihydroxy-5a-androstan-17-one; 7a-Adiol-17-one, 3/?,7a-dihydroxy-5a-androstane-17-one; 5a-d 9 O 2 , refers to 5a-DHT, 3a-Adiol, 3/3-Adiol, 5aA, An, and IsoAn; 5a-Ci9O3, refers to 6a-Atriol, 7a-Atriol, 6a-Adiol17-one, and 7a-Adiol-17-one; 17-oxo-Ci9O2, refers to A, 5a-A, An, and IsoAn; 17/3-OH-Ci9O2, refers to T, 5a-DHT, 3a-Adiol, and 3/3-Adiol; 5a-R, A4-3-ketosteroid-5a-reductase (EC 1.3.1.4); 3a-HSOR, 3a-hydroxysteroid oxidoreductase (EC 1.1.1.50), 3/3-HSOR, 30-hydroxysteroid oxidoreductase (EC 1.1.1.51); 170-HSOR, 170-hydroxysteroid oxidoreductase (EC 1.1.1.63); subscripts Ox or red after HSOR indicate enzyme activities in oxidative or reductive directions, respectively. 6aH, 6a-hydroxylase; 7a-H, 7a-hydroxylase; 6a/7a-H, mixture of 6a-H and 7a-H activities; HBSS, HEPES-sodium hydroxide-buffered saline solution; Bes, iV,iV-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid.
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874
ANDROGEN METABOLISM IN PROSTATE CELLS
rat prostate or lung homogenate in 0.1 M Tris-HCl buffer (pH 7.4) as the source of 3a-HSORred activity. [1,2-3H]3(8-Adiol, [414 C]3/3-Adiol, and [4-14C]IsoAn were prepared from [l,2-3H]5aDHT, [4-14C]5a-DHT, and [4-14C]5a-A, respectively, by reduction with NaBH4, as previously described (20). [4-14C]6a-Adiol, [4-14C]7a-Atriol, [4-14C]6a-Atriol-17-one, and [4-14C]7a-Adiol17-one were prepared in our laboratory by hydroxylation of [414 C]3/3-Adiol using rat ventral prostate epithelial cell cultures as the source of the 6a/7a-H activity. The hydroxylated metabolites of [4-14C]3j8-Adiol were isolated and identified by their capacity factors on HPLC (21). Authentic reference steroids were not available commercially for confirmation of their identification. All radioactive steroids were at least 97% pure, as determined by TLC and paper chromatography (20). 14C-Labeled steroids for monitoring recovery and assessing purity were prepared in methanol and contained approximately 5000 dpm of the appropriate 14C-labeled steroid and 25 ng radioinert steroid/100 pi. Chemicals. Sodium chloride, potassium chloride, sodium bicarbonate, sodium dihydrogen phosphate monohydrate, sodium citrate, citric acid, and anhydrous dextrose were of reagent grade and obtained from Canadian Laboratory Supplies (Toronto, Ontario, Canada). Benzene, chloroform, hexane, heptane, and methylene chloride were obtained from Fisher Scientific Co. (Ville St. Pierre, Quebec, Canada). HPLC grade acetonitile and methanol were purchased from Fisher Scientific Co. (Pittsburgh, PA). Hoescht 33258 stain was purchased from Calbiochem-Behring (La Jolla, CA). Collagenase type IV (140 U/mg), beef pancreatic DNase-I, HEPES, Bes, Trizma base (Tris), phenol red, insulin, transferrin, retinoic acid, dexamethasone, and highly polymerized calf thymus DNA were obtained from Sigma Chemical Co. Percoll was purchased from Pharmacia Fine Chemicals (Montreal, Quebec, Canada). Fetal bovine serum, chicken serum, F-12 medium (powder) with sodium bicarbonate (F12), Dulbecco's Modified Eagle's Medium (DME; powder) with sodium bicarbonate, and trypan blue exclusion dye were purchased from Flow Laboratories Ltd. (Mississauga, Ontario, Canada). Lyophilyzed penicillin (10,000 U/ml), streptomycin (10,000 Mg/ml), fungizone (250 Mg/ml), trypsin (1:250; Difco certified), and 0.05% trypsin-0.02% EDTA solution were obtained from Gibco (New York, NY). Methods Establishment of primary cultures of prostate epithelial and stromal cells. Primary cell cultures were established by enzymatic dissociation of the ventral prostate lobes from immature rats (21-22 days old), as described previously (22). The epithelial and stromal cells were separated by isopycnic centrifugation (22) and selective attachment of fibroblast-type cells to plastic culture flask surfaces (22, 23). Both cell types were maintained in F12-DME (1:1) culture medium supplemented with 10% fetal bovine serum, HEPES (20 mM), NaHCO3 (10 mM), insulin (5 Mg/ml), transferrin (5 /ig/ml), T (50 nM), dexamethasone (50 nM), penicillin (100 U/ ml), streptomycin (100 /xg/ml), and fungisone (1 ng/n\\) at 37 C in a gas phase of 98%:2% (vol/vol) air to carbon dixoide ratio. The culture medium was changed every 48 h. Cultures of both cell types were monitored daily using a Leitz
Endo • 1991 Vol 128 • No 2
(Wetzlar, Rockleigh, NJ) inverted phase contrast light microscope. Representative cultures were fixed in 100% methanol for 5 min and stained with 2% Geisma stain for 5-30 min. Cell culture conditions for quantitation of androgen metabolism. Primary monolayer cultures of prostate epithelial and stromal cells were cultured to confluency in 10-cm2 culture dishes. The culture medium was removed and replaced with serum-free and steroid-free culture medium for 24 h. After this time, the medium was replaced with fresh serum and steroid-free culture medium to which 50 nM 3H-labeled androgens (T, 5«-DHT, 3a-Adiol, 3/3-Adiol, and A; 0.1-0.2 /iCi/lOO pmol steroid-2 ml culture medium) in an ethanol medium were added; the final ethanol concentration of the medium (95%) of rat ventral prostate epithelial and stromal cells were obtained. Epithelial cells in culture reached confluency in approximately 7 days. As previously described (18, 22), cells examined by light microscopy were polygonal in shape and closely associated with each other, which is characteristic of this cell type in culture.
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ANDROGEN METABOLISM IN PROSTATE CELLS The stromal cells were also confluent after approximately 7 days. They were elongated, spindle-shaped, and loosely associated with each other. This is characteristic of fibroblast-type cells in culture. In the stromal cell cultures, confluency generally was attained at a lower cell population density than epithelial cells. The appearance of both cell types was not altered when they were grown in the absence or presence of androgen. The functional integrity of the epithelial cells was further demonstrated by their capability to express secretory acid phosphatase activity, a well characterized androgenregulated secretory protein of the rat ventral prostate (25). Androgen metabolism by epithelial and stromal cell cultures To determine the presence of steroid-metabolizing enzymes in the two cell types, 3H-labeled androgens (T, 5a-DHT, 3a-Adiol, 3/3-Adiol, and A) at supraphysiological concentrations (50 nM; 0.1-0.2 juCi/lOOpmol steroid2 ml) were incubated for 3-24 h with confluent monolayers. The radioactive androgen metabolite profiles described below have been obtained by HPLC of androgens extracted from serum-free culture medium plus cells. Extraction recoveries of unchanged radioactive substrate from 24-h control incubations in the absence of cells were 90-95%. In the presence of cells, recovered radioactive substrate and metabolites accounted for 85-95% of the added radioactivity for both cell types. 14C-Labeled androgen standards were added to the organic solvent containing medium plus cells just before steroid extraction to monitor losses. This allowed for correction of recovered radioactivity to 98-102% of the starting levels. Undefined aqueous radioactive metabolites not extracted from the culture medium by the organic solvent with the various androgen substrates accounted for 1-2% of the total radioactivity and remained constant throughout the 24-h incubation period, with the exception of cultures using 3/?-Adiol as substrate. For the latter substrate, the undefined aqueous radioactive metabolites increased gradually with incubation time from approximately 2.8% to 3.5% in epithelial and 5.0% to 10.0% of the total radioactivity in stromal cell cultures. The aqueous radioactive metabolites were not analyzed further.
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period, 5a-A was the major metabolite, accounting for 55.1% of the total radioactive androgens. Increases were observed in the amounts of 5a-Ci9O3 polar metabolites (12.3%) and IsoAn (7.1%). 3/3-Adiol (2.3%), 3a-Adiol (2.1%), and An (0.7%) were present in smaller amounts. These results indicated that epithelial cells have high 5a-R and 17/3HSOROX activities. In contrast, stromal cells revealed a less complex metabolic pattern (Fig. 2B). Very little metabolism of T (21.0%) to 5«-reduced products occurred during the 24h incubation period. Stromal cells appeared to have lower 5a-R activity than epithelial cells. Stromal cells also produced small amounts of 3a-Adiol as well as an unidentified androgen metabolite (H05a-DHT; 6.2%) that eluted in the polar region of the HPLC profile. This was the only polar metabolite found. Based on the capacity factor (K) of H05a-DHT on HPLC, this metabolite did not comigrate with any of the other polar androgen metabolites previously reported for the prostate (21). Also, 17-oxo-Ci9O2 metabolites were not detected, suggesting that 17/?-HSORox activity in the stromal cells was negligible. Localization of 3a-HS0RTe
