Pharmacological and Molecular Evidence for theExpression of the Two Steroid 5a-ReductaseIsozymes in Normal and Hyperplastic Human

Prostatic Cells in Culture

Isabelle Berthaut,1 Chidi Mestayer,2 Marie-Claire Portois,1 Olivier Cussenot,3and Irene Mowszowicz1*

1Laboratoire de Biochimie B, Hopital Necker-Enfants Malades, Paris, France2Service de Biochimie Medicale, Faculte de Medecine Pitie-Salpetriere, Paris, France

3Service d’Urologie, Hopital Saint-Louis, Paris, France

BACKGROUND. Whereas the embryological development of the human prostate is clearlydependent on steroid 5a-reductase (5a-R) type 2 expression, the respective expression of thetwo known isoforms (types 1 and 2) of 5a-R in the adult human prostate remains unclear.METHODS. 5a-R isoform mRNA expression (Northern blots and reverse transcriptase-polymerase chain reaction [RT-PCR]) and enzyme activity were studied in immortalizedepithelial cells (NE) and in fibroblasts from normal (NF) or hyperplastic (BPHF) humanprostates.RESULTS. 5a-R activity (fmol/mg DNA/hr) was 1.43 ± 0.5 in NE, 10.7 ± 4.7 in NF, and 79 ±37 in BPHF. mRNAs for both 5a-R isoforms were expressed in the three cell types, as shownby Northern blot and RT-PCR analysis. LY306089, a selective 5a-R type 1 inhibitor, stronglyinhibited 5a-R activity in all cell types (IC50: 10 nM), confirming the predominant expressionof 5a-R type 1 in these cells. Finasteride, a 5a-R type 2 inhibitor, was less efficient (IC50: 45,35, and 65 nM in NE, NF, and BPHF, respectively). In addition, the inhibition by finasteridedecreased with serial subculture in NF only, suggesting an effect of age in culture on theexpression of 5a-R type 2 in these cells. SKF105657, also a 5a-R type 2 inhibitor, was a poorinhibitor in this system.CONCLUSIONS. These studies demonstrate that human prostate cells in culture expressboth isoforms of 5a-R and suggest a balance in the expression of the two isoforms as afunction of various regulatory factors. Prostate 32:155–163, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: steroid 5a-reductases; isozyme expression; prostate cells in culture; nor-mal and hyperplastic prostate

INTRODUCTION

The prostate depends on androgens for both itsdifferentiation during embryogenesis and its furthergrowth during adult life [1]. In addition, androgensare responsible for the development of benign ormalignant prostate pathology, neither of which devel-ops in castrated males. Dihydrotestosterone (DHT),the 5a-reduced metabolite of testosterone (T), is theactive molecule triggering androgen action. 5a-reductase (5a-R), the enzyme converting T to DHT, istherefore a key step in this mechanism [2]. 5a-R is anNADPH-dependent enzyme located in the endoplas-

Abbreviations: 5a-R, 5a-reductase; BPH, benign prostatic hyperpla-sia; NE, human normal immortalized epithelial cells; NF, human nor-malprostate fibroblasts; BPHF, human hyperplastic prostate fibro-blasts; PBS, phosphate-buffered saline; Adiols, (3a and 3b)-androstanediols; DHT, dihydrotestosterone; T, testosterone;RT-PCR, reverse transcriptase-polymerase chain reaction; TLC,thin-layer chromatography; FCS, fetal calf serum; EGF, epidermalgrowth factor.These data were partially presented at the IIIrd European Congressof Endocrinology, Amsterdam, The Netherlands, 1994.*Correspondence to: Irene Mowszowicz, Laboratoire de BiochimieB, Hopital Necker-Enfants Malades, 149 rue de Sevres, 75743 ParisCedex 15, France.Received 11 March 1996; Accepted 30 May 1996

The Prostate 32:155–163 (1997)

© 1997 Wiley-Liss, Inc.

mic reticulum and the nuclear membrane of the cell[3,4].

Two genes encoding for 5a-R enzymes have beencloned recently. They are located on chromosomes 5and 2, respectively [5,6]. They encode two hydropho-bic enzymes, called 5a-R type 1 and 2, with approxi-mately 50% homology in amino acid sequence [6].Both genes have a similar structure with five exonsand four introns each [7,8]. The determinants of an-drogen binding are located in both the N- and car-boxyl-terminal ends of the molecule [9] whereas de-terminants of NADPH binding are in the carboxyl-terminal half of the enzyme [9]. The two isoforms canbe distinguished on the basis of biochemical, pharma-cological, and genetic criteria [6,10]. In particular, type2 isoenzyme is very sensitive to finasteride whereastype 1 is relatively insensitive. From studies of pa-tients with pseudohermaphroditism due to 5a-R defi-ciency, it is clearly established that 5a-R type 2 is theisoform expressed in the urogenital sinus during malesexual differentiation and that it plays a determinantrole in this process [6,11]. The distribution and role of5a-R type 1 are less clear. In rat prostate, both isoformsare present [12] with a predominant expression of type1 in the epithelium and of type 2 in the stroma [13]. Inthe human prostate in contrast, whereas the expres-sion of mRNAs for both isoforms has been reported[14], several authors have failed to find evidence forthe expression of the type 1 protein or enzyme activity[15,16]. In contrast, expression of the type 1 isozymehas been reported in human prostatic adenocarcinomacell lines [17]. The high level of expression of 5a-Rtype 2 in prostate tissues has led to the hypothesis thatthis isoform was implicated in the development of be-nign prostatic hyperplasia (BPH) and the 5a-R 2 in-hibitor finasteride has been proposed in the treatmentof this pathology [18]. In the present study, we haveused an experimental model allowing the separatestudy of epithelial cells and fibroblasts from normal orhyperplastic prostate. This model allowed the study ofthe isoforms (type 1 or 2) expressed in the different celltypes of the human prostate in culture and of the po-tential differences between normal fibroblasts and fi-broblasts derived from prostates of patients sufferingfrom BPH. The data, including mRNA analysis withNorthern blot or reverse transcriptase-polymerasechain reaction (RT-PCR) techniques and studies withspecific type 1 or 2 inhibitors, suggest that the twoisoforms can be expressed in human prostatic cells.

MATERIALS AND METHODS

Materials

1,2[3H]-testosterone (SA 40–56 Ci/mmol), [14C]-testosterone (SA 57 mCi/mmol), [14C]-DHT (SA 52–58

mCi/mmol), and [14C]-androstenedione (SA 52–54mCi/mmol) were purchased from DuPont deNemours (Cedex, France) and their purity checked be-fore use by thin-layer chromatography (TLC, Merck 60F254; Merck, Parmstadt, Germany). [14C]-androstane-diols were prepared in the laboratory as previouslydescribed [19]. Unlabeled steroids were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Stock solu-tions (4 mg/ml) were prepared in ethanol and storedat −20°C. All solvents were reagent grade or better.Tissue culture media and reagents were purchasedfrom Life Technologies (Cergy-Pontoise, France). Fetalcalf serum (FCS) was from D. Dutscher, (Brumath,France) and penicillin and streptomycin were fromAssistance Publique (Paris, France) and Sigma Chemi-cal Co., respectively.

Cell Culture

The epithelial cells used in this study (NE1, NE2,NE3) are three cell lines derived from normal humanprostatic epithelium and immortalized by transfectionof large SV 40 T antigen [20]. Normal human prostates(n = 4) were obtained from brain-dead organ trans-plant donors and hyperplastic human prostates (n = 3)were obtained, with informed consent, from patientsundergoing transurethral resection of the prostate forBPH. Fibroblasts were derived from explants as pre-viously described [21]. The cells were maintained inRPMI 1640 medium supplemented with 8% FCS, glu-tamin (2%), penicillin (100 IU/ml), and streptomycin(100 mg/ml). For immortalized epithelial cells, thisbasal medium was supplemented with hydrocortisone2 × 10 −7 g/l, bovine insulin 10−2 g/l, spermine 2 × 10−4

g/l, cholera toxin 10−4 g/l, O-phosphoethanolamine,2H+ 10−4 g/l, bovine albumin 10−2 g/l (Sigma Chemi-cal Co.), and epidermal growth factor (EGF) 2 × 10−6

g/l (Boehringer Chemical Co., Indianapolis, IN).Cells were maintained at 37°C in a humid atmosphereof 95% air/5% CO2 and were routinely trypsinizedevery week.

Fibroblasts and epithelial cells have been character-ized by the expression of vimentin and cytokeratinKL1, respectively, and T metabolism has been charac-terized in each cell type. In particular, we have shownthat this metabolism was reproducible in each cat-egory of cells: epithelial cells (NE) or fibroblasts fromnormal prostate (NF) or fibroblasts from BPH (BPHF)[22].

5a-R Assay

For enzymatic assays, cells originating from thesame 100 mm diameter dish were plated in the appro-priate number of 60 mm diameter culture dishes (Fal-

156 Berthaut et al.

con, Lincoln Park, NJ) at day D0. T 5a-R was assayedin intact cell monolayers as previously described [21].Briefly, 1 day after plating, cells were placed in serum-free medium for 24 hr. The next day (day 2), the me-dium was replaced by 2 ml of serum-free mediumcontaining 5 nM of [3H]-testosterone and cells werefurther incubated in normal culture conditions for 1 or2 hr. Additional dishes containing medium and sub-strate but no cells were always included to account forthe nonenzymatic conversion of substrate. At the endof incubation, cells were rapidly cooled on ice. Extrac-tion and chromatography of the medium were per-formed as previously described [21]. 5a-R activity wasexpressed in fmol of 5a-reduced metabolites (DHT +androstanediols)/mgDNA/hr. These two metabolitesplus D4-androstenedione and unmetabolized sub-strate always accounted for 98–100% of the total ra-dioactivity.

Inhibition Studies

Three specific inhibitors were used: Finasteride(MK906; Merck Sharp & Dohme, Chibret, France); thismolecule is a high-affinity inhibitor of 5a-R type 2 [6]and a slow binding, low-affinity inhibitor of 5a-R type1 [23]. LY306089, a selective type 1 5a-R inhibitor [17],was obtained from Eli Lilly Laboratories (Indianapo-lis, IN). SKF105657 (Epristeride; SmithKline BeechamPharmaceuticals, King of Prussia, PA) is an uncom-petitive inhibitor of 5a-R type 2 [24]. Stock solutions(10−3 M) were dissolved in absolute ethanol and storedat −20°C.

Increasing concentrations of the inhibitor (0–500nM) were added to culture plates, together with thesubstrate in a volume of ethanol that never exceeded1/1,000 of the total incubation medium. Control platesreceived the same amount of ethanol. Cells were pro-cessed as above. In each case, one dish represents anindividual point and each point was run in duplicateor triplicate. IC50 is the concentration of inhibitor re-quired to produce 50% inhibition during the assay pe-riod.

mRNA Studies

Total RNA extraction and poly (A)+ RNA prepara-tion. Confluent cells were harvested in trypsin-EDTA. Cell pellets were washed twice with saline andeither processed immediately or kept at −80°C untilextraction. Total RNA was extracted from cell pelletsin a one-step procedure [25] using TRIZol reagent(Life Technologies) and quantified by absorption at260 nm. The integrity of the extracted RNA was moni-tored by electrophoresis on 1% agarose gel. Poly (A)+

RNAs were purified with a polyA mRNA isolation kit

(Promega, Madison, WI) according to the manufactur-er’s instructions.

Northern blot analysis. Types 1 and 2 human steroid5a-R cDNAs in pBluescript vector were generous giftsfrom Dr. D.W. Russell (Dallas, TX). The vectors werelinearized with BamH I (type 1) and Sal I (type 2) andused as templates to synthesize [32P]-riboprobes in thepresence of [32P]-aCTP (NEN-Life Science Products, leBlancmesnil, France) using T3 RNA polymerase (Pro-mega). Poly (A)+ RNAs were separated on 1% agarose,2.2 M formaldehyde gel, and transferred onto a Gene-screen membrane (DuPont de Nemours,) in 10X SSC(0.75 M NaCl, 0.075 M sodium citrate). A 0.24–9.5 kbRNA ladder (Life Technologies) was used as sizemarker. Prehybridization and hydbridization werecarried out at 65°C in 50% formamide, 1% SDS, 1 mMEDTA, 0.005% Ficoll, 0.005% polyvinyl pyrolidone,0.005% BSA, 5X SSPE (0.9 M NaCl, 0.05 M NaH2PO4,0.005 M EDTA) containing 200 mg/ml yeast RNA. Hy-bridization with [32P]-labeled riboprobes was carriedout overnight at 65°C. Membranes were washed threeto five times in 0.1X SSC, 0.1% SDS at 65°C beforebeing exposed with an intensifying screen to Fuji X-Ray films (Fuji Medical System, Clichy, France) at−80°C for periods of time ranging from 24 hr to 1week. Because the mRNA for the two isoforms is simi-lar in size (2.4 kb), the two riboprobes could not beused simultaneously. Membranes were always hy-bridized first with the 5a-R 2 riboprobe, then dehy-bridized and checked before rehybridization with the5a-R 1 riboprobe.

RT-PCR Studies

They were conducted according to a previously de-scribed technique [26]. Specific primers were designedaccording to exonic DNA sequences published for5a-R 1 [7] or 5a-R 2 [8]. The oligonucleotides se-quences were as follow:5a-R 1: Primer 1: 58GGAATCGTCAGACGAACTCA-

GTGT38

Primer 2: 58GCATAGCCACACCACTCCAT-GATT38

5a-R 2: Primer 3: 58 CACTGGCCTTGTACGTCGCG-AA 38

Primer 4: 58 GGCAGTGCCTCTGAGAAT-GAGTAT38

The primers were commercially synthesized byGenset (Paris, France).

RT-PCR reactions. RNA was first reverse transcribedinto cDNA: 1–3 mg of total RNA were incubated at42°C for 1 hr in a final volume of 20 ml of 1× first-strand buffer containing 1 mM each of dNTP, 10 mM

5a-Reductase Isoforms in Prostate Cells 157

DTT, 0.2 mg random hexamers (Pharmacia Biotech,Orsay, France), and 200 U of murine leukemia virusreverse transcriptase (Superscript RNase H- RT, LifeTechnologies). Reverse transcriptase was inactivatedby heating to 95°C for 5 min and the cDNA mixturewas quick chilled on ice and kept at −20°C.

The amplification of cDNA was carried out in afinal volume of 50 ml, using 3 ml of the RT reactionmixture in 1× PCR buffer (20 mM Tris-HCl, pH 8.4, 50mM KCl), 2 mM MgCl2, 0.2 mM each of the 4 dNTP,25 pmol each of the suitable primer set for 5a-R type1 or type 2, and 2.5 U of Taq DNA polymerase. Allreagents were from Life Technologies. PCR incuba-tions were carried out in a PTC-100™ thermal cycler(programmable thermal controller, MJ Research, Inc.,Watertown, MA). After an initial step at 95°C for 4min, amplification was for 40 cycles, each consisting of1 min denaturation at 94°C, 1 min primer annealing at62°C, and 2 min primer extension at 72°C followed bya 8-min extension at 72°C after the last cycle. Type 1and type 2 cDNA subclones were used as positivecontrol templates and a sample without DNA wasused as negative control. The PCR products wereseparated on 2% agarose gel in TAE 1× (40 mM Tris-acetate and 2 mM EDTA, pH 8) and stained withethidium bromide.

DNA assay. DNA was assayed according to themethod of Burton [27], after perchloric acid extractionof the cell pellet.

RESULTS

5a-R Activity in Human Prostate Epithelial andStromal Cells in Culture

5a-R activity was measured in immortalized epi-thelial cells obtained from NE, NF, or BPHF. 5a-R ac-tivity was 1.43 ± 0.5 fmol/mg DNA/hr (mean of threecell lines, n = 11 experiments) in NE, 10.7 ± 4.7 fmol/mg DNA/hr (mean of four cell strains, n = 10 experi-ments) in NF, and eight times higher (79 ± 37 fmol/mgDNA/hr, mean of three cell strains, n = 7 experiments)in BPHF (Fig. 1). The 5a-reduced metabolites obtainedwere essentially DHT and 3a- and 3b-androstanediols;little if any 5a-androstanedione was produced, as 98–99% of total substrate radioactivity was always recov-ered between T, DHT, 3a- and 3b-androstanediols,and D4-androstenedione. However, the overall meta-bolic profile was strikingly different in the three celltypes as 17b-hydroxysteroid dehydrogenase activitywas much higher in NF than in NE and even 275-foldhigher in BPHF relative to NF [22].

The basal 5a-R activity did not vary significantly in

the course of subculture, either in NE (studied frompassage 6 to 107) or in NF (studied from passage 5 to15) (Fig. 2).

mRNA Studies

Northern blot analysis. To determine which of thetwo known isoforms of the 5a-R enzyme was ex-pressed in cultured epithelial and stromal cells,polyA+ RNAs were extracted from NE, NF, and BPHFand analyzed by Northern blots using specific ribo-probes for 5a-R types 1 and 2. We were unable todemonstrate the presence of 5a-R type 2 mRNA in NE(Fig. 3, lane 2) even after long exposition times (8days); a very faint band of 2.4 kb, not visible on allblots, suggested the possible expression of very lowlevels of 5a-R type 2 in NF and BPHF (Fig. 3, lanes 3and 4); genital skin fibroblasts, used as positive con-trols, expressed a high level of 5a-R type 2 mRNA(Fig. 3, lane 1), thus validating both the probe and themethod. In contrast, 5a-R type 1 mRNA was presentin all four cell types and visible after an expositiontime of 24 hr. Upon long exposition times as used totry and visualize 5a-R type 2 mRNA, a band of ap-proximately 4.4 kb was also observed in all cell types.A band of this size has been reported previously in

Fig. 1. 5a-R activity in human prostatic cells in culture. NE,epithelial cells (three cell lines, n = 11 experiments); NF, fibro-blasts from normal prostate (four cell strains, n = 10 experiments);BPHF, fibroblasts from hyperplastic prostate (three cell strains, n= 7 experiments). In each experiment, 5a-R activity was deter-mined as the mean of 6 to 8 time points and each column repre-sents the mean ± SD of the different experiments.

158 Berthaut et al.

mRNA analysis of 5a-R [15]. Alternatively, it couldrepresent rRNA not completely eliminated duringPolyA+ RNA preparation.

RT-PCR analysis. The absence or low level of 5a-Rtype 2 mRNA on Northern blot analysis of culturedprostatic cells could reflect the weak sensitivity of thistechnique. We therefore developed an assay using thehighly sensitive RT-PCR technique. After 40 cycles ofPCR (Fig. 4), both the amplified 5a-R type 2 (289 bp)and 5a-R type 1 (543 bp) fragments were present, con-firming that all cell types could express mRNAs ofboth isoforms. It is important to note that this assay isabsolutely not quantitative as we voluntarily extendedthe PCR to its plateau stage in order to amplify what-ever messenger was present.

Inhibition Studies

Dose-dependence studies. To further characterizethe enzymatic activity expressed in each cell type, weevaluated the dose-dependent inhibition of 5a-R pro-duced by three specific inhibitors: LY306089 (type 1inhibitor), MK906, and SKF105657 (type 2 inhibitors)(Fig. 5). In all three cell types, LY306089 was by far thestrongest inhibitor with an IC50 of about 10 nM, con-sistent with the mRNA studies and confirming the

expression of 5a-R 1 in these cells. Finasteride inhib-ited 5a-R activity in NE and NF with comparable IC50of 45 and 35 nM, respectively. It was less efficient inBPHF (IC50: 65 nM). Surprisingly, SKF105657 was apoor inhibitor in all cell types (IC50 > 100 nM). None ofthe inhibitors affected the formation of androstenedi-one in any of the cell types (not shown).

Effect of serial subcultures on 5a-R inhibition byfinasteride. These initial studies were performed on‘‘young’’ cells (5th or 6th subculture). However, whilecells were serially subcultured, we noticed some vari-ability in the inhibition profile by finasteride, contrast-ing with the extreme reproducibility of LY306089 in-hibition (not shown). We therefore designed experi-ments to systematically study the effect of serialsubcultures on 5a-R inhibition in all three cell types.Cells were serially subcultured once a week and 5a-Rwas assayed every three to five passages in the pres-ence of a constant dose (100 nM) of finasteride. In NEand BPHF, the inhibition was constant (75 and 55%,respectively) throughout the time course tested. In NFin contrast, inhibition by finasteride steadily de-creased from 75% at passage 5 to less than 10% atpassage 15. No apparent modifications of the inhibi-tion profile of LY306089 or SKF105657 were observedin the same time course (not shown).

Fig. 2. 5a-R activity in epithelial cells (NE) andfibroblasts (NF) from normal prostate in the courseof serial subculture (passage number). None of theapparent variations were significant.

Fig. 3. Typical Northern blot analysis profile of5a-R type 1 (top) and 2 (bottom) mRNAs. Poly(A)+ RNAs were extracted from genital skin fi-broblasts (lane 1), epithelial cells (NE, lane 2),fibroblasts from normal prostate (NF, lane 3),and fibroblasts from hyperplastic prostate (BPHF,lane 4) and successively hybridized with specificriboprobes. Hybridization conditions were as de-scribed in the text; exposition was 24 hr for 5a-Rtype 1 and 8 days for 5a-R 2.

5a-Reductase Isoforms in Prostate Cells 159

DISCUSSION

The prostate depends on androgens for its devel-opment and the maintenance of its differentiated

structures and functions. DHT, the 5a-reduced me-tabolite of T, is the active molecule in this tissue asshown by the absence of prostate development in pa-tients with 5a-R deficiency [11]. Whereas it is clearlyestablished that the 5a-R type 2 isoform is responsiblefor the embryogenesis of the prostate, the respective

Fig. 4. RT-PCR analysis of 5a-R type 1 (lanes 1–6) and 2 (lanes 8–13). The molecular weight marker (lane 7) is a Hint I, Rsa I, andSin I digestion of a PGEM plasmid. Lanes 1 and 8 are negative controls (water); lanes 2 and 9 are positive controls (plasmids containing 5a-Rtype 1 and type 2 cDNA, respectively). The following lanes are from genital skin fibroblasts (lanes 3 and 10), epithelial cells (NE, lanes 4and 11), fibroblasts from normal prostate (NF, lanes 5 and 12), and fibroblasts from hyperplastic prostate (BPHF, lanes 6 and 13). Theamplified fragments specific for 5a-R type 1 (543 bp) and type 2 (289 bp) are indicated by arrows.

Fig. 5. Dose-dependent inhibition of 5a-R activity by LY306089(5a-R type 1 inhibitor), finasteride (5a-R type 2 inhibitor), andSKF105657 (5a-R type 2 inhibitor) in prostatic cells in culture. NE,epithelial cells; NF, fibroblasts from normal prostate; BPHF, fibro-blasts from hyperplastic prostate. Each point represents the mean± SD of at least three separate experiments on different cellstrains, each point in each experiment being run in triplicate.

Fig. 6. Inhibition of 5a-R activity by a constant dose of finaste-ride (100 nM) in the course of serial subculture (passage number).NE, epithelial cells; NF, fibroblasts from normal prostate; BPHF,fibroblasts from hyperplastic prostate.

160 Berthaut et al.

expression and roles of the two isoforms in the adulttissue are still subject to controversy. The aim of thisstudy was to provide some clues toward the answer tothis question. Based on mRNA and specific inhibitorstudies, we propose that the adult prostate is capableof a versatile expression of both type 1 and type 2 5a-Risoforms, controlled by age, cellular, and hormonalenvironment of the concerned cells.

These studies have been performed on separate cul-tures of epithelial cells and fibroblasts derived fromnormal or hyperplastic prostates. Whereas it is wellestablished that in vivo, interactions between the epi-thelium and the stroma play an essential role in thedevelopment and maintenance of the prostate [28],separate cultures of epithelial and stromal cells allowthe study of the intrinsic properties of each cell typeirrespective of these interactions. Such knowledgeshould provide a strong basis for the further study ofstroma-epithelium interactions.

We have used epithelial cells obtained from normalprostate immortalized by transfection of the large TSV 40 antigen. These cells are indeed not normal andif they have retained most of the characteristics ofprostatic epithelial cells [20], they also present some ofthe genetic alterations described in prostatic tumors.However, they do not induce the formation of tumorswhen injected into nude mice [29]. In contrast to nor-mal epithelial cells, they can be easily subcultured,allowing the production of sufficient material for re-producible studies. Fibroblasts have been derivedfrom the periurethral zone of normal or hyperplasticprostates known to be involved in the development ofBPH [30]. We have previously characterized the cellstrains used as concerns their expression of vimentinand keratin to establish their stromal or epithelial na-ture and we have shown that human epithelial andstromal cells in culture maintain their main metaboliccharacteristics [22].

5a-R has been assayed in intact cell monolayersrather than in a cell-free system. We thus explore func-tionally intact cells using their own endogenousequipment of cofactors. This does not, however, allowthe classical distinction between the two 5a-R iso-zymes based on their optimal pH difference: 5a-R 1with a broad optimal neutral pH, 5a-R 2 with a sharpoptimal pH peak at 5.5 [6]. However, a recent study[31] has shown that this difference was due to differ-ential extraction of the two isozymes from their mem-brane locations and that both enzymes had an optimalneutral pH in intact cells. It is therefore likely that inour assay conditions we explore the whole 5a-R po-tential of a given cell and that the use of specific in-hibitors allows the distinction of the two isoforms.

It is currently considered that, whereas the rat pros-tate expresses both 5a-R isozymes [12,13], 5a-R type 2

is the predominant or even the only isoform expressedin the human prostate. Conflicting data exist in theliterature about the presence and expression of 5a-Rtype 1: whereas several studies bearing on the mRNA[15,32] or the protein [16] have failed to reveal theexpression of the type 1 isoform, others brought evi-dence for the expression of 5a-R type 1 [10,14,33].However, the ratio, individual physiological role, andcontribution toward DHT production in the gland ofthe two isoforms remain unknown.

Our data, demonstrating the expression of both iso-forms, are consistent with these later studies. Indeed,5a-R type 1 mRNA was clearly expressed in all threecell types on analysis on Northern blots. Only occa-sionally have we been able to find slight evidence ofthe presence of 5a-R type 2 in NF and BPHF and neverin NE with this technique. However, the very sensitiveRT-PCR technique has confirmed the expression ofboth isoforms in all cell types. Although we do notknow with certainty the reasons for these differencesbetween our data and data found in the literature, wecan suggest two hypotheses: 1) Dilution of one iso-form expression by the other in conditions where therelative abundance of the two compartments of theprostate tissue (stroma and epithelium) is highly dif-ferent. Indeed, the studies which failed to report thepresence of 5a-R type 1 have generally been per-formed on whole homogenates of prostate tissue inwhich the stromal fraction is by far predominant. If, asin the rat [12], the 5a-R type 1 is mainly expressed inthe epithelium, it could have been diluted out in thesestudies. 2) Another possibility could be the modifica-tion of isozyme expression due to disruption of theprostate tissue. It has been reported that in homog-enates of fresh prostate tissue, essentially, if not only,the 5a-R type 2 isozyme was expressed and that theappearance of 5a-R type 1 was only due to ‘‘cultureconditions’’ [34]. In a recent study [35], Delos et al.reported that BPH and prostate adenocarcinoma tis-sues expressed the 5a-R type 2 mRNA, whereas epi-thelial cells derived from these tissues expressed the5a-R type 1 and fibroblasts neither type 1 nor 2. Thesestudies emphasize the differences between fresh tissueand cultured cells. However, culture conditionsmainly reflect dissociation between epithelium andstroma and different environmental and hormonalregulation. It thus appears that prostate cells are ca-pable of expressing both isozymes depending on asyet unknown regulatory factors.

To further establish the nature of the 5a-R isoformsexpressed in cultured human prostatic cells, we haveused three different inhibitors: finasteride, SKF105657,and LY306089. Finasteride is a strong competitive in-hibitor of type 2 5a-R [6] and a slow inhibitor of 5a-Rtype 1 [23]. SKF105657 (epristeride) is an uncompeti-

5a-Reductase Isoforms in Prostate Cells 161

tive type 2 inhibitor [24] and LY306089 is a noncom-petitive type 1 inhibitor [17]. Consistent with theabove mRNA studies, LY306089 was by far the strong-est inhibitor of 5a-R activity, further demonstratingthe expression of 5a-R type 1 in the three cell typesstudied. The specificity of this inhibitor and the valid-ity of the methodology are supported by our recentstudies on skin fibroblasts [26]. These studies clearlyshow that in genital skin (skin from the external geni-talia) where the predominant expression of 5a-R type2 is clearly established [36], LY306089 is a poor inhibi-tor (IC50 > 500 nM). In contrast, in pubic skin (imme-diate suprapubic area) where the expression of 5a-Rtype 1 is dominant [32], LY306089 is a strong inhibitor.

Finasteride also inhibited 5a-R activity in the threecell types, though less efficiently, with an IC50 consis-tent with data in the literature [36,37], confirming theconcomitant expression of 5a-R type 2. Surprisingly,SKF105657, also a 5a-R type 2 inhibitor, exhibited avery different inhibition profile as compared to finas-teride in all three cell types. One possible explanationcould be a poor penetration of this molecule in theintact cell system used. Alternatively, this differencecould be due to differences in the mechanism of actionof the two inhibitors. Finasteride was first reportedto be a competitive inhibitor of 5a-R activity [38] anda sequence of four amino acids has been shown tobe responsible for the sensitivity of the enzyme tothe inhibitor [39]. Recent kinetics studies, however,suggest that finasteride can compete with T for thebinding to 5a-R with an affinity such that a pseudoir-reversible inhibition of the enzyme activity can be pre-dicted [40]. In addition, finasteride is also a slow, time-dependent inhibitor of 5a-R type 1 with kinetic char-acteristics suggesting a covalent modification of theenzyme [23]. In this respect, it is of interest to note thatin pubic skin fibroblasts, where 5a-R type 1 is pre-dominant, a high dose of finasteride (500 nM) resultsin an 80% inhibition [26]. It is therefore possible thatfinasteride inhibits to some degree 5a-R type 1, ac-counting for the different actions of these two 5a-Rtype 2 inhibitors.

Another factor which could possibly modify the ex-pression profile of 5a-R is the age of the cells. In thisrespect, the slow decline of finasteride inhibition ob-served with time in culture in NF is noteworthy. In-deed, it was not observed in NE, which are immortal-ized cells, and as such, probably not subject to theaging process, nor was it observed in BPHF. IndeedBPHF, which come from older donors, are already en-gaged in the aging process, and, because of theirslower doubling time, they have spent, after seven oreight passages, the same amount of time in culture asNF after 14 to 15 passages. The modification of theinhibition profile with age in culture was not observed

in genital skin fibroblasts (not shown), suggesting thatit is prostate specific.

Finally, it remains to be determined why there aretwo isozymes and what unique and/or overlappingroles they play in androgen action and steroid metabo-lism, especially in a DHT-dependent organ like theprostate. Initial studies on substrate affinities and tis-sue distribution of the two isozymes had led to thesuggestion that 5a-R type 2 plays an anabolic rolewhereas 5a-R type 1 is involved in androgen andother steroid metabolism [12]. Our studies could alter-natively suggest a balance in the expression of the twoisoforms as a function of various regulatory factors.

CONCLUSION

It appears that in different conditions, the prostategland can modulate in stromal and epithelial cells therelative expression of one or the other 5a-R isozymes.Whether this can occur in vivo, under particular con-ditions, or is specific to in vitro conditions, the regu-latory factors responsible for the switch in enzymeactivity and the implication of these findings in thetherapeutic use of 5a-R inhibitors in prostate pathol-ogy remain to be determined.

ACKNOWLEDGMENTS

The financial support of ARTP (Association de Re-cherche sur les Tumeurs de la Prostate) and the Com-mission Scientifique de la Faculte de MedecineNecker-Enfants Malades is gratefully acknowledged.The inhibitors were kindly provided by Merck Sharp& Dohme-Chibret (Paris, France), Eli Lilly Laborato-ries, and Smith Kline Beecham Pharmaceuticals.

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