Rat Ventral Prostate Epithelial Cells Research Articles

APP-081-PM Clusterin As a Novel Mediator of Benign Prostatic Hyperplasia in Rat Ventral Prostate Epithelial Cells(The 97th Annual Meeting of the Japanese Urological Association)

APP-081-PM Clusterin As a Novel Mediator of Benign Prostatic Hyperplasia in Rat Ventral Prostate Epithelial Cells(The 97th Annual Meeting of the Japanese Urological Association)

Regulation of calcium homeostasis by S100RVP, an androgen-regulated S100 protein in the rat ventral prostate

S100RVP was previously identified as an androgen-response gene in the rat ventral prostate (RVP). Characterization of S100RVP is important for elucidating the function of S100 proteins in androgen action. The expression and subcellular localization of S100RVP were determined by Northern blot, in situ hybridization, and fluorescent microscopy. Calcium overlay and calcium ionophore sensitivity assays were performed to investigate the calcium binding and function of S100RVP. S100RVP is abundantly expressed in the RVP epithelial cells. A green fluorescent protein(GFP)-S100RVP fusion protein is present in both the cytoplasm and nucleus of transfected cells. A GST-S100RVP fusion protein bound calcium in vitro at levels similar to known S100 proteins. Furthermore, GFP-S100RVP transfected LNCaP and PC3 cells exhibited reduced sensitivity to calcium ionophore-induced cell death, but not to UV-induced cell death. The results of this study argue for a role of S100RVP in calcium homeostasis in the prostate.

Androgens Regulate the Immune/Inflammatory Response and Cell Survival Pathways in Rat Ventral Prostate Epithelial Cells

A major hurdle in understanding the role of androgens is the heterogeneity of androgen receptor (AR) expression in the prostate. Because the majority of prostate cancer arises from the AR-positive secretory luminal epithelial cells, identifying the androgen-mediated pathways in the prostate epithelium is of great significance to understanding their role in prostate pathogenesis. To meet this objective, the current study was designed to identify immediate-early genes expressed in response to the synthetic androgen R1881 in cultured rat ventral prostate epithelial cells. Rat ventral prostate epithelial cells, purified from 20-d-old rats, were cultured, and the presence of AR and the response to androgen were established. The cells were then treated with R1881 for 2 and 12 h to capture immediate-early genes in an Affymetrix-based gene chip platform. A total of 66 nonredundant genes were identified that were responsive to R1881. The functional androgen response elements were identified in the proximal promoter to determine possible molecular mechanism. Cluster analysis identified five distinct signatures of R1881-induced genes. Pathway analysis suggested that R1881 primarily influences cell proliferation/differentiation and inflammatory/immune response pathways. Androgens appear to regulate cell renewal by regulating differentiation, cell proliferation, and apoptosis. Two mutually exclusive inflammatory response pathways were observed. The interferon pathway was up-regulated, and the ILs were down-regulated. The data identified novel androgen-regulated genes (e.g. Id1, Id3, IL-6, IGF-binding protein-2 and -3, and JunB). The loss of androgen regulation of these genes can have important consequences for cellular transformation and transition to androgen-independent growth and survival.

Ca2+-activated Cl- channel currents in rat ventral prostate epithelial cells.

In many epithelial tissues, the Cl(-) efflux via Ca(2+)-activated Cl(-) channels (Cl(Ca)) play a key role for the fluid secretion. To elucidate the mechanism of prostatic fluid secretion, the properties of whole-cell chloride conductance were investigated. Rat prostate secretory epithelial cells (RPSECs) were isolated by collagenase treatment, and were used for the whole-cell voltage clamp. Both extra- and intracellular monovalent cations were replaced by N-methyl-D-glucamate to record the Cl(-) current selectively. A bath application of Ca(2+)-ionophore, ionomycin (0.2 micro M), increased the membrane conductance with outwardly rectifying voltage-dependence. On step-like depolarization from -60 to +80 mV (500 msec), the ionomycin-induced current showed slowly activating kinetics, a known property of Cl(Ca) current (I(Cl(Ca))) of other tissues. The relative permeability of Cl(Ca) to various anions was calculated from the reversal potentials measured under a total replacement of extracellular Cl(-) with various anions, and the relative order of permeability was SCN(-)>I(-)>Br(-)>Cl(-)>>gluconate. The amplitude of I(Cl(Ca)) was decreased by various anion channel blockers: niflumic acid (100 micro M), DPC (100 micro M), DIDS (1 mM), and NPPB (200 micro M). RPSECs have Cl(Ca) that may provide Cl(-) efflux pathways for the exocrine secretions of the prostate.

K+ channel currents in rat ventral prostate epithelial cells.

Electrophysiological function of the normal prostate has not been extensively studied. In particular, ion channel currents and their regulation have not been studied in freshly-isolated prostate cells. Rat prostate secretory epithelial (RPSE) cells were isolated by collagenase treatment. Columnar epithelial cells were used for nystatin-perforated, whole-cell voltage clamp, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. Step-like depolarizing pulses (900 msec) starting from - 90 mV induced outwardly rectifying K(+) currents without inactivation. ACh (10 microM) or ATP (100 microM) increased the outward current and hyperpolarized the cell membrane potential. Ionomycin (0.1 microM), a Ca(2+) ionophore, induced a similar increase in the outward current. TEA (5 mM), charybdotoxin (50 nM), and iberiotoxin (30 nM) inhibited the effect of ACh (or ATP) on the outward current, whereas apamin (100 nM) had no effect. The [Ca(2+)](i) of RPSE cells was increased by ACh, ATP, and UTP. RPSE cells have iberiotoxin-sensitive Ca(2+)-activated K(+) channels that may play an important role in the exocrine secretions of the prostate.

Mitochondrial aconitase gene expression is regulated by testosterone and prolactin in prostate epithelial cells .

m-aconitase catalyzes the first step leading to the oxidation of citrate via the Krebs cycle. It is a constituitive enzyme in virtually all mammalian cells, found in excess, and is considered to be a regulatory or regulated enzyme. In contrast to these general relationships, prostate secretory epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase which minimizes the oxidation of citrate. This permits the unique prostate function of accumulating and secreting extraordinarily high levels of citrate. Previous animal studies demonstrated that testosterone and prolactin regulate the level of m-aconitase specifically in citrate-producing prostate cells. The present studies were conducted to determine if testosterone and prolactin regulated the expression of the m-aconitase gene in prostate cells, and to determine the effect of the hormones on human prostate cells. The studies were conducted with freshly prepared rat ventral, rat lateral, and pig prostate epithelial cells, and with the human malignant cell lines LNCaP and PC-3. The effects of 1 nM testosterone and 3 nM prolactin on the level of m-aconitase mRNA and on the transcription rate of m-aconitase were determined. The studies revealed that both prolactin and testosterone increase the levels of m-aconitase mRNA and the transcription rates of m-aconitase in rat ventral prostate cells, pig prostate cells, and human malignant prostate cells (LNCaP and PC-3). In contrast, both hormones decreased the level of m-aconitase mRNA and repressed m-aconitase gene transcription in rat lateral prostate cells. The hormonal regulation of m-aconitase corresponded with the levels of m-aconitase enzyme, m-aconitase activity, and citrate oxidation. In addition to the constitutive expression of m-aconitase, the m-aconitase gene is testosterone- and prolactin-regulated in specifically targeted prostate cells. The hormonal regulation of m-aconitase gene expression and biosynthesis of m-aconitase provide a regulatory mechanism for the oxidation of citrate, and consequently, the level of net citrate production by prostate. The hormonally increased expression and biosynthesis of m-aconitase in human malignant cells might be involved in the increased citrate oxidation associated with the development of true malignant cells in prostate cancer.

Zinc causes a shift toward citrate at equilibrium of the m-aconitase reaction of prostate mitochondria

Prostate secretory epithelial cells have the unique function and capability of accumulating and secreting extraordinarily high levels of citrate. To achieve this, these cells possess a uniquely limiting mitochondrial (m)-aconitase activity that minimizes the oxidation of citrate via the Krebs cycle. The steady-state citrate/isocitrate ratio of mammalian tissues is generally maintained at about 10–11/1, independent of the concentration of citrate, which is the result of the chemical equilibrium reached in the presence of m-aconitase. In contrast, the citrate/isocitrate ratio of prostate tissue is about 30–40/1. Zinc, which is also accumulated in prostate cells at much higher levels than in other cells, inhibits m-aconitase activity thereby minimizing citrate oxidation. This current report is concerned with an effect of zinc on the equilibrium of the reaction catalyzed by m-aconitase. Studies were conducted with mitochondrial extract preparations from rat ventral prostate epithelial cells. With citrate as the initial substrate, the addition of zinc (7–10 μM) to the prostate mitochondrial preparation resulted in a change in the citrate/isocitrate ratio at equilibrium from an average of 10.5/1 to 13.5/1. In contrast, the identical treatment of kidney mitochondrial preparations resulted in no zinc-induced change in the citrate/isocitrate ratio. When either cis-aconitate or isocitrate was employed as the initial substrate, the addition of zinc did not alter the citrate/isocitrate ratio of prostate or kidney preparations. Partial purification of the prostate preparation revealed that the prostate mitochondrial extract contained a putative protein (which we have designated as ‘citrate factor protein’) that is required for the zinc-induced increase in the citrate/isocitrate ratio. This novel effect of zinc provides another mechanism by which it is assured that the accumulation of citrate is maximized in citrate-producing prostate epithelial cells.

Zinc Inhibition of Mitochondrial Aconitase and Its Importance in Citrate Metabolism of Prostate Epithelial Cells

Prostate epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase activity that minimizes their ability to oxidize citrate. These cells also possess uniquely high cellular and mitochondrial zinc levels. Correlations among zinc, citrate, and m-aconitase in prostate indicated that zinc might be an inhibitor of prostate m-aconitase activity and citrate oxidation. The present studies reveal that zinc at near physiological levels inhibited m-aconitase activity of mitochondrial sonicate preparations obtained from rat ventral prostate epithelial cells. Corresponding studies conducted with mitochondrial sonicates of rat kidney cells revealed that zinc also inhibited the kidney m-aconitase activity. However the inhibitory effect of zinc was more sensitive with the prostate m-aconitase activity. Zinc inhibition fit the competitive inhibitor model. The inhibitory effect of zinc occurred only with citrate as substrate and was specific for the citrate --> cis-aconitate reaction. Other cations (Ca2+, Mn2+, Cd2+) did not result in the inhibitory effects obtained with zinc. The presence of endogenous zinc inhibited the m-aconitase activity of the prostate mitochondrial preparations. Kidney preparations that contain lower endogenous zinc levels exhibited no endogenous inhibition of m-aconitase activity. Studies with pig prostate and seminal vesicle mitochondrial preparations also revealed that zinc was a competitive inhibitor against citrate of m-aconitase activity. The effects of zinc on purified beef heart m-aconitase verified the competitive inhibitor action of zinc. In contrast, zinc had no inhibitory effect on purified cytosolic aconitase. These studies reveal for the first time that zinc is a specific inhibitor of m-aconitase of mammalian cells. In prostate epithelial cells, in situ mitochondrial zinc levels inhibit m-aconitase activity, which provides a mechanism by which citrate oxidation is limited.

Testosterone stimulates the biosynthesis of m-aconitase and citrate oxidation in prostate epithelial cells

Mitochondria (m-)aconitase is a rate-limiting regulatory enzyme in prostate epithelial cells which minimizes citrate oxidation by these cells. This unique metabolic characteristic is responsible for the ability of the prostate to accumulate and secrete extraordinarily high levels of citrate. Testosterone is a major regulator of prostate growth and function, and stimulates citrate oxidation. Therefore, an important action of testosterone might be its stimulation of m-aconitase in prostate epithelial cells. Studies were conducted with rat ventral prostate (VP) epithelial cells to establish the effect of testosterone on the level of m-aconitase and corresponding citrate oxidation. Physiological concentrations (10 −7–10 −10 M) of testosterone in vitro markedly increased the level of m-aconitase in freshly prepared isolated prostate epithelial cells. This increase was apparent within 3 h of exposure to the hormone. The stimulatory effect of testosterone on m-aconitase was abolished by actinomycin D and by cycloheximide. Both the level of m-aconitase enzyme and the level of m-aconitase activity were similarly increased by testosterone treatment. Correspondingly, testosterone increased the rate of mitochondrial citrate oxidation while having no effect on the rate of isocitrate oxidation, thereby demonstrating that the action of testosterone is specifically targeted at the m-aconitase reaction. In vivo studies revealed that castration markedly decreased and testosterone administration increased the m-aconitase level of prostate epithelial cells. In contrast, neither liver nor kidney m-aconitase level was altered by castration. These studies demonstrate that testosterone regulates the biosynthesis of m-aconitase in prostate epithelial cells. It appears that this is a cell-specific effect since neither kidney nor liver m-aconitase was affected. The studies reveal that prostate epithelial cells contain a constitutive and an androgen-induced m-aconitase component; whereas, kidney and liver contain a constitutive but no androgen-induced m-aconitase. Unlike essentially all other cells, m-aconitase is a regulatory and regulated enzyme in prostate epithelial cells.

Testosterone regulates mitochondrial aspartate aminotransferase gene expression and mRNA stability in prostate

The effect of testosterone on the precursor mitochondrial aspartate aminotransferase (pmAAT) gene and on pmAAT-mRNA was studied in rat ventral prostate (VP) and pig prostate epithelial cells. Castration significantly decreased the level of nuclear pmAAT transcripts in VP; whereas testosterone treatment of castrated animals restored the level of pmAAT transcripts. Correspondingly, castration resulted in a marked decrease in the transcription rate of the pmAAT gene; whereas testosterone treatment markedly increased the transcription rate. In vitro studies with isolated pig prostate epithelial cells demonstrated that testosterone directly and rapidly induced a transient increase in the transcription rate of the pmAAT gene. The increase in transcription was associated with an increase in the steady-state level of pmAAT-mRNA. Similar in vitro effects were observed with isolated VP epithelial cells. In addition to its stimulatory effect on transcription of the pmAAT gene, testosrone also increased the half-life of pmAAT-mRNA from 2 h in the absence of hormone to 16 h in its presence. Consequently, testosterone appears to stabilize the pmAAT-mRNA. The combination of its immediate effect on stimulating the transcription of the pmAAT gene and its stabilizing effect on pmAAT-mRNA would account for the increase in the steady-state level of pmAAT-mRNA by testosterone. These studies support our proposal that, through these effects, testosterone increases the biosynthesis of mAAT thereby increasing the transamination of aspartate to oxaloacetate and ultimately increasing the synthesis of citrate. This appears to provide at least one of the mechanisms by which testosterone regulates prostate citrate production.

Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.

Isolation and Characterization of Transcripts Induced by Androgen Withdrawal and Apoptotic Cell Death in the Rat Ventral Prostate

A variety of stimuli have been identified which initiate transcription-dependent programmed cell death (apoptosis) in specific target cells. Since the withdrawal of androgens induces regression and apoptosis in rat ventral prostate (RVP) epithelial cells, and it is known that the androgen receptor is a transcriptional regulator, we used subtraction cDNA cloning to isolate differentially expressed transcripts from the RVP of androgen ablated rats. In addition to sulfated glycoprotein-2 and glutathione S-transferase (GST), which had been previously described, several other transcripts were found to be elevated 3- to 8-fold in the regressing RVP. DNA sequencing revealed that two of these cDNA clones encode matrix carboxyglutamic acid and gamma-actin, respectively. A third cDNA contained novel sequence information and was named RVP.1. The RVP.1 transcript is expressed at very low levels in the RVP and epididymis of normal adult rats (less than 0.01% of the total mRNA) and is undetectable in other tissues, such as kidney, liver, and muscle. RVP.1 encodes a putative 280-amino acid protein, which shares no significant homology with previously described protein functional domains. We examined the expression of these transcripts in serum-starved NIH 3T3 cells to determine whether any of them are elevated in cells that are growth arrested. It was found that only GST mRNA levels are increased under these conditions. These data may suggest that induction of some genes, such as RVP.1, could be associated with apoptosis, whereas other transcripts, such as GST, may be up-regulated in response to altered rates of cellular metabolism.

Epithelial- Stromal Interactions in the Regulation of Rat Ventral Prostate Function: Identification and Characterization of Pathways for Androgen Metabolism in Isolated Cell Types*

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, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha(and 3 beta), 17 beta-diols, and delta 4-androstene-3,17-dione), and the metabolites were analyzed at several time points over a 24-h period. The metabolism studies indicated that 5 alpha-reductase activity, the oxidative reactions of 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid oxidoreductases, and the reductive reaction of 3 beta-hydroxysteroid oxidoreductase were expressed at significantly higher levels in epithelial cells compared to stromal cells. The reductive reactions of 3 alpha- and 17 beta-hydroxysteroid oxidoreductases were similar in both cell types. In contrast, stromal cells exhibited substantially higher levels of 6 alpha/7 alpha-hydroxylase activity. In addition, stromal cells were capable of metabolizing 5 alpha-dihydrotestosterone directly to a new unidentified polar androgen metabolite (HO5 alpha-DHT). Overall, epithelial cells were approximately 29 times more capable than stromal cells of forming the biologically active androgen 5 alpha-dihydrotestosterone. Conversely, stromal cells were more capable of forming biologically inactive polar androgen metabolites.

Secretion of inhibin‐like material by rat ventral prostate epithelial cells in culture

Monolayer cultures of epithelial as well as stromal components of the rat ventral prostate were established, taking advantage of differences in the requirement of sera supplementation and also in the temporal span for attachment. These monolayers were characterized and used to identify the cellular source of prostatic inhibin. The spent medium obtained from epithelial cell monolayer alone had significant inhibin-like activity.

Differential Androgen Modulation of Acid Phosphatase Isozymes in Primary Cultures of Rat Ventral Prostate Epithelial and Stromal Cells*

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.

Does the lack of regression-associated mRNA expression render a rat ventral prostate epithelial cell line androgen independent?

A series of rapidly dividing epithelial (RDE) cell lines have been isolated from primary cultures of rat ventral prostate (RVP) epithelial cells. Unlike androgen-dependent secretory epithelial cells, the RDE cells in culture do not express the androgen-dependent secretory proteins, nor do they express the androgen-repressed cell death sequences (TRPM-2) found in the epithelial cells during prostatic regression. Screening of a cDNA clone library established from RDE cell mRNA has yielded a number of RDE cell-specific sequences. One of these, RDE-.25 is a 250-base mRNA. The sequence of RDE-.25 shows considerable homology with the rat growth hormone gene and two murine oncogene sequences. We believe that the absence of androgen-repressed cell death sequence expression confers androgen independence for survival and growth, while the expression of RDE-.25 may represent an autocrine growth stimulus which greatly increases the rate of cell division in these cells.

Separation of mature rat ventral prostate epithelial and fibroblast cells

Several techniques for the separation of rat ventral prostate cells, using density gradient centrifugation or mechanical means have been published, yielding fibroblast and epithelial cell populations of varying purities and viability. These techniques are often tedious, yield relatively limited numbers of cells, demand considerable technical expertise, and result in the isolation of cells of limited viability. Two techniques for the isolation and establishment of epithelial and fibroblast cell cultures from mature rat ventral prostate are described here. Collagenase/trypsin digestion of the tissue yields a single-cell suspension of both cell types that are separated on Percoll isopycnic centrifugation gradients. A continuous gradient system allows for the separation of a greater number of cells with very high degrees of purity. A second technique based on a step-gradient system produces reproducible subfractionation of the epithelial cell component of the prostate within a considerable shorter period of time. An improved medium for plating epithelial and fibroblast cells has also been developed. The separated cells are plated on collagen and/or fibronectin-coated dishes in a serum-free plating medium that is later replaced with a serum containing growth medium. The plating medium greatly increases the plating efficiency of the isolated cell types, particularly the epithelial cells.

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride

Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl2) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95-100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl2 in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl2 in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.

Estrogen metabolism by primary cultures of rat ventral prostate epithelial and stromal cells

Estrogen metabolism was examined in primary cultures of rat ventral prostate epithelial and stromal cells developed from young (~3 weeks old) animals. Supraphysiologic concentrations (50 nM) of tritium-labelled estradiol (E 2) and estrone (E 1) were incubated separately with each cell type and the metabolites formed were measured at selected time points over a 24 h period. The metabolites were analyzed using high performance liquid chromatography. Epithelial cells exhibited an equal capability to interconvert E 2 and E 1 thus demonstrating the presence of similar oxidative and reductive activities for 17β-hydroxysteroid oxidoreductase (17β-HSOR) [0.45 and 0.40 pmol/3 h/μg DNA respectively]. In contrast, stromal cells showed a 6-fold lower rate of oxidation of E 2 to E 1 (0.08 pmol/3 h/μg DNA) but exhibited an approx 5-fold higher rate of reduction of E 1 to E 2 (1.81 pmol/3 h/μg DNA). Estriol (E 3) formation from either substrate was not detected in the two cell types. The results demonstrate that rat ventral prostate epithelial cells have similar capabilities to form or remove biologically active E 2. In contrast, prostate stromal cells exhibited a preferential capability to form and possibly maintain high levels of biologically active E 2. These findings are discussed with reference to the actions of estrogens on prostate epithelial-stromal cellular interactions.

Effect of a 4-methyl-4-aza steroid on androgen metabolism by rat ventral prostate epithelial and stromal cell cultures: selective inhibition of 5 alpha-reductase activity.

The effect of a potent steroid metabolic inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA), on androgen metabolism was investigated in primary monolayer cultures of rat ventral prostate epithelial and stromal cells. Using testosterone (T) as substrate, 5 alpha-reductase (5 alpha-R) activity in both cell types was inhibited by greater than 98% at an inhibitor concentration of 1000 nM. The concentrations required to produce a 50% inhibition (IC50) were 7.4 and 9.0 nM for epithelial and stromal cells, respectively. To examine the specificity of this compound, its effect on other steroid-metabolic enzymes was examined. DMAA at a concentration of 1,000 nM had no effect on 3 alpha-hydroxysteroid oxidase (3 alpha-HSORox), 3-ketosteroid reductase (3 alpha-HSORred), and 6/7-hydroxylase (6/7-HSH) activities in both cell types; 17 beta-hydroxysteroid oxidase (17 beta-HSORox) activity, located primarily in epithelial cells, also was not influenced by DMAA. In contrast, epithelial 3 beta-hydroxysteroid oxidase (3 beta-HSORox) and 3-ketosteroid reductase (3 beta-HSORred) activities were inhibited by 65% (P less than .001) and 58% (P greater than .05), respectively, albeit the latter result was not statistically significant. Stromal 3 beta-HSORox and 3 beta-HSORred activities were negligible; hence the effect of the inhibitor of these enzymes could not be assessed. In conclusion, DMAA is a relatively selective and potent inhibitor of 5 alpha-R activity in primary cultures of rat ventral prostate epithelial and stromal cells and should be a useful compound for antagonizing androgen-mediated actions in the prostate and other androgen target tissues.