An organ culture system for the study of programmed cell death in the rat ventral prostate

Glandular epithelial cells of the rat ventral prostate undergo programmed cell death in vivo following androgen ablation. Fragmentation of the prostatic DNA is an irreversible commitment step in this programmed cell death process. The amount of prostatic DNA fragmentation thus is a quantitative measure of the number of androgen-dependent prostatic glandular cells undergoing programmed death. An in vitro organ culture system was devised for determining rates of prostatic programmed cell death based upon the daily percentage of prostatic DNA fragmentation. To do this, rats were castrated and 2 weeks later treated in vivo for 3 days with exogenous androgen replacement to maximally stimulate DNA synthesis (i.e. proliferation) of the ventral prostatic glandular cells. In vitro organ cultures were established from these ventral prostates and the DNA of these explants was 125I-labeled by incubation in media containing [125I]iododeoxyuridine [( 125I]IDU). Using this in vivo-in vitro DNA labeling technique, greater than 85% of the [125I]IDU radioactivity was incorporated into DNA of the prostatic explants glandular cells. The decrease in 125I-radioactivity from prostatic explants was determined for over a 10-day period of organ culture. Using regression analysis of these data, the daily rate of programmed cell death of the glandular cells was determined. To test the validity of the method, organ cultures were maintained in media capable of inducing either necrotic (i.e. HgCl2-containing media) or programmed cell death (i.e. media lacking testosterone) and the daily decrease in the percentage of [125I]IDU retained in the tissue determined. In addition, the morphologic appearance of necrotic vs apoptotic cell death (i.e. programmed) was quantitated and compared to the [125I]IDU data. These studies demonstrated that this [125I]IDU labeled rat prostatic organ culture system can be used as an in vitro screen to quantitate the ability of various test agents to activate the programmed cell death pathway in prostatic glandular cells.     

Identification of a cellular receptor for transforming growth factor-beta in rat ventral prostate and its negative regulation by androgens

Scatchard analyses of the binding of transforming growth factor-beta (TGF beta) to membranes from rat ventral prostate revealed the presence of high affinity (Kd = 140 pM) saturable binding sites for [125I]TGF beta. The binding of [125I]TGF beta to prostatic membranes, while displaced in the presence of excess unlabeled TGF beta, is unaffected by epidermal growth factor, nerve growth factor, fibroblast growth factor, or insulin, indicating the specificity of binding. Affinity labeling of these membrane receptors by covalent attachment to [125I]TGF beta with bis-(sulfosuccinimidyl)suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I]TGF beta to a macromolecule that predominantly migrates as a 260,000 mol wt band in 7.5% acrylamide gels. Castration-induced androgen deprivation produced a significant increase in [125I]TGF beta binding to prostatic membranes with no apparent change in the affinity of membrane receptors for TGF beta. TGF beta receptor levels per total gland increased approximately 2-fold by 3 days after castration, reached a peak value by day 4, and then declined during the subsequent 10 days. Androgen administration to 4-day castrated animals decreased the number of TGF beta receptors to a value similar to that in the intact controls. These results demonstrate the presence of specific binding sites for TGF beta in the rat ventral prostate. Furthermore, the TGF beta receptor levels seem to be under negative androgenic regulation, indicating a potential role for this growth factor in the mechanism of activation of castration-induced death of androgen-dependent epithelial cells in the ventral prostate.     

Androgens and transforming growth factor beta modulate the growth response to epidermal growth factor in human prostatic tumor cells (LNCaP)

Treatment of LNCaP human prostatic cancer cells with 0.1 nM of the synthetic androgen, R1881, resulted in a three-fold stimulation of growth in 6 days. Of several growth factors tested (epidermal growth factor (EGF), platelet-derived growth factor, insulin-like growth factor, and insulin) only EGF (1 ng/ml) stimulated cell growth (two-fold). This stimulatory effect of EGF was inhibited for approximately 70% by 0.02 ng transforming growth factor beta (TGF beta)/ml. EGF (1 ng/ml) acted synergistically with R1881 (0.1 nM) on LNCaP cells to induce cell proliferation (seven-fold increase in cell growth). The synergistic effect of androgen and EGF was inhibited by TGF beta (0.05 ng/ml). In conclusion: human prostatic LNCaP cells are sensitive to EGF. Androgen increases and TGF beta decreases the growth response to EGF. This effect of TGF beta on an androgen-responsive system has not been observed before.     

Prostatic involution in rats induced by a novel 5 alpha-reductase inhibitor, SK&F 105657: role for testosterone in the androgenic response.

Within the prostate, androgen stimulates glandular cell secretion and proliferation while inhibiting glandular cell death. Due to its predominant nuclear localization, higher affinity for the androgen receptor, and more than 10-fold higher intracellular concentration than testosterone, dihydrotestosterone (DHT), not testosterone, appears to be the active intracellular androgen within the prostate of intact male hosts. The issue has remained unanswered, however, whether testosterone itself, without irreversible conversion to DHT by the 5 alpha-reductase enzyme, is capable of androgenic effects in the prostate. To address this issue, a novel dead end (i.e. product) inhibitor of the 5 alpha-reductase enzyme, SK&F 105657, was administered to intact or castrated male rats treated with either exogeneous testosterone or DHT. When administered twice a day orally at 25 mg/kg.dose, SK&F 105657 reduced the prostatic DHT content of either intact or castrated rats maintained with exogeneous testosterone to the same low level as that produced by surgical castration. Unlike castration, however, such SK&F 105657 treatment increased the prostatic testosterone content by more than 5-fold. The decrease in prostatic DHT coupled with a raise in testosterone are specifically due to the in vivo inhibition of the 5 alpha-reductase activity, since they were not observed in castrated rats maintained with exogeneous DHT. Treatment of intact or castrated male rats with exogeneous testosterone and oral SK&F 105657 (25 mg/kg, twice daily) resulted in a substantial inhibition of prostatic secretion, an inhibition of prostatic glandular cell proliferation, and an increase in prostatic glandular cell death. The magnitude of the changes, however, was not as great as that observed after surgical castration. The results are, however, specific for 5 alpha-reductase inhibition, since they were not observed in castrated rats given exogeneous DHT. These results demonstrate that if the prostatic testosterone content is elevated to sufficient levels, androgenic effects are induced without a requirement for an elevation in prostatic DHT content. Thus, the conversion of testosterone to DHT appears to function as a means of amplifying androgenic stimulation in the prostate.     

Inhibition of spontaneous and androgen-induced prostate growth by a nonhypercalcemic calcitriol analog

We have recently found that analog V (BXL-353, a calcitriol analog) inhibits growth factor (GF)-stimulated human benign prostate hyperplasia (BPH) cell proliferation by disrupting signal transduction, reducing Bcl-2 expression, and inducing apoptosis. We now report that BXL-353 blocks in vitro and in vivo testosterone (T) activity. BPH cells responded to T and dihydrotestosterone (DHT) with dose-dependent growth and reduced apoptosis. Exposure of BPH cells to BXL-353 significantly antagonized both T- and DHT-induced proliferation and induced apoptosis, even in the presence of T. To verify whether BXL-353 reduced prostate growth in vivo, we administered it orally to either intact or castrated rats, supplemented with T enanthate. Nonhypercalcemic doses of BXL-353 time- and dose-dependently reduced the androgen effect on ventral prostate weight, similarly to finasteride. Comparable results were obtained after chronic administration of BXL-353 to intact rats. Clusterin (an atrophy marker) gene and protein were up-regulated by BXL-353 in rat prostate, and nuclear fragmentation was widely present. The antiandrogenic properties of BXL-353 did not interfere with pituitary and testis function, as assessed by serum determination of rat LH and T. BXL-353 did not compete for androgen binding to BPH homogenates and failed to inhibit 5alpha-reductase type 1 and type 2 activities. In conclusion, BXL-353 blocks in vitro and in vivo androgen-stimulated prostate cell growth, probably acting downstream from the androgen receptor, without affecting calcemia or sex hormone secretion. BXL-353 and other vitamin D(3) analogs might thus represent an interesting class of compounds for treating patients with BPH.     

Castration-induced apoptotic cell death in the Brown Norway rat prostate decreases as a function of age

Growth and differentiation of the prostate gland depends upon androgens, yet overgrowth of the human prostate occurs later in life when serum levels of testosterone are declining. We have reported a similar phenomenon in the Brown Norway rat, but the age-dependent overgrowth of the prostate is confined to the dorsal and lateral lobes and, hence, is lobe specific. Because tissue growth depends upon the balance between proliferation and death of cells, the present study was designed to investigate whether cell death differed in the various prostatic lobes of Brown Norway rats as a function of age. Apoptosis of cells in the ventral, dorsal, lateral, and anterior lobes of the prostate was examined in young (4-month-old) and old (24-month-old) Brown Norway rats after castration. Whereas castration caused tissue weights of all four prostatic lobes to decrease over the course of 10 days, this occurred more rapidly and to a greater magnitude in the ventral than in the dorsal, lateral, and anterior lobes. Tissue DNA content, a measure of cell number, decreased only in the ventral lobe after castration. DNA fragmentation, indicative of apoptotic cell death, was detected by in situ labeling using the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling method and as intranucleosomal cleavage of genomic DNA analyzed by agarose gel electrophoresis. Both methods demonstrated the correlation between loss of DNA content and apoptotic cell death in the ventral lobe, whereas only the highly sensitive terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method revealed relatively few dying cells in the dorsal, lateral, and anterior lobes after castration. Moreover, when examined as a function of age, less cell death occurred in all four lobes of old rats compared with young rats. In both young and old rat prostates, cell death was observed in epithelial and stromal cells within the ventral lobe where apoptotic cells were detected throughout the branched ductal network and were not restricted to a particular region. Taken together, these studies demonstrate the marked differences in cell death and survival between the different rat prostatic lobes in response to castration and further suggest that the androgen-sensitive apoptotic response is age dependent. Hence, the lower rates of cell death observed for the dorsal and lateral lobes, accompanied by the further decline that occurs with increasing age, are important components of the age-dependent and lobe-specific overgrowth observed for these lobes. Moreover, the age-dependent decline in apoptotic cell death observed in the prostates of old rats suggests that prostatic cells develop androgen independence as a function of age, and survival of these cells does not require androgen.     

Androgen-regulated expression of a novel member of the aldo-keto reductase superfamily in regrowing rat prostate

The rat prostate is dependent on androgen for normal growth and differentiation. In addition, the organ undergoes rapid cell death upon withdrawal of androgen on castration, and the atrophied tissue is capable of regrowth after androgen replacement in adult animals. In our search for novel factor(s) that participate in this androgen-induced proliferation of adult rat prostate cells, we have generated a complementary DNA (cDNA) library enriched in cDNAs transiently up-regulated after androgen stimulation in castrated rat ventral prostate using a PCR-based subtractive hybridization technique. Sequence analysis of about one hundred clones in the library showed that approximately 70% of them are identical or closely related to genes of known function, the remaining ones showing no or very low similarity to any genes characterized previously. Among the former a new member of the rat aldo-keto reductase superfamily that is closely related to aflatoxin, B1 aldehyde reductase has been identified. The newly identified protein (androgen-inducible aldehyde reductase, AIAR) and rat aflatoxin B1 aldehyde reductase (AFAR) exhibit 80% amino acid sequence homology. The enzymatic activity toward 4-nitrobenzaldehyde of recombinant AIAR expressed in Escherichia coli was about 16% of that of rat AFAR. Northern blot analysis revealed AIAR expression in various adult rat tissues in addition to the ventral and dorsolateral prostates, which differs from the highly restricted expression of AFAR in the kidney and liver. The AIAR messenger RNA (mRNA) content of the ventral prostate was low in normal and castrated rats, transiently increased after androgen administration to castrated rats, attaining a peak 12-24 h after the treatment. Although the physiological substrate(s) of AIAR has not been identified, the current results suggest that AIAR expression is associated with some growth-related processes in regrowing rat prostate.     

Identification and characterization of an androgen-responsive gene encoding an aci-reductone dioxygenase-like protein in the rat prostate

The ALP1 [aci-reductone dioxygenase (ARD)-like protein 1] gene was identified in a comprehensive cDNA subtraction aimed at identifying genes regulated by androgens in the rat ventral prostate. ALP1 is homologous to the ARD/ARD' that were discovered in Klebsiella pneumoniae as enzymes that have the same polypeptide sequence and differ only in their metal content. This family of proteins is evolutionarily conserved from bacteria to humans and is involved in the methionine salvage pathway. Northern and Western blot confirmed the regulation of ALP1 by androgens in the rat ventral prostate. ALP1 mRNA is expressed in a variety of tissues; however, its regulation by androgens was specific to the prostate. ALP1 is expressed by the glandular epithelial cells of the rat prostate, with little or no expression in the stromal cells. ALP1 is down-regulated in the different rat Dunning tumor cell lines compared with the normal or castrated rat prostate. Expression studies showed that ALP1 overexpression is not tolerated by AT6.1 cells. Further studies demonstrated that ALP1 is also down-regulated in the human prostate cancer cell lines LNCaP, PC3, and DU145, and overexpression induces cell death in these cells. Taken together, our observations suggest that ALP1 may have an important role in androgen regulated prostate homeostasis as well as in prostate cancer progression by regulating cell death of prostate cancer cells.     

Increased levels of clusterin (SGP-2) mRNA and protein accompany rat ventral prostate involution following finasteride treatment

Finasteride is a well-known inhibitor of the prostatic enzyme 5 alpha-reductase type 2 which prevents conversion of testosterone into 5 alpha-dihydrotestosterone, the active intraprostatic androgen, which causes prostate involution through a combination of cell atrophy and cell death. The drug is widely used to improve symptoms of benign prostatic hyperplasia in man. Clusterin, a glycoprotein which is generally up-regulated under conditions inducing cell atrophy or organ involution, is produced at a high level in the regressing rat ventral prostate following androgen ablation. According to several authors, clusterin does not respond to finasteride treatment, suggesting a different action of testosterone and 5 alpha-dihydrotestosterone. We show here that, under our conditions, finasteride was capable of inducing production of both clusterin mRNA and protein in the rat ventral prostate. In fact, by using different and converging techniques, such as Northern hybridization, in situ hybridization histochemistry and immunohistochemistry, we were able to show a strong induction of the clusterin gene in the epithelial cell population of the gland. The response to finasteride, which was similar to that seen with castration, occurred with a delay of a few days. In situ and immunohistochemistry experiments indicated that both orchidectomy and finasteride administration resulted in increased transition of the epithelial cells from the columnar to the cuboidal (atrophic) shape, and this was accompanied by an increased intensity of the signal for clusterin. Thus, it appears that induction of clusterin is part of the molecular process leading to prostate involution caused by either orchidectomy or finasteride administration.     

Increased androgen receptor expression correlates with development of age-dependent, lobe-specific spontaneous hyperplasia of the brown Norway rat prostate

Androgens are essential for development and differentiated function, as well as proliferation and survival of cells within the prostate gland. Age-related changes in the hormonal milieu, marked by a decrease in the serum androgen to estrogen ratio may contribute to the evolution of pathological changes, such as benign prostatic hyperplasia and carcinoma of the prostate gland, in older men. A similar phenomenon occurs in Brown Norway rats, in which the serum testosterone to estradiol ratio declines with age, and despite the lower serum testosterone level, age-dependent prostatic hyperplasia develops in the dorsal and lateral lobes, but not in the ventral lobe. To evaluate a role for changes in androgen action in the evolution of prostatic hyperplasia, we compared the immunostaining intensity of androgen receptor in the different prostate lobes from young (4 months of age) and old (24 months of age) Brown Norway rats. Androgen receptor immunostaining was present in the nuclei of all epithelial cells and some stromal cells throughout the prostatic ducts of each lobe from both young and old rats. Whereas androgen receptor immunostaining intensity decreased in luminal epithelial cells of the ventral prostate from old rats, it increased in luminal epithelial cells of the dorsal and lateral lobes from old rats, when compared with young rats. To validate immunocytochemical studies, Western blot analyses were performed. The total tissue level of androgen receptor decreased by 30% in the ventral lobe of old rats, whereas tissue levels of androgen receptor increased 2.7-fold and 1.3-fold in the dorsal and lateral lobes, respectively, of old rats. Similarly, the percentage of epithelial cells staining positive for the proliferation marker, proliferating cell nuclear antigen, was increased approximately 2-fold in the dorsal and lateral lobes as a function of older age. The presence of higher levels of androgen receptor and increased number of proliferating cell nuclear antigen-positive cells in the dorsal and lateral lobes of old rats suggest that changes in androgen receptor levels may be related to the lobe-specific proliferation of cells that occurs with increasing age. Additional evidence for lobe-specific regulation of androgen receptor expression was obtained from Western blots and by immunocytochemistry following castration. Androgen receptor levels in the ventral and dorsal lobes, but not the lateral lobe, of young and old rats were down-regulated in the absence of testicular androgen. However, nuclear immunostaining of androgen receptor returned by 7-10 d after castration in the ventral and dorsal lobes in the continued absence of androgen. Moreover, up-regulation of the androgen receptor level occurred more rapidly in the ventral and dorsal lobes of old, compared with young, castrated rats. Taken together, these results suggest that lobe-specific and age-dependent differences in the regulation of androgen receptor expression might lead to changes in tissue androgen responsiveness and the consequent development of lobe-specific hyperplasia in the Brown Norway rat prostate gland.     

Tumor suppression and apoptosis of human prostate carcinoma mediated by a genetic locus within human chromosome 10pter-q11.

Prostate cancer is the second leading cause of male cancer deaths in the United States. Yet, despite a large international effort, little is known about the molecular mechanisms that underlie this devastating disease. Prostate secretory epithelial cells and androgen-dependent prostate carcinomas undergo apoptosis in response to androgen deprivation and, furthermore, most prostate carcinomas become androgen independent and refractory to further therapeutic manipulations during disease progression. Definition of the genetic events that trigger apoptosis in the prostate could provide important insights into critical pathways in normal development as well as elucidate the perturbations of those key pathways in neoplastic transformation. We report the functional definition of a novel genetic locus within human chromosome 10pter-q11 that mediates both in vivo tumor suppression and in vitro apoptosis of prostatic adenocarcinoma cells. A defined fragment of human chromosome 10 was transferred via microcell fusion into a prostate adenocarcinoma cell line. Microcell hybrids containing only the region 10pter-q11 were suppressed for tumorigenicity following injection of microcell hybrids into nude mice. Furthermore, the complemented hybrids undergo programmed cell death in vitro via a mechanism that does not require nuclear localization of p53. These data functionally define a novel genetic locus, designated PAC1, for prostate adenocarcinoma 1, involved in tumor suppression of human prostate carcinoma and furthermore strongly suggest that the cell death pathway can be functionally restored in prostatic adenocarcinoma.     

Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and growth factors to determine those factors that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal growth factor, insulin-like growth factor 1 (IGF1), and fibroblast growth factor did not influence DNA synthesis whereas transforming growth factor beta (TGF beta) alone had a significant effect. Neither estradiol-17 beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal growth factor or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the growth promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell growth in addition to exerting pronounced effects on cytodifferentiation.     

Rat prostate cancer cells contain functional receptors for transforming growth factor-beta

Clonally derived C-, D-, and T-family AXC/SSh rat prostate cancer cell lines contain transforming growth factor-beta (TGF beta) receptors. The content in C3, D1, T1, and T5 cells, respectively, was 8,560 +/- 1,450, 13,160 +/- 1,240, 2,425 +/- 490, and 10,540 +/- 1,025 sites/cell (mean +/- SEM). Respective Kd values were 160 +/- 48, 200 +/- 53, 24 +/- 3, and 115 +/- 15 pM (mean +/- SEM). T1 cell TGF beta receptor site content and Kd differed significantly from those of other prostate cancer cell lines (P less than 0.05). TGF beta is a bifunctional concentration-dependent modulator of T1 and T5 cell thymidine incorporation. At low concentrations, thymidine incorporation was inhibited, whereas as the medium TGF beta content was increased, T1 and T5 cell thymidine incorporation was stimulated. The concentrations of TGF beta causing half-maximum inhibition of T1 or T5 cell thymidine incorporation, respectively, were 0.11 and 0.24 pM, whereas the respective TGF beta concentrations causing half-maximum stimulation of thymidine incorporation were 14.4 and 134 pM. These findings establish that rat prostate cancer cell sensitivity to TGF beta inhibition of function is at least 2 orders of magnitude greater than that of most other mammalian cells. In contrast, the sensitivity of rat prostate cancer cells to TGF beta enhancement of function is comparable to that of other mammalian cells. TGF beta inhibited basic fibroblast growth factor (bFGF) stimulation of T1 and T5 cell thymidine incorporation. Because the concentration of bFGF required for half-maximum increase of T5 cell thymidine incorporation was independent of medium TGF beta content, the effect of TGF beta is distal to the T5 cell bFGF receptor. In contrast, the concentration of bFGF required for half-maximum increase in T1 cell thymidine incorporation increased 5-fold as the medium TGF beta content was increased; suggesting that the effect of TGF beta in T1 cells is proximal to the T1 cell bFGF receptor. Our studies establish that rat prostate cancer cells contain functional TGF beta receptors, imply the presence of functional bFGF receptors, and demonstrate that mitogen modulation of prostate cancer cell function is multifactorial. The finding that TGF beta is a bifunctional effector of prostate cancer cell DNA synthesis provides some insight into the potential complexity of mitogen modulation of prostate cancer cell proliferation. The mechanism by which these mitogens interact is unknown; however, our studies suggest that some interactive effects may be cell line specific.     

The metaplastic effects of estrogen on mouse prostate epithelium: proliferation of cells with basal cell phenotype

The exogenous administration of estrogens to male mice alters the hypothalamic-pituitary-gonadal axis and reduces androgen levels, leading to a regression of the prostatic epithelium. As well, a specific direct response to estrogens is the induction of epithelial squamous metaplasia. The aims of this study were to identify the process by which the prostatic epithelium is transformed in intact adult male mice using the synthetic estrogen, diethylstilbestrol. A comparison of the effects of diethylstilbestrol in the three lobes revealed a hierarchy of response, with the anterior lobe being the most responsive, the dorsolateral lobe less responsive, and the ventral lobe the least responsive. The effect of castration was used to distinguish between the epithelial responses to estrogen administration and androgen deprivation. The results demonstrate that transformation of the epithelium involved proliferation of cells with a basal cell phenotype, the onset of cytokeratin 10 expression, up-regulation of progesterone receptor expression, and loss of the cell cycle inhibitor, p27(Kip1) expression; none of these changes was observed after castration. Mice lacking functional estrogen receptor alpha failed to respond, demonstrating a requirement for estrogen receptor alpha in the epithelium and/or stroma to mediate the proliferative response to estrogen in the prostate gland.     

The culture of hormone-dependent epithelial cells from the rat ventral prostate

This paper describes a method for obtaining cultures of rat ventral prostate epithelial cells. The prostate is first perfused with a collagenase solution before removal from the animal; subsequent mincing and incubation in vitro produces a suspension of alveolar cell clumps. Upon incubation, these clumps attach to the surface of the culture dish and spread into discrete epithelial cell colonies, which both retain differentiated morphology, and secrete a species of plasminogen activator that is characteristic of prostatic tissue. These properties were not observed in cultures prepared from single cell suspensions of the same organ.Maintenance of epithelial colony integrity and secretory activity specifically required the continued presence of stromal cells, glucocorticoids and insulin. Androgenic steroids were much less effective than glucocorticoids in stimulating plasminogen activator secretion and in maintaining colony integrity, in spite of the well-established androgen dependence of prostatic tissue morphology in vivo and in organ culture. Furthermore, no effects of prolactin were observed, either when this hormone was tested alone or in conjunction with steroid hormones. Of 3 retinoids tested, retinal was highly cytotoxic at concentrations in the range of 1 μM, whereas retinol and retinoic acid were without detectable effect.     

The production of transforming growth factor-beta in the porcine ovary and its secretion in vitro

Porcine granulosa cells isolated from small (1-3 mm in diameter) follicles proliferate rapidly in culture in response to 10% fetal bovine serum (FBS) and epidermal growth factor (EGF) (10 ng/ml). Transforming growth factor-beta (TGF beta) inhibits FBS/EGF-stimulated proliferation in a dose-dependent manner. We have used this proliferation inhibitory property of TGF beta to assay qualitatively, the presence of this growth factor in conditioned medium from cultured follicle cells as well as in partially purified preparations from porcine ovarian compartments. In addition, the concentration of TGF beta in the theca cell conditioned medium was quantitatively estimated by generating a TGF beta-dose-response curve (inhibition of FBS/EGF-stimulated proliferation of granulosa cells in monolayer culture) using authentic human TGF beta-1. Ovarian thecal cells isolated from small and large size follicles in the pig ovary secrete TGF beta-like activity in vitro. Medium conditioned by thecal cells in primary monolayer culture contains a latent form of TGF beta which can be activated by heat or acid treatment. In contrast, and unlike rat granulosa cells, porcine granulosa cells in primary monolayer culture do not secrete detectable levels of TGF beta-like activity in the medium. Incubation of heat-activated thecal cell conditioned medium with a TGF beta-neutralizing antibody (which recognizes TGF beta-1 and 2) but not nonimmune serum attenuated the TGF beta-like activity in thecal cell conditioned medium suggesting that this activity is due to authentic TGF beta. Since many cell types secrete latent TGF beta in the medium when cultured in vitro, we next investigated whether thecal cell secretion of latent TGF beta was a function of cell culture or whether the ovarian thecal compartment actually contained detectable levels of TGF beta-like activity. To this end, we used an acid-ethanol extraction procedure to isolate thecal proteins from fresh-frozen tissue. The acid-ethanol extracted protein fraction was mixed with trace amounts of 125I-TGF beta for detection and chromatographed on Bio-Gel P-60 column under acidic conditions. Elution of TGF beta bioactivity from the Bio-Gel P-60 column as measured by inhibition of granulosa cell proliferation correlated with the elution of radioiodinated authentic TGF beta. Preincubation of TGF beta-like activity-containing fractions with TGF beta-neutralizing antibody attenuated the proliferation-inhibitory activity in these fractions. TGF beta activity was also observed in fractions extracted from porcine corpora lutea.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction between prostatic fibroblast and epithelial cells in culture: role of androgen

We have established and characterized two cell lines from the ventral prostate gland of normal Nb rats: NbE-1 (prostatic epithelial) and NbF-1 (prostatic fibroblast) cell lines. To identify the direct mitogenic action of dihydrotestosterone (DHT), we incubated these cell lines alone and together (in the presence and absence of cell contact) with various concentrations of DHT (0.1-10,000 ng/ml) for 24-72 h and assayed for the rate of DNA synthesis and the total number of cells in tissue culture at specified time periods. Results demonstrate that the primary target for DHT mitogenic action is the prostatic fibroblasts. DHT inhibited the growth of prostatic epithelial cells by themselves, but stimulated prostatic epithelial cell growth when the epithelial cells were cocultured with prostatic fibroblasts. Furthermore, the cell-conditioned medium obtained from either the fibroblast or the epithelial cells stimulated in an autocrine or a paracrine manner the growth of prostatic cells in culture. These results are consistent with the concept that DHT stimulates the growth of prostatic epithelial cells indirectly via its direct mitogenic action on the prostatic fibroblasts. Because epithelial cells are the cell type principally responsible for converting testosterone to DHT, and the fibroblasts respond to the mitogenic action of DHT, our results support the concept that tight metabolic cooperation exists between prostatic epithelial and fibroblast cells. These data are in agreement with previous in vivo studies in which we have demonstrated that androgen receptors in the mesenchyme are obligatory for androgen-induced prostate growth and development.     

Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate

Binding sites of [3H]testosterone and [3H]dihydrotestosterone in the rat fetal urogenital sinus and postnatal prostate and vagina grown in vitro were examined by steroid autoradiography. Distinct nuclear incorporation of both androgens appeared between 14.5 and 16.5 days of gestation in rat fetuses. Nuclear labelling in the sinus was restricted to the mesenchyme surrounding the epithelium which showed no nuclear labelling. A similar distribution of labelled cells was observed in male and female sinuses up to 18.5 days of gestation. By 20.5 days of gestation, the labelling in the ventral mesenchyme of female urogenital sinuses became less intense but persisted in the mesenchyme of the dorsal sinus wall from which the vagina is formed. In the postnatal prostate, the epithelium showed nuclear [3H]testosterone labelling at 10 days coinciding with the onset of its functional differentiation. Epithelial labelling became more intensive at 4 weeks post partum while that of the mesenchyme declined. The results suggest two phases of androgen action: formation of the prostatic buds mediated by the androgen-activated mesenchyme of the fetal urogenital sinus and the differentiation of the postnatal prostatic epithelium directly stimulated by androgens.