Autocrine effect of DHT on FGF signaling and cell proliferation in LNCaP cells: role of heparin/heparan-degrading enzymes.

BACKGROUND: LNCaP cells are androgen-sensitive human prostate cancer cells. They are characterized by a bell-shaped growth curve in response to increasing doses of dihydrotestosterone (DHT) in culture. At a low concentration of DHT (0.1 nM), these cells show an increase in proliferation, but their growth is arrested at a high concentration (100 nM) of DHT. Results of our previous study demonstrated that the inhibitory effect of DHT at a high concentration was mediated through the action of TGF-beta1. The objective of the present study was to elucidate the mechanism of the proliferative effect of DHT in LNCaP cells. METHODS AND RESULTS DHT stimulated LNCaP proliferation only when cells were cultured in the presence of serum. In serum-free cultures, the characteristic DHT-induced proliferation was not observed. The addition of neutralizing antibody against FGF-2 (basic fibroblast growth factor) was able to inhibit this DHT-induced proliferation. These results suggest that the proliferative effect of DHT was mediated through the action of FGF-2. However, results of the reverse transcriptase polymerase chain reaction indicated that LNCaP cells did not express FGF-2 message. As a result, the source of FGF-2 in these cultures must be the serum supplemented in the culture media. FGF-2 can bind to heparin sulfate chains within the extracellular matrix (ECM). In cultures treated with exogenous heparin, the proliferative effect of DHT was abolished. These results led to the development of the hypothesis that DHT treatment mediates the release of FGF-2 entrapped in the ECM through increased heparinase activity. The addition of heparinase to cultures of LNCaP cells, in the absence of DHT, was able to stimulate cell proliferation. Moreover, 0.1 nM DHT caused a significant increase in heparinase activity. CONCLUSIONS: These results provide a possible mechanism for DHT action in LNCaP cells. In the absence of DHT, FGF-2 in culture was trapped in the extracellular matrix and was not available to interact with LNCaP cells. However, in the presence of 0.1 nM DHT, heparinase activity in the culture was elevated and, as a result, it liberated the trapped FGF-2 which, in turn, stimulated proliferation in LNCaP cells.

Prostatic ductal system in rats: changes in regional distribution of extracellular matrix proteins during castration-induced regression.

BACKGROUND: The extracellular matrix (ECM) is an intricate network composed of an array of molecules that play an integral role in the regulation of cell function, differentiation, and tissue-specific gene expression in various epithelia. In the present study, we examined the distribution of collagen type IV and laminin along the rat ventral prostatic duct before and after castration. METHODS: Mature Sprague-Dawley rats were castrated and their prostates processed for immunocytochemistry of ECM proteins, laminin, and collagen type IV. Tissue sections were also processed for apoptosis staining, using the 3' end-labeling technique. To examine the effect of ECM proteins on epithelial growth, rat ventral epithelial cells were cultured on ECM-coated surfaces. RESULTS: In the intact rat, laminin was localized in the basement membrane along all regions of the ventral prostate ductal system. Collagen type IV was found to be distributed evenly in the basement membrane of the distal and intermediate regions but was absent or poorly organized in the proximal region, where apoptosis in the epithelium occurs at a high rate. In the regressing prostate after castration, there was a shift in apoptosis from the proximal region to the distal intermediate regions of the prostatic duct. Associated with the shift was a remodeling of basement membrane proteins due to the specific loss of collagen type IV in the distal and intermediate regions. Collagen type IV reappeared underneath the epithelium 7 days after castration, when apoptosis in the epithelium stopped. In vitro, collagen type IV enhanced the growth of ventral prostatic epithelial cells, as assessed by cell number. CONCLUSIONS: Collagen basement membrane type IV mediates growth of rat ventral prostate epithelium, and its loss during tissue remodeling after castration is associated with cell death.

Transforming growth factor-beta1-induced proliferation of the prostate cancer cell line, TSU-Pr1: the role of platelet-derived growth factor.

The results of our previous study revealed that transforming growth factor-beta1 (TGFbeta1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGFbeta usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGFbeta1 induces this proliferation. We postulate that TGFbeta1 action is mediated through an indirect mechanism by inducing the expression of platelet-derived growth factor (PDGF), which, in turn, stimulates proliferation. The TGFbeta1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGFbeta1 resulted in an increase in PDGF secretion. These results indicate that TGFbeta1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF.

Transforming growth factor-beta in benign and malignant prostate.

BACKGROUND: The present review summarizes the cellular action of TGF-beta in benign and malignant growth of the prostate. METHODS: TGF-beta is a pleiotropic growth factor. It plays an important role in the regulation of growth and differentiation in many cells. In benign prostatic epithelia, its action is mediated through a paracrine mechanism. It inhibits proliferation and induces apoptosis in prostatic epithelia. It provides a mechanism to maintain epithelial homeostasis in the prostate. In prostatic stroma, its continual action leads to smooth muscle differentiation. This effect of TGF-beta may regulate the development of prostatic smooth muscle nodules in benign prostatic hyperplasia. RESULTS: As prostatic epithelial cells undergo malignant transformation, two major events occur regarding TGF-beta action. These include the loss of expression of functional TGF-beta receptors and overproduction of TGF-beta in malignant cells. The loss of expression of functional TGF-beta receptors provides a growth advantage to cancer cells over their benign counterparts. The overproduction of TGF-beta by cancer cells has a multitude of adverse consequences. TGF-beta can promote extracellular matrix production, induce angiogenesis, and inhibit host immune function. The biological consequence of these activities is an enhanced tumorigenicity in prostate cancer. Results of our recent studies with a rat prostate cancer model suggest that the immunosuppressive effect of TGF-beta seems to be the primary cause of tumor progression. This is because, if these cancer cells were engineered to reduce the production of TGF-beta, tumor growth was inhibited in syngeneic hosts but not in immune compromised hosts. CONCLUSIONS: Our future research should take advantage of this knowledge to devise therapeutic strategies aimed at eradicating prostate cancer.

Cytotoxic sensitivity to tumor necrosis factor-alpha in PC3 and LNCaP prostatic cancer cells is regulated by extracellular levels of SGP-2 (clusterin).

BACKGROUND: SGP-2 is a ubiquitous secreted glycoprotein that prevents cellular apoptosis. This study was carried out to determine the extracellular action of SGP-2 in a model of tumor necrosis factor-alpha (TNF)-induced cytotoxicity using two human prostatic cancer lines, LNCaP and PC3. These two lines were selected because LNCaP cells are highly sensitive to the cytotoxic effect of TNF, while PC3 cells are resistant to TNF at 24 hr. METHODS: Cells were cultured in the presence or absence of TNF (10 ng/ml). LNCaP cells were treated with varying concentrations of exogenous SGP-2, while PC3 cells were treated with antisera to SGP-2 with and without exogenous SGP-2. Following a 24-hr treatment, cultures were assessed by counting of cell number and by the trypan blue exclusion assay. RESULTS: Western blot analysis of conditioned media revealed that PC3 secreted more SGP-2 than did LNCaP. The sensitivity to TNF in LNCaP cells was reduced by the addition of exogenous SGP-2. PC3 cells became sensitive to TNF when SGP-2 antibody was added to the culture. The effect of SGP-2 antibody on PC3 cells was reversed by the addition of exogenous SGP-2 to the culture. CONCLUSIONS: These results suggest that SGP-2 can act as an extracellular mediator of anti-TNF-induced cytotoxicity.

Differential growth rates in stromal cultures of human prostate derived from patients of varying ages.

BACKGROUND: This study was undertaken to attempt to characterize changes in in vitro growth rates and cellular phenotypes of human prostatic stroma associated with aging and/or development of benign prostatic hyperplasia (BPH). METHODS: Prostate stromal cell strains were established from 12 tissue donors of varying age. Culture growth rate was determined by cell counts over a 6-day period. Cell phenotype was assessed by immunocytochemical staining for smooth muscle alpha-actin, smooth muscle myosin, and prolyl-4-hydroxylase. RESULTS: Growth rates of prostate stromal strains in vitro varied. Stromal cells derived from aged males with BPH had significantly slower growth rates than cells from younger donors. A positive reaction for prolyl-4-hydroxylase, a mesenchymal cell marker, was present in all cell cultures regardless of donor age. Expression of smooth muscle-specific actin, a nonspecific smooth muscle cell marker, was present in 48-79% of prostate stromal cultures. Staining for smooth muscle myosin, a specific smooth muscle cell marker, was found to vary significantly with age. The percentage of smooth muscle myosin-positive cells derived from males aged 15, 45, 57, and 72 years were 0.70 +/- 0.14%, 2.12 +/- 0.30%, 4.20 +/- 0.89%, and 26.25 +/- 1.0%, respectively. The shape and size of actin- and/or myosin-positive stromal cells from a 72-year-old donor culture were also usually larger and polygonal in shape as compared to thin and elongated shapes in 15-year-old donor cultures. The shape of actin- and/or myosin-positive cells from a 45-year-old donor culture demonstrated both phenotypes. CONCLUSIONS: These results suggest that in human prostate stromal cells cultured as described, the growth rate decreases, the percent of smooth muscle cells increases, and the cellular shape changes with increasing donor age and/or development of BPH.

Synergistic action of steroids and spermatocele fluid on in vitro proliferation of prostate stroma.

PURPOSE: Our goal is to understand human prostate growth phenomena potentially important to BPH development and growth. The objective of the present study is to characterize in vitro prostate stromal proliferative factors in testis epididymal secretions. MATERIALS AND METHODS: Human spermatocele fluids were used as a source of testicular epididymal plasma (STEP). Primary cultures of human prostate stromal cells were routinely grown in RPMI-1640 with 10% fetal bovine serum. During a 6-day experimental period, cells were cultured in RPMI-1640 in the absence of serum but supplemented with ITS. Whole STEP, ether stripped STEP, or heparin affinity column treated STEP was included in the culture medium with and without the addition of testosterone (T), dihydrotestosterone (DHT), or estradiol (E). Results of these treatments were assessed by cell counts. Antibodies against smooth muscle myosin heavy chain, smooth muscle alpha actin, and prolyl-4-hydroxylase were utilized in immunocytochemical characterization of cultured cells. RESULTS: Whole STEP stimulated prostatic stromal cells derived from prostates of 15, 45, 70 and 72-year-old men. Treatment of STEP by ether stripping or heparin affinity column exposure did not result in a significant reduction in cell counts. With the exception of the 15-year-old specimen, addition of T or DHT to ether stripped STEP resulted in a significant increase in cell counts over that of ether stripped STEP treatment alone. Preliminary immunocytochemical evaluation indicated the presence of variable mixture of fibroblasts, myofibroblasts, and smooth muscle cells in these cultures. CONCLUSIONS: These in vitro observations indicate that testis epididymal secretions contain androgen/STEP synergistic and androgen independent STEP factors promoting prostate stromal growth. These factors are not heparin binding. These observations are consistent with the concept that, in addition to the production of steroids, the testis produces non-androgenic factors that act in concert with, as well as independently of, androgen to stimulate prostatic growth.

Prostatic ductal system in rats: tissue-specific expression and regional variation in stromal distribution of transforming growth factor-beta 1.

BACKGROUND: Regional variations in stromal-epithelial interactions, mediated through soluble growth factors, may be responsible for differences in epithelial growth and death observed between regions of the rat prostatic ductal system. Since transforming growth factor-beta 1 (TGF-beta 1) can induce prostatic epithelial cell death in vitro and in vivo, we examined the localization and production of TGF-beta 1 with respect to the functional regions of the rat prostatic ductal system. METHODS: The distribution of TGF-beta 1 in the rat ventral prostate was examined by immunohistochemistry. Cell type-specific expression of TGF-beta 1 was determined using RT-PCR analysis of prostate epithelial and stromal cell fractions separated by Percoll gradient centrifugation. RESULTS: Immunohistochemical staining of normal prostate revealed regional variations in stromal TGF-beta 1 protein, which was most abundant in the stroma surrounding the degenerative proximal ducts. TGF-beta 1 staining was also tightly associated with the prostatic smooth muscle. Results of RT-PCR experiments confirmed the major source of TGF-beta 1 mRNA in normal rat prostate to be the stroma, with lesser expression by the epithelium. CONCLUSIONS: Stromal TGF-beta 1 was associated with cell death in the adjacent epithelial cell compartment in the prostatic ductal system, and alpha-smooth muscle actin-positive stromal cells may play a negative growth-regulatory role in the rat ventral prostate through production of TGF-beta 1.

Captopril (an inhibitor of angiotensin converting enzyme) inhibits obstructive changes in the neonatal rabbit bladder.

OBJECTIVES: To investigate whether angiotensin II has a role in the regulation of bladder smooth muscle growth and function, we developed a model of bladder neck obstruction (BNO) in the neonatal rabbit and investigated the effect of captopril (angiotensin converting enzyme inhibitor) on the obstructive changes in the developing bladder. METHODS: Partial BNO was induced in a group of 2-day-old rabbits (n = 8) by placing a loose 2-0 silk ligature around the vesicourethral junction. A second group of rabbits subjected to the identical partial BNO procedure (n = 8) was given captopril (1 mg/kg/day). Twelve days postobstruction, bladders from these animals, along with paired controls (n = 8), were harvested and assayed for total protein, DNA, and collagen content. RESULTS: Partial BNO resulted in a 170% increase in wet weight (P < 0.05), 132% increase in protein/deoxyribonucleic acid (DNA) ratio (P < 0.05), 75% increase in total DNA (P < 0.05), and 115% increase in total collagen (P < 0.05). When compared with obstructed animals, captopril administration significantly inhibited the increase in total DNA (P < 0.05) and reduced the amount of total collagen (P = 0.054). Examination of histology specimens demonstrated that captopril inhibited the serosal hyperplasia and collagen deposition associated with obstruction. CONCLUSIONS: These data demonstrate that captopril partially inhibits the changes in the neonatal rabbit bladder associated with obstruction, supporting the hypothesis that angiotensin II is involved in the regulation of bladder smooth muscle growth and collagen production.

Two-dimensional gel electrophoresis detects prostate-specific antigen-alpha1-antichymotrypsin complex in serum but not in prostatic fluid.

We investigated the interaction between prostate-specific antigen (PSA) and 1-antichymotrypsin (ACT) in prostatic secretions, identifying PSA and ACT in human serum, prostatic fluid, and seminal plasma by two-dimensional gel electrophoresis (2-D PAGE). Both PSA and ACT were detected in all three body fluids, but PSA-ACT complex was detected only in serum. Moreover, the 2-D PAGE Western blot staining profile for ACT from serum differed from that for prostatic fluid or seminal plasma. Incubation of prostatic fluid with purified ACT led to formation of PSA-ACT complex. Incubation of prostatic fluid with purified PSA, however, failed to form the complex, suggesting that the ACT in prostatic fluid was inactive or inhibited. Given that physiological concentrations of zinc inhibited the formation of PSA-ACT complex, we consider zinc a possible physiological inhibitor of the formation of the PSA-ACT complex. These results indicate that the failure to detect the PSA-ACT complex in prostatic fluid could be related to the inactivation of ACT, the presence of inhibitors (e.g., zinc), or simply the PSA:ACT ratio in the fluid.

Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer.

Our previous observations in LNCaP cells in vitro demonstrated an association between apoptotic cell death resistance and SGP-2 (Clusterin) overexpression. Accordingly, we hypothesized that high levels of cellular SGP-2 would aid in identifying biologically aggressive prostate cancer cells with unique survival advantages. To test this hypothesis, 40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1-3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior.

Transforming growth factor-beta1 is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line, LNCaP.

LNCaP is an androgen-responsive prostatic cancer cell line that exhibits a bell-shaped growth response to increasing doses of dihydrotestosterone (DHT) in culture. Although the precise mechanism responsible for this growth response to androgen stimulation remains unclear, many studies have suggested that androgen modulates the level of various growth factors. In the present study, the role of transforming growth factor-beta (TGF-beta) in mediating the androgen-regulated growth arrest of LNCaP cells was investigated. The following concentrations of DHT were used: 0, 10(-12), 10(-10), and 10 (-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth arrest dose, respectively. Results of RT-PCR showed that LNCaP cells express TGF-beta1 but not -beta2 and -beta3 messenger RNA. Competitive quantitative RT-PCR demonstrated that the level of TGF-beta1 messenger RNA increased approximately 7-fold when cells were treated with 10(-7) M DHT. Results of Western blot analysis showed a dramatic increase in the level of latent TGF-beta1 protein in cell lysates with increasing concentrations of DHT. In addition, results of enzyme-linked immunoadsorbent assay for TGF-beta1 indicated that treatment of LNCaP cells with DHT led to a dose-dependent increase in both total and biologically active TGF-beta1 in the conditioned media. To determine the role of TGF-beta1 in regulating LNCaP proliferation, the action of TGF-beta1 was blocked by two different but complementary approaches. First, TGF-beta1 neutralizing antibody was added to the culture medium with varying concentrations of DHT. Second, mannose-6-phosphate, which has been demonstrated to inhibit the activation of latent TGF-beta1, was added in a similar manner to the culture. Results demonstrated that the characteristic bell- shaped growth response following treatment with increasing doses of DHT was converted to a linear dose-response curve as the growth of inhibition seen at the high dose by DHT was abolished. These observations, taken together, indicate that TGF-beta1 mediates at least in part the growth arrest observed at the high concentration of DHT in LNCaP cells.

Stromal cells of the human prostate: initial isolation and characterization.

The present study was conducted to isolate and to characterize stromal cells from the human prostate and to study the effects of androgen and different growth factors in this model system. Benign prostatic hyperplasia (BPH) tissue samples were obtained from transurethral resection of the prostate (TURP). Tissue specimens were mechanically and enzymatically dissociated by treatment with DNAse and collagenase. Epithelial cells were separated from stromal cells by discontinuous Percoll gradient centrifugation. The stromal cells obtained were cultured in phenol red-free RPMI-1640 supplemented with 10% fetal bovine serum. Immunocytochemical analysis revealed that the stromal cell cultures were composed of both smooth muscle cells and fibroblasts. The short and broad, smooth muscle cells wee identified by using an antibody directed against alpha-smooth muscle actin. The thin and elongated fibroblasts stained positively for prolyl 4-hydroxylase. Smooth muscle cells were the predominant cell type in the present investigation. Typical cultures contained up to 99% of cells staining positively for alpha-smooth muscle actin. The prostate smooth muscle cultures were treated with dihydrotestosterone (DHT), bovine pituitary extract (BPE), basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). When cells were cultured in serum free RPMI-1640 supplemented with ITS+ (insulin, transferrin, and selenious acid) no significant (P > 0.05) mitogenic effect in medium supplemented with ITS+. In the presence of 10% charcoal-stripped fetal bovine serum (cFBS) DHT, at a concentration of 0.1 nM, was able to cause a slight but significant (P < 0.05) mitogenic effect on BPH smooth muscle cells growth. Basic FGF was able to stimulate BPH smooth muscle cells in a concentration-dependent fashion. The combination of DHT and 0.1 ng/ml bFGF was able to increase the proliferation of prostate smooth muscle cells above either agents alone. Addition of BPE to serum free RPMI-1640 caused a significant (P < 0.05) stimulation of cell proliferation in a concentration-dependent fashion. Addition to TGF-beta to serum or BPE containing RPMI-1640 caused a significant (P < 0.05) inhibition to cell proliferation in a concentration-dependent fashion. TGF-beta was cytostatic to the benign prostatic smooth muscle cells only in the presence of media containing growth stimulating factors found in charcoal-stripped serum or in bovine pituitary extract. These results demonstrated that stromal fraction isolated from BPH specimens was composed of both fibroblasts and smooth muscle cells. These cells could be cultured and were able to respond to various growth stimulatory and inhibitory agents.

Modulation of sensitivity to transforming growth factor-beta 1 (TGF-beta 1) and the level of type II TGF-beta receptor in LNCaP cells by dihydrotestosterone.

Transforming growth factor-beta 1 (TGF-beta 1) and androgen are potential physiological regulators of prostate cancer cells. In the present study, we have used LNCaP cells as a model of androgen-responsive prostate cancer to investigate the effects of dihydrotestosterone (DHT) on the sensitivity to TGF-beta 1. The ability of LNCaP cells to respond to TGF-beta has been controversial. In some studies, LNCaP cells were insensitive to TGF-beta 1 while, in others, they were sensitive to the growth inhibitory effect of TGF-beta 1. The present study was carried out to establish androgenic conditions that rendered LNCaP cells sensitive to TGF-beta 1. Cells were cultured in phenol-red-free RPMI 1640 medium supplemented with 10% charcoal-stripped fetal bovine serum. DHT was added at the following concentrations: 0, 10(-12), 10(-10), and 10(-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth-arrest dose, respectively. The effects of TGF-beta 1 observed on LNCaP cells included inhibition of cell proliferation, decrease in cell viability, alteration in cell morphology, and enhancement of gene transcriptional activity through activation of a TGF-beta responsive promoter. Of the various DHT concentrations investigated in this study, these effects of TGF-beta 1 on LNCaP cells were consistently demonstrated only at 10(-10) M. At other concentrations, the effects of TGF-beta 1 were either minimal or undetectable. Accompanying these effects of TGF-beta 1, a low but statistically significant level of TGF-beta 1-specific binding and an increased protein level of TGF-beta receptor type II were detected by a competitive binding assay and Western blot analysis respectively. These results indicate that LNCaP cells can be induced by DHT to respond to TGF-beta 1 and that DHT modulates the sensitivity to TGF-beta 1 and the level of TGF-beta receptor type II in these cells.

Genetic change in transforming growth factor beta (TGF-beta) receptor type I gene correlates with insensitivity to TGF-beta 1 in human prostate cancer cells.

Transforming growth factor beta 1 (TGF-beta 1), a potential regulator of growth of prostate cancer cells, exerts its effects through interaction with membrane receptors. In the present study, an attempt was made to establish a correlation between TGF-beta 1 sensitivity and TGF-beta receptor expression in three prostate cancer cell lines (PC3, DU145, and LNCaP). In a dose-dependent manner, TGF-beta 1 inhibited the proliferation of PC3 and DU145 cells but not LNCaP cells. Since TGF-beta signals through a heteromeric complex composed of TGF-beta receptors type II and type I, the expression of these receptors was investigated by Western blot analysis and reverse transcriptase-PCR. These studies demonstrated that all three prostate cancer cell lines express type II receptor. In contrast, type I receptor was detected only in the TGF-beta 1-sensitive PC3 and DU145 cells but not in the TGF-beta 1-insensitive LNCaP cells. To investigate the possibility that the undetectable expression of type I receptor in LNCaP cells is due to a change in the respective gene, Southern blot analysis was performed. The result demonstrated that there was a genetic change in type I receptor gene in these cells. Subsequently, when LNCaP cells were transiently transfected with T beta R-I cDNA, sensitivity to TGF-beta 1 was restored. These observations indicate that LNCaP cells contain a defective T beta R-I gene which rendered these cells insensitive to the action of TGF-beta 1.

Expression and localization of transforming growth factor-beta receptors type I and type II in the rat ventral prostate during regression.

Of the three ubiquitously expressed transforming growth factor-beta (TGF beta) receptors, only type I and type II receptors contain serine/threonine kinase activity and have a direct role in TGF beta signal transduction. In the prostate, it has been reported that the level of type III receptor expression increases transiently after castration. However, the relationship between the TGF beta signaling receptors, type I and type II, and androgen is currently unclear. Thus, in the present study, we made an initial attempt to elucidate the effect of androgen on type I and type II receptor expression in the rat ventral prostate by measuring the levels of messenger RNA (mRNA) and protein at specific time points after castration up to 10 days. Within 3 days after castration, an increase in type II receptor mRNA was observed in the prostate, and the level continued to rise until 7 days postcastration (approximately 8-fold increase). Between days 7-10 postcastration, no significant change in the level of type II receptor mRNA was observed. Testosterone administration immediately after castration abolished the induction of type II receptor mRNA during the same 10-day period. Western blot analysis performed for type II receptor showed a similar result, in that the level of type II receptor protein increased approximately 5-fold by day 10 postcastration. In a similar manner to the expression of type II receptor mRNA, the level of type I receptor mRNA increased steadily until day 7 postcastration (approximately 6-fold increase). Between days 7-10 postcastration, the level of type I receptor mRNA did not change significantly. As with type II receptor mRNA, the induction of type I receptor mRNA was suppressed when testosterone was administered immediately after castration. To localize the expression of TGF beta receptor type II, immunohistochemical studies were performed. The results of these studies demonstrated a preferential localization of type II receptor in the prostatic epithelial cells and an increased staining intensity for the receptor after castration. Taken together, these data indicate that TGF beta signaling receptors, type I and type II, are under negative androgenic regulation at the transcriptional level and that TGF beta may be an important regulator of a stromal-epithelial interaction in the rat ventral prostate.

Effect of TGF-beta 1, TGF-alpha, and EGF on cell proliferation and cell death in rat ventral prostatic epithelial cells in culture.

A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Analysis of cell death and cell proliferation in embryonic stages, normal adult, and aging prostates in human and animals.

Homeostasis in the prostate is recognized to be maintained by a complex interplay between the opposing actions of cell proliferation and cell death. Growth regulatory factors that promote or inhibit cell proliferation and promote cellular death have been identified in the prostate. The integration of these forces involves cellular cooperation between the prostatic stroma and epithelium. Hormone-regulated production of growth regulatory factors by one cell type may determine growth stimulation, inhibition, or cell death in a reciprocal cell partner. Imbalance between net cell proliferation and net cell death rates may result in abnormal growth leading to BPH. Additional study of the growth regulatory factors associated with distal vs. proximal epithelial cells and stroma and comparison of growth factor expression by the neonatal, postnatal growing, adult quiescent, and aging prostates will likely provide further insight into the regulation of prostate cell division and death.

Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone.

LNCaP is an androgen-sensitive human prostatic cancer cell line. The effect of androgen on these cells is characterized by a bell-shaped growth response and a dose-dependent induction of prostate-specific antigen (PSA) production. The present study was carried out to gain further insight into the effect of androgen on LNCaP. Cells were cultured in phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped fetal bovine serum, with concentrations of dihydrotestosterone (DHT) ranging from 0-10(-7) M, in a 4-day culture system. A bell-shaped growth response was reproduced with a peak level of cell count at 10(-10) M DHT. PSA secretion from these cells did not increase significantly until the DHT level in the medium reached 10(-9) M. A progressive increase in PSA secretion was observed at higher DHT concentrations accompanied with a progressive decline in cellular proliferation. The results of immunocytochemical analysis of PSA localization indicated that the proportion of cells with positive staining for PSA also increased with increasing concentrations of DHT. Analysis of androgen receptors, as determined by both immunocytochemistry and Western blot analysis, showed a decline in nuclear androgen receptor at low concentrations of DHT and an increase in the amount of receptor protein at high concentrations. These results indicated that the androgen-induced bell-shaped growth response in LNCaP cells represented the manifestation of two different cellular events in dose-related manner: cellular proliferation at low DHT concentrations and increased production of PSA at high DHT concentrations.