Characterization and localization of nitric oxide synthase isoforms in the BB/WOR diabetic rat.

PURPOSE: Erectile dysfunction is a common pathological development in individuals with diabetes mellitus. Nitric oxide synthase (NOS) is essential for regulation of normal penile erection and NOS protein activity has been shown to be altered with diabetes. Several different isoforms and subtypes of NOS exist. However, little is known about how the distribution and abundance of these isoforms are altered with diabetes. We characterized the distribution and abundance of NOS isoforms and explored how they are altered with diabetes and result in erectile failure. MATERIALS AND METHODS: In situ hybridization and quantitative reverse transcriptase-polymerase chain reaction were done to measure the abundance and distribution of NOS-Ia, NOS-Ib, NOS-Ic, NOS-II and NOS-III in control and diabetic (BB/WOR) rats. Protein was localized by immunohistochemical analysis and alterations in protein abundance with diabetes were examined by Western blot analysis. RESULTS: NOS-I, NOS-II and NOS-III were observed in the endothelium lining the cavernous spaces and in the epithelium of the urethra. NOS-I protein was also present in the nerves of control and diabetic penes. We observed an increase in NOS-II expression around the dorsal nerves of diabetic penes, a decrease in NOS-III expression in diabetic pelvic ganglia and a decrease in NOS-Ib expression in the diabetic penis. NOS-I protein abundance was significantly decreased in diabetic pelvic ganglia. CONCLUSIONS: To our knowledge this is the first report of regional differences in the distribution of NOS-III in the urethra and altered NOS-Ib gene expression with diabetes.

Analysis of NOS isoform changes in a post radical prostatectomy model of erectile dysfunction.

Optimal treatment of erectile dysfunction (ED) following radical prostatectomy remains a subject of much controversy and is a significant concern for prostate cancer patients requiring surgical intervention. Neural stimulation involving nitric oxide synthase (NOS) is a crucial aspect of the normal erection process. In this study NOS isoform interaction was evaluated to improve our understanding of molecular changes pertaining to erection post radical prostatectomy. Bilateral cavernous nerve (CN) resected and control adult male Sprague-Dawley rats were killed 7, 14 and 21 days after injury. RT-PCR, in situ hybridization, Western blot and immunohistochemical analysis were used to evaluate changes in NOS isoform expression and distribution. NOS-I protein was dramatically decreased after CN injury while NOS-III and NOS-II remained unchanged. A profound decrease in smooth muscle and endothelium was observed in the corpora. To our knowledge this is the first report of differential altered NOS isoform protein abundance under conditions which mimic radical prostatectomy. These results show the importance of maintaining at least partial innervation of the penis after surgical intervention and that endothelial and smooth muscle changes resulting from loss of innervation may account for the ED observed in prostatectomy patients.

Prostate cancer cell growth inhibition by tamoxifen is associated with inhibition of protein kinase C and induction of p21(waf1/cip1).

BACKGROUND: Inhibition of protein kinase C (PKC) and modulation of transforming growth factor-beta (TGF-beta) are both associated with tamoxifen treatment, and both appear to be important in the regulation of prostate cancer cell growth. Investigations were performed which sought to measure the efficacy, and to elucidate the mechanism of growth inhibition by tamoxifen, in hormone-refractory prostate cancer. METHODS: Growth assays were performed on PC3, PC3-M, and DU145 prostate cancer cells. TGF-beta was measured by ELISA; p21(waf1/cip1) and retinoblastoma (Rb) protein levels were measured by Western blot; PKC activity was measured by kinase assay; and effects upon cell cycle were measured by flow cytometric analysis. RESULTS: IC50s for growth inhibition ranged from 5.5-10 microM, and were not affected by estrogen. Tamoxifen-mediated growth inhibition was not associated with induction of TGF-beta. However, tamoxifen treatment was associated with inhibition of PKC, which was followed by induction of p21(waf1/cip1), Rb dephosphorylation, and G1/S phase cell cycle arrest. Similar effects were observed with the known PKC inhibitor, Ro31-8220. CONCLUSIONS: These data suggest that micromolar concentrations of tamoxifen inhibit prostate cancer cell growth by inhibition of PKC, resulting in induction of the p21(waf1/cip1) protein.

The conventional transforming growth factor-beta (TGF-beta) receptor type I is not required for TGF-beta 1 signaling in a human prostate cancer cell line, LNCaP.

LNCaP is an androgen-responsive human prostate cancer cell line that has a defective gene for ALK-5, the conventional TGF-beta receptor type I. Yet, these cells respond to exogenous TGF-beta 1 under appropriate concentrations of dihydrotestosterone (DHT). Because a heteromeric complex composed of type I and type II receptor is required for TGF-beta signaling, the expression of these receptors was investigated in LNCaP cells at following concentrations of DHT-0, 0.1, and 100 nM. These concentrations were selected because they represent the zero DHT control in which LNCaP cells are not sensitive to TGF-beta 1, the proliferative dose of DHT in which these cells are sensitive to exogenous TGF-beta 1, and the growth-arrest dose of DHT in which LNCaP exhibits signs of TGF-beta signaling but are insensitive to exogenous TGF-beta 1, respectively. Results of Western blot analysis showed that LNCaP cells express an increased level of type II receptor at 0.1 nM DHT, the TGF-beta 1-sensitive dose. However, results of competitive quantitative RT-PCR demonstrated that DHT did not significantly change the level of type II receptor mRNA, suggesting that DHT modulates the level of type II receptor at the posttranscriptional level. In contrast, ALK-5 was not detected in these cells by either Western blot analysis or RT-PCR at all concentrations of DHT used in this study. Subsequently, the expression of ALK-1, -2, and -4 in LNCaP cells was examined because these proteins have been shown to bind TGF-beta 1 in vitro. ALK-1 and -2 were detected in these cells. Further analysis by competitive quantitative RT-PCR and Western blot demonstrated that DHT did not affect the level of expression of ALK-1 and -2 in LNCaP cells. These observations, taken together, demonstrate that ALK-5 is not required for TGF-beta 1 signaling and that there may be alternative mechanism(s) for TGF-beta 1 signal transduction in some systems.

Keratinocyte growth factor in the rat ventral prostate: androgen-independent expression.

Keratinocyte growth factor (KGF/FGF-7) is a stromally derived factor which exerts proliferative and differentiating effects on a variety of epithelial cells. Results of recent studies utilizing in vitro methods such as tissue culture and organ culture have suggested that KGF may act as a paracrine mediator of androgen-induced growth and development of the prostate and seminal vesicle. We undertook the present study to determine the distribution of KGF in relation to the functional regions of the rat prostatic ductal system, and whether KGF expression is influenced by androgen in vivo. Immunohistochemical staining revealed KGF to be present in the stroma throughout the prostate, regardless of the functional region, and staining for KGF remained high through 21 days post-castration. Message for KGF could also be detected by reverse transcriptase-PCR analysis of prostate stromal cells isolated from 4- and 21-day castrated animals, and no gross change in message level was observed following castration. Furthermore, no significant change in either stromal staining or message for KGF was observed in newborn rat prostates 10 days after castration, suggesting a similar regulatory mechanism for KGF in the adult and immature prostate. Epithelial staining for KGF decreased following castration, and greatly increased upon androgen replacement, possibly indicating a change in KGF internalization. These observations suggest that the presence of KGF protein is not related to functional differences in the prostate epithelium, and that expression of KGF in vivo is not greatly influenced by androgen.

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.

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.

Loss of expression of transforming growth factor beta type I and type II receptors correlates with tumor grade in human prostate cancer tissues.

Transforming growth factor beta1 (TGF-beta1) is a potential regulator of prostate cancer cell growth that signals through a heteromeric complex composed of type I and type II receptors. In the present study, an attempt was made to establish a correlation between expression of TGF-beta receptors and tumor grade in archival human prostate cancer tissues. To this end, immunohistochemical studies for TGF-beta receptors were carried out on 32 cases of human prostate cancer and 8 samples of benign human prostate. In both benign and malignant human prostate tissues, immunoreactivity for both type I and type II receptors was detected predominantly in epithelial cells. In addition, there was an inverse correlation between the loss of expression of TGF-beta1 type I and type II receptors and the tumor grade. Of the 32 prostate cancer cases screened, staining was completely absent in four samples for type II receptor (P < 0.05) and eight samples for type I receptor (P < 0.025). In contrast, all eight samples of benign prostate tissues investigated in this study showed strong staining for both type I and type II receptors. These results, taken together, indicate that human prostate cancer cells frequently have loss of expression of TGF-beta type I and/or type II receptors. Furthermore, these observations provide a potential mechanism for prostate cancer cells to escape the growth-inhibitory effect of TGF-beta.

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.

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.

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.

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.