Expression of G1 cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors in androgen-induced prostate proliferation in castrated rats

Androgen induces prostate cell proliferation in the castrated rat. We hypothesized that G1 cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors mediate this cellular response to mitogenic signals. In this study, induction of cyclins D1, D2, D3, E, and cdks 2, 4, and 6 expression was observed at various time points during testosterone replacement in the ventral prostate of castrated rats. The induction followed prostate epithelium proliferation, which peaked at 48 h and decreased at 120 h during the treatment. The study of cyclin/cdk complex formation revealed that more cyclin D1/cdk4 and cyclin D1/cdk6 complexes were formed at 48 h than at 120 h of treatment, but cyclin D1/cdk2 complexes remained the same. Furthermore, both hyperphosphorylated and hypophosphorylated forms of Rb were detected at 48 h, but only the hypophosphorylated form was detected at 120 h of treatment. p21Cip1, which was very abundant in the ventral prostate of castrated and intact rats, was not detected when the prostate started proliferation and increased gradually as proliferation decreased during the androgen treatment. Meanwhile, p27Kip1 dramatically increased after androgen treatment, and the induction levels were less at the peak of prostate proliferation and higher when proliferation was low. The results presented here suggest that expression of G1 cyclins and their related kinases and kinase inhibitors are well regulated after androgen replacement in the ventral prostate of castrated rats. The cooperation between these cell cycle regulators leads to a well-controlled prostate regeneration.     

Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment

In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.     

Synthesis and biological activity of a novel series of nonsteroidal, peripherally selective androgen receptor antagonists derived from 1,2-dihydropyridono[5,6-g]quinolines

A new nonsteroidal antiandrogenic pharmacophore has been discovered using cell-based cotransfection assays with human androgen receptor (hAR). This series of AR antagonists is structurally characterized by a linear tricyclic 1,2-dihydropyridono[5,6-g]quinoline core. Analogues inhibit AR-mediated reporter gene expression and bind to AR as potently as or better than any known AR antagonists. Several analogues also showed excellent in vivo activity in classic rodent models of AR antagonism, inhibiting growth of rat ventral prostate and seminal vesicles, without accompanying increases in serum gonadotropin and testosterone levels, as is seen with other AR antagonists. Investigations of structure-activity relationships surrounding this pharmacophore resulted in molecules with complete specificity for AR, antagonist activity on an AR mutant commonly observed in prostate cancer patients, and improved in vivo efficacy. Molecules based on this series of compounds have the potential to provide unique and effective clinical opportunities for treatment of prostate cancer and other androgen-dependent diseases.     

FMRFamide-related gene family in the nematode, Caenorhabditis elegans

The pharmacological profile of RU 58642, a new non-steroidal antiandrogen was investigated both in vitro and in vivo. In vitro, the compound displays a strong and specific affinity for androgen receptor. In vivo, its antiandrogenic activity was evaluated in castrated rat supplemented with testosterone propionate and in intact animals on prostate, seminal vesicles weight and serum levels of testosterone by oral and subcutaneous route. In castrated rats RU 58642 induced a significant decrease in prostate weight at a dose as low as 0.3 mg/kg whatever the route of administration. In intact rats its activity was compared to that of other non-steroidal antiandrogens such as flutamide, nilutamide and bicalutamide. RU 58642 proved to be significantly more potent than the reference compounds in reducing prostate weight: 3-30 times orally and 3-100 times subcutaneously, and thus the most potent antiandrogen to date to our knowledge. These results suggest that this compound may be very useful in the treatment of systemic androgen-dependent diseases.     

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.     

Changing patterns of gene expression identify multiple steps during regression of rat prostate in vivo

The rat ventral prostate is an androgen-dependent organ that undergoes dramatic cell death upon removal of testosterone by surgical castration. Several well characterized criteria, such as nuclear condensation, organelle blebbing, and DNA fragmentation, have been used to demonstrate that most of this cell loss is due to programmed cell death, or apoptosis, of the secretory epithelial cells. In addition to changes in morphology, it is well known that cells undergoing apoptosis show alterations in gene expression, and it is widely assumed that many of these genes are directly involved in the mechanism of programmed cell death. Using poly A+ RNA derived from normal rat prostate as well as from the regressing prostates of castrated rats, we have used a PCR-based subtractive hybridization approach to generate complementary DNA (cDNA) libraries greatly enriched in cDNAs strongly regulated during rat prostate regression. Several hundred of the genes represented in these libraries appear to be strongly regulated during prostate regression and most of these are prostate specific. Sequence analysis indicates that up to 30% of these clones are similar or identical to genes of known function, approximately 20% are similar to expressed sequence tags (ESTs), and as many as 50% of these clones have not been characterized previously. Analysis of selected clones using in situ hybridization indicates that they are expressed specifically in prostate epithelial cells, and that certain of these clones are regulated temporally in a pattern consistent with apoptosis. The patterns of gene expression include: 1) genes whose expression decreases uniformly after removal of androgen, indicative of androgen sensitive genes; 2) genes whose expression increases in apoptotic prostate cells and in other tissues, suggesting a class of genes generally involved in apoptosis; 3) and genes whose expression increases in individual regressing prostate epithelial cells, suggesting a class of prostate specific genes associated with apoptosis.     

Identification of an androgen-induced C3-P4 DNA binding protein in the cytosol of rat ventral prostate

A DNA binding protein C3-P4 was detected in the rat ventral prostate cytosol by gel retardation assay using a 32P-labeled 31 base pair synthetic oligomers (sequence deduced from the rat prostatic steroid binding protein C3-1 gene promoter -149 to -119) as a probe. The DNA binding activity of C3-P4 DNA binding protein is sequence specific, with preference for single strand and coding strand exclusive. This protein can be detected in many androgen target tissues and controlled well by androgen in the rat ventral prostate, we speculate that this DNA binding protein may function as an accessory factor to androgen receptor (AR) for the regulation of the C3-1 gene expression.     

The androgenic regulation of prostate proteins with a high affinity for deoxyribonucleic acid. Evidence for a prostate deoxyribonucleic acid-unwinding protein

1. When testosterone is injected into castrated rats in vivo, a significant increase in the incorporation of [35S]methionine into prostate proteins may be detected under conditions in vitro. 2. Studies based on DNA-cellulose chromatography show that the synthesis of prostate proteins with a high affinity for DNA is particularly enhanced by androgenic stimulation. 3. These changes in protein synthesis are negated when the anti-androgen, cyproterone acetate, is administered concomitantly with testosterone in vivo. 4. Two assays were developed for measuring the strand separation of prostate DNA; first, the retention of 3H-labelled native DNA on nitrocellulose membranes, and second, the activation of native DNA as a template for 9S prostate DNA polymerase. On the basis of these criteria, DNA-unwinding activity is present in the prostate gland and it is regulated by androgens in a steroid-and tissue-specific manner. 5. The results are discussed in the context of the mechanism of action of androgens, particularly since the changes provoked in DNA-unwinding activity by androgens precede the onset of DNA replication and mitosis.     

Effect of estradiol-17beta & estriol-3-methyl ether on spermatozoa & genital organs of rats

The effect of estradiol-17beta (E2) and estriol-3-methyl ether on spermatozoa and genital organs was investigated in rats. The motility pattern of spermatozoa in epididymis and vas deferens was adversely affected by both treatments in intact rats. The number of spermatozoa in cauda epididymis was significantly (p less than .05) reduced after treatment with estriol-3-methyl ether, however, the combined treatment had a more severe effect. Sperm transport was accelerated after both treatments in castrated rats. No effect was observed on weight and gross histology of testis. Epididymis weight was reduced only after combined treatment. The weight of seminal vesicles, ventral prostate, and vas deferens was reduced after both treatments. However, in castrated rats there was a transient increase in the weight of vas deferens and seminal vesicles after 7 days of E2 treatment while ventral prostate weight was reduced.     

Stromal development in the ventral prostate, anterior prostate and seminal vesicle of the rat

The prostate and seminal vesicle (SV) are androgen-dependent secretory glands of the male genital tract. They produce the bulk of the seminal secretions. The object of the present study was to examine and document the ontogeny of stromal maturation in the rat anterior and ventral prostate and SV. These organs have a loosely organized cellular mesenchyme during fetal development. During prostatic development the mesenchyme condensed to form smooth muscle sheaths immediately surrounding the epithelium, with looser connective tissue between individual ducts. In the SV, a loose connective tissue layer called the lamina propria lies between the epithelium and developing muscle. Smooth muscle alpha-actin, myosin, desmin, laminin, vinculin, vimentin and androgen receptor (AR) expression were examined by immunocytochemical methods during the pre- and postnatal developmental periods. The first marker to be detected was vimentin, which was initially found throughout the mesenchyme. During development vimentin became mostly restricted to the interductal tissue of the prostate and the lamina propria of the SV. Smooth muscle markers were expressed in an orderly sequence in a proximal to distal manner along prostatic ducts, from the urethra towards the tips. Expression of alpha-actin was followed by vinculin, myosin, desmin, and laminin. These markers became localized to the developing smooth muscle sheaths and were not expressed in the interductal tissue of the prostate or the lamina propria of the SV. Organ culture experiments demonstrated that androgens were required for the differentiation of smooth muscle sheaths. Castration of adult rats demonstrated that androgens were required to maintain smooth muscle differentiation. In castrates, the stroma was relatively thicker but less dense than in intact animals. Following castration, expression of the smooth muscle markers was lost sequentially in the reverse order of their expression during development. In long-term castrates alpha-actin, vimentin and a small amount of vinculin were detected. AR were first detected in the urogenital sinus mesenchyme immediately surrounding the epithelium at 16 days of gestation. As development progressed expression of AR became more widespread, and postnatally was found throughout the mesenchyme. As maturation of smooth muscle occurred, stromal expression of AR became localized to the muscular sheath immediately surrounding the epithelium. In the prostate the interductal connective tissue displayed very low levels of AR expression. In the SV, AR were also observed in the lamina propria. In summary, stromal differentiation and dedifferentiation in the rat prostate and SV were found to be androgen-dependent processes with ordered sequential ontogenic expression of specific markers.     

Castration-induced apoptosis in the rat ventral prostate is associated with increased expression of genes encoding insulin-like growth factor binding proteins 2,3,4 and 5

Insulin-like growth factor binding proteins (IGFBPs) have recently been demonstrated to act as regulators of apoptosis in vitro in both prostate and breast cancer cell lines. We show here that gene expression of IGFBP-2,-3,-4 and -5 increase rapidly in the rat ventral prostate following castration. Increases in IGFBP mRNA levels were detectable by Northern blotting by 6 hours and reached 5 to 10 fold of control levels at 72 hours after castration. Apoptosis in the ventral prostate, as detected in situ by the TUNEL method, was also induced as early as 6 hours after castration. TRPM-2/clusterin, a gene known to be associated with involution of the prostate, was not detected in sham castrated controls but was expressed by 24 hours following androgen ablation. IGF-I mRNA levels increased to 160% of control values within 6 hours following castration, then decreased gradually over the next 72 hours to 35% of control. Affinity labelling experiments demonstrated that IGF-I receptor levels increased initially after castration with peak binding at 24 hours, then declined to levels lower than control. These results suggest that rapid induction of IGFBPs in the rat ventral prostate following androgen ablation may play a role in apoptosis and involution of the prostate gland.     

Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.