Androgen interactions with intact nuclear envelopes from the rat ventral prostate

Intact nuclear envelopes containing nuclear pore complexes have been prepared from the rat ventral prostate. The polypeptide profile of the nuclear envelopes from the rat prostate resembled that of nuclear envelopes prepared from the male rat liver. Isolation of the nuclear envelopes after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More dihydrotestosterone is bound after incubations at 22 degrees C for 18 h than at 2 degrees C for 18 h or 22 degrees C for 2 h. Scatchard analysis revealed a class of binding sites with an apparent Kd of 46 nM. Dihydrotestosterone, testosterone, cyproterone acetate and methyltrienolone were effective as competitors of labelled dihydrotestosterone binding to the nuclear envelopes, while estradiol did not compete. Castration of the rats 24, 48 and 96 h prior to preparation of nuclei resulted in loss of androgen binding to the membranes. Extraction with 0.6 M NaCl resulted in the loss of 72% of the androgen binding.     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.     

Androgen receptor expression in the rat prostate is down-regulated by dietary phytoestrogens

Background  

It is well established that the growth of the prostate gland is a hormone-dependent phenomenon involving both androgenic and estrogenic control. Proliferation of prostate cells is, at least in part, under control of estrogen receptor beta (ER-beta). Phytoestrogens bind ER-beta with high affinity and therefore may have antiproliferative effects in the prostate.     

Aging in the rat prostate. Reduction in detectable ventral prostate androgen receptor content.

Aging in the rat is associated with a reduction in the detectable androgen receptor content of the ventral prostate. The reduction in cytoplasmic receptor content did not appear to be attributable to an aging-associated production of a receptor-inactivating factor or to an aging-associated change in the sedimentation properties of the androgen receptor of young and aged animals.Saturation analysis of cytoplasmic extracts prepared from two different breeds of similar albino rats and a genetically distinct strain of inbred brown rats demonstrated quantitative aging-associated reductions in the androgen-receptor content per cell of the ventral prostate. The reduction in receptor content per cell appeared to increase progressively in magnitude with increasing age. The mean value for the cytoplasmic androgen receptor sites per cell for the oldest animals (mean age 884 days) was only 14% of the mean value for the young mature animals (mean age 185 days) of the same breed. The binding affinities of the detectable androgen receptor of the young mature and aged animals were essentially identical. This observation does not eliminate the possibility that the observed reduction results from an aging-associated production of defective receptor. Evaluation of the total DNA content of the ventral prostate did not provide evidence for an aging-associated selective loss of receptor-containing cells. These data in toto were consistent with the interpretation that aging is associated with a mean reduction in the androgen-receptor content per receptor-containing cell.Both cytoplasmic and nuclear androgen retention were evaluated in vivo. These experiments provided qualitative confirmation of the in vitro saturation analyses as there was a highly significant aging-associated reduction in the amount of androgen specifically bound by these prostatic compartments. Total specific androgen retention by the ventral prostate of aging adults was reduced by 55% relative to young mature animals. This result was nearly identical to that obtained for the same breed and age category of animals when evaluated by in vitro saturation analysis.Preliminary in vitro experiments revealed a diminution in the uptake of androgen receptor by purified nuclei from aged animals relative to purified nuclei from young mature animals. The magnitude of the diminution in nuclear acceptor capacity was insufficient to account for the reduction in nuclear retention of androgen determined in vivo. The data were consistent with the interpretation that the cytoplasmic receptor is the major determinant of nuclear androgen retention in the ventral prostate.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Androgen receptors in ventral prostate glands of zinc deficient rats

Androgen binding has been studied in the prostate cytosol of zinc deficient rats by charcoal assays. Rats were housed individually in plastic cages and maintained on a zinc deficient diet for 3 months. The cytosol fraction of prostate gland was incubated with various concentrations of tritiated methyltrienolone (3H-R1881, a synthetic androgen) alone or in the presence of 500-fold excess of radioinert R1881. Scatchard analysis of the data revealed that the number of androgen binding sites in the cytosol fraction of the zinc deficient rat prostate was 31 +/- 5.2 fmol/mg cytosol protein. This was significantly lower than that (84 +/- 11.5 fmol/mg protein) of the controls. Their dissociation constant (Kd = 1.6 +/- 0.6 nM) on the other hand was not different from that (1.7 +/- 0.7 nM) of control animals. This decrease in the concentration of cytosol receptor sites in the zinc deficient state suggests that this metal is involved in the androgen-binding process in the target cells.     

Androgen repression of the production of a 29-kilodalton protein and its mRNA in the rat ventral prostate

The regression of the ventral prostate, after a rat is deprived of androgens by castration, is accompanied by a marked decrease in the prostate's ability to synthesize RNA and major proteins. Surprisingly, in vitro translation of prostate RNA, isolated from rats 2 days after castration, detects four proteins with Mr of approximately 29,000, 37,000, 46,000, and 49,000 whose message levels increased 4- to 12-fold compared to results from normal rats. According to cDNA dot hybridization analysis, the increase after castration in the level of the 29-kDa protein-mRNA (per unit amount of DNA) was reversed within 6 h by androgen treatment of castrated rats. In contrast, the level of a mRNA in male rat liver, which hybridized to a cloned probe for the prostate 29-kDa protein-mRNA was reduced by castration and increased by androgen treatment. During an in vitro incubation, the ventral prostates of normal rats were much less efficient than the prostates of rats castrated 2 days earlier in synthesizing a 29-kDa protein. Despite the fact that androgenic manipulation of rats induced very rapid and significant changes in the production of the 29-kDa protein and in the level of its mRNA, the cellular level of this protein in the prostate, as determined by radioimmunoassay, was maintained at near normal values throughout the 2-week experimental period. Thus, the prostate appears to have a mechanism, based on androgen repression of certain genes, to maintain the cellular levels of the 29-kDa protein and possibly other structurally or functionally important proteins during both the periods of androgen-dependent growth and the castration-induced regression. The loss of such a regulatory mechanism may result in androgen-independent abnormal prostate growth.     

Mechanisms of replenishment of nuclear and rogen receptor in rat ventral prostate

1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2μg of [³H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [³H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [³H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5–60min interval after the administration of [³H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.     

Androgen receptors in rat and human prostate

In intact adult rats almost all androgen receptor (AR) sites of the rat ventral prostate (RVP) are occupied by endogenous dihydrotestosterone, and about 80% of these sites are nuclear. Nuclear AR disappears rapidly after castration (half-life of 3 h). The amount of cytosolic AR does not change within the initial 36 h, then markedly decreases during the next 2-5 days. An early and specific action of androgen is a remarkable increase of its own receptor. RVP also contains an estradiol receptor (ER) which rapidly disappears after castration and which, contrary to AR, is predominantly localized in the cytosol of stromal elements. The published procedures for steroid receptors grossly underestimate receptors concentrations in normal (NHP) and hyperplastic (BPH) human prostate. We have recently established a reliable method for the measurement of total AR, and we have found no difference in AR concentrations between NHP and BPH. BPH also contains a progesterone receptor and an elusive ER. Finally, we have used specific immunoglobulins in sex hormone binding plasma protein (SBP) for the demonstration of SBP-like immunoreactivity by the indirect immunofluorescence technique. The specific antigenic material was exclusively localized in the cytoplasm of BPH epithelial cells.     

Androgen receptor gene mutations in human prostate cancer

To investigate the structural abnormality of the androgen receptor (AR) in human prostate cancers, exons B-H encoding DNA- and hormone-binding domains were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products using originally designed oligoprimers. Tissues from 7 cases of untreated stage B prostate cancer surgically removed and from 8 cases of endocrine therapy-resistant cancers obtained at autopsy were used in the study. Two different mutations were identified in exons D and H in the different cancer foci of the same cancer death patient. One mutation in exon D (at codon 701, Leu to His) was detected in the prostate, and the other in exon H (at codon 877, Thr to Ala) was found in metastatic tissues. In untreated cancer tissues and the other autopsy samples, no mutations were detected. The mutation in exon H was identical to that reported in LNCaP cells. These results indicate that AR gene mutations occur in relation to endocrine therapy-resistance, although the mutation was found in 1 out of 8 resistant cases (12.5%) at autopsy.