Effects of dutasteride on prostate growth in the large probasin-large T antigen mouse model of prostate cancer.

PURPOSE: We evaluated the effects of dutasteride for preventing or delaying prostate growth and neoplastic changes in a transgenic model of prostate cancer. MATERIALS AND METHODS: Large probasin-large T antigen mice were treated for 4 or 8 weeks with dutasteride. The prostate and seminal vesicles were compared with those from intact and castrated large probasin-large T antigen mice and WT mice. RESULTS: Dutasteride greatly decreased the transgene induced increase in prostate weight but castration caused greater reduction. Dutasteride inhibited type 1 and 2, 5alpha-reductase activities, decreased DNA and protein, and increased apoptotic bodies and TUNEL staining in the dorsolateral prostate. No evidence of poorly differentiated cancer was seen. Dutasteride did not decrease the weight of the androgen dependent levator ani or bulbocavernosus muscle. CONCLUSIONS: Dutasteride inhibited type 1 and 2, 5alpha-reductase activities, and decreased DNA and protein content of the dorsolateral prostate without affecting androgen responsive muscle weight in large probasin-large T antigen mice. These studies provide support for the hypothesis that a 5alpha-reductase inhibitor inhibits the initiation and/or progression of clinical prostate cancers.

Comparison of the growth-promoting effects of testosterone and 7-alpha-methyl-19-nor-testosterone (MENT) on the prostate and levator ani muscle of LPB-tag transgenic mice.

BACKGROUND: 7-alpha-methyl-19-nortestosterone (MENT) is being considered for androgen replacement in testosterone deficient men and as a male contraceptive. Because androgenic effects on the prostate are a major concern, we have evaluated MENT in a transgenic model of prostate cancer. METHODS: LPB-Tag mice were castrated and infused with testosterone (T; 5 or 30 microg/day) or MENT (5 or 30 microg/day) for 4 weeks. Prostate, seminal vesicle, and levator ani muscle (LAM) weights were compared. RESULTS: At an equivalent dose, MENT maintained or stimulated the mean weights of these organs more than T. However, the dorsolateral prostate/LAM ratio of weights did not favor MENT, but DNA/mg tissue and Ki 67 immunostaining suggested that MENT may increase DNA less than T. CONCLUSIONS: MENT is more potent than T in maintaining or stimulating prostate, seminal vesicle, and LAM. Using doses that resulted in comparable stimulation of the levator ani muscle, MENT had similar effect on prostate weight, but increased DNA/mg prostate less than T in this transgenic mouse model of prostate cancer.

Induction of apoptosis in BPH stromal cells by adenoviral-mediated overexpression of caspase-7.

PURPOSE: We hypothesized that expression/activity of critical components of the apoptotic pathway can be used to induce apoptosis of a human prostate cell line derived from benign prostatic hyperplasia (BPH) tissue. MATERIALS AND METHODS: We analyzed the apoptotic pathway in BPH cells treated with the powerful inducer of apoptosis, staurosporine (STS), and adenoviruses overexpressing caspase-3, -7, or the control gene lacZ. RESULTS: Twelve hours post-STS, most BPH cells were floating in the culture medium, TUNEL staining was widespread, and DEVDase activity (the catalytic activity of type II caspases) was increased. The pan-caspase inhibitor, Z-VAD-FMK, prevented STS-induced apoptosis. Based on these observations, we performed immunoblot analysis for the three known group II caspases (that is caspase-2, -3 and -7), but none of them was detected with three commercially available antibodies. Nevertheless, in view of the presence of increased DEVDase activity, we reasoned that a group II caspase must be a critical mediator of apoptosis in this model. If correct, we postulated that overexpression and activation of a type II caspase should cause apoptosis. To test this hypothesis, we coupled the cDNAs encoding caspase-3 and caspase-7 to adenoviral vectors and obtained constructs AvC3 and AvC7. Cells infected with AvC3 or AvC7 overexpressed the protein for caspase-3 or -7 within 24 to 48 hours. Caspase-3 overexpression did not cause apoptosis above that observed in cells receiving the control adenovirus expressing the lacZ cDNA (AvLac-Z). In contrast, caspase-7 overexpression induced massive apoptosis. BPH cells were then infected with increasing multiplicity of infection (MOI) of AvC7 and AvlacZ. A positive correlation was found between the amount of caspase-7 expressed and the level of DEVDase activity measured. AvC7 at MOIs of 25:1 and 50:1 induced apoptosis in about 50% of BPH cells at 72 hours post-infection. This effect was AvC7 specific, because the same MOIs of AvlacZ were not apoptogenic. CONCLUSIONS: Adenoviral-mediated overexpression of caspase-7 induces apoptosis of BPH-derived cells.

Lovastatin-induced apoptosis in prostate stromal cells.

Benign prostatic hyperplasia (BPH) is a common disease of aging men. Current medical treatment for this condition is only partially effective, therefore many patients must undergo surgery for symptomatic relief. BPH is caused by an increase in prostate epithelial and stromal cells, especially the latter. Since BPH stromal cells have a long life span and are not very responsive to androgen withdrawal, cultured BPH stromal cells were used to explore the feasibility of pharmacologically inducing apoptosis in these cells. We obtained BPH tissue during surgery, and stromal cells were isolated and maintained in culture. After cells achieved confluence, we induced apoptosis with the HMGCoA reductase inhibitor, lovastatin (30 micromol/L). The effects of testosterone (100 micromol/L), dihydrotestosterone (DHT; 100 micromol/L) and finasteride (100 micromol/L) on lovastatin-induced apoptosis were studied on cells grown in media containing charcoal stripped serum. Similarly, we examined the effect of the cholesterol pathway metabolites, mevalonic acid (30 micromol/L), geranyl geraniol (30 micromol/L), farnesol (10 micromol/L), squalene (30 micromol/L) and 7-ketocholesterol (3 micromol/L) on lovastatin-induced apoptosis. We demonstrated apoptosis by DNA laddering in agarose gels, by fluorescence microscopy following acridine orange staining, and by flow cytometry after end-labeling of DNA strand breaks with biotin-16-dUTP using deoxynucleotidyl exotransferase (TdT). Lovastatin at 30 micromol/L, but not at lower concentrations, induced apoptosis in BPH prostate stromal cells. This was seen (by flow cytometry) in 16.6 +/- 7.3% (mean +/- SD) of BPH cells treated with lovastatin at 72 h vs. 2.5 +/- 1.2% of cells treated with ethanol. Lovastatin-induced apoptosis was not increased in stripped serum or by the addition finasteride, and was not inhibited by testosterone or DHT. Only mevalonate and geranyl geraniol, prevented lovastatin-induced apoptosis whereas farnesol, squalene, or 7-ketocholesterol did not. We conclude that lovastatin can induce apoptosis in BPH stromal cells in vitro, and this is not affected by androgen withdrawal or stimulation. It is unlikely that lovastatin, per se, will be an effective treatment for BPH in vivo, but it does provide a means for inducing apoptosis in vitro. Understanding the apoptotic process in BPH stromal cells ultimately may lead to new therapeutic strategies for BPH.

Anti-androgen effects of the aromatase inhibitor, atamestane.

Prostatic hyperplasia can be induced in both intact and castrated dogs and in intact cynomolgus monkeys by the administration of androgenic steroids. Estrogenic steroids potentiate this effect in dogs. These changes also can be induced by androstenedione, which increases androgen and estrogen levels. Atamestane (ATA; 1-methyl-3,17-dione-androsta-1,4-diene), a potent aromatase inhibitor, inhibits some of the androstendione-induced effects; however, the nonsteroidal aromatase inhibitor, CGS-16949A, has been reported to decrease serum estradiol levels in adult rats but to have no effect on androgen-dependent organ weights. To examine the mechanisms by which ATA affects the rat prostate, in vivo and in vitro studies were conducted using adult rat ventral prostate (VP). Intact Sprague-Dawley rats were injected daily for 14 days with sesame seed oil, ATA (70 mg/kg/day), finasteride (FIN; 5 mg/kg/day), a 5 alpha-reductase inhibitor, or the combination of FIN plus ATA. A fifth group was castrated (CASTR) on day 1. The mean +/- standard error VP weight of the controls was 350 +/- 19 mg. It was reduced 17% (P < 0.05) by ATA, 29% (P < 0.001) by FIN, 48% (P < 0.001) by FIN plus ATA, and 86% (P < 0.001) by CASTR. The DNA/VP was reduced 22% (not significant) by ATA, 18% by FIN (not significant), 35% (P < 0.01) by FIN plus ATA, and 60% (P < 0.001) by CASTR. More significant changes were observed in RNA and protein. The mRNA for prostatein C3 was reduced by each of the treatments, but only CASTR increased the mRNA for TRPM-2, a marker of apoptosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 4-MAPC, a 5 alpha-reductase inhibitor, and cyproterone acetate on regrowth of the rat ventral prostate.

Inhibitors of 5 alpha-reductase activity cause less involution of the rat ventral prostate (VP) than does castration. Studies were conducted in adult Sprague Dawley rats to evaluate the effects of a potent 5 alpha-reductase inhibitor, 4-MAPC, and the antiandrogen, cyproterone acetate (CA), on DNA synthesis and apoptosis. In experiment 1, VP weight fell 33%, 53%, and 83%, and DNA per ventral prostate was reduced 24%, 46%, and 71%, by 4-MAPC, CA, and castration, respectively. In experiment 2, adult rats were castrated, and the VP involuted for 7 days prior to 3 daily injections of testosterone propionate (TP; 1 mg/kg/d) +/- 10 mg/kg/d of 4-MAPC or CA. 3H-thymidine incorporation into VP DNA was increased in castrated animals treated with TP, and 4-MAPC and CA reduced uptake. In experiment 3, animals were treated for 14 days with the same protocol as that used in experiment 2. VP weight was increased in all animals treated with TP when compared with castration, and was reduced by both 4-MAPC and CA. DNA in rats treated with TP was similar to that in intact animals. DNA was not reduced by 4-MAPC, but was reduced by CA. The mRNA for TRPM-2, a marker of apoptosis, was increased only in untreated castrated rats. It appears that CA has a greater inhibitory effect than 4-MAPC on DNA synthesis. A major reason why castration reduces DNA more than either 4-MAPC or CA is that neither of these agents was able to increase programmed cell death to the degree seen with castration.

Effects of finasteride on the rat ventral prostate.

The objectives of this study were to compare changes in the ventral prostate (VP) of young adult Sprague Dawley rats after 28 days of treatment with finasteride (F), a potent 5 alpha-reductase inhibitor, with those caused by castration (Cx). VP concentrations of DHT were reduced to 20.2% and 6.6% of controls (1,947 +/- 207 pg/VP, mean +/- SE) by F (5 or 20 mg/kg/day) and to 2.6% of controls by Cx. VP weights were reduced 49% and 54% by F and 88% by Cx. DNA/VP fell 25% and 15% with F treatment and 72% after Cx, whereas RNA and protein/VP were reduced 37-51% by F and 91-93% by Cx. The RNA/DNA and the protein/DNA ratios fell to 30-36% of controls after F treatment and to 70% of controls after Cx. The mRNA concentrations of the C3 subunit of prostatein 28S ribosomal RNA fell after treatment with F (5 mg/kg/day) and after Cx, whereas the mRNA for TRPM-2, an androgen-suppressed protein associated with apoptosis, was increased only after castration. To examine further the effects of F on the rate of DNA synthesis, 7-day regressed adult rats were treated for 3 days with testosterone propionate +/- F, and incorporation of 3H-thymidine in minced ventral prostates was determined. F inhibited 3H-thymidine incorporation. We conclude that Cx causes a greater reduction in cell number/VP and a greater reduction in RNA and protein cell than F and that the differences between F treatment and castration probably result from differences in prostatic concentrations of T.(ABSTRACT TRUNCATED AT 250 WORDS)

4-MAPC, a 5 alpha-reductase inhibitor, reduces rat ventral prostate weight, DNA, and prostatein concentrations.

Several compounds, such as 4-MAPC (4-methyl-3-oxo-4-aza-5 alpha-pregnane-20- carboxylate), that inhibit conversion of testosterone (T) to dihydrotestosterone (DHT) by 5 alpha-reductase have been demonstrated to reduce prostate size in rats and dogs. The current studies were undertaken to determine if this effect is due to a reduction in cell number, in epithelial cell synthetic activity, or both. Eight-week-old intact rats were treated daily for 14 days with sesame seed oil, 4-MAPC (10 mg/kg), 4-MAPC + testosterone propionate (TP, 1 mg/kg), or 4-MAPC + TP (3 mg/kg). Rats were killed 24 hours after the last injection. In the animals treated only with 4-MAPC, ventral prostate weight was reduced 37%, but the 14% reduction in total DNA was not significant. The mean intraprostatic concentration of prostatein, a major secretory protein, was reduced 45% (P less than 0.05). The 3 mg/kg dose of TP increased ventral prostate weight, prostatein concentrations, and acid phosphatase activity, even though DNA/ventral prostate was similar to that in control animals. These observations indicate that the reduction in ventral prostate weight in adult rats is due in part to a reduction in cell number, but the primary effect was due to a reduction in synthetic activity, and possibly atrophy of the epithelial cells. Furthermore, TP in pharmacologic doses increased ventral prostate weight and synthetic activity without increasing DNA.

Prostatein C3-mRNA: a sensitive marker of androgen-responsiveness in prostate explant cultures.

Prostatein is an androgen-dependent protein which is secreted by the rat ventral prostate. To determine if prostatein or its mRNA were responsive to androgen in vitro, prostate explants were cultured in media containing 0 or 25 nM dihydrotestosterone (DHT), estradiol (E2), or cortisol (F). Prostatein concentrations in medium were measured by radioimmunoassay at 2 and 4 days and in homogenates at 4 days. They were not changed significantly by any of these steroids. The concentration of the mRNA for the C3-subunit of prostatein was determined by dot hybridization at 0, 2, 4, 6, and 8 days. It was decreased significantly by 2 days when compared with explants cultured in the presence of DHT and significant differences persisted through 8 days. In conclusion, quantitation of the mRNA for the C3-subunit of prostatein in short-term cultures of ventral prostate explants appears to be more sensitive to changes in androgen concentration than does measurement of prostatein, per se. Prostatein C3-mRNA may be a useful marker for in vitro studies of androgen agonists and antagonists.

Acid phosphatase activity: a marker of androgen action in prostate explant cultures.

Acid phosphatase activity in rat ventral prostate explants has been assayed to determine if this parameter could serve as a specific and quantitative marker of androgen action in this in vitro model. Dihydrotestosterone (10 nM) caused an absolute increase in both total (42.5 +/- 2.9 vs control 27.1 +/- 4.0 nmoles p-nitrophenol generated in 30 min/micrograms DNA, P less than .01) and tartrate-resistant acid phosphatase activity (34.1 +/- 1.5 vs control 17.2 +/- 2.8 U/micrograms DNA, P less than .05), and this effect was maximal on the 4th day of culture. This was the time when explant weight and DNA content tended to fall or only to be maintained by androgen. Similar changes were observed with the potent synthetic androgen, mibolerone. The addition of either the antiandrogen cyproterone acetate or flutamide in a 100-fold excess to that of androgen caused significant inhibition in acid phosphatase activity. No significant change was observed at low concentrations of estradiol or progesterone, and only minimal and inconsistent increases in activity were noted at high concentrations. No increase was noted when cortisol, cyproterone acetate, or flutamide was added to the media. We conclude that measurement of acid phosphatase activity in cultured explants of rat ventral prostate provides a biochemical marker of androgenicity that is more specific than measurement of [3H]-thymidine incorporation.

Age dependency of androgen and estrogen effects on incorporation of (3H)-thymidine by rat prostates in organ culture.

Organ culture of the rat ventral prostate has been evaluated as a model for studying the biological effects of androgen agonists, androgen antagonists, and estrogens. Explants were cultured for up to 8 days, and incorporation of (3H)-thymidine and (3H)-uridine by the explants was measured. Dihydrotestosterone (DHT) increased the incorporation of (3H)-thymidine/microgram DNA when compared with the untreated controls at 4 days, P less than .05; and at 6 days, P less than .01. Enhanced uptake in explants from 6 to 8-week old rats also was observed with 10 nM estradiol (P less than .05) and 10 nM cyproterone acetate (P less than .02). DHT (10 nM) caused greater enhancement of uptake in explants from 3-week-old rats than in explants from 6- or 12-week-old rats. In contrast, estradiol (E2) increased incorporation only in prostates from the 6-week-old rats. Since both DHT and E2 can enhance (3H)-thymidine uptake even though they are associated with strikingly different effects on prostate morphology, it suggests that their effects on (3H)-thymidine incorporation are mediated by different cells.

Characterization of steroid binding specificity of the androgen receptor in human foreskin fibroblasts.

Our objective was to evaluate a convenient in vitro model for measuring steroid affinities to the human androgen receptor. The ability of unlabeled analogues of dihydrotestosterone (DHT) to compete with [3H]DHT for binding to the receptor in human fibroblasts was measured and expressed relative to DHT. The C-3 ketone group and the planar configuration of the A and B rings were critical for binding. Absence of the 10 beta-methyl group increased affinity of the androstane compounds for the receptor. The 17 beta-hydroxyl group was also essential for high affinity binding and addition of a 17 alpha-methyl group enhanced binding. Binding of steroids with a delta 4 double bond was consistently less than that of the 5 alpha-reduced steroids. This was true of both the androstene and estrene series. We conclude that human foreskin fibroblasts offer a useful model for in vitro studies characterizing the effects of steroid structural modifications on binding to the human androgen receptor.

Thyroid hormone nuclear receptor levels are influenced by the acetylation of chromatin-associated proteins.

The thyroid hormone receptor is a chromatin-associated protein which appears to mediate the actions of the thyroid hormones in mammalian cells. Unlike steroid hormone receptors, a cytoplasmic form of the receptor has not been identified, and the factors which govern the nuclear concentrations of the receptor are poorly understood. Using cultured GH1 cells, a rat pituitary cell line, we having previously demonstrated that thyroid hormones reduces the concentration of its receptor by a mechanism which involves the association of the ligand with the receptor binding site (Samuels, H.H., Stanley, F., and Shapiro, L.E. (1977) J. Biol. Chem. 252, 6052-6060). In this study, we demonstrate that n-butyrate and other aliphatic carboxylic acids elicit a reduction of thyroid hormone nuclear receptor levels without altering total cell protein synthetic rates. In contrast, the nuclear association and total cell level of the glucocorticoid receptor is not altered by n-butyrate. Evidence is presented that the aliphatic carboxylic acid-mediated reduction of thyroid hormone nuclear receptor levels is secondary to the inhibitory effect of these compounds on chromatin-associated deacetylases which is reflected as an increase in the acetylation of the nucleosome core histones. Isokinetic gradient centrifugation of chromatin solubilized from GH1 cell nuclei by micrococcal nuclease indicates that the receptor exists as a form associated with high molecular weight chromatin, as a 12.5 S form that sediments slightly faster than the bulk of the mononucleosomes, and as a 6.5 S form which appears to remain associated with low molecular weight chromatin components. Exclusive of the receptor associated with the high molecular weight chromatin, the 6.5 S form represents 80% and the 12.5 S form 10% of the receptor resolved in the gradient. n-Butyrate decreases both forms to the same degree suggesting that they are generated from the same "entity" of chromatin structure. Studies on the reappearance of receptor after restoration of the chromatin to the "normal" acetylated state are consistent with a model in which the affinity of chromatin for newly synthesized receptor is diminished in the "hyperacetylated" state.

Selective retention and formation of a delta5-androstenediol-receptor complex in cell nuclei of the rat vagina.

Cellular protein binding of a number of androstene and androstane derivatives that promote the growth of the vagina in rats has been studied. It was found that cell nuclei of the rat vagina contain a tissue-specific protein that binds 3beta,17beta-dihydroxy-androst-5-ene (delta5-androstenediol), a unique steroid causing growth and keratinization of the vaginal epithelium. The formation of the steroid-protein complex can be demonstrated by the administration of delta5-[3H]androstenediol to ovariectomized rats or by the incubation of minced vagina with the radioactive steroid. The steroid can interact with purified vaginal cell nuclei even in the absence of a cytosol preparation, forming the same steroid-protein complex. The formation of the complex is temperature-dependent; it occurs much more readily at 37 degrees than at 0 degrees. The delta5-[3H]androstenediol-protein complex migrated as about 4 S in a sucrose gradient medium containing 0.4 M KCl. A similar complex can be detected when nuclei of vaginal cells are incubated with 3alpha,17beta-dihydroxy-5alpha-androstane, 3beta,17beta-dihydroxy-5alpha-androstane, and 3beta-hydroxy-androst-5-en-17-one which also have the capability of stimulating vaginal epithelium, although in somewhat different ways. These steroids may bind to different groups of chromatin-bound receptor proteins in various layers of vaginal epithelium. The delta5-androstenediol binding protein is not found in the vaginal cytosol fraction that contains receptor proteins for estrogens and progestins, nor in the cytosol or nuclei of rat uterus cells, but not in muscle, brain, kidney, or liver. Testosterone and 5alpha-dihydrostestosterone bind weakly to the protein, whereas cortisol, androstenedione, 17beta-estradiol, and progesterone do not bind to the same protein by any significant extent.