Neutralizing VEGF bioactivity with a soluble chimeric VEGF-receptor protein flt(1-3)IgG inhibits testosterone-stimulated prostate growth in castrated mice

BACKGROUND: Recent studies show that testosterone-stimulated growth of the glandular tissue in the ventral prostate in adult castrated rats is preceded by increased epithelial VEGF synthesis, endothelial cell proliferation, vascular growth, and increased blood flow. These observations suggest that testosterone-stimulated prostate growth could be angiogenesis dependent, and that VEGF could play a central role in this process. METHODS: Adult male mice were castrated and after 1 week treated with testosterone and vehicle, or with testosterone and a soluble chimeric VEGF-receptor flt(1-3)IgG protein. RESULTS: Treatment with testosterone markedly increased endothelial cell proliferation, vascular volume, and organ weight in the ventral prostate lobe in the vehicle groups, but these responses were inhibited but not fully prevented by anti-VEGF treatment. The testosterone-stimulated increase in epithelial cell proliferation was unaffected by flt(1-3)IgG, but endothelial and epithelial cell apoptosis were increased in the anti-VEGF compared to the vehicle-treated groups. CONCLUSIONS: This study suggests that testosterone stimulates vascular growth in the ventral prostate lobe indirectly by increasing epithelial VEGF synthesis and that this is a necessary component in testosterone-stimulated prostate growth.     

Systemic treatment with epidermal growth factor but not insulin-like growth factor I decreases the involution of the prostate in castrated rats

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are strong inducers of proliferation to prostate cells cultured in serum-free medium. Accordingly we wanted to study the growth of the prostate gland in castrated rats after treatment with EGF, IGF-I and testosterone. Castrated Wistar rats were treated with growth factors (EGF 35 μg/rat per day; IGF-I 350 μg/rat per day) or testosterone (2 mg/rat per day) for 3 days either immediately after or 10 days after castration. Prostate tissue was examined by stereological and immunohistochemical techniques and by enzyme-linked immunosorbent assay (ELISA). Treatment with EGF inhibited the involution of the prostate (P < 0.05), whereas treatment with IGF-I did not affect the prostate involution as compared to castrated controls. EGF treatment significantly increased the endogenous rat EGF in the ventral prostate, but cellular proliferation was not affected. Testosterone treatment increased the weight of the prostate, by increase of all tissue components of the prostate, and significantly increased cellular proliferation. Systemic administration of EGF but not IGF-I decreased the involution of the rat prostate induced by castration. Compared with testosterone, the effects of EGF treatment on the prostate involution were moderate, and the effects of EGF were not related to cellular proliferation. Received: 20 January 1999 / Accepted: 8 October 1999     

Castration rapidly decreases local insulin-like growth factor-1 levels and inhibits its effects in the ventral prostate in mice

BACKGROUND: The mechanisms by which castration induces prostate involution are largely unknown. METHODS: Early responses to castration in mouse ventral prostate (VP) were explored by quantitative microscopy, cDNA array expression, quantitative RT-PCR, and Western blot analysis. As several changes occurred in the insulin-like growth factor (IGF) system this was studied in more detail. Laser micro-dissection was used to localize sites of IGF-1 and IGF-1 receptor (IGF-R1) production. IGF-1 protein levels and IGF-R1 mediated signaling via insulin regulated substrate 1 and 2 (IRS-1 and 2) were examined. IGF-1 was injected into the VP in intact, and castrated mice and effects studied 1 day later. RESULTS: IGF-1 and IGF binding protein 2 (IGFBP-2) mRNA were rapidly reduced whereas IGFBP-3 and IGF-R1 mRNA were increased after castration. IGF-1 was principally produced in the stromal compartment, while IGF-R1 was produced in both epithelial and stromal cells. IGF-1 and IRS-1 protein levels were decreased 1 and 3 days after castration, respectively, while IRS-2 was unchanged. Inactivating phosphorylation of IRS-1 at serine 307 was increased 1 day after castration, and activating phosphorylation at tyrosine 612 was decreased 2 days later. These changes were accompanied by decreased cell proliferation and increased cell death in the glandular and vascular compartment. Local injection of IGF-1 increased vascular density and epithelial cell proliferation in intact mice, but had no effect in castrated animals. CONCLUSION: Decreased IGF-1 levels and action may mediate some of the key features of castration-induced prostate involution.     

雄激素对大鼠腹叶前列腺GDNF mRNA表达的影响

目的 :探讨雄激素对大鼠腹叶前列腺中胶质细胞源性神经营养因子 (GDNF)mRNA表达的影响。　方法 :2 4只SD大鼠分为 3组 ,其中A组 (n =8)为假手术对照组 ,B组 (n =8)为去势组 ,C组 (n =8)为雄激素替代组 (去势后肌注十一酸睾酮 5 0mg/kg) ;术后 3d处死 ,通过半定量RT PCR检测GDNFmRNA在去势前后和雄激素替代组大鼠腹叶前列腺中的表达变化。　结果 :去势后大鼠前列腺的体积萎缩变小 ;雄激素替代组出现前列腺增生变大 ;对照组正常的大鼠前列腺有GDNFmRNA表达 ,去势组GDNFmRNA表达量减少 ,雄激素替代组GDNFmRNA表达量增加。与正常对照组比较 ,去势组的GDNFmRNA表达量显著减少 (P <0 .0 5 ) ,雄激素替代组的GDNFmRNA表达量显著增加(P <0 .0 5 )。　结论 :雄激素可增加GDNFmRNA表达 ,促进前列腺细胞生长。     

Serum testosterone plays an important role in the metastatic ability of castration resistant prostate cancer

Purpose

Prostate cells are dependent on androgens for growth and proliferation. Androgen deprivation therapy is the recommended treatment for advanced/metastatic prostate cancer. Under this therapy, prostate cancer will inevitably progress to castration resistant prostate cancer (CRPC). Despite putative castration resistance, testosterone might still play a crucial role in the progression of CRPC. The goal of this study was to determine the role of testosterone in the formation of metastases of CRPC in both in vitro and in vivo settings.

Methods

In vitro, the effect of testosterone and the non-aromatizable androgen methyltrienolone on migration, invasion and proliferation of a castration-resistant prostate cancer rat cell line (Dunning R3327-MATLyLu) was assessed using a transwell assay and a sulforhodamine B assay and immunohistochemical detection of ki67. Androgen receptor status was determined using Western blot. In vivo, Copenhagen rats were divided in four groups (males, females, castrated males and females with testosterone suppletion) and inoculated with MATLyLu cells. Tumor size was assessed daily.

Results

Testosterone increased cell migration and invasion in a concentration-dependent manner in vitro. Testosterone did not affect in vitro cell proliferation. No difference was shown between the effect of testosterone and methyltrienolone. In vivo, in groups with higher levels of circulating testosterone, more rats had (micro)metastases compared with groups with low levels of testosterone. No effect was observed on primary tumor size/growth.

Conclusions

Despite assumed castration resistance, progression of prostate cancer is still influenced by androgens. Therefore, continuous suppression of serum testosterone in patients who show disease progression during castration therapy is still warranted.     

Increase in amphiregulin and epiregulin in prostate cancer xenograft after androgen deprivation-impact of specific HER1 inhibition

BACKGROUND: We investigated the expression of the epidermal growth factor (EGF) network before and after castration in the prostate cancer xenograft CWR22 implanted in nude mice, and examined the effects of gefitinib (Iresssa, ZD1839), a new drug directed towards the EGF tyrosine kinase receptor (HER1) of the EGF network. METHODS: CWR22 prostate cancer xenografts were propagated in immunodeficient male mice. The expression of the growth factors and receptors in the EGF network was examined by real-time PCR analysis and ELISA at 0, 7, 14, and 30 days after castration, and the tumor growth was examined after treatment with gefitinib or placebo. RESULTS: A fraction of growth factors showed a steady increase in the mRNA expression reaching between fourfold and eightfold 30 days after castration, including amphiregulin (P < 0.005) and epiregulin (P < 0.001). ELISA for amphiregulin showed a fivefold increase 30 days after castration. Tumor bearing mice were castrated and treated with or without the HER1 tyrosine kinase inhibitor gefitinib. Tumor involution was significantly increased by castration plus gefitinib as compared to castration alone. CONCLUSIONS: Castration leads to adaptive increase in the concentrations of a subset of growth factors from the EGF network in the androgen sensitive CWR22 prostate cancer xenograft. Specific inhibition of the HER1 tyrosine kinase receptor significantly increases the tumor involution, and suggests that HER1 targeted drugs could be of therapeutic relevance in the treatment of advanced prostate cancer.     

Androgen-insensitive prostate cancer cells transiently respond to castration treatment when growing in an androgen-dependent prostate environment

BACKGROUND: Castration-induced involution of the normal prostate is caused by primary effects in the prostate stroma and vasculature, but if this is the case also in tumors is unknown. METHODS: Androgen-independent AT-1 prostate tumor cells were therefore injected into the ventral prostate (VP) in Copenhagen rats. Seven days later when the growing tumor was surrounded by normal VP tissue the rats were castrated and the effect examined 3 and 7 days later. RESULTS: Castration reduced vascular density in the surrounding VP tissue and this was accompanied by tumor cell hypoxia, apoptosis, and temporarily retarded tumor growth. Castration-induced VP tissue regression occurred more rapidly in the contra-lateral than in the tumor-bearing lobe. CONCLUSIONS: Androgen-independent tumor cell respond to castration when growing in an androgen-dependent environment. The presence of a tumor influences the castration response in the surrounding normal tissue. The microenvironment determines how prostate epithelial cells respond to castration.     

Vascular endothelial growth factor and angiopoietin are required for prostate regeneration

BACKGROUND: The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens. METHODS: Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2. RESULTS: Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells. CONCLUSION: VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens.     

Testosterone induces vascular endothelial growth factor synthesis in the ventral prostate in castrated rats

PURPOSE: Recent studies suggest that the vasculature is important for the control of prostate growth. Castration induces an involution of the prostate gland and its vasculature. Replacement of testosterone stimulates endothelial cell proliferation and normalizes vascular volumes and blood flow several days before organ regrowth. Antiangiogenesis treatment inhibits the growth of prostate tumors. Understanding the regulation of the prostate vasculature may therefore provide important knowledge of the mechanisms responsible for the growth of non-malignant and malignant prostate tissue. Castration induced regression and testosterone stimulated regrowth of the prostatic vasculature have here been used to study the involvement of the angiogenic factor vascular endothelial growth factor (VEGF) and its receptors flt-1 and flk-1/KDR in the regulation of the prostatic vasculature. MATERIALS AND METHODS: VEGF, flt-1, and flk-1/KDR levels were quantified in the rat ventral prostate following castration and testosterone replacement. Methods used were competitive RT-PCR, Western blot and immunohistochemistry. RESULTS: VEGF mRNA and protein levels were significantly decreased by castration and testosterone treatment induced VEGF synthesis in the rat ventral prostate epithelium. Flt-1 and flk-1/KDR receptor levels were unaffected by castration and testosterone treatment. CONCLUSIONS: Castration down regulates VEGF and testosterone induces VEGF synthesis in epithelial cells in the rat ventral prostate.     

After radiotherapy testosterone stimulation is unable to increase growth in the Dunning R3327-PAP prostate tumour

A study was carried out to investigate whether testosterone treatment is able to influence tumour growth in a rat prostatic adenocarcinoma previously treated with castration and high-dose fractionated irradiation. Copenhagen × Fisher rats bearing the androgen-sensitive, well-differentiated Dunning R3327-PAP tumour were castrated and thereafter treated with external beam radiation with photons from a 4-MV linear accelerator. One month after irradiation, substitution with subcutaneous testosterone was started. Tumour volumes and rat weights were monitored up to 256 days after castration, and at the end of the study a microscopic analysis of the tumours was performed. Irradiation delayed tumour growth as compared with untreated tumours. Castration delayed tumour growth, but a hormone-refractory relapse to doubled tumour volume was seen within 45 days. If testosterone was added after castration, the tumours grew rapidly. However, testosterone failed to increase tumour growth when given to rats treated with orchiectomy and irradiation. Histological examination showed that the irradiated tumours still contained tumour epithelial cells, but these cells apparently do not respond to testosterone stimulation. The well-differentiated and androgen-sensitive rat prostatic adenocarcinoma did not grow after irradiation despite stimulation with testosterone. This indicates that the malignant cells lose their androgen sensitivity after high-dose irradiation. Received: 7 October 1998 / Accepted: 11 March 1999     

Induction of proliferative lesions of ventral prostate, seminal vesicle, and other accessory sex glands in rats by N-methyl-N-nitrosourea: effect of castration, pretreatment with cyproterone acetate and testosterone propionate and rat strain.

Wistar (Cpb:WU), F344 or Sprague-Dawley rats were sequentially treated with cyproterone acetate (CA) for 21 days, testosterone propionate (TP) for 3 days, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU). One group of Wistar rats was castrated 4 weeks after MNU injection, and another group 58 weeks after MNU, when the first prostatic carcinoma was detected. Control groups received only CA + TP, CA, MNU, or they remained untreated. Early or late castration inhibited the development of atypical hyperplasia of the ventral prostate in Wistar rats. This lesion was induced by the CA + TP + MNU treatment in F344 rats, but not Sprague-Dawley rats; in Wistar rats, it was induced by CA + TP treatment, irrespective of whether MNU was given. Hypertrophic-hyperplastic lesions of the seminal vesicle were induced by MNU, irrespective of pretreatment, and their development was prevented by early castration and inhibited by late orchiectomy. Dorsolateral prostate carcinomas and preneoplasia occurred only in low incidence in Wistar and Sprague-Dawley rats. These lesions were absent in F344 rats that had received treatment with CA + TP + MNU. No dorsolateral prostate (pre)neoplasia was found in Wistar rats subjected to early orchiectomy, but rats castrated at 58 weeks had an incidence similar to that for the intact group treated with CA + TP + MNU. This finding supports the contention that androgens are required for the development of MNU-induced prostatic cancer in rats but that advanced carcinomas are androgen insensitive. Differences in incidence and localization of prostatic proliferative lesions between F344 and Wistar rats and between dorsolateral and ventral prostate could not be explained by differences in epithelial cell proliferative responses to CA + TP treatment at the time of MNU injection, since they were similar in ventral and dorsolateral prostate and were more prominent in F344 rats than in Wistar rats. DNA damage as estimated by MNU-induced unscheduled DNA synthesis also did not differ between dorsolateral and ventral prostate.     

缺氧诱导因子-1α在去势前后大鼠腹叶前列腺中表达的变化

目的探讨与缺氧相关的缺氧诱导因子-1α(HIF-1α)是否参与去势后前列腺萎缩过程.方法24只SD大鼠分为3组,其中A组(n=8)为假手术对照组,B组(n=8)为去势组,C组(n=8)为雄激素替代组(去势后肌注十一酸睾酮50mg/kg);术后3天处死,通过半定量RT-PCR检测与HIF-1α在去势前后前列腺表达变化.结果去势后大鼠前列腺的体积萎缩变小;雄激素替代组出现前列腺增生变大;对照组正常的大鼠前列腺有HIF-1 α mRNA低水平表达,去势组HIF-1α mRNA表达量增加,雄激素替代组HIF-1αmRNA表达量减少,与正常对照组比较,去势组的HIF-1α mRNA的表达量显著增加(P＜0.05),雄激素替代组的HIF-1αmRNA的表达量显著减少(P＜0.01).结论前列腺组织的缺氧参与去势后大鼠前列腺的早期萎缩过程.     

Potentiating Effect of Buserelin Acetate, an LHRH Agonist, on the Proliferation of Ventral Prostatic Epithelial Cells in Testosterone-Treated Castrated Rats

Background: We used buserelin acetate ([D-Ser(But)6] LHRH), an LHRH agonist and strong blocker of LH secretion, as a treatment for prostatic cancer. It is possible that this LHRH agonist has a proliferative effect on the prostate in addition to suppressing LH secretion. The purpose of this study was to examine the proliferative effect of LHRH agonist on rat prostatic epithelial cells.
Methods: We determined the optimal dose of testosterone necessary to maintain a positive level of proliferating cell nuclear antigen (PCNA) in the ventral prostatic epithelial cells of castrated Wistar rats. Testosterone-treated rats then received various doses of buserelin acetate. Castrated rats without exogenous testosterone also received buserelin acetate. The PCNA positivity was determined by immunohistochemistry with anti-PCNA monoclonal antibody.
Results: The optimal dose of testosterone enanthate was 4mg at 0 and 28 days after castration. Administration of buserelin acetate on day 0 and 28 in doses of 0.1 6mg to 1.28mg significantly increased PCNA positivity in a dose-dependent manner. Administration of buserelin acetate to castrated rats without testosterone also increased PCNA positivity but there was no statistical significance. Conclusions: Buserelin acetate has a potentiating effect on the proliferation of ventral prostatic epithelial cells of castrated rat in the presence of a physiological level of exogenous testosterone. This effect may slightly influence the result of hormonal therapy by LHRH agonist.     

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

Inhibitors of 5α-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 5α-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. © 1994 Wiley-Liss, Inc.     

Castration rapidly results in a major reduction in epithelial cell numbers in the rat prostate,but not in the highly differentiated dunning R3327 prostatic adenocarcinoma

Rats with implanted highly differentiated Dunning R3327PAP prostatic tumors were castrated, and at 3, 7, and 14 days thereafter, the effects on tumor volume, epithelial cell numbers, and sizes were quantified using morphometrical methods. The castration response on these parameters was also examined in the normal prostate of the same rats. Castration resulted in a rapid decrease in organ volume, epithelial cell number, and size in the normal prostate, and morphological signs of epithelial cell death (apoptosis) were observed. Tumor growth and mitotic index were reduced, but there were no signs of increased apoptosis, and cell numbers remained fairly constant in the Dunning tumors during the study period. It is concluded that the castration-induced inhibition of tumor growth is caused by factors other than tumor cell death. © 1993 Wiley-Liss. Inc.     

Antagonistic effect of androgen on prostatic cell death

Androgen, besides having the well-established agonistic ability to stimulate prostate cell proliferation, also has an antagonistic ability to inhibit prostatic cell death. This statement is based upon the observations that 1) only 2.1% of the total prostatic cells die per day when serum testosterone level is sufficient for chronic maintenance of the gland; 2) 3 days following castration, when serum testosterone level is less than 10% of the intact value, the percentage of total prostatic cells now dying per day is increased tenfold to a value of 20.8%; and 3) this high rate of prostatic cell death can be inhibited following castration if serum androgen level is appropriately maintained by exogenous testosterone treatment. The serum testosterone level needed to antagonistically inhibit prostatic cell death (ie, 1.4 +/- 0.1 ng/ml) is more than twofold lower than that needed to antagonistically stimulate prostatic cell proliferation (3.3 +/- 0.4 ng/ml). Due to this dose difference, it is experimentally possible in castrated rats to inhibit prostatic cell death selectively without simultaneously stimulating cell proliferation and still completely prevent the rapid involution of the prostate following castration. These results suggest that the rapid involution of the prostate following castration is predominantly due to a decreased antagonistic effect of androgen on prostatic cell death rather than to a decreased agonistic effect of androgen on prostatic cell proliferation and that these two androgenic effects are distinct processes in the prostate.     

Characterization of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) as a model to study effects of castration therapy

BACKGROUND: In order to learn more about short- and long-term effects of castration therapy, relevant model systems for prostate cancer are required. In this study, we examined whether the transgenic adenocarcinoma of the mouse prostate (TRAMP) tumor response to castration in C57BL/6 mice mimics that seen in patients. METHODS: Transgenic animals were examined before and 3 days after castration, at the ages of 17, 24, and 36 weeks. Moreover, 24-weeks old animals were castrated and followed for 6 months. Immunohistochemistry (IHC) and stereology were used to evaluate epithelial cell proliferation and death, blood vessel volume, androgen receptor (AR) expression, and transgenic expression of SV40 large T. RESULTS: Cancer developed preferentially in the dorso-lateral prostate lobe. Tumor burden and incidence of metastases increased with age. The majority of tumors were well differentiated, while poorly differentiated, large tumors and macroscopic metastases developed in 8% of the animals. Well and moderately differentiated tumors responded to castration with cessation of proliferation and induction of apoptosis. Poorly differentiated tumors and metastases did not respond. Castration prevented local tumor growth for at least 6 months in 82% of the cases. Although, 45% of the treated animals developed wide-spread metastatic disease suggesting that castration may enhance growth of distant metastases. CONCLUSIONS: The C57Bl/6 TRAMP tumor in several ways mimics how prostate cancer in patients responds to castration both in the short and long term, but some differences may also exist. This model can preferably be used to elucidate how this treatment works, and to test how it can be improved by additional therapies.     

Effects of 6-methylene progesterone on growth, morphology, and blood flow of the Dunning R3327 prostatic adenocarcinoma.

Copenhagen x Fisher F1 rats were implanted with the androgen-dependent Dunning R3327 prostatic adenocarcinoma. When the tumors had median volumes of ca 470 mm3, the rats were castrated and/or treated with 6-methylene-4-pregnene-3,20-dione (6MP) in different doses. Tumor growth inhibition occurred in all castrated and treated groups, with decrease in volume of the epithelial compartment in the intact group. Tumor volumes at the highest dose level of 6MP equalled those observed in the castrate group. Plasma levels of testosterone were within the normal range. The administration of 6MP surprisingly induced an increment of tumor blood flow in the castrate group. Also in castrated and testosterone-supplemented animals, 6MP induced a reduction of prostatic tumor growth. Through the castration-like effect on tumor growth, the use of 6MP may represent an attractive alternative to castration for treatment of androgen-responsive prostate cancer.     

Fas antigen/CD-95 upregulation and activation during castration-induced regression of the rat ventral prostate gland.

BACKGROUND: Fas antigen/CD 95 is a 45-kDa transmembrane protein that can initiate intracellular signaling pathways, leading to apoptosis when it is clustered on the cell surface. A recent report claiming that the ventral prostate glands of lpr -/- mutant mice (lacking functional fas antigen) do not regress following castration prompted our analysis of the regressing rat ventral prostate gland for evidence that fas antigen might participate in the molecular process leading to prostate cell apoptosis after castration. METHODS: An RNase protection assay and Western blotting analysis were used to quantify fas antigen mRNA and protein expression in the regressing rat ventral prostate gland. Immunoprecipitates of fas antigen from membrane preparations made from control or castrated rat prostates were analyzed for coprecipitation of FADD and RIP proteins to assess the activation state of the fas antigen before and after castration. Finally, prostate tissues obtained from two different strains of lpr -/- mutant mice were analyzed for induced apoptosis after castration by the TUNEL staining method. RESULTS: Rat ventral prostate gland fas antigen mRNA and protein expression was upregulated approximately 3-5-fold in the 3-day castrated rat as compared to hormonally intact rats. Immunoprecipitates of fas antigen from membranes of ventral prostates from castrated rats contained significantly increased amounts of both FADD and RIP proteins when compared to those of intact or control operated rats. However, counts of TUNEL-labeled cells in the ventral prostate glands of castrated lpr -/- mice were not significantly different from those in castrated, genetically normal controls. Likewise, the morphology of apoptotic bodies formed in the prostates of castrated lpr -/- mice was indistinguishable from that in control animals. CONCLUSIONS: Fas antigen/CD-95, a protein that is involved in some forms of apoptosis, is upregulated during regression of the rat ventral prostate gland and becomes functionally "activated." However, our inability to distinguish any difference in the apoptosis rate or in the morphology of the apoptotic bodies formed in response to castration between lpr -/- mice and genetically normal controls indicates that, contrary to the prior report, functional fas protein is not required for castration-induced prostate cell apoptosis.     

Experimental cryptorchidism inhibited growth of the rat ventral prostate

Adult Sprague-Dawley male rats, weighing about 350 g, were rendered cryptorchid by suturing the testes to the lateral abdominal wall. Twenty-eight days later, cryptorchidism resulted in a significant decline in testis weight and suppressed spermatogenesis. The ventral prostate was significantly smaller in cryptorchid rats. There was no significant difference in serum testosterone levels between the normal and cryptorchid rats. Charcoal-stripped aqueous extracts of the testis from intact and cryptorchid animals were tested on primary cultures of rat prostatic stromal cells. Cultures treated with extract from the intact testis had a significantly increased cell proliferation as assessed by cell count and by the rate of 3H-thymidine incorporation. Additionally, extracts of seminiferous tubules significantly increased prostate stromal cell proliferation compared to extracts of testicular interstitial components. Furthermore, this proliferative effect of testicular extracts is specific to the prostate as extract of both normal and cryptorchid testis stimulated proliferation of rat footsole fibroblasts in culture, but only extracts from intact testis stimulated proliferation of prostate stromal cells. These observations demonstrate that the testis produces nonandrogenic substances that can promote growth of prostatic stromal cells and that these substances were eliminated in the cryptorchid testis.