Prostatic binding protein,polyamine, and dna synthesis in rat ventral prostate cells

Prostatic binding protein (PBP), polyamine, and DNA synthesis were examined in primary cultures of rat ventral prostate cells. Soon after aggregates of prostate cells were placed in culture, PBP synthesis fell dramatically and DNA synthetic activity in the epithelial cells increased. The amount of polyamines that were labeled with [³H] from [³H]ornithine fell transiently, but rose again at or before the peak of DNA synthesis. Individual ³H-labeled polyamines in cells and medium were dansylated and separated by thin-layer chromatography. The ratio of [³H]Spermidine plus [³H]Spermine to [³H]Putrescine in the culture medium declined as DNA synthesis increased. Ornithine decarboxylase (ODC) activity fell dramatically along with PBP synthesis even as DNA synthesis and ³H-labeled polyamines increased in the prostate-cell cultures. These results support others that suggest that high ODC activity in prostate epithelial cells is a correlate of prostate epithelial cell function rather than proliferation. However, prostate epithelial cells retain the capacity to synthesize significant levels of polyamines from ornithine (especially Putrescine) during proliferation even when ODC activity is low.     

Androgen Interaction with the Polyamine System of the Rat Prostate

The ventral and dorsal lobes of the rat prostate contain larger quantities of the aliphatic amines putrescine, spermidine and spermine and higher activities of the enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) than other accessory sex glands. In contrast, the coagulating glands and the seminal vesicles contain only small quantities of the amines but the highest activities of the arginase (ARG; EC 3.5.3.1). Lineweaver-Burk plots indicated that the Km-values for ARG in the coagulating gland and ODC in the ventral prostate lobe were 20 mM and 0.2 mM, respectively. Castration decreased ODC and ARG activities to 3 and 50% of control levels, respectively, after 3 days, whilst the Km-values were unaffected. Daily administration of 3 mg dihydrotestosterone (DHT) prevented these castrational changes. Oestrogen treatment alone had no effect on the activities of the enzymes, but appeared to exert a synergistic effect with androgen on the ODC. Administration of androgen to intact rats for 7 days caused a dose-related alteration in the ratios of the various amines, particularly the spermine: putrescine ratio. A minor but significant decrease was also recorded in the activity of the ODC, which was mirrored by an increase in the levels of putrescine in the tissue. The data suggest that androgen control of the polyamine pathway is biphasic, first stimulatory and later inhibitory with lesions occurring at the ODC, possibly via short loop feedback of its product putrescine, but also at subsequent enzymic steps in spermidine and spermine biosynthesis.     

Identification and characterization of PLZF as a prostatic androgen-responsive gene

BACKGROUND: Promyelocytic leukemia zinc finger protein (PLZF) was initially identified by virtue of its fusion with RARalpha as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients. PLZF has been reported to have pro-apoptotic and anti-proliferative activity both in vivo and in vitro. METHODS: Using a modified subtractive hybridization, we identified PLZF as an androgen-responsive gene in the rat ventral prostate. Northern blot and Western blot were used to characterize the regulation of PLZF by androgens in LNCaP cells. Stable transfections of PLZF in LNCaP cells were performed to assay the effect of PLZF overexpression on LNCaP cell proliferation. RESULTS: PLZF mRNA was transiently up-regulated by androgens in the regressed ventral prostate of castrated adult rat. PLZF was also up-regulated by androgens, at both mRNA and protein levels, in the androgen-responsive human prostate cancer cell line LNCaP. Androgen induction of PLZF mRNA was not inhibited by protein synthesis inhibitor cycloheximide but inhibited by androgen receptor antagonist bicalutamide, indicating that PLZF is a direct androgen-responsive gene. To study the functions of PLZF in androgen action, LNCaP sublines stably overexpressing PLZF were generated. PLZF overexpression inhibited LNCaP proliferation either in the presence or absence of androgen, which is consistent with the reported anti-proliferative activity of PLZF. CONCLUSIONS: The above observations indicate that PLZF is an androgen-responsive gene with anti-proliferative activity in prostate cancer cells.     

Androgen-independent epithelial cells of the rat ventral prostate

Androgen-independent cell lines have been clonally selected from primary cultures of androgen-dependent epithelial cells from the rat ventral prostate. These rapidly dividing epithelial-like cells (RDE) have altered morphology and adherence characteristics. Unlike normal prostate epithelial cells, the RDE cell lines do not require androgens for cell division or cell survival. In the presence of physiological concentrations of testosterone, the isoelectric focusing patterns of prostatic acid phosphatases are abnormal in these RDE cells, and the prostate steroid-binding protein genes are not expressed. The loss of androgen dependence is not due to the inability of RDE cells to metabolize testosterone to 5α-dihydrotestosterone, the active androgen, since the RDE cell lines metabolize testosterone in a manner similar to normal androgen-dependent epithelial cells. When RDE cells are grown on collagen matrices, the cells assume ductlike structures, similar to prostatic acini, although PSBP gene expression is not induced. When seeded into soft agar these cell lines form distinct foci, suggesting that they are potentially tumorigenic.     

Androgen regulation of polyamine synthesis in seminal vesicle and in different lobes of the rat prostate

Endogenous levels of the polyamines putrescine, spermidine, and spermine have been examined in the 10(6) m/s2 supernatant of different lobes of the rat prostate and in the seminal vesicles of castrates, androgen-stimulated castrates, and intact controls. Content of the polyamines varied between the lobes, with spermidine highest in intact animals. After castration, the content of polyamines fell significantly in all lobes but the coagulating gland (CG). Spermidine levels were highest, except in the lateral prostate (LP) and CG, where the content of putrescine was highest. In castrated animals treated with testosterone propionate for 72 h, the amount of the three polyamines examined increased dramatically in the ventral prostate (VP) and moderately in the CG and seminal vesicle (SV). Concerning individual polyamines, spermidine increased significantly in all lobes, while putrescine increased significantly only in the two saccular parts of rat prostate, i.e., CG and SV. Spermidine content decreased significantly in the DP. Major differences in the content of the three polyamines--putrescine, spermidine, and spermine--in the various tissues studied have been found. Moreover, distinct differences among intact, castrated, and testosterone-treated castrated animals have been revealed.     

Androgen-dependent regulation of medium and long chain fatty acids uptake in prostate cancer

BACKGROUND: Epidemiological and experimental studies suggest that both fatty acids and androgens have a role in the development and progression of prostate cancer (PC). Plasma membrane fatty acid binding protein (FABP(pm)) is a transporter of medium and long chain fatty acids (MCFA and LCFA) across the plasma membrane, and is identical to the mitochondrial protein aspartate aminotransferase (mAAT) that is regulated by testosterone only in prostate epithelial cells, a site where PC initially develops. We therefore hypothesized that FABP(pm) is also regulated by androgens. METHODS: We examined the effect of a synthetic androgen, R1881, and that of androgen receptor (AR) blocker, bicalutamide, on the expression of FABP(pm) and mAAT and on the uptake of fatty acids in the androgen-sensitive LNCaP, androgen responsive 22rv1 and androgen-independent CL1 human PC cells. This was done using immunofluorescence and confocal microscopy, Western blot, flow cytometry, and (3)H-oleate uptake studies. RESULTS: Androgen supplementation increased the cellular and surface expression of FABP(pm) and mAAT and increased the uptake of fluorescently labeled MCFA and LCFA and that of (3)H-oleate only in PC cells that express the AR. Bicalutamide inhibited this phenomenon. CONCLUSIONS: The uptake of MCFA and LCFA into PC cells is androgen regulated as well as the expression of FABP(pm) and mAAT.     

Relaxin becomes upregulated during prostate cancer progression to androgen independence and is negatively regulated by androgens

BACKGROUND: Relaxin is a potent peptide hormone normally secreted by the prostate. This study characterized relaxin expression during prostate cancer progression to androgen independence (AI), and in response to androgens. METHODS: The prostate cancer cell line, LNCaP, was assayed by microarrays and confirmatory Northern analysis to assess changes in relaxin levels due to androgen treatment and in LNCaP xenografts following castration. Relaxin protein levels were examined by immunohistochemistry (IHC) in tissue microarrays of human prostate cancer samples following androgen ablation. RESULTS: Relaxin levels decreased in a time and concentration-dependent manner due to androgens in vitro, and increased in xenografts post-castration. Relaxin increased in radical prostatectomy specimens after 6 months of androgen ablation and in AI tumors, was highest in bone metastases. CONCLUSIONS: Relaxin is negatively regulated by androgens in vitro and in vivo, which correlates to clinical prostate cancer specimens following androgen ablation. The role of relaxin in angiogenesis and tissue remodeling suggests it may contribute to prostate cancer progression.     

High fat diet increases the weight of rat ventral prostate

BACKGROUND: Understanding the mechanisms by which diet influences the prostate may eventually lead to novel approaches for preventing prostate cancer. The objective of this research is to examine the impact of dietary fat, vitamin D, and genistein on prostate weight, serum and intraprostatic androgen levels, and the expression of several androgen-response genes. METHODS: Sprague-Dawley rats were fed, beginning at 21 days of age, for 1 or 3 months of experimental diets with high saturated fat (32.2% calories from fat), low saturated fat (3.6% calories from fat), genistein plus (20 mg/kg), genistein deficient, vitamin D surplus (4,000 U/kg), or vitamin D deficient. The body weight, food intake, the weights of the ventral prostate and dorsolateral prostate, and the levels of testosterone and dihydrotestosterone (DHT) in the serum and in the prostate were determined. The expression of androgen-response genes was characterized by Northern blot analysis. RESULTS: The pilot experiments showed that high dietary fat appeared to consistently increase the weight of the ventral prostate, while vitamin D or genistein did not have a consistent effect on prostate weight. Further analysis confirmed that the ventral prostate is 15% (P < 0.001) heavier in the rat on a high fat diet as compared to a low fat diet. Dietary fat had no significant influence on the levels of serum and intraprostatic androgens and the expression of androgen-response genes. CONCLUSIONS: Our results suggested that the ventral prostate weight of the rat is increased without affecting the androgen axis by feeding the animals with high fat diet beginning at 21 days of age. This observation is potentially important since epidemiological data suggest that saturated fat consumption is a major risk factor associated with prostate cancer incidence rate.     

Androgens modulate the balance between VEGF and angiopoietin expression in prostate epithelial and smooth muscle cells

BACKGROUND: The vasculature of the prostate responds to androgens. Androgens most likely affect the vasculature indirectly by modulating the expression of angiogenic factors in the cells of the prostate. Most studies to date have examined the production of angiogenic factors by the prostate luminal epithelium. Here we examine the effects of androgen on production of three angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2, by the three major cell types in the prostate. METHODS: The ability of androgen to modulate VEGF, angiopoietin-1, and angiopoietin-2 production in cultured mouse prostate luminal epithelial, basal epithelial, and smooth muscle cells (SMCs) was assessed by Western blot and RT-PCR. RESULTS: The production of VEGF was modulated by androgens in both luminal epithelial and prostate SMCs but not in basal epithelial cells. However, in prostate luminal epithelial cell cultures, VEGF was predominately secreted apically, suggesting that in vivo most of the epithelium-derived VEGF is unavailable to the underlying blood vessels. In addition, prostate luminal epithelial cells produced angiopoietin-2, an angiogenesis inhibitor. In contrast, prostate SMCs produced angiopoietin-1, a positive modulator of angiogenesis. Synthesis of the angiopoietins did not respond to androgen treatment. CONCLUSIONS: Prostate smooth muscle may play an important role in regulating vascular responses to androgen.     

KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that is expressed at lower levels in prostate cancer cells than in normal prostate cells

BACKGROUND: Fifteen human tissue kallikrein (KLK) genes have been identified as a cluster on chromosome 19. KLK expression is associated with various human diseases including cancers. Noncoding RNAs such as PCA3/DD3 and PCGEM1 have been identified in prostate cancer cells. METHODS: Using massively parallel signature sequencing (MPSS) technology, RT-PCR, and 5' rapid amplification of cDNA ends (RACE), we identified and cloned a novel gene that maps to the KLK locus. RESULTS: We have characterized this gene, named as KLK31P by the HUGO Gene Nomenclature Committee, as an unprocessed KLK pseudogene. It contains five exons, two of which are KLK-derived while the rest are "exonized" interspersed repeats. KLK31P is expressed abundantly in prostate tissues and is androgen regulated. KLK31P is expressed at lower levels in localized and metastatic prostate cancer cells than in normal prostate cells. CONCLUSIONS: KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that may play a role in prostate carcinogenesis or maintenance.     

Androgens upregulate aquaporin 9 expression in the prostate

Objectives: Aquaporins (AQP) function as selective pores allowing water, glycerol and other small solutes to pass through the cell membrane. We previously reported that AQP are expressed in the prostates of both humans and rats. The present study investigated the androgen‐dependent expression of AQP9 in the prostate in vivo and in vitro. Methods: Rat ventral prostate tissue specimens and a normal human prostatic epithelial cell line (PNT2) were used. AQP9 messenger ribonucleic acid (mRNA) and protein expression were examined using real‐time polymerase chain reaction, Western blotting and immunohistochemical methods. Androgen modulation was achieved by surgical castration, treatment with testosterone propionate (5 µg/g bodyweight) or bicalutamide (20 µg/g bodyweight), and the ribonucleic acid interference (RNAi) method of the androgen receptor (AR). The synthetic small interfering RNA was transfected into PNT2 at a concentration of 10 nM, and the RNAi effect was evaluated using a Western blotting analysis. Results: AQP9 mRNA and protein were expressed in the rat prostate. Surgical castration or bicalutamide treatment significantly decreased their expression. In addition, the treatment with testosterone propionate after castration restored the expression to the level of the controls. An RNAi experiment in PNT2 also decreased the expression. Conclusions: AQP9 expression in the prostate is controlled by androgens.     

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.     

Androgens induce CD-9 in human prostate tissue

Based on microarray analyses of LNCaP and LNCaP-r prostatic cell-lines we tentatively identified CD-9 as an androgen sensitive protein. This prompted us to characterize the androgen sensitivity and the correlation to malignancy of CD-9 at the protein level. Using Western blot, RT-PCR and immunohistochemistry the expression of CD-9 was analysed in LNCaP cells stimulated during increasing time by the synthetic androgen R1881 and also in 88 specimens of human prostate cancer tissues. Expression of CD-9 was induced by R1881 in LNCaP. CD-9 was immunolocalized in human prostate tissue sections representing non-malignant tissue as well as tumour areas. In non-malignant glands CD-9 immunoreactivity was observed at the apical and lateral cell borders of luminal epithelial cells. Basal epithelial cells were largely unstained. In tumour areas CD-9 staining intensity was variable and apparently not related to primary Gleason grade. In prostate tissue from a patient under androgen ablation therapy no staining was observed in luminal epithelial cells or in the tumour areas, but some staining was observed in basal epithelial cells. CD-9 is regulated by androgens in LNCaP and present in human prostate specimens. The expression is variable and changes in expression levels. These and earlier studies using other tissues indicate that CD-9 and its cellular localization could have an important role in prostate cancer cell development.     

Regulation of calcium homeostasis by S100RVP, an androgen-regulated S100 protein in the rat ventral prostate

BACKGROUND: S100RVP was previously identified as an androgen-response gene in the rat ventral prostate (RVP). Characterization of S100RVP is important for elucidating the function of S100 proteins in androgen action. METHODS: The expression and subcellular localization of S100RVP were determined by Northern blot, in situ hybridization, and fluorescent microscopy. Calcium overlay and calcium ionophore sensitivity assays were performed to investigate the calcium binding and function of S100RVP. RESULTS: S100RVP is abundantly expressed in the RVP epithelial cells. A green fluorescent protein(GFP)-S100RVP fusion protein is present in both the cytoplasm and nucleus of transfected cells. A GST-S100RVP fusion protein bound calcium in vitro at levels similar to known S100 proteins. Furthermore, GFP-S100RVP transfected LNCaP and PC3 cells exhibited reduced sensitivity to calcium ionophore-induced cell death, but not to UV-induced cell death. CONCLUSION: The results of this study argue for a role of S100RVP in calcium homeostasis in the prostate.     

Development of the VCaP androgen-independent model of prostate cancer

Prostate epithelial cell growth is dependent on the presence of androgens, and transition of prostate cancer to an androgen-independent phenotype results in a highly aggressive, currently incurable cancer. We have developed a new preclinical model of androgen-independent prostate cancer derived from the VCaP prostate cancer epithelial cell line. VCaP cells were subcutaneously implanted and serially passaged in castrated male severe combined immunodeficient mice. Androgen independence was confirmed by WST-1 (a tetrazolium salt) cell proliferation assay in the absence or presence of dihydrotesterone (1-100 nM). VCaP androgen-sensitive cells responded dose dependently to dihydrotesterone, whereas VCaP androgen-independent cells did not alter their proliferation in response to dihydrotesterone. Gene expression of androgen receptor, B-cell lymphoma-2, prostate cancer antigen 3, prostate acid phosphatase, 6 transmembrane epithelial antigen of the prostate, and survivin was determined by polymerase chain reaction amplification. B-cell lymphoma-2 expression was up regulated in the VCaP androgen-independent lines compared to the VCaP androgen-sensitive, suggesting a possible mechanism of androgen independence. Furthermore, tumor-associated angiogenesis was assessed by immunofluorescence confocal microscopy of CD31. VCaP androgen-independent tumors showed enhanced angiogenesis compared to VCaP androgen-sensitive tumors. These results illustrate the development of a novel model of prostate cancer androgen independence and provide a new system to study angiogenesis and the transition to androgen independence.