Effects of castration and replacement of androgen on the separation of rat ventral prostate cells by Ficoll gradient centrifugation

When regressing or growing (hypertrophic) cells from collagenase-digested ventral prostates were centrifuged on isokinetic Ficoll gradients for 6-8 min, they distributed into four fractions. Because of changes in epithelial cell morphology and density following castration to induce regression and replacement of androgens to cause cell growth, and contrary to results with normal rat ventral prostate, stromal cell fraction 2 was contaminated to a greater extent with regressing epithelial cells, as judged by their morphology and binding of radioactive androgens. However, centrifugation for 3 min increased the purity of epithelial cell fraction 4, although the yield of desired cells was reduced. Most cells from endocrine-manipulated rats were viable, as judged by exclusion of trypan blue and the initial incorporation of 3H-uridine. Cells centrifuged on a similar gradient of Percoll separated by a 'sieving' effect, which inverted the order of cellular fractions and removed red blood cells from fraction 2. Metrizamide offered no advantages, compared with Ficoll or Percoll. Neither physiologic nor pharmacologic amounts of testosterone returned the morphology of isolated epithelial cells to normal. To obtain consistent results with prostates from normal or hormone-manipulated rats, one should take care to select an active preparation of collagenase, avoid the use of very old animals, cool the tissue after it is dissociated, and do not apply undigested clumps of cells or overload the gradient. If attention is paid to these details, populations enriched in viable regressing or growing prostate epithelial or stromal cells can be obtained from hormonally manipulated rats.     

Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

Internucleosomal DNA fragmentation is not obligatory for castration induced rat ventral prostate cell apoptosis in vivo

Castrated male rats were treated with the reversible S1-phase cell cycle blocking drug, mimosine, and the effects of this drug on prostate cell apoptosis was characterized. At a single dose of mimosine (25 mg/kg/day), we found that the internucleosomal DNA fragmentation associated with apoptosis was partially suppressed in the rat ventral prostate at all early time points (24, 48 and 72 h) analyzed post-castration. This suppression was dose-dependent, and treatment with mimosine up to 150 mg/kg/day was sufficient to reduce the internucleosomal DNA fragmentation in the prostate by 90% at 72 h post-castration. Intriguingly, this drug did not suppress the induction of mRNAs for several apoptosis-associated gene products in the ventral prostate gland (bcl-2, p53, TGF-beta and SGP-2/clusterin). Moreover, this treatment did not suppress the histological appearance of apoptotic bodies in the ventral prostate detectable by fast green staining of thin sections of tissue. The apoptotic bodies present in mimosine-treated regressing ventral prostate tissues, however, were refractory to labeling by the in situ gap labeling method, further demonstrating lack of nuclear DNA fragmentation in the condensed nuclei of apoptotic cells. In summary, the cell cycle-blocking drug mimosine does not appear to affect the rate of apoptosis in the regressing rat ventral prostate gland. However, this drug was capable of suppressing the nuclear DNA fragmentation associated with androgen-regulated prostate cell apoptosis. These results support the concept that nuclear DNA fragmentation is not obligatory for apoptosis. Additionally, they imply that cell cycle movement from the G1/S-phase boundary might be important for the terminal DNA degradation associated with androgen-regulated prostate cell apoptosis.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M_r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M_r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M_r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.     

In-vivo expression of a C-type RNA virus in rat ventral prostate epithelial cells.

During evaluation of a procedure for separating rat ventral prostate epithelial from connective tissue cells, isolated fractions were examined by transmission electron microscopy. Characteristic C-type RNA viruses were seen budding from or in close proximity to the plasma membranes of isolated epithelial cells. Dissociation of rat prostate cells and concentration of the epithelial fraction facilitated detection of this virus.     

Regional cellular heterogeneity and DNA synthetic activity in rat ventral prostate during postnatal development.

Previous studies have provided evidence that the rat ventral prostate grows primarily, if not exclusively, at its distal tips. However, as yet there have been no analyses in which individual cells in defined regions of the prostatic ductal system have been resolved and quantified. Moreover, the possibility that the prostate might grow differently at different times of postnatal development has received little attention. Our objectives were to identify and quantify the proliferating epithelial and stromal cells in defined regions of the rat ventral prostate during its postnatal development. To this end, 3H-thymidine was administered in vivo to rats of ages 10-60 days. A dissection technique was then used by which the distal, intermediate, and proximal segments of the prostatic ductal system were physically isolated from each other without removing the stromal tissue. Longitudinal sections of these segments were examined for cellular composition and DNA synthetic activity. Regional heterogeneity with respect to cell composition and cell proliferation was seen. In rats of all ages, DNA synthetic activity was seen in epithelial and stromal cells throughout the prostate, rather than only in the distal segment. At Days 10 and 20, significantly higher percentages of epithelial and stromal cells were labeled in the distal than in the proximal segments; but at Days 45 and 60, the percentages of labeled epithelial and stromal cells in the distal, intermediate, and proximal segments were similar. Thus, in all segments, and at all ages, substantial labeling was seen throughout the prostate. These data suggest that the prostate grows in both length and width throughout postnatal development, reminiscent of the growth of a tree.     

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.     

5alpha-Dihydrotestosterone binding protein in rat ventral prostate; purification, nuclear incorporation, and subnuclear localization.

Treatment of cytosol from the rat ventral prostate with cold acetone (-20 degrees C) evoked a 8 approximately 10-fold increase in the binding capacity with 5alpha-dihydrotestosterone (DHT). Starting from the extract of acetone-dried prostate cytosol, some 400 approximately 600-fold purification of the DHT-binding protein complex was acieved by (NH4)2504 fractionation, DEAE-cellulose chromatography and gel- filtration with Sephadex G-200. The purified 3H-DHT-binding protein complex was incorporated into the nuclei from the ventral prostate in a temperature dependent manner. The similar incorporation was also observed in nuclei from the liver and the kidney...     

5 Alpha-reductase in the non-secretory cells of the rat ventral prostate epithelium

Kinetic constants for the 5 alpha-reductase were determined in freshly isolated epithelial cells from the rat ventral prostate. Studies were also performed on stromal tissue but not isolated stromal cells for comparison. Secretory and non-secretory epithelial cells were separated by centrifugation in a Percoll gradient. Both epithelial cell populations metabolized testosterone to predominantly 5 alpha-dihydrotestosterone (5 alpha-DHT), although when expressed per cell the capacity for conversion was 3-4-fold higher for secretory cells (7.4 pmol/min/10(6) cells) than for non-secretory cells (2.3 pmol/min/10(6) cells; P less than 0.01 in 4 separate studies). When compared per mg cytosol protein this difference became non-significant. Stromal tissue contained a 5 alpha-reductase Vmax (expressed) per mg protein) which was comparable to the non-secretory cell enzyme. Lineweaver-Burke plots revealed different Km values for the different cell populations (12.5, 5.9 and 4.7 microM for secretory, non-secretory and stromal cells, respectively) suggesting the presence of different isoforms of the enzyme, or differences in the intracellular concentrations of enzyme antagonists.     

Inhibition of human erythrocyte Ca++-transport ATPase by phenothiazines and butyrophenones

Indirect immunofluorescent staining revealed that Prostatic Binding Protein, the major androgen-dependent protein in rat ventral prostate $\text{in}\text{vivo}$, is associated specifically with the epithelial cells in primary cell cultures derived from rat ventral prostate. The epithelial cells release Prostatic Binding Protein into the medium during primary culture. $\text{De}\text{novo}$ synthesis of Prostatic Binding Protein is demonstrable during early phases of cell culture. Prostatic Binding Protein is an excellent marker for the identification of functional prostate epithelial cells and for the study of regulation at the cellular level of the synthesis and secretion of a major androgen-dependent prostate protein.     

The release of radioactive arachidonate from lipids of red blood cells during prolonged incubations in vitro

Red blood cells were isolated from rat blood and incubated in the presence of [3H]arachidonate. A sizeable quantity (18%) of the radioactivity was incorporated into red cell lipids, of which phosphatidylcholine was the most highly labelled. Radioactive arachidonate was found at position 2 of this phospholipid. Free fatty acids were removed by washing the cells in solutions containing fatty-acid-free bovine serum albumin. The labelled red cells were then incubated for up to 16 h at 37 degrees C. After 16 h of incubation in saline-buffer-glucose or rat serum, 20 and 26%, respectively, of the total radioactivity was found in free fatty acids, and there were corresponding declines in the percentage radioactivities found in phosphatidylcholine. In the presence of serum, there was a more rapid release of radioactive fatty acid over the 2- to 16-h time course. There was not a significant drop in the phosphate levels of the total red cell phospholipids or phosphatidylcholine after 16 h of incubation and, as a result, there were large declines in the specific radioactivities of phosphatidylcholine. Diacylglycerols were not highly labelled and the action of phospholipase A2 on labelled phosphatidylcholine was indicated. When white blood cells were added to labelled red cells, there was little evidence of white cell involvement in the release of radioactive fatty acid, suggesting that the red cells themselves may be involved in arachidonate release. Red cells may serve as sources of arachidonate, released following hemorrhage in brain and metabolized to form various biologically active eicosanoids.     

Distribution of the vacuolar H+ atpase along the rat and human male reproductive tract

Luminal acidification in parts of the male reproductive tract generates an appropriate pH environment in which spermatozoa mature and are stored. The cellular mechanisms of proton (H+) secretion in the epididymis and the proximal vas deferens involve the activity of an apical vacuolar H+ ATPase in specialized cell types, as well as an apical Na+/H+ exchanger in some tubule segments. In this study we used Western blotting and immunocytochemistry to localize the H+ ATPase in various segments of the male reproductive tract in rat and man as a first step toward a more complete understanding of luminal acidification processes in this complex system of tissues. Immunoblotting of isolated total cell membranes indicated a variable amount of H+ ATPase in various segments of the rat reproductive tract. In addition to its known expression in distinct cell types in the epididymis and vas deferens, the H+ ATPase was also localized at the apical pole and in the cytoplasm of epithelial cells in the efferent duct (nonciliated cells), the ampulla of the vas deferens and the ventral prostate (scattered individual cells), the dorsal and lateral prostate, the ampullary gland, the coagulating gland, and all epithelial cells of the prostatic and penile urethra. Both apical and basolateral localization of the protein were found in epithelial cells of the prostatic ducts in the lateral prostate and in periurethral tissue. Only cytoplasmic, mostly perinuclear localization of the H+ ATPase was found in all epithelial cells of the seminal vesicles and in most cells of the ventral prostate and coagulating gland. No staining was detected in the seminiferous tubules, rete testis, and bulbourethral gland. In human tissue, H+ ATPase-rich cells were detected in the epididymis, prostate, and prostatic urethra. We conclude that the vacuolar H+ ATPase is highly expressed in epithelial cells of most segments of the male reproductive tract in rat and man, where it may be involved in H+ secretion and/or intracellular processing of the material endocytosed from the luminal fluid or destined to be secreted by exocytosis.     

Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate.

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.     

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates

Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca⁺⁺-and Mg⁺⁺-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis. This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from the National Cancer Institute, National Institutes of Health, Bethesda, MD.     

Cyclic AMP-stimulating effect of vasoactive intestinal peptide in isolated epithelial cells of rat ventral prostate

Vasoactive intestinal peptide (VIP) has been shown to increase cyclic AMP content in isolated epithelial cells of rat ventral prostate. The stimulatory effect of VIP was dependent on time and temperature and was potentiated by a phosphodiesterase inhibitor. At 15 degrees C, the response occurred in the 1 X 10(-10)-10(-7)M range of VIP concentrations. Half-maximal stimulation of cellular cyclic AMP was obtained at 1.4 nM and maximal stimulation (3-fold basal level) at about 100 nM VIP. Chicken VIP and porcine secretin were agonists of porcine VIP but exhibited a 2-times higher and a 170-times lower potency, respectively. A high concentration (1 X 10(-6)M) of glucagon, somatostatin, neurotensin, substance P, Met-enkephalin or Leu-enkephalin did not modify cAMP levels. The finding of a VIP-stimulated cAMP system in rat prostatic epithelial cells together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, as well as the presence of VIP-containing neurones innervating the male genitourinary tract, strongly suggest that VIP may be involved in prostatic growth regulation and function.     

原代培养大鼠前列腺细胞建立前列腺增生筛药模型

目的建立原代培养大鼠前列腺上皮细胞体外筛药模型。方法无菌状态下取雄性SD大鼠腹侧叶前列腺,称量后,剪成1 mm3小块,经Ⅱ型胶原酶消化1 h后,过滤、离心获取前列腺细胞,接种于24孔板培养并对细胞进行形态学和免疫组织化学鉴定。获取的大鼠前列腺上皮细胞分别接种于96孔板和24孔板培养12 d后给予系列剂量(0.1～1000μmol/L)的他莫昔芬和阳性药物爱普列特处理72 h,利用CCK-8法检测前列腺上皮细胞存活率并计算药物IC50值,并进一步通过Giemsa染色后观察细胞生长及形态变化。结果细胞鉴定结果显示,获取的细胞具典型上皮细胞形态特征,细胞角蛋白和前列腺特异性抗原表达阳性,提示所获细胞为前列腺上皮细胞。爱普列特与前列腺上皮细胞孵育72 h后,CCK-8法检测结果显示能够明显抑制前列腺上皮细胞生长,其IC50值为42.7μM,进一步镜下观察结果显示,爱普列特未明显改变大鼠前列腺上皮细胞形态,但能导致存活细胞数减少。他莫昔芬则对大鼠前列腺上皮细胞生长无明显抑制作用,镜下观察前列腺上皮细胞数量和形态未见明显改变。结论利用原代培养SD大鼠前列腺上皮细胞可以成功建立前列腺增生体外筛选模型。