Cells Of Rat Ventral Prostate Research Articles

Immunoelectron microscope localization of androgen receptors and proliferating cell nuclear antigen in the epithelial cells of albino rat ventral prostate

Androgen receptor (AR) and proliferating cell nuclear antigen (PCNA) play a crucial role in development and progression of various prostatic diseases including prostatic carcinoma that is a leading cause of death in males. Previous studies have evaluated the expression pattern of AR and PCNA in prostate epithelial cells using immunohistochemistry (IHC). However, this technique has limited ability to identify their precise subcellular localization. Therefore, the aim of this study was to localize, subcellularly, AR and PCNA in the secretory epithelial cells of rat ventral prostate using post embedding immunogold-electron microscopy. The ventral lobes were dissected from six adult male albino rats after being perfused with paraformaldehyde. Some specimens were immuno-labeled with AR or PCNA and others were processed for immunoelectron microscope of AR and PCNA using 15-nm gold conjugated secondary antibodies. The results showed that, by immunoperoxidase reaction, AR and PCNA were localized diffusely throughout the nuclei of the epithelial cells of prostatic acini without visible cytoplasmic expression. However, the higher resolution immuno-electron microscopy was able to detect AR and PCNA in the nucleus and some cytoplasmic organelles. In conclusion, this study emphasizes the importance of immuoelectron microscopy in precise localization of AR and PCNA at the subcelullar levels in the secretory epithelial cells of the rat prostatic acini. These findings will help to further understand the mechanism of action of these receptors under normal and pathological conditions that could have future clinical application after careful human investigation.

Dehydroepiandrosterone-induced proliferation of prostatic epithelial cell is mediated by NFKB via PI3K/AKT signaling pathway

Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. In this study, rat ventral prostatic epithelial cells were used to determine whether DHEA-modulated proliferation and prostate-specific antigen (PSA listed as KLKB1 in the MGI Database) production were mediated via the androgen receptor (AR) and its potential mechanism. We demonstrated that proliferation of prostatic epithelial cells and increase of PSA expression induced by DHEA were neutralized by Casodex or Ar siRNA, two specific AR blockers. DHEA stimulated Nfkb DNA binding activity, with this effect being blunted by Casodex or Ar siRNA. Moreover, the inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT nullified the effects of DHEA on NFKB activation. These findings suggested that DHEA stimulated normal prostatic epithelial cell proliferation, and AR is involved in DHEA-induced PSA expression in normal prostatic epithelial cells. This stimulation effect induced by DHEA is mediated by the activation of NFKB via PI3K/AKT pathway.

Stromal and epithelial cells from rat ventral prostate during androgen deprivation and estrogen treatment--regulation of transcription.

To identify the functional capacities of prostatic tissue, the expression of steroid hormone receptors, growth factors, oncogenes and particular enzymes was studied at the RNA level in isolated stromal and epithelial cells of rat ventral prostate (RVP) under different hormonal conditions (androgen deprivation, estrogen treatment). Slot blot and Northern blot analyses of isolated RNA resulted in characteristic changes: In the control prostate, androgen receptor (AR) mRNA was high in epithelium of intact prostate, but low in stroma. Its level was increased after castration in the epithelium during the initial 24 hours, whereas an only slight increase occurred in stroma after one week castration. The AR signal was not altered by estradiol treatment in epithelium and stroma. Conversely, the estrogen receptor (ER) mRNA, predominant in stroma and very low in epithelium, decreased after castration in stroma and epithelium within 24 hours and was absent one week later. After estrogen treatment the ER signal increased considerably in stroma. mRNA of both basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF-beta) were exclusively found in stroma. Both were low in controls and responded in a different way to castration and estrogen treatment within 24 hours. bFGF was increased in estrogen-treatment animals, while TGF-beta was induced by castration. Shortly after castration (2 hours) v-fos expression increased and reached a maximum after 6 hours, but was no more detectable after 12 hours in epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Pneumadin in the ventral prostate of rats during postnatal development: A radioimmunological and immunocytochemical study

We have recently demonstrated that the ventral prostate of adult rats contains high levels of pneumadin (PNM), a decapeptide originally isolated from mammalian lung, and that testosterone is needed for the maintenance of a normal level of the peptide in the prostate. Hence, we have investigated, by radioimmunoassay (RIA) and light ultrastructural immunocytochemistry (ICC), PNM concentration and localization in the rat ventral prostate during postnatal development. RIA showed that PNM content increased steadily from day 20 to day 90 of postnatal life, parallel to the increase in the prostate weight. In contrast, PNM concentration remained rather stable, although it showed a marked rise at day 40 when rat testes are known to reach their full maturation. ICC demonstrated that PNM immunoreactivity was mainly located in the apical pole of epithelial cells of rat ventral prostate, especially in the subcellular organelles involved in protein secretion, i.e. rough endoplasmic reticulum cisternae, vacuoles and granules. Taken together our study suggests the involvement of PNM in the functional control of rat prostate during postnatal maturation, although its exact role remains to be elucidated.

Immunocytochemical Localization of Glutathione-Peroxidase (GSH-PO) and Bcl-2 in the Rat Ventral Prostate

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) and Bcl-2, we have studied the immunocytochemical localization of GSH-PO, Bcl-2, androgen receptor (AR) and apoptosis in the rat ventral prostatic cells in the presence and absence of testosterone. Male Crj:CD(SD) IGS rats were divided into four experimental groups. Group 1 consisted of untreated controls. In group 2, rats were sacrificed two days after castration. In groups 3 and 4, rats were subcutaneously administered 1 mg/animal of testosterone daily for 3 or 7 days after two days of castration, respectively. In group 1, GSH-PO was predominantly observed in the glandular epithelial cells. Bcl-2 was localized exclusively in the cytoplasm of the glandular epithelial cells. AR was localized in the nuclei of the glandular epithelial cells. In group 2, the intensity of the staining of GSH-PO, Bcl-2 and AR markedly decreased. However, the number of apoptotic cells (apoptotic index) markedly increased. In groups 3 and 4, the immunoreactivities of GSH-PO, Bcl-2 and AR clearly recovered. These findings strongly suggest that the expression of GSH-PO and Bcl-2 in the glandular epithelial cells of the rat ventral prostate is testosterone-dependent. Furthermore, co-expression of GSH-PO and Bcl-2 in the prostatic cells are considered to be normal or adaptive aspects of the cells.

Mitochondrial aconitase gene expression is regulated by testosterone and prolactin in prostate epithelial cells .

m-aconitase catalyzes the first step leading to the oxidation of citrate via the Krebs cycle. It is a constituitive enzyme in virtually all mammalian cells, found in excess, and is considered to be a regulatory or regulated enzyme. In contrast to these general relationships, prostate secretory epithelial cells possess a uniquely limiting mitochondrial (m-) aconitase which minimizes the oxidation of citrate. This permits the unique prostate function of accumulating and secreting extraordinarily high levels of citrate. Previous animal studies demonstrated that testosterone and prolactin regulate the level of m-aconitase specifically in citrate-producing prostate cells. The present studies were conducted to determine if testosterone and prolactin regulated the expression of the m-aconitase gene in prostate cells, and to determine the effect of the hormones on human prostate cells. The studies were conducted with freshly prepared rat ventral, rat lateral, and pig prostate epithelial cells, and with the human malignant cell lines LNCaP and PC-3. The effects of 1 nM testosterone and 3 nM prolactin on the level of m-aconitase mRNA and on the transcription rate of m-aconitase were determined. The studies revealed that both prolactin and testosterone increase the levels of m-aconitase mRNA and the transcription rates of m-aconitase in rat ventral prostate cells, pig prostate cells, and human malignant prostate cells (LNCaP and PC-3). In contrast, both hormones decreased the level of m-aconitase mRNA and repressed m-aconitase gene transcription in rat lateral prostate cells. The hormonal regulation of m-aconitase corresponded with the levels of m-aconitase enzyme, m-aconitase activity, and citrate oxidation. In addition to the constitutive expression of m-aconitase, the m-aconitase gene is testosterone- and prolactin-regulated in specifically targeted prostate cells. The hormonal regulation of m-aconitase gene expression and biosynthesis of m-aconitase provide a regulatory mechanism for the oxidation of citrate, and consequently, the level of net citrate production by prostate. The hormonally increased expression and biosynthesis of m-aconitase in human malignant cells might be involved in the increased citrate oxidation associated with the development of true malignant cells in prostate cancer.

The Pyruvate Dehydrogenase E1 Alpha Gene Is Testosterone And Prolactin Regulated In Prostate Epithelial Cells

The prostate gland of humans and other animals has the unique function of accumulating and secreting extraordinarily high levels of citrate. The prostate secretory epithelial cells synthesize citrate which, due to a limiting mitochondrial (m-) aconitase, accumulates rather than being oxidized. Thus citrate is essentially an end product of metabolism in prostate. For continued net citrate production, a continual source of oxaloacetate (OAA) and acetyl CoA is required. Glucose via pyruvate oxidation provides the source of Acetyl CoA. In prostate cells, citrate production is regulated by testosterone and/or by prolactin. Both hormones selectively regulate the level and activity of pyruvate dehydrogenase E1 alpha (E1 a) in animal prostate cells; thereby regulating the availability of acetyl CoA for citrate synthesis. Studies were conducted to determine if testosterone and prolactin might regulate the expression of the E1a gene in prostate epithelial cells. Prolactin treatment of rat ventral and lateral prostate cells and human PC3 cells increased the levels of E1a mRNA and the rates of transcription of the E1a gene. Testosterone also increased the mRNA level and transcription of E1a in rat ventral prostate cells, and in PC3 cells transfected with androgen receptor. However, testosterone treatment resulted in a repression of E1a gene expression in lateral prostate cells. Evidence is presented which supports the view that prolactin regulation of E1a is mediated via PKC. The rapidity of the effects of both hormones is representative of an immediate-early gene response. To our knowledge this represents the first report in any mammalian cells that, in addition to its constitutive expression in all mammalian cells, the E1a gene is a hormonally-regulated gene in specifically targeted prostate epithelial cells.

EVIDENCE FOR A LOCAL ACTION OF MELATONIN ON THE RAT PROSTATE

Purpose Melatonin, secreted by the pineal gland at night, inhibits pubertal development in rats and possibly humans. We have recently found functional specific binding sites for 125I-labeled melatonin (sup 125 I-melatonin) in human benign prostate tissue, localized in the microsomal fraction of the glandular epithelium. The aim of the present study was to set up an animal (rodent) model for the growth inhibitory effects of melatonin on the prostate. Materials and Methods Putative melatonin receptors in the rat ventral prostate were explored by means of autoradiography and receptor binding assays and the ability of melatonin to inhibit stimulated prostate growth was tested in vivo. Results In vitro autoradiography and equilibrium binding experiments demonstrated specific binding sites for 125I-labeled melatonin (sup 125 I-melatonin) associated with the microsomal fraction of the rat ventral prostate cells (apparent dissociation constant 0.9 nM). 125I-melatonin binding was inhibited by 2-iodomelatonin>6-hydroxy-melatonin>melatonin=N-(2,4 dinitrophenyl)-5-methoxytryptamine whereas similar concentrations of serotonin, 5-methoxytryptamine, and tryptamine were less potent. The guanine nucleotide analogs, guanosine 5′-0-[3-thiotriphosphate] and guanosine 5′-0-[2-thio-diphosphate], inhibited specific 125I-melatonin binding whereas 5′-guanylyl imido-diphosphate was less potent. Daily injections of testosterone to castrated rats induced regrowth of the prostate and increased the weight of the seminal vesicles. Administration of melatonin to the rats through drinking water prevented the testerone-mediated regrowth of the prostate but had no effect on the seminal vesicles' weight. Conclusions The results demonstrated putative melatonin receptors in the rat prostate and suggest a direct suppression by melatonin of testosterone-dependent prostate growth.

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.

Prolactin specifically regulates citrate oxidation and m-aconitase of rat prostate epithelial cells

The prostate gland of many animals, including humans, produces and secretes extremely high levels of citrate. To achieve this function, prostate secretory epithelial cells possess unique metabolic properties that permit accumulation and ultimate secretion (net citrate production) of citrate. Mounting evidence continues to support the concept that prostate epithelial cells possess a limiting mitochondrial (m)-aconitase activity that minimizes citrate oxidation and results in the accumulation of citrate synthesized by the cells. Recent studies have revealed that prolactin (PRL) stimulates net citrate production of rat lateral prostate (RLP). The mechanism of this PRL effect has not been established. The current studies were concerned with the possibility that PRL might be involved in the regulation of citrate oxidation and m-aconitase of prostate cells. Studies were conducted with RLP, RVP (rat ventral prostate), RDP (rat dorsal prostate), and kidney cells. The results showed that PRL in vitro and in vivo decreased citrate utilization and the level of m-aconitase in RLP cells, and conversely increased citrate utilization and m-aconitase in RVP cells. Furthermore, PRL had no effect on either RDP or kidney cells. The effects of PRL on both citrate utilization and m-aconitase of RLP and RVP were abolished by cycloheximide and actinomycin. Mitochondrial studies revealed that PRL decreased citrate oxidation of RLP and increased citrate oxidation of RVP, but had no effect on isocitrate oxidation. In conclusion, these studies establish that PRL has a physiological role in the regulation of citrate oxidation in prostate, and that this action is associated with PRL regulation of the biosynthesis of m-aconitase. Furthermore, the effects of PRL are cell-specific and targeted at m-aconitase.

Increased Expression of Early Growth Response-1 Messenger Ribonucleic Acid in Prostatic Adenocarcinoma

Purpose We isolated genes whose expression differs between normal and malignant prostate. Materials and Methods Differential display polymerase chain reactions revealed a messenger ribonucleic acid (mRNA) with higher expression in prostatic adenocarcinoma versus age matched normal tissue. Deoxyribonucleic acid sequencing identified this mRNA as the product of the early growth response-1 gene. Results Early growth response-1 mRNA levels were elevated in 12 of 12 intraprostatic adenocarcinomas but not in breast or ovarian cancers, or in rapidly dividing rat ventral prostate cells. Early growth response-1 mRNA was detected in epithelial and stromal cells at tumor margins but not in lymph node metastases. Conclusions Early growth response-1, a nuclear transcription factor, is implicated in the growth and invasion of intraprostatic cancers.

Effect of TGF-beta 1, TGF-alpha, and EGF on cell proliferation and cell death in rat ventral prostatic epithelial cells in culture.

A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.

Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.

Immunoelectron microscopic localization of glutathione-peroxidase (GSH-PO) in rat ventral prostate. Effect of anti-androgens.

In order to confirm the relationship between glutathione-peroxidase (GSH-PO) and testosterone action in rat ventral prostate, we have studied the immunocytochemical localization of GSH-PO in glandular epithelial cells of rat ventral prostate after treatment of TZP-4238 or chlormadinone acetate (CMA) as antiandrogens. In the control rat ventral prostate, GSH-PO was predominantly observed in the glandular epithelial cells and intracellular localization of GSH-PO was mainly observed in the cytoplasmic matrix near the rough endoplasmic reticulum and it was occasionally noted as a small granular structure at the supranuclear region. In TZP-4238-or CMA-administered rats, the glandular epithelial cells of the ventral prostate were markedly atrophic. The intensity of GSH-PO staining in the glandular epithelial cells was markedly decreased. Immunoelectron microscopically, GSH-PO-positive granules were hardly seen in the atrophic glandular epithelial cells. These findings strongly suggest that loss of GSH-PO staining in the glandular epithelial cells of the rat ventral prostate treated with TZP-4238 or CMA as anti-androgens is thought to be caused by inhibition of testosterone action, and that its staining pattern is a useful for the effect of antiandrogens on prostate.

Effect of Transforming Growth Factor-β1on Proliferation and Death of Rat Prostatic Cells*

The ability of transforming growth factor B1 (TGF beta 1) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGF beta 1/day directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen-stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1-20 ng/ml TGF beta 1. In contrast in vivo administration of 50 ng TGF beta 1/day directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGF beta 1 treatment did not lower serum testosterone, nor did it affect the size or DNA content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in vitro, there was a dose-response relationship between glandular cell death and TGF beta 1 concentration in the medium. These results demonstrate that TGF beta 1 can induce the death of androgen-dependent prostatic glandular cells even when physiological levels of androgen are present. Previous studies have demonstrated that both the receptor and the mRNA for TGF beta 1 increase rapidly in the ventral prostate after castration. Taken with the present data, these results suggest that TGF beta 1 may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation.

Functional Characterizations of the Androgen Receptor Confirm that the Molecular Basis of Androgen Action Is Transcriptional Regulation

In an effort to understand the molecular basis of androgen action in the prostate, we isolated androgen receptor (AR) cDNA from rat ventral prostate cells and analyzed the transcriptional regulatory activity of the encoded protein in a cotransfection assay. We found that AR is capable of inducing chloramphenicol acetyltransferase activity more than 20-fold using the mouse mammary tumor virus LTR as a source of androgen response elements. This induction was observed in both monkey CV1 cells and human HeLa cells, neither of which contains endogenous functional AR, and was entirely dependent on added androgens. Deletion mapping studies showed that carboxy-terminal deletions of approximately 250 amino acids convert AR into a constitutive activator of transcription. In addition, a chimeric receptor protein containing the amino-terminus and DNA-binding domains of AR fused to the previously defined ligand domain of the glucocorticoid receptor was found to be fully functional based on dexamethasone-induced chloramphenicol acetyltransferase activity. Our results support the prediction that androgens modulate rates of transcriptional initiation, suggesting that posttranscriptional effects of androgens are secondary responses. Moreover, these data reveal that, like other steroid receptors, AR contains a number of distinct regulatory regions important for normal activity. The isolation and characterization of fully functional AR sequences will facilitate the use of molecular genetics to study complex androgen responses in target tissues such as the prostate.

Regulation of aspartate aminotransferase messenger ribonucleic acid level by testosterone

The effect of testosterone on precursor mitochondrial aspartate aminotransferase (pmAAT) mRNA was studied in rat ventral prostate and primary cell cultures of mini-pig prostate. Testosterone induced a 2–3-fold increase in pmAAT mRNA level in both rat ventral prostate and mini-pig prostate cultures. The pmAAT mRNA induction occurred 30 min after testosterone treatment and was maximal by 1.5 h. Prostatic mAAT activity was also induced by testosterone with a 1–2 h lag period. The time-course of induction of pmAAT mRNA, pmAAT activity and mAAT activity was consistent with stimulation of mRNA synthesis followed by increased synthesis and import of pmAAT into mitochondria. The effect of testosterone on pmAAT mRNA was specific because the increase in pmAAT mRNA was at least 2-fold greater than the increase in poly (A +) RNA. These results suggest that testosterone stimulated mAAT activity by induction of pmAAT mRNA. This continues to support our proposal that a major physiological effect of testosterone is increased pmAAT mRNA steady-state levels which result in increased pmAAT synthesis and increased mAAT activity. These changes ultimately result in increased citrate production by prostate epithelial cells.

An Isocratic Reversed-Phase High Performance Liquid Chromatographic Analysis of 5α-Reduced Androgen Metabolites Formed by Rat Ventral Prostate Cells in Culture

Abstract Androgen actions in the rat prostate gland are complicated because of extensive metabolism of the androgens. Traditional methods for separating and quantitating the androgens and their metabolites involved thin layer and paper chromatographic steps which are laborious and time-consuming. An isocratic reversed-phase high performance liquid chromatographic system has been developed which separates 11 androgens. This system has been applied to the analysis of the androgen metabolites formed by rat ventral prostate cells in culture and these compounds quantitatively account for all significant metabolism. Using a flow-through radioactivity detector experiments are performed with 3H-substrates. Previously reported systems did not achieve separation of all the compounds.

Separation of mature rat ventral prostate epithelial and fibroblast cells

Several techniques for the separation of rat ventral prostate cells, using density gradient centrifugation or mechanical means have been published, yielding fibroblast and epithelial cell populations of varying purities and viability. These techniques are often tedious, yield relatively limited numbers of cells, demand considerable technical expertise, and result in the isolation of cells of limited viability. Two techniques for the isolation and establishment of epithelial and fibroblast cell cultures from mature rat ventral prostate are described here. Collagenase/trypsin digestion of the tissue yields a single-cell suspension of both cell types that are separated on Percoll isopycnic centrifugation gradients. A continuous gradient system allows for the separation of a greater number of cells with very high degrees of purity. A second technique based on a step-gradient system produces reproducible subfractionation of the epithelial cell component of the prostate within a considerable shorter period of time. An improved medium for plating epithelial and fibroblast cells has also been developed. The separated cells are plated on collagen and/or fibronectin-coated dishes in a serum-free plating medium that is later replaced with a serum containing growth medium. The plating medium greatly increases the plating efficiency of the isolated cell types, particularly the epithelial cells.

Oncogenicity of rat prostate cells transformed in vitro with cadmium chloride

Three rat ventral prostate (RVP) cell lines transformed after in vitro treatment with cadmium chloride (CdCl2) and one control untreated cell line were tested for tumorigenicity in newborn rats. All three cadmium-transformed RVP cell lines induced tumors at the site of inoculation in 95-100% of animals. The fibroblastoid RVP56Cd cell line induced sarcomas, whereas the epithelial cell lines RVP47-3G and RVP47-3F produced highly differentiated squamous cell carcinomas. About 20% of animals injected with RVP47-3G developed lung and splenic metastases. The tumors could be further passaged into young rats. The sarcomas had a hyperdiploid modal chromosome number similar to that of the RVP56Cd cell line. Carcinomas induced by the RVP47-3G cell line had a large proportion of stromal metaphases. The modal chromosome number of these carcinomas was in the hypertriploid-hypotetraploid range, similar to that of the parental cell line. These results demonstrate that treatment of RVP cells with CdCl2 in vitro results in neoplastic transformation. Since both fibroblastoid and epithelial prostate cells have undergone transformation, it seems possible that cadmium acted as a carcinogen without cell specificity. The susceptibility of these cells to the carcinogenic effect may be related to their resistance to cadmium. In the process of neoplastic transformation induced by CdCl2 in RVP epithelial cells changes of squamous metaplasia occur, and probably precede acquisition of tumorigenicity.