Ventral Prostate Epithelial Cells Research Articles

Environmental Toxicant Induced Epigenetic Transgenerational Inheritance of Prostate Pathology and Stromal-Epithelial Cell Epigenome and Transcriptome Alterations: Ancestral Origins of Prostate Disease

Prostate diseases include prostate cancer, which is the second most common male neoplasia, and benign prostatic hyperplasia (BPH), which affects approximately 50% of men. The incidence of prostate disease is increasing, and some of this increase may be attributable to ancestral exposure to environmental toxicants and epigenetic transgenerational inheritance mechanisms. The goal of the current study was to determine the effects that exposure of gestating female rats to vinclozolin has on the epigenetic transgenerational inheritance of prostate disease, and to characterize by what molecular epigenetic mechanisms this has occurred. Gestating female rats (F0 generation) were exposed to vinclozolin during E8-E14 of gestation. F1 generation offspring were bred to produce the F2 generation, which were bred to produce the transgenerational F3 generation. The transgenerational F3 generation vinclozolin lineage males at 12 months of age had an increased incidence of prostate histopathology and abnormalities compared to the control lineage. Ventral prostate epithelial and stromal cells were isolated from F3 generation 20-day old rats, prior to the onset of pathology, and used to obtain DNA and RNA for analysis. Results indicate that there were transgenerational changes in gene expression, noncoding RNA expression, and DNA methylation in both cell types. Our results suggest that ancestral exposure to vinclozolin at a critical period of gestation induces the epigenetic transgenerational inheritance of prostate stromal and epithelial cell changes in both the epigenome and transcriptome that ultimately lead to prostate disease susceptibility and may serve as a source of the increased incidence of prostate pathology observed in recent years.

Effects of low dose of bisphenol A on the proliferation and mechanism of primary cultured prostate epithelial cells in rodents.

Bisphenol A (BPA) is a well-known endocrine disruptor compound (EDC) that aggravates testosterone-induced benign prostate hyperplasia by increasing the relative weight of the ventral and dorsolateral prostate in rats. This phenomenon is primarily attributed to the exogenous estrogen effect of BPA. However, the direct effect of BPA on prostate cells has not been characterized. The present study investigated the proliferative effect and possible mechanisms of action of BPA on the prostatic epithelium of rats. The ventral prostate epithelial cells were cultured in vitro and the proliferation effects of BPA on cells were studied. The cells were identified as prostatic epithelial cells, and cell viability, cell apoptosis and the expressions of androgen receptors (AR) and estrogen receptors (ER), were detected. It was observed that 0.01–1 nM BPA promoted cell growth, with 1 nM BPA inducing the greatest increase in the rate of cell growth. However, BPA-treated cells exhibited no marked morphological changes compared with the control group. The cell apoptosis rate in each BPA-treated group was lower compared with the control group. The expression levels of ERα and ERβ increased, but the expression of AR decreased. The present study demonstrated that environmental exposure to BPA directly promoted the proliferation of prostate cells in rats through increasing the expression of estrogen receptors, reducing the expression of androgen receptors of the cells and decreasing apoptosis-induced cell death.

Insulin Affects Tissue Organization and the Kinetics of Epithelial Cell Death in the Rat Ventral Prostate After Castration

Prostate growth and physiology are regulated by steroid hormones and modulated by multiple endocrine factors. We investigated the action of insulin on the tissue organization and kinetics of epithelial cells in the rat ventral prostate (VP) in response to castration up to 120 hours after surgery by using an acute protocol of alloxan-induced diabetes. Diabetes caused a reduction in volume density (V(v%)) and volume of the epithelium. The effects of castration on the epithelium were accelerated in the diabetic animals, as determined by changes in V(v%) and volume. The smooth muscle cells became atrophic and apparently relaxed in response to castration, in contrast to the spinous aspect observed in nondiabetic castrated rats. Counting of apoptotic nuclei in the epithelium showed the classical apoptosis peak at 72 hours in nondiabetic rats and an advance of the apoptosis peak to 48 hours after castration in diabetic rats. Insulin restored the time of the peak to 72 hours. These results were confirmed after immunostaining for cleaved caspase-3 and suggest a survival and antiapoptotic effect on VP epithelial cells in both the presence and absence of androgen stimulation. This idea is supported by the observation that insulin also reduced the overall rate of apoptosis at all experimental points analyzed before and after castration.

Inhibitor of differentiation 1 (Id1) promotes cell survival and proliferation of prostate epithelial cells

Id1 (inhibitor of differentiation 1) is a member of the bHLH protein family. Consistent with its role in promoting proliferation and inhibiting differentiation, Id1 expression is low or negligible in normal prostate epithelial cells but is high in prostate cancer. Ectopic expression of Id1 in normal prostate epithelial cells could therefore provide a model for understanding early events involved in initiation of prostate cancer. Over-expression of Id1 immortalized but did not transform ventral prostate epithelial cells (Id1-RPE). Immortalization was associated with decreased Cdkn2a, Cdkn1a, androgen receptor and increased Tert expression. Gene expression profiling over successive doublings was used to identify transcriptomic changes involved during immortalization (Tieg, Jun, alpha actin, Klf10, Id2) and in maintaining the immortalized phenotype (Igfbp3, Igfbp5, Mmp2, Tgfb3). Network analysis indicated that Id1 promotes cancer/tumor morphology, cell cycle and epithelial to mesenchymal transition by influencing AP1, tnf, tgfβ, PdgfBB and estradiol pathways. During immortalization, the expression of majority of differentially expressed genes reduced over progressive doublings suggesting a decline in transcriptional regulatory mechanisms. The associated molecular/gene expression profile of Id1-RPE cells provides an opportunity to understand the molecular pathways associated with prostate epithelial cell survival and proliferation.

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.

APP-081-PM Clusterin As a Novel Mediator of Benign Prostatic Hyperplasia in Rat Ventral Prostate Epithelial Cells(The 97th Annual Meeting of the Japanese Urological Association)

APP-081-PM Clusterin As a Novel Mediator of Benign Prostatic Hyperplasia in Rat Ventral Prostate Epithelial Cells(The 97th Annual Meeting of the Japanese Urological Association)

Transgenerational effects of the endocrine disruptor vinclozolin on the prostate transcriptome and adult onset disease

The ability of an endocrine disruptor exposure during gonadal sex determination to promote a transgenerational prostate disease phenotype was investigated in the current study. Exposure of an F0 gestating female rat to the endocrine disruptor vinclozolin during F1 embryo gonadal sex determination promoted a transgenerational adult onset prostate disease phenotype. The prostate disease phenotype and physiological parameters were determined for males from F1 to F4 generations and the prostate transcriptome was assessed in the F3 generation. Although the prostate in prepubertal animals develops normally, abnormalities involving epithelial cell atrophy, glandular dysgenesis, prostatitis, and hyperplasia of the ventral prostate develop in older animals. The ventral prostate phenotype was transmitted for four generations (F1-F4). Analysis of the ventral prostate transcriptome demonstrated 954 genes had significantly altered expression between control and vinclozolin F3 generation animals. Analysis of isolated ventral prostate epithelial cells identified 259 genes with significantly altered expression between control and vinclozolin F3 generation animals. Characterization of regulated genes demonstrated several cellular pathways were influenced, including calcium and WNT. A number of genes identified have been shown to be associated with prostate disease and cancer, including beta-microseminoprotein (Msp) and tumor necrosis factor receptor superfamily 6 (Fadd). The ability of an endocrine disruptor to promote transgenerational prostate abnormalities appears to involve an epigenetic transgenerational alteration in the prostate transcriptome and male germ-line. Potential epigenetic transgenerational alteration of prostate gene expression by environmental compounds may be important to consider in the etiology of adult onset prostate disease.

235. Activin C antagonises activin A in vitro and over-expression leads to prostate pathologies in vivo

Activin A is a well characterised inhibitor of proliferation in most epithelial cells. The actions of activin A on cell growth are mediated through Smad-dependent pathways. Activin A is potent at low levels, therefore its synthesis and bioactivity must be tightly regulated. Follistatin binding or inhibin subunit heterodimerisation block access to the activin receptor and/or receptor activation. We postulate that another mechanism of regulating activin A bioactivity is through the activin-βC subunit. In order to test our hypothesis produced recombinant activin C and mice overexpressing activin-βC. Recombinant activin C abrogated activin A-induced growth inhibition in vitro and the mechanism of action was downregulation of activin A-induced Smad signalling molecules. In the prostate overexpression of activin-βC increased epithelial cell proliferation while there was no significant difference in apoptotic epithelial cells. This imbalance between proliferation and apoptosis led to a significant increase in ventral prostate weight, prostatic hypertrophy and epithelial cell hyperplasia. A significant decrease in nuclear localisation of Smad-2 was associated with activin-βC overexpression in the prostate which implies antagonism of activin signalling also occurs in vivo. This is the first study to provide evidence that activin-βC is an antagonist of activin A in vitro and in vivo and implicates a role for the activin-βC subunit in maintenance of tissue homeostasis in the prostate.

Androgens Regulate the Immune/Inflammatory Response and Cell Survival Pathways in Rat Ventral Prostate Epithelial Cells

A major hurdle in understanding the role of androgens is the heterogeneity of androgen receptor (AR) expression in the prostate. Because the majority of prostate cancer arises from the AR-positive secretory luminal epithelial cells, identifying the androgen-mediated pathways in the prostate epithelium is of great significance to understanding their role in prostate pathogenesis. To meet this objective, the current study was designed to identify immediate-early genes expressed in response to the synthetic androgen R1881 in cultured rat ventral prostate epithelial cells. Rat ventral prostate epithelial cells, purified from 20-d-old rats, were cultured, and the presence of AR and the response to androgen were established. The cells were then treated with R1881 for 2 and 12 h to capture immediate-early genes in an Affymetrix-based gene chip platform. A total of 66 nonredundant genes were identified that were responsive to R1881. The functional androgen response elements were identified in the proximal promoter to determine possible molecular mechanism. Cluster analysis identified five distinct signatures of R1881-induced genes. Pathway analysis suggested that R1881 primarily influences cell proliferation/differentiation and inflammatory/immune response pathways. Androgens appear to regulate cell renewal by regulating differentiation, cell proliferation, and apoptosis. Two mutually exclusive inflammatory response pathways were observed. The interferon pathway was up-regulated, and the ILs were down-regulated. The data identified novel androgen-regulated genes (e.g. Id1, Id3, IL-6, IGF-binding protein-2 and -3, and JunB). The loss of androgen regulation of these genes can have important consequences for cellular transformation and transition to androgen-independent growth and survival.

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.

Ca2+-activated Cl- channel currents in rat ventral prostate epithelial cells.

In many epithelial tissues, the Cl(-) efflux via Ca(2+)-activated Cl(-) channels (Cl(Ca)) play a key role for the fluid secretion. To elucidate the mechanism of prostatic fluid secretion, the properties of whole-cell chloride conductance were investigated. Rat prostate secretory epithelial cells (RPSECs) were isolated by collagenase treatment, and were used for the whole-cell voltage clamp. Both extra- and intracellular monovalent cations were replaced by N-methyl-D-glucamate to record the Cl(-) current selectively. A bath application of Ca(2+)-ionophore, ionomycin (0.2 micro M), increased the membrane conductance with outwardly rectifying voltage-dependence. On step-like depolarization from -60 to +80 mV (500 msec), the ionomycin-induced current showed slowly activating kinetics, a known property of Cl(Ca) current (I(Cl(Ca))) of other tissues. The relative permeability of Cl(Ca) to various anions was calculated from the reversal potentials measured under a total replacement of extracellular Cl(-) with various anions, and the relative order of permeability was SCN(-)>I(-)>Br(-)>Cl(-)>>gluconate. The amplitude of I(Cl(Ca)) was decreased by various anion channel blockers: niflumic acid (100 micro M), DPC (100 micro M), DIDS (1 mM), and NPPB (200 micro M). RPSECs have Cl(Ca) that may provide Cl(-) efflux pathways for the exocrine secretions of the prostate.

The clinical implications of the difference between castration, gonadotrophin releasing-hormone (GnRH) antagonists and agonist treatment on the morphology and expression of GnRH receptors in the rat ventral prostate.

To examine the effects of castration and continuous gonadotrophin releasing-hormone (GnRH) agonist and antagonist therapy on the expression of GnRH-receptors type I (GnRH-RI) in rat ventral prostate (VP) and pituitary tissue, and to compare the effects on prostate morphology. Mature Sprague-Dawley rats were assigned to four treatment groups: group 1, vehicle only; group 2, GnRH agonist goserelin (100 microg/rat/day); group 3, GnRH antagonist cetrorelix (100 microg/rat/day); and group 4, orchidectomy. After 4 weeks the body weights were recorded and VP and pituitary tissue analysed for GnRH-RI expression using a competitive reverse transcriptase-polymerase chain reaction and immunohistochemistry. GnRH antagonist and orchidectomy decreased testosterone secretion and VP volume similarly, but the effects were not identical. The involution of the glandular lumina was more pronounced after orchidectomy while the antagonist therapy was more effective in suppressing epithelial cell proliferation. In the VP, GnRH-RI mRNA levels were increased after GnRH analogue therapy, but were unaffected by orchidectomy. In the pituitary, GnRH-RI mRNA expression was higher in response to orchidectomy and decreased after GnRH analogue treatment. Treatment with a GnRH antagonist for 4 weeks is more effective than an agonist in suppressing testosterone and inducing VP involution. The GnRH antagonist was more effective in suppressing VP epithelial cell proliferation than was castration, suggesting that it induces reduced proliferation by interfering with effects of locally produced GnRH. These results suggest that different regulatory mechanisms may operate in the rat VP than in the pituitary to control GnRH-RI mRNA expression.

K+ channel currents in rat ventral prostate epithelial cells.

Electrophysiological function of the normal prostate has not been extensively studied. In particular, ion channel currents and their regulation have not been studied in freshly-isolated prostate cells. Rat prostate secretory epithelial (RPSE) cells were isolated by collagenase treatment. Columnar epithelial cells were used for nystatin-perforated, whole-cell voltage clamp, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. Step-like depolarizing pulses (900 msec) starting from - 90 mV induced outwardly rectifying K(+) currents without inactivation. ACh (10 microM) or ATP (100 microM) increased the outward current and hyperpolarized the cell membrane potential. Ionomycin (0.1 microM), a Ca(2+) ionophore, induced a similar increase in the outward current. TEA (5 mM), charybdotoxin (50 nM), and iberiotoxin (30 nM) inhibited the effect of ACh (or ATP) on the outward current, whereas apamin (100 nM) had no effect. The [Ca(2+)](i) of RPSE cells was increased by ACh, ATP, and UTP. RPSE cells have iberiotoxin-sensitive Ca(2+)-activated K(+) channels that may play an important role in the exocrine secretions of the prostate.

Mixed lineage kinase (MLK) family members are not involved in androgen regulation of prostatic proliferation or apoptosis*

Once paracrine growth factors are secreted by androgen receptor expressing prostatic stromal cells, they diffuse across the basement membrane of glandular acini, where they bind to epithelial cell surface receptors. This binding stimulates signaling pathways that regulate both the rate of proliferation and apoptosis of prostate epithelial cells. In the present studies, the role of mixed lineage kinases (MLKs) in these signaling processes were studied using a pharmacological approach. The indolocarbazole CEP-1347 (KT 7515) is a potent inhibitor of kinase activity of MKLs. Male rats were treated with CEP-1347 (1 mg/kg of body weight/day) to determine whether inhibition of the MLKs can prevent androgen ablation (i.e. castration) induced apoptosis of prostatic epithelial cells, using as indexes total ventral prostatic DNA content and the percentage of ventral prostatic epithelial cells whose DNA can be terminal transferase end-labeled. In addition, animals previously castrated a week earlier were treated daily with either vehicle or CEP-1347 and exogenous androgen replacement to induce the proliferative re-growth of the prostatic epithelial cells. After 1 week of treatment, the total ventral prostatic DNA content in the vehicle vs. CEP-1347 groups was compared. Using the National Center for Bio-Informatics data bank, MLK2, MLK3, and DLK members of the MLK family are expressed by the normal prostate. Inhibition of the MLKs with CEP-1347 did not affect the kinetics of apoptosis of prostatic epithelial cells induced by androgen ablation. In addition, such MLK inhibition did not prevent androgen replacement induced proliferative regrowth of the prostate epithelium in castrated animals. Signaling through the MLK family is not involved in either the androgen-induced proliferation or the androgen ablation-induced apoptosis of prostatic epithelial cell in the rat.

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation and androgen responsiveness of the ventral prostate without inhibiting prostatic 5alpha-dihydrotestosterone formation or testicular androgen production in rat offspring.

In utero and lactational exposure to a single maternal dose of 1 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg causes some overt toxicity and impairs prostate growth in male offspring. As similar effects on the ventral prostate can be caused by decreased testosterone production during perinatal development, we determined whether intratesticular testosterone content, testicular responsiveness to gonadotropin stimulation, or plasma testosterone concentrations were reduced in fetal and newborn rats. Because these endpoints were not affected, the ability of TCDD exposure to inhibit synthesis of the proximal androgen in prostate development, 5alpha-dihydrotestosterone (DHT), from the circulating precursor testosterone and 5alpha-androstane-3alpha,17ss-diol (3alpha-Diol), was studied on postnatal days (PNDs) 14, 21, and 32. The ability of the ventral prostate to form DHT from 3alpha-Diol was slightly impaired on PND 14, but this transient effect was not statistically significant, and recovery was evident by PND 21. Subsequent experiments used organ culture to study the effects of in vivo TCDD exposure on androgen metabolism, androgen responsiveness, androgen receptor expression, and luminal epithelial cell differentiation after in vitro exposure to graded androgen concentrations. In utero and lactational TCDD exposure had no effect on DHT formation in organ culture, but transiently reduced the androgen -induced expression of prostatic-binding protein subunit C3, decreased ventral prostate epithelial cell androgen receptor expression, and inhibited the formation of androgen responsive luminal epithelial cells. These results suggest that TCDD exposure impairs prostate growth and androgen responsiveness by inhibiting prostatic epithelial cell differentiation.

Prostatic neoplasia in transgenic mice with prostate‐directed overexpression of the c‐myc oncoprotein

BACKGROUND Promoter elements within the 5′ DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration. METHODS The modified promoter and 5′ region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities. RESULTS Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes. CONCLUSIONS Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice. Prostate 43:278–285, 2000. © 2000 Wiley-Liss, Inc.

Prostatic ductal system in rats: changes in regional distribution of extracellular matrix proteins during castration-induced regression.

The extracellular matrix (ECM) is an intricate network composed of an array of molecules that play an integral role in the regulation of cell function, differentiation, and tissue-specific gene expression in various epithelia. In the present study, we examined the distribution of collagen type IV and laminin along the rat ventral prostatic duct before and after castration. Mature Sprague-Dawley rats were castrated and their prostates processed for immunocytochemistry of ECM proteins, laminin, and collagen type IV. Tissue sections were also processed for apoptosis staining, using the 3' end-labeling technique. To examine the effect of ECM proteins on epithelial growth, rat ventral epithelial cells were cultured on ECM-coated surfaces. In the intact rat, laminin was localized in the basement membrane along all regions of the ventral prostate ductal system. Collagen type IV was found to be distributed evenly in the basement membrane of the distal and intermediate regions but was absent or poorly organized in the proximal region, where apoptosis in the epithelium occurs at a high rate. In the regressing prostate after castration, there was a shift in apoptosis from the proximal region to the distal intermediate regions of the prostatic duct. Associated with the shift was a remodeling of basement membrane proteins due to the specific loss of collagen type IV in the distal and intermediate regions. Collagen type IV reappeared underneath the epithelium 7 days after castration, when apoptosis in the epithelium stopped. In vitro, collagen type IV enhanced the growth of ventral prostatic epithelial cells, as assessed by cell number. Collagen basement membrane type IV mediates growth of rat ventral prostate epithelium, and its loss during tissue remodeling after castration is associated with cell death.

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.

Zinc causes a shift toward citrate at equilibrium of the m-aconitase reaction of prostate mitochondria

Prostate secretory epithelial cells have the unique function and capability of accumulating and secreting extraordinarily high levels of citrate. To achieve this, these cells possess a uniquely limiting mitochondrial (m)-aconitase activity that minimizes the oxidation of citrate via the Krebs cycle. The steady-state citrate/isocitrate ratio of mammalian tissues is generally maintained at about 10–11/1, independent of the concentration of citrate, which is the result of the chemical equilibrium reached in the presence of m-aconitase. In contrast, the citrate/isocitrate ratio of prostate tissue is about 30–40/1. Zinc, which is also accumulated in prostate cells at much higher levels than in other cells, inhibits m-aconitase activity thereby minimizing citrate oxidation. This current report is concerned with an effect of zinc on the equilibrium of the reaction catalyzed by m-aconitase. Studies were conducted with mitochondrial extract preparations from rat ventral prostate epithelial cells. With citrate as the initial substrate, the addition of zinc (7–10 μM) to the prostate mitochondrial preparation resulted in a change in the citrate/isocitrate ratio at equilibrium from an average of 10.5/1 to 13.5/1. In contrast, the identical treatment of kidney mitochondrial preparations resulted in no zinc-induced change in the citrate/isocitrate ratio. When either cis-aconitate or isocitrate was employed as the initial substrate, the addition of zinc did not alter the citrate/isocitrate ratio of prostate or kidney preparations. Partial purification of the prostate preparation revealed that the prostate mitochondrial extract contained a putative protein (which we have designated as ‘citrate factor protein’) that is required for the zinc-induced increase in the citrate/isocitrate ratio. This novel effect of zinc provides another mechanism by which it is assured that the accumulation of citrate is maximized in citrate-producing prostate epithelial cells.

Prostatic neoplasia in transgenic mice with prostate-directed overexpression of the c-myc oncoprotein.

Promoter elements within the 5' DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration. The modified promoter and 5' region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities. Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes. Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice.