Prostatic Glandular Cells Research Articles

The impact of bcl-2 expression and bax deficiency on prostate homeostasis in vivo

Prostatic glandular epithelial cells undergo apoptosis in response to androgen-deprivation. The molecular determinants of androgen-responsiveness in these cells are incompletely understood. Recent evidence suggests that bcl-2 gene family members may be important in this context. We used the probasin promoter to target a human bcl-2 transgene specifically to the prostate in order to assess its impact on conferring resistance to androgen withdrawal in, otherwise sensitive, prostatic glandular epithelial cells in vivo. We examined the contribution of bax to mediating androgen-responsiveness in prostatic glandular epithelial cells using bax knockout mice. The histologic appearance of the prostates from probasin-bcl-2 transgenic mice or bax-/- mice did not differ from those of control littermates. There was no evidence of hyperplastic or neoplastic growth. There was no difference between probasin bcl-2 transgenic mice, bax-/- mice, and control littermates in steady-state levels of apoptosis. Following castration our findings suggest that both bax and bcl-2 may each contribute to the androgen-responsiveness of prostatic glandular epithelial cells. It is apparent from these results, however, that bax is not required to mediate cell death in prostatic glandular epithelial cells following castration. A comparison between the apoptotic indices in the ventral prostate from the probasin-bcl-2 and bax-/- mice following castration suggests that the presence of bcl-2 may be a more important indicator of androgen-sensitivity than a deficiency of bax.

Expression of macrophage migration inhibitory factor in the human prostate.

The expression of macrophage migration inhibitory factor (MIF) in prostate tissue was investigated by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and Northern blot analysis using a prostate tissue bank. MIF expression was examined in each of the following established prostate tissue categories: prepubertal, pubertal, adult normal, benign hyperplastic (BPH), focal carcinoma within the prostate, and metastatic prostate cancer. IHC showed that all samples tested were positive for MIF protein, which localized to the glandular epithelial cells with no apparent staining of stroma. The most intense staining was observed in the metastatic prostatic adenocarcinoma and the human prostatic adenocarcinoma cell line LNCaP. Using quantitative ELISA, MIF expression was found to be at least three times higher in metastatic adenocarcinoma than in normal, BPH, or focal carcinoma in the adult prostate. This study is the first to report that prostate glandular epithelial cells express MIF. The exact role of MIF in prostate development and disease progression requires further study.

Expression and function of the complement membrane attack complex inhibitor protectin (CD59) in human prostate cancer.

Protectin (CD59) inhibits homologous complement-mediated cytolysis by preventing formation of the membrane attack complex at the point of insertion and polymerization of C9 into cell membranes. The present study investigated the expression and function of CD59 on human prostatic tumor cells in situ and on 5 human prostate cell lines in vitro originating from either metastatic tumors or benign prostate hypertrophy epithelial cells. Immunohistochemical staining of prostate carcinoma tissue with monoclonal antibody (MAb) MEM43 revealed weak to moderately strong expression of CD59 by prostate glandular epithelial cells. Flow cytometry with MEM43 demonstrated that the 5 prostate cell lines expressed different relative quantities of CD59. Indirect immunofluorescence analysis revealed uniform membrane staining of DU145 and PC3 cell lines with no membranous granularity in the staining pattern. Western immunoblots with MAb BRIC 229 showed that PC3 and DU145 cells express CD59 with a m.w. of 18-25 kDa. Treatment of DU 145 and PC3 cells with phosphatidylinositol-specific phospholipase C caused a significant decrease of CD59 expression indicating that the CD59 expressed by prostate cancer cells is anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. PC3 and DU145 cells were completely resistant to human complement-mediated cytolysis but became sensitive to killing in the presence of the CD59-neutralizing MAb YTH53.1. We conclude that malignant and benign human prostate cells express CD59 that is GPI-linked to the cell surface and that CD59 may regulate the immunological response to cancerous prostate cells by protecting the cells from the cytolytic activity of complement.

Incidence of apoptosis and cell proliferation in prostate cancer: relationship with TGF-beta1 and bcl-2 expression.

The incidence of programmed cell death (apoptosis) and cell proliferation was investigated in the normal and malignant human prostate to define the significance of their potential deregulation in human prostate cancer. The incidence of "spontaneous" apoptosis was analyzed using an in situ end-labeling procedure for detection of nucleosomal DNA fragmentation, as well as the pattern and topological localization of expression of the 2 proteins regulating apoptosis, TGF-beta1, and bcl-2, in 40 primary prostatic adenocarcinomas with varying tumor grades, 17 lymph nodes positive for metastatic prostate cancer and 9 normal prostate specimens. The basal level of cell proliferation of the different prostatic cell populations in the same specimens was determined, utilizing the Ki-67 nuclear antigen. Localized prostate cancer cells exhibited a relatively low rate of apoptosis, which was significantly lower than the apoptotic index of normal prostate glandular epithelial cells. Metastatic prostate tumor cells, however, exhibited a significantly higher apoptotic index compared with localized prostate cancer cells. A significant increase in the proliferative index was detected in prostatic tumors compared with the normal gland (5-fold), and there was an even more marked elevation in the proliferative index of the metastatic prostate tumor cells compared to the normal prostate epithelial cells (approximately 24-fold). Immunohistochemical analysis of normal and malignant prostate specimens revealed a predominant TGF-beta immunoreactivity in the glandular epithelial cells, while the stromal component was totally negative. There was a significant increase in the levels of TGF-beta in primary prostatic tumors compared to the normal prostate. Bcl-2 expression was detected among certain populations of tumor epithelial cells in a mutually exclusive topological distribution pattern for apoptosis. In marked contrast, neither TGF-beta1 nor bcl-2 immunoreactivity was detected in metastatic prostate tumor cells, despite their high proliferative and apoptotic rates. Balancing the prostatic growth equation for the prostatic tumor epithelial cell populations revealed a substantial net increase in cell number in both primary and metastatic prostate cancers. This loss of apoptotic control in favor of cell proliferation may be responsible for prostate cancer initiation and progression.

Androgen receptor modulation in benign human prostatic tissue and prostatic adenocarcinoma during neoadjuvant endocrine combination therapy

Modulation of androgen receptor (AR) expression during neoadjuvant endocrine therapy in human prostates of patients with localized prostate cancer was investigated by immunohistochemistry. In 8 of 15 untreated prostatectomy specimens, the majority of prostatic glandular cells displayed nuclear immunostaining for AR, whereas only 1 of 26 pretreated cases displayed a similar glandular AR expression. Expression of AR in the prostatic stromal compartment of nontreated cases proved to be quite heterogeneous, since 4 of the 15 (27%) examined specimens did not show stromal AR expression. After preoperative neoadjuvant therapy, this value was 68%, although this difference did not reach statistical significance. Prostatectomy specimens of the treated patients contained carcinomas with a higher Gleason score than those of untreated patients. AR expression in carcinomas of treated patients was diminished (P=0.05), which may be attributed to their relatively lower differentiation grade. The data strongly suggest that neoadjuvant hormone therapy reduces AR expression by nonneoplastic prostatic glandular cells and carcinoma cells by a selective, but incomplete, elimination of AR-positive cells. © 1996 Wiley-Liss, Inc.

Immunolocalization of Glutathione-Peroxidase (GSH-PO) and Bc1-2 in the Rat Ventral Prostate. Effects of Castration and Administration of Testosterone.

Immunolocalization of glutathione-peroxidase (GSH-PO), apoptosis and bcl-2 protein in the rat ventral prostate was investigated under the presence or absence of androgen. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact 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-propionate daily for three or seven days after two days of castration. The intensity of GSH-PO staining in the glandular epithelial cells of the ventral prostate was remarkably decreased after castration (Group 2), and it was clearly recovered by testosterone-administration (Groups 3 and 4) to the castrated rats. The prostatic GSH-PO mRNA levels were diminished in the castrated rat ventral prostate but greatly increased by testosterone (Groups 3 and 4). Furthermore, castration (Group 2) induced apoptosis in the prostatic glandular epithelial cells and apoptosis was reduced by testosterone-administration (Groups 3 and 4) to the castrated rats. In groups 3 and 4, expression of bcl-2 protein was clearly detected in the glandular epithelial cells of the ventral prostate. These findings strongly suggest that expression of GSH-PO and bcl-2 protein in the glandular epithelial cells of the rat ventral prostate is considered to be testosterone-dependent.

Androgen receptors in untreated and treated prostatic intraepithelial neoplasia.

The effect of androgen deprivation therapy on the expression of androgen receptors (AR) and the proliferation-associated MIB-1 antigen in prostatic intraepithelial neoplasia (PIN) was investigated. Immunohistochemistry was performed on tissue sections of prostatectomy specimens of patients with prostate cancer who either were not treated prior to surgery or received androgen blockade therapy for 3 months. Immunohistochemistry on slides, selected for the presence of PIN was performed with an AR-specific monoclonal antibody and MIB-1 using a microwave antigen-retrieval method. AR expression was present in a majority of cells in PIN of both untreated and treated prostatectomy specimens. In addition, a proportion of normal prostatic glandular cells retained AR expression. The basal cells in both untreated and treated PIN did not express AR. MIB-1-positive cells were identified in PIN of untreated and treated cases. PIN lesions that persist after 3 months of androgen deprivation therapy continue to express AR and may therefore retain the potential to expand after cessation of this therapy.

Androgen ablation-induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction.

Proliferating cells characteristically undergo programmed (i.e. apoptotic) death if their progression through the cell cycle is sufficiently perturbed. To determine whether androgen ablation-induced programmed death of prostatic glandular cells involves apoptosis triggered by recruitment of nonproliferating cells into a perturbed cell cycle, rat ventral prostates were assessed temporally after castration for several stereotypical molecular stigmata of entry into the proliferative cell cycle. Northern blot analysis was used to assess levels of transcripts from genes characteristically activated 1) during the transition from quiescence (G(0)) into G1 of the proliferative cell cycle (cyclin-D1 and cyclin-C), 2) during the transition from G1 to S (cyclin-E, cdk2, thymidine kinase, and H4-histone), and 3) during progression through S (cyclin-A). Although levels of each of these transcripts increased as expected in prostatic glandular epithelial cells stimulated to proliferate by the administration of exogenous androgen to previously castrated rats, levels of the same transcripts decreased in prostatic glandular cells induced to undergo apoptosis after androgen withdrawal. Northern and Western blot analyses also demonstrated that there was no increase in prostatic p53 messenger RNA or protein content per cell after androgen ablation. Likewise, after castration, there was no enhanced prostatic expression of the WAF1/CIP1 gene, a gene whose expression is known to be induced in both a p53-dependent and -independent manner during recruitment from G0 into G1. In addition, androgen ablation-induced apoptosis of prostatic glandular cells was not accompanied by retinoblastoma protein phosphorylation, which is characteristic of progression into late G1. Nuclear run-on assays demonstrated that there was no increase in the prostatic rate of transcription of the c-myc and c-fos genes after castration. These results demonstrate that prostatic glandular cells undergo programmed death in G(0) without recruitment into the G1 phase of a defective cell cycle, and that an increase in p53 protein or its function is not involved in this death process.

Androgen ablation-induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction

Androgen ablation-induced programmed death of prostatic glandular cells does not involve recruitment into a defective cell cycle or p53 induction

Differential gene regulation during programmed death (apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation.

By manipulating the circulating blood level of androgen, it is possible to induce either the programmed death (apoptosis) or proliferation of prostatic glandular cells. To examine the role of differential gene regulation in these two procedure, the expression of the mRNA of a series of genes was quantitated on a per cell basis during the androgen ablation-induced programmed death of rat prostatic glandular cells. These results were then compared to quantitative analysis of the mRNA expression of these same series of genes during the proliferative regrowth of prostatic glandular cells induced in rats castrated for 1 week before being treated with exogenous androgen replacement. These comparisons demonstrated that androgen ablation-induced programmed death of prostatic glandular cells share several (i.e. c-myc, H-ras, and tissue transglutaminase), but not most, of the epigenetic changes associated with androgen-stimulated proliferation of these cells. No enhancement of the mRNA expression of several genes required for entrance of prostatic glandular cells into the S-phase of the proliferative cycle (i.e. histone-H4, c-fos, p53, and ornithine decarboxylase) occurred during androgen ablation-induced programmed death of these cells. These results demonstrated that neither entrance into the S-phase nor progression through a defective proliferative cell cycle is involved in androgen ablation-induced programmed death of prostatic glandular cells. This was further supported by the observation that there is a set of genes (i.e. TRPM-2, transforming growth factor-beta 1, alpha-prothymosin, and calmodulin) in which mRNA expression is only enhanced during programmed cell death and not during proliferation of prostatic glandular cells induced by androgen replacement. These results demonstrate that prostatic programmed cell death is a distinct pathway from cell proliferation involving differential gene regulation.

Differential gene regulation during programmed death (apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation

Differential gene regulation during programmed death (apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation

Cell proliferation, DNA repair, and p53 function are not required for programmed death of prostatic glandular cells induced by androgen ablation.

Androgen ablation induces programmed death of androgen-dependent prostatic glandular cells, resulting in fragmentation of their genomic DNA and the cells themselves into apoptotic bodies. Twenty percent of prostatic glandular cells undergo programmed death per day between day 2 and 5 after castration. During this same period, < 1% of prostatic glandular cells enter the S phase of the cell cycle, documenting that > 95% of these die in G0. During the programmed death of these G0 glandular cells, a futile DNA repair process is induced secondary to the DNA fragmentation. This futile DNA repair is not required, however, since inhibition of this process by > 90% with an appropriately timed hydroxy-urea dosing regimen had no effect upon the extent of the programmed death of these cells after castration. Likewise, p53 gene expression is not required since the same degree of cell death occurred in prostates and seminal vesicles after castration of wild-type and p53-deficient mice.

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.

An Organ Culture System for the Study of Programmed Cell Death in the Rat Ventral Prostate*

Glandular epithelial cells of the rat ventral prostate undergo programmed cell death in vivo following androgen ablation. Fragmentation of the prostatic DNA is an irreversible commitment step in this programmed cell death process. The amount of prostatic DNA fragmentation thus is a quantitative measure of the number of androgen-dependent prostatic glandular cells undergoing programmed death. An in vitro organ culture system was devised for determining rates of prostatic programmed cell death based upon the daily percentage of prostatic DNA fragmentation. To do this, rats were castrated and 2 weeks later treated in vivo for 3 days with exogenous androgen replacement to maximally stimulate DNA synthesis (i.e. proliferation) of the ventral prostatic glandular cells. In vitro organ cultures were established from these ventral prostates and the DNA of these explants was 125I-labeled by incubation in media containing [125I]iododeoxyuridine [( 125I]IDU). Using this in vivo-in vitro DNA labeling technique, greater than 85% of the [125I]IDU radioactivity was incorporated into DNA of the prostatic explants glandular cells. The decrease in 125I-radioactivity from prostatic explants was determined for over a 10-day period of organ culture. Using regression analysis of these data, the daily rate of programmed cell death of the glandular cells was determined. To test the validity of the method, organ cultures were maintained in media capable of inducing either necrotic (i.e. HgCl2-containing media) or programmed cell death (i.e. media lacking testosterone) and the daily decrease in the percentage of [125I]IDU retained in the tissue determined. In addition, the morphologic appearance of necrotic vs apoptotic cell death (i.e. programmed) was quantitated and compared to the [125I]IDU data. These studies demonstrated that this [125I]IDU labeled rat prostatic organ culture system can be used as an in vitro screen to quantitate the ability of various test agents to activate the programmed cell death pathway in prostatic glandular cells.

Role of calcium in the programmed death of rat prostatic glandular cells

By using a newly developed and validated rat ventral prostatic organ culture system in which prostatic glandular cells can be induced to undergo programmed cell death, the role of an elevation in the intracellular free Ca2+ concentration in this death process was studied. By using this organ culture system, ventral prostatic glandular epithelial cells can be maintained in culture for a period of more than 14 days with a low daily rate of cell death (i.e., approximately 5% die per day) if androgen is included in the media. In contrast, if androgen is not included in the media, the daily rate of prostatic glandular cell death increases approximately 3-fold (i.e., approximately 15% die per day). With this organ culture system it has been demonstrated that the daily rate of programmed death of the glandular epithelial cells can be shifted from 5% to 15% of the cells dying per day when testosterone and 10 microM of the Ca2+ ionophore A23187 are both present in the media. Thus, when the intracellular free Ca2+ is elevated within prostatic cells by means of ionophore treatment, the daily rate of glandular cell death in the presence of testosterone is identical to that induced when testosterone is not present in the media. If the organ cultures are maintained in media lacking testosterone but containing 10 microM of the Ca2+ channel blocker nifedipine to inhibit elevations in the intracellular free Ca2+ derived from the extracellular pools, the rise in the daily rate of cell death from 5 to 15% of the cells dying per day induced by androgen ablation can be inhibited by approximately 70%. These results suggest that an increase within prostatic glandular cells in their intracellular free Ca2+ derived from extracellular Ca2+ pools is a critical early event involved in triggering the subsequent process of programmed cell death (i.e., specifically DNA fragmentation) in these cells following androgen ablation.

γ-Seminoprotein に対するモノクローナル抗体の研究

Gamma seminoprotein (gamma Sm), a glycoprotein isolated from human seminal plasma with a molecular weight of 29,000 and possibly a serine protease, has been demonstrated to be one of the prostate organ-specific antigens. We established a murine monoclonal antibody (MoAb) to gamma-Sm in order to prove the presence and localization of this protein in the prostate. The hybrid clones were obtained by fusing mouse SP2/O-Ag-14 myeloma cells with splenocytes from Balb/c mouse immunized with the major fractions of gamma-Sm. The enzyme-linked immunosorbent assay was done for antibody screening. After cloning twice in soft agarose, the stable clone, termed 43-21-1-1, was finally chosen. This MoAb, IgG1(kappa), recognized gamma-Sm specifically, which was verified by an immunoblotting assay. The specificity of the MoAb was further evaluated by immunohistochemical study by the avidin biotin complex method. Periodate-lysine-paraformaldehyde-fixed surgical specimens, including the prostate associated with fibromuscular hyperplasia, seminal vesicles, bladder, testis and epididymis, were examined. Formaldehyde (10%)-fixed surgical specimens from patients with adenocarcinoma of the prostate and primary transitional cell carcinoma arising from the periurethral prostatic ducts were also examined. Positive reactions of gamma-Sm were recognized only in the cytoplasm of prostatic glandular epithelial cells and along the luminal surface. Fibrous and muscular tissues always given negative staining. Neither nonprostatic tissues nor transitional cell carcinoma of the prostate were stained positively for gamma-Sm. These results show that this MoAb (43-21-1-1) is quite specific to gamma-Sm and may be useful for the immunohistochemical study with prostatic tissue.

Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration

Previous studies have demonstrated that the rapid involution of the rat ventral prostate following castration involves the death of the androgen-dependent epithelial cells present within the gland and that this death is the result of a series of discrete biochemical steps. The degradation of genomic DNA into nucleosomal-sized fragments is an early event in this process and is catalyzed by calcium magnesium-dependent endonuclease activity. The morphologic correlation of the involution process involves a series of structural changes which are collectively referred to as apoptosis. The apoptotic process describes the earliest apparent signs of morphologic change exhibited by the dying cells through their eventual complete destruction and deletion from the tissue. The temporal relationship between these recently described biochemical events and the morphologic changes of the apoptotic process were compared in the present study, in order to test the cause versus effect nature of DNA fragmentation in the programmed death of androgen dependent prostatic cells following castration. These studies demonstrated that the early elevation of the Ca+2 Mg+2-dependent endonuclease activity and the fragmentation of DNA into nucleosomal oligomers occurs within prostatic glandular epithelial cells and probably does not involve the direct participation of extraprostatic cells which may subsequently migrate into the gland. Once the DNA is initially cleaved into the nucleosomal oligomers, the subsequent participation of lysosomal enzymes act in a less restricted fashion to degrade both the nucleosomal DNA as well as the cytoplasmic elements and the cell becomes morphologically apoptotic. As the elevations in Ca+2 Mg+2-dependent endonuclease activity and DNA fragmentation are initiated at a time well before the cell is morphologically dead, as defined by apoptosis, these changes in DNA metabolism must not be the consequences of cell death but instead are early causal events in an active process of programmed cell death.

Squamous Metaplasia of the Prostate: An Immunohistochemical Study

Immunoperoxidase strains for prostate-specific antigen (PSA), prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA), and cytokeratins (MAK 6 and CK-KES) were performed on 1 case of squamous cell carcinoma of the prostate and on 13 cases of squamous metaplasia of prostatic epithelium in an effort to demonstrate prostatic origin of the neoplastic and metaplastic cells and to differentiate them from primary or metastatic well-differentiated squamous cell carcinoma. The authors found no specific staining of the metaplastic or neoplastic cells for PSA and only focal single cell PAcP positivity in three cases of squamous metaplasia. All cases showed strong staining of surrounding normal glandular epithelium for both antigens. In all but one case, both the metaplastic and glandular epithelium had positive results for MAK 6 and CK-KES. EMA was expressed strongly in ten cases, was weak or variable in two, and had negative results in two cases of squamous metaplasia. In only four cases did the glandular epithelium have positive results for EMA. The remaining cases showed no staining. PSA and PAcP marking, therefore, may not be useful for separating atypical squamous metaplasia from well-differentiated squamous cell carcinoma or even primary prostatic from metastatic squamous cell carcinoma. These findings suggest that although prostatic glandular epithelial cells retain their ability to express some prostate-associated antigens, this ability is greatly reduced, lost, or not developed in cells that undergo metaplasia into squamous cells or that develop into squamous cell carcinoma.