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Endocrinology Vol. 143, No. 5 1825-1832
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Bcl-2 Protein Expression Correlates with Cell Survival and Androgen Independence in Rat Prostatic Lobes

Partha P. Banerjee1, Subhadra Banerjee1 and Terry R. Brown

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins University, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Terry R. Brown, Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, The Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. E-mail: . ppb{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Castration of young and old male Brown Norway rats induces apoptosis in the ventral, but not in the dorsal and lateral, lobes of the prostate gland, and apoptosis in old rats is diminished by 50% compared with that in young rats. In this study we examined the lobe-specific and age-dependent expression of Bcl-2 and Bax proteins. Bcl-2 levels in the ventral lobe were 5-fold lower compared with expression in the dorsal and lateral lobes. Bax expression in the ventral lobe was 2- and 20-fold higher than that in the lateral and dorsal lobes, respectively. In all three lobes, Bcl-2 was detected in epithelial cells, but not in stromal cells, whereas Bax protein was localized in both cell types. After castration, Bcl-2 expression in the ventral lobe decreased significantly from the control level after 2–3 d, but increased significantly by 7–10 d. By contrast, Bax expression increased significantly by d 1, gradually decreased by 2–4 d, and was nearly undetectable by 7–10 d postcastration. In the dorsal and lateral lobes, neither Bcl-2 nor Bax expression was significantly altered after castration. In the ventral lobe of old rats after castration, Bcl-2 followed a pattern of expression similar to that observed in young rats. However, Bax levels were 50% lower in old rats compared with those in young rats on d 1 after castration. Therefore, cell death follows the down-regulation of Bcl-2 expression in the ventral lobe of young and old rats. Moreover, the higher relative levels of Bcl-2 expression in the dorsal and lateral lobes of intact animals and in the ventral lobe by 7–10 d after castration serve to protect cells from apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOTIC CELL death is a physiological process critical for organ development, tissue homeostasis, and elimination of defective or potentially dangerous cells in complex organisms (1, 2, 3, 4). Defects in normal apoptotic cell death mechanisms play a major role in the pathogenesis of various cancers. Therefore, attempts to activate apoptosis provide a therapeutic approach to the treatment of these malignancies (4, 5, 6). Androgen ablation forms a cornerstone in the clinical management of prostate cancer (7, 8), but tumors that initially are sensitive to hormonal therapy eventually progress to androgen independence (9, 10, 11). Presently, our understanding of cell death or survival after androgen ablation in a tissue that is normally androgen responsive is incomplete.

We previously reported the lobe-specific incidence of apoptotic cell death in the prostate gland of Sprague Dawley (12) and Brown Norway (13) rats after castration. In both rat strains apoptosis was largely confined to epithelial cells in the ventral lobe, and only minimal cell death was observed in the lateral and dorsal lobes. This occurs despite the fact that AR are expressed in epithelial and stromal cells of the three prostatic lobes along the entire proximal to distal axis of the luminal ducts (14, 15, 16) and even though maintenance of homeostasis in the prostate gland is androgen dependent (17, 18). These findings suggest that some cells in the androgen-responsive rat prostate require androgen for survival, whereas the survival of other cells is androgen independent. We also observed that the sensitivity of cells to undergo apoptosis after androgen ablation changes with age, represented most dramatically in the ventral lobe of Brown Norway rats (13). These results suggested that epithelial cells in each of the prostatic lobes are not equally dependent upon androgen for their survival and that other factors may confer survival of androgen-independent epithelial cells.

Recent evidence suggests that bcl-2 gene family members may be important in the context of cell survival and apoptosis. Bcl-2 is a member of a multigene family consisting of interacting prosurvival and proapoptotic family members (19). Over the past several years, the Bcl-2 protein has been characterized as a prosurvival, apoptosis-suppressing factor, whereas the Bax protein, another member of the bcl-2 gene family, was identified as an apoptosis-promoting factor (20, 21). The apparent antagonistic action of these two proteins has been described as a cellular rheostat of apoptosis sensitivity, such that the intracellular levels of the Bcl-2 and Bax proteins can direct the death and survival responses of a cell to an apoptotic signal (22). According to this concept, a cell with a higher ratio of Bax/Bcl-2 protein expression will be more sensitive to a given apoptotic stimulus than a similar cell type with a comparatively lower ratio of Bax/Bcl-2.

Therefore, the Bcl-2 family members, in particular the Bcl-2 and Bax proteins, have emerged as critical regulators of apoptosis in a variety of cell systems. The objective of this study was to characterize changes in Bcl-2 and Bax proteins in different prostatic lobes after androgen withdrawal. Regression of the rat ventral prostate lobe after castration is a well documented model for in vivo apoptosis. Moreover, we compared the prototypic events in the ventral lobe with those in the dorsal and lateral lobes where the level of apoptotic cell death is relatively insignificant after androgen ablation. In this model we examined whether the expression and/or abundance of Bcl-2 and Bax proteins are lobe specific and are modulated during the onset of apoptosis in the regressing ventral lobe. In addition, because cell death in the ventral lobe decreases with age, we also compared the expression of Bcl-2 and Bax in this lobe from young and old rats after castration. Our findings indicate that the expression of Bcl-2 and Bax differs in the three lobes of the rat prostate gland and that their expression is differentially regulated during castration-induced regression. Our results confirm previous findings that the relative proportions of Bcl-2 and Bax are important in cell death and survival, such that the higher expression of Bcl-2 can protect cells against death and promote their survival, thereby allowing cells to exhibit androgen independence. Moreover, the correlation between Bcl-2 expression and reduced cell death and/or enhanced cell survival may contribute to the lobe-specific and age-dependent epithelial cell hyperplasia that is present in the dorsal and lateral lobes of aging Brown Norway rats (23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young (4-month-old) and old (24-month-old) male Brown Norway rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), under special arrangement with the NIA (NIH, Bethesda, MD). The rats were housed under standard conditions, with food and water ad libitum. Castration was performed via the abdominal route under ether anesthesia. Epididymides were removed along with the testes. Rats were killed at 1, 2, 3, 4, 7, and 10 d after castration. Animal protocols were approved by the animal care and use committee of The Johns Hopkins University Bloomberg School of Public Health.

Dissection of prostatic lobes
The entire urogenital complex was removed from rats, and the ventral, dorsal and lateral lobes were separated under a dissection microscope as previously described (23). Each lobe was divided into left and right portions to preserve the distal and proximal aspects and weighed. Prostatic fluid was expressed from the left portion of each lobe before snap-freezing the tissue in liquid nitrogen for subsequent determination of Bcl-2 and Bax levels by Western blot analysis. The right portion of each lobe was fixed in neutral buffered paraformaldehyde and embedded in paraffin for Bcl-2 and Bax immunolocalization.

Immunohistochemical localization of Bcl-2 and Bax
The immunocytochemical staining for Bcl-2 and Bax were performed as described previously (24). In brief, tissue sections were deparaffinized, rehydrated, digested with 0.025% pronase, treated to remove endogenous peroxidase activity, and blocked for nonspecific binding. Bcl-2 (BD Transduction Laboratories, San Diego, CA) and Bax (BD PharMingen, San Diego, CA) antisera were diluted to 5 µg/ml and 1:250, respectively, in 1% crystalline grade BSA in PBS, and slides were incubated in a humidified chamber at 4 C for 16–18 h. The reaction sites were then visualized by incubating the tissue sections with biotinylated second antibody, avidin-biotin-peroxidase complex, and diaminobenzidine reagent (Vector Laboratories, Inc., Burlingame, CA). Negative control slides were prepared in an identical manner and consisted of 1) replacement of the primary antibody with similar concentrations of mouse or rabbit IgG, and 2) neutralization of the primary antibody with synthetic peptide for each antigen (Bcl-2, PP52 or Bax, PP51 from Oncogene Research Products, Boston, MA). The localization of immunoreactive Bcl-2 and Bax was observed by light microscopy (Carl Zeiss, Oberkochen, Germany), and the microscopic images were recorded on Kodak 64T color slide film (Rochester, NY) and scanned in Adobe Photoshop Version 5.5 (Adobe Systems Inc., San Jose, CA).

Preparation of tissue extracts for Western blot analysis
Frozen tissue samples were homogenized in tissue lysis buffer [10 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, 0.28 U/ml aprotinin, 50 µg/ml leupeptin, and 0.7 µg/ml pepstatin] as described previously (24). DNA was quantified in an aliquot of tissue homogenate by the Hoechst fluorometric method described previously (25). The tissue homogenates were clarified by centrifugation at 14,000 x g for 20 min at 4 C. The clarified supernatants were mixed (1:1) with 2 x Laemmli buffer (100 mM Tris-HCl, pH 6.8, containing 10% 2-mercaptoethanol, 4% SDS, and 20% glycerol), transferred to a boiling water bath for 5 min, rapidly frozen on dry ice, and stored at -70 C until use.

Samples of protein prepared from prostate tissues were adjusted to reflect equivalent amounts of tissue DNA. This was important because of sample to sample variations in the amount of residual luminal secretory protein, particularly after castration. These samples were subjected to SDS-PAGE on a 10% acrylamide gel and run under reducing conditions, according to the method described by Laemmli (26). After separation, proteins were electrophoretically transferred at 500 mA for 2 h at 4 C to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the method of Towbin et al. (27). The membrane was initially incubated for 1 h with TBS (20 mM Tris-HCl, pH 7.6, with 137 mM NaCl) containing 0.1% Tween 20 and 5% nonfat dry milk to block nonspecific binding. Subsequently, the membrane was incubated for an additional 2 h at room temperature in the presence of Bcl-2 (1:400 dilution) or Bax (1:1500 dilution) antibody or anti-ß-actin (1:8000 dilution; clone AC-15, Sigma) with frequent agitation. The membrane was washed with TBS-Tween 20, incubated with horseradish peroxidase-labeled secondary antibody (1:3000 dilution; Amersham Pharmacia Biotech) for 1 h at room temperature, and washed with TBS-Tween 20. Antibody binding sites were visualized on Hyperfilm (Amersham Pharmacia Biotech) by exposure for either 5 min (Bcl-2 or Bax) or 1 min (ß-actin) using the ECL detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Mol wt markers (Life Technologies, Inc., Grand Island, NY) were run on each gel to confirm the molecular size of the immunoreactive proteins.

Statistical analysis
Data are expressed as the mean ± SEM. Statistical differences within treatment groups were determined by one-way ANOVA. Differences between individual groups were determined with Scheffé’s F test (P < 0.05). Statistical differences between young and old groups of rats were compared by t test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Quantification of Bcl-2 and Bax levels in ventral, dorsal, and lateral prostatic lobes
We knew from our previous studies that cell death occurs predominantly in the ventral lobe and not in the dorsal and lateral lobes. Therefore, we first compared the lobe-specific expression and abundance of Bcl-2 and Bax proteins in the prostatic lobes from young adult (4-month-old) Brown Norway rats. Western blot analysis of Bcl-2 expression revealed a single immunoreactive protein of approximately 26 kDa in all rat prostate tissue extracts (Fig. 1Go). When Bcl-2 levels were compared in the three lobes (Fig. 1Go, top panel) relative to levels of ß-actin (Fig. 1Go, middle panel), the level was 5-fold lower in the ventral than in the dorsal and lateral lobes (Fig. 1Go, bottom panel). By contrast, when Bax protein levels (Fig. 2Go, top panel) were compared relative to levels of ß-actin (Fig. 2Go, middle panel), the level was 20- and 2-fold higher in the ventral than in the dorsal and lateral lobes, respectively (Fig. 2Go, bottom panel). Although Bax protein expression in the dorsal lobe is not evident in Fig. 2Go, a longer exposure time revealed a low, but detectable, level of Bax protein on the Western blots (data not shown).



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  		Figure 1. Western blot analysis of Bcl-2 (top panel) and ß-actin (middle panel) proteins from young rat ventral, dorsal, and lateral prostate tissue extracts. Lanes 1–3 represent the same lobe from three different rats. Tissue extracts of proteins were adjusted to contain equivalent amounts (5 µg) of tissue DNA for samples loaded in each lane. The relative positions on the gel of the molecular mass markers (kilodaltons) are indicated. The bottom panel shows the quantitative analysis of Bcl-2 protein normalized for ß-actin levels in the same tissue samples. Values are the mean ± SEM from three different rats.

 


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  		Figure 2. Western blot analysis of Bax (top panel) and ß-actin (middle panel) proteins from young rat ventral, dorsal, and lateral prostate tissue extracts. Lanes 1–3 represent the same lobe from three different rats. Tissue extracts of proteins were adjusted to contain equivalent amounts (5 µg) of tissue DNA for samples loaded in each lane. The relative positions on the gel of the molecular mass markers (kilodaltons) are indicated. The bottom panel shows the quantitative analysis of Bax protein normalized for ß-actin levels in the same tissue samples. Values are the mean ± SEM from three different rats.

 
Immunolocalization of Bcl-2 and Bax in the ventral, dorsal, and lateral prostatic lobes
Based upon the differences observed for the lobe-specific expression and abundance of Bcl-2 and Bax proteins in the Western blot analyses (Figs. 1Go and 2Go), we examined their immunolocalization in tissue sections from the ventral, dorsal, and lateral lobes. As expected, intense staining for Bcl-2 protein was observed in the dorsal and lateral lobes (Fig. 3Go, B and C) compared with the ventral lobe (Fig. 3AGo). Bcl-2 immunoreactivity was localized exclusively within the epithelial compartment, whereas the stromal compartment was devoid of any immunostaining. By contrast, Bax protein expression was detected by immunostaining in both epithelial and stromal cells of all three lobes (Fig. 3Go, E–G). Moreover, the staining intensity for Bax was much greater in the ventral lobe (Fig. 3EGo) than in the dorsal and lateral lobes. (Fig. 3Go, F and G). When the Bcl-2 (Fig. 3DGo) and Bax (Fig. 3HGo) antisera were neutralized with synthetic peptides for their respective antigens, no immunostaining was detected in prostate tissue sections from any of the lobes, as shown for the lateral lobe where both proteins are expressed.



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  		Figure 3. Immunohistochemical localization of Bcl-2 and Bax in tissue sections of ventral, dorsal, and lateral prostatic lobes from young Brown Norway rats. A, B, and C show immunostaining specific for Bcl-2 protein expression in the ventral, dorsal, and lateral prostate lobes, respectively. D, E, and F show immunostaining specific for Bax protein expression in the ventral, dorsal, and lateral prostate lobes, respectively. Immunostaining was specific for epithelial cells (Epi) or stromal cells (St). Negative controls for Bcl-2 (D) and Bax (H) immunostaining were the result of preabsorption of each antisera with synthetic peptides for their respective antigens before incubation with tissue sections of the lateral lobe. All photomicrographs were obtained at the same magnification, as represented by the bar in D equal to 50 µm.

 
Regulation of Bcl-2 levels in young and old ventral, dorsal, and lateral prostatic lobes
To understand the androgen regulation of Bcl-2 expression in the ventral, dorsal, and lateral lobes from young and old rats after castration, we used Western blot analyses to quantify the relative levels of Bcl-2 expression in tissue extracts (Fig. 4Go). All samples were normalized for ß-actin expression within each sample and for cell number based upon DNA content to compensate for differences in apoptotic cell death between individual lobes that was also dependent on age of the animal and time after castration. The Bcl-2 level was down-regulated in the ventral lobe of young rats within 1 d after castration, coincident with the fall in serum T concentration and the increase in apoptotic cell death (Fig. 4AGo). The Bcl-2 levels in the ventral lobe decreased significantly by 2–3 d in castrated rats. However, by 7 d after castration, when serum T levels were undetectable, the Bcl-2 protein level was significantly up-regulated compared with levels observed between 1–4 d postcastration and compared with the level in the ventral lobe of intact animals. Similarly, in the ventral lobe of old rats (Fig. 4DGo), the Bcl-2 level was also down-regulated by 2–3 d postcastration, but up-regulation of Bcl-2 expression was detected as early as 4 d postcastration. Moreover, the Bcl-2 level was up-regulated by 7–10 d postcastration. In contrast to the ventral lobe of young and old rats, the levels of Bcl-2 expression in the dorsal (Fig. 4BGo) and lateral (Fig. 4CGo) lobes were down-regulated in young rats as well as in old rats (data not shown) during 1–10 d postcastration. However, the relative decrease in Bcl-2 protein expression was not as dramatic as that observed for the ventral lobe.



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  		Figure 4. Western blot analysis of Bcl-2 protein expression in prostate lobes from intact and castrated rats. Intact (0 d) and castrated animals were killed on various days after castration (1, 2, 3, 4, 7, and 10 d). Bcl-2 (top panels) and ß- actin (middle panels) protein expression was determined in tissue extracts from the ventral (A), dorsal (B), and lateral (C) lobes from young rats and from the ventral (D) lobe of old rats. Tissue extracts of proteins were adjusted to contain equivalent amounts (5 µg) of tissue DNA for samples loaded in each lane. The relative positions on the gel of the molecular mass markers (kilodaltons) are indicated. The bottom panels in each section show the quantitative analysis of Bcl-2 protein normalized for ß-actin levels in the respective tissue samples. Values are the mean ± SEM from three different rats. *, Significantly different from intact controls (P < 0.05).

 
Regulation of Bax levels in young and old ventral, dorsal, and lateral prostatic lobes
Similarly, we examined androgen regulation of Bax protein expression by Western blot analyses (Fig. 5Go). The relative level of Bax expression was normalized for ß-actin expression within each sample and for cell number based upon DNA content to compensate for differences in apoptotic cell death between individual lobes that was also dependent upon the age of the animal and time after castration. In the ventral lobe of young rats, the Bax protein level was maximally up-regulated on d 1 after castration (Fig. 5AGo) and began to decline by 2 d after castration, although the relative level of Bax expression remained greater than that in intact control animals. Bax expression returned to the control level by 3 d postcastration and was significantly down-regulated between 4–10 d postcastration. By contrast, in the ventral lobe of old rats (Fig. 5DGo), the level of Bax expression was not significantly increased on d 1 after castration, but was significantly decreased thereafter. In striking contrast to the ventral lobe, Bax protein expression was undetectable in the dorsal lobe of young (Fig. 5BGo) and old (data not shown) rats. In the lateral lobe of young (Fig. 5CGo) and old (data not shown) rats, Bax protein levels did not change immediately after castration; however levels were down-regulated significantly by 7–10 d postcastration.



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  		Figure 5. Western blot analysis of Bax protein expression in prostate lobes from intact and castrated rats. Intact (0 d) and castrated animals were killed on various days after castration (1, 2, 3, 4, 7, and 10 d). The Bax (top panels) and ß-actin (middle panels) protein expression was determined in tissue extracts from the ventral (A), dorsal (B), and lateral (C) lobes from young rats and from the ventral (D) lobe of old rats. Tissue extracts of proteins were adjusted to contain equivalent amounts (5 µg) of tissue DNA for samples loaded in each lane. The relative positions on the gel of the molecular mass markers (kilodaltons) are indicated. The bottom panels in each section show the quantitative analysis of Bax protein normalized for ß-actin levels in the respective tissue samples. Values are the mean ± SEM from three different rats. *, Significantly different from intact controls (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented herein demonstrate that lobe- specific cell death and its age-dependent decrease can be associated with lobe-specific differences in the expression of the Bcl-2 protein family members, Bcl-2 and Bax. We are interested in the regulation of apoptosis in the ventral, dorsal, and lateral lobes of the Brown Norway rat prostate as a contributing factor to the spontaneous and androgen-stimulated, age-dependent epithelial cell hyperplasia that occurs in the lateral and dorsal, but not the ventral, lobes (23, 28). The current study was conducted to compare the expression levels of Bcl-2 and Bax proteins in the different prostate lobes and to determine the effect of castration and influence of aging on their expression.

Previous studies have determined the expression of Bcl-2 and/or Bax in relation to prostate cancer in human and rat models. Furuya et al. (29) examined Bcl-2 protein expression in a series of progressive human and rat prostate cancers that exhibited androgen-sensitive, nonmetastatic or androgen-insensitive, metastatic phenotypes. They observed Bcl-2 protein expression in some, but not all, tumors and concluded that the development of androgen independence and/or metastatic ability was associated with but did not require the expression of Bcl-2. In another study pathological low and high grade prostatic intraepithelial neoplasia was induced in the ventral lobe of Noble rats treated with a combination of T and E2 for 3 and 5 months, respectively (30). Interestingly, Bax, but not Bcl-2, protein expression was detected in the ventral lobe of rats before hormonal treatment. After induction of prostatic intraepithelial neoplasia lesions, both Bcl-2 and Bax proteins were expressed in association with a higher index of both cell proliferation and apoptosis. In our studies the higher relative expression of the antiapoptotic protein, Bcl-2, correlates with the development of age-dependent hyperplasia in the dorsal and lateral lobes of the Brown Norway rat prostate (23).

In the human prostate Bcl-2 protein expression was localized primarily to basal, but not differentiated, secretory epithelial cells (31). In the Brown Norway rat prostate Bcl-2 protein expression was detected in both basal and secretory epithelial cells throughout all segments of the prostatic ducts. Perlman et al. (32) also reported the primary localization of Bcl-2 expression to epithelial cells in the rat ventral prostate by in situ mRNA hybridization. Whereas our studies show that Bax is expressed in epithelial cells of the ventral lobe from Brown Norway rats, it is expressed at relatively lower levels and primarily in stromal cells of the dorsal and lateral lobes. The higher level of Bax expression in the ventral lobe of intact Brown Norway rats is similar to observations reported for the Noble rat (30), although the turnover of cells is normally low in the presence of androgen. The significance of Bax protein expression in the stromal compartment is unknown, as few stromal cells die after androgen ablation. However, the general absence of epithelial cell death in the dorsal and lateral lobes after castration suggests that this may be due to low or absent expression of Bax by these cells. The death effector function of Bax is also dependent upon its cleavage by calpain to the proapototic p18 peptide (33, 34).

To understand the regulation of Bcl-2 and Bax protein expression in different prostatic lobes and to correlate cell survival and cell death with the relative abundance of each protein, we analyzed protein expression after castration of young and old rats. Our results show that the induction of cell death in the ventral lobe of young rats is associated with an increase in Bax and a decrease in Bcl-2 protein expression. Perlman et al. (32) reported a transient increase in Bax mRNA levels in the ventral lobe that peaked at 3 d after castration and declined thereafter. By contrast, Bcl-2 mRNA expression was continuously elevated over a 7-d period after castration. Bax and Bcl-2 protein levels were similar to changes in their respective mRNAs over the time course after castration. These results infer that the relative abundance of Bcl-2/Bax expression in ventral prostate epithelial cells is important for the regulation of cell death and survival. Interestingly, Woolveridge et al. (35) reported that neither Bcl-2 nor Bax expression was altered in the ventral prostate lobe despite the induction of apoptosis after androgen ablation provoked by the testicular Leydig cell toxicant, ethane dimethanesulfonate. Instead, these investigators suggested that changes in the Fas/Fas ligand signal system were responsible for cell death in the ventral lobe. We also observed changes in the relative expression of Bcl-2 and Bax that were consistent with our previous report of diminished cell death in the ventral lobe of old compared with young Brown Norway rats after castration (13). Similarly, our findings that Bcl-2 protein levels in the dorsal and lateral lobes did not decrease by the same magnitude as in the ventral lobe were consistent with the absence of apoptosis in the dorsal and lateral lobes. Moreover, Bax protein levels were unchanged in the dorsal and lateral lobes after androgen ablation.

An intriguing aspect of our study is the survival of numerous epithelial cells in the ventral lobe after castration despite an absence of androgen. These cells expressed levels of Bcl-2 that exceeded levels present in epithelial cells from intact rats. Coincidentally, the levels of Bax protein expression decreased to almost undetectable levels. We observed a similar pattern of expression in the ventral lobe of old rats. In the dorsal and lateral lobes, where no significant cell death occurred after castration, we observed higher relative levels of Bcl-2 protein expression throughout the period after castration. These results confirm the earlier observations by other laboratories that overexpression of Bcl-2 protected human prostate cancer and benign prostatic hyperplasia cells after androgen deprivation (31, 36, 37, 38).

Taken together, we conclude that the relative abundance of Bcl-2 and Bax protein expression is critical in directing cells toward or away from the apoptotic pathway. Moreover, our results corroborate other studies in which the expression of Bcl-2 correlated with a reduction of cell death and androgen independence. Further studies will be required to define additional intracellular factors, including other members of the bcl-2 gene family that affect the apoptotic and/or survival pathways. In summary, our study highlights several findings. First, this is the first report to show that Bcl-2 and Bax proteins are differentially expressed in the three prostate lobes and in epithelial and stromal cells of these lobes. Second, castration experiments show that the expression of Bcl-2 and Bax proteins is differentially regulated in a lobe-specific pattern. Third, the down-regulation of Bcl-2 and the up-regulation of Bax expression are coincident with the induction of apoptosis in the ventral lobe over the time course after castration. Fourth, the age-dependent decrease in cell death correlates with a reduced stimulation of Bax expression. Finally, the relative abundance of Bcl-2 protein correlates with the survival of prostatic epithelial cells. Based on these results, we suggest that lobe-specific differences in apoptosis of cells in the rat prostate play a significant role in the age-dependent epithelial hyperplasia of the lateral and dorsal lobes. Moreover, the rat prostate lobes offer an attractive model to understand the molecular mechanisms of cell death and survival that are pertinent to the androgen dependence of prostate epithelial cells in normal and pathological conditions.


   Footnotes
 
This work was supported by NIH Grant PO1-AG-08321.

1 Present address: Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, D.C. 20007. E-mail: . Back

Received September 18, 2001.

Accepted for publication January 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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