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Cell Proliferation and Expression of Cell Cycle Regulatory Proteins that Control the G1/S Transition Are Age Dependent and Lobe Specific in the Brown Norway Rat Model of Prostatic Hyperplasia

Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Terry R. Brown, Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Room W3606, 615 North Wolfe Street, Baltimore, Maryland 21205-2103. E-mail: tbrown{at}jhsph.edu.

	Abstract

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Age-dependent epithelial cell hyperplasia in the dorsal and lateral lobes of Brown Norway rats is analogous to benign prostatic hyperplasia in aging men. A major question is whether differential lobe-specific and age-dependent proliferation of cells, rather than cell survival, contributes to the hyperplasia. Although serum testosterone (T) levels decline in aged rats, active cell proliferation was detected as Ki67-positive cells in the dorsal and lateral lobes. We determined whether androgens differentially affect cell proliferation and cell-cycle regulatory proteins in the prostate lobes of young and aged rats. Castrated rats were treated with different doses of T to restore serum levels to those of intact young or aged rats. Rates of cell proliferation, measured by 5-bromodeoxyuridine labeling, peaked after 3-d T treatment in all lobes. 5-bromodeoxyuridine-labeling indices were higher in the dorsal and lateral lobes of aged than of young rats with equivalent serum T levels. No age-dependent difference was seen in the ventral lobe. Cell proliferation was marked by increased levels of cyclins D1 and E and cyclin-dependent kinases 4 and 6, decreased p27 and increased phosphorylation of Rb. Levels of cyclins D1 and E were higher in the dorsal and lateral lobes of intact and T-treated aged than young rats. Confocal immunofluorescent microscopy documented changes in cyclin-dependent kinase 4 and cyclin D1 subcellular localization. Cyclin D1 nuclear localization correlated with the time frame for cell proliferation. In conclusion, rates of cell proliferation and levels of cell-cycle regulatory proteins that control the G1/S transition exhibit lobe-specific and age-dependent differences in response to androgens.

	Introduction

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THE HUMAN PROSTATE is homologous to the dorsal and lateral lobes of the rat prostate based upon common aspects of their embryological origins, biochemical similarities, and susceptibilities to pathophysiological conditions (1). Benign prostatic hyperplasia (BPH) is a pathological overgrowth of the human prostate that develops in a majority of aging men beyond the sixth decade of life. Similarly, spontaneous age-dependent epithelial cell hyperplasia develops in the lateral and dorsal lobes of the Brown Norway rat prostate (2). By comparison, the ventral lobe of the rat prostate has no identifiable homology with human prostate and does not develop hyperplasia in the Brown Norway rat. Thus, the ventral lobe can serve as a comparative control to the dorsal and lateral lobes. Ongoing studies in our laboratory are directed toward the identity of the molecular mechanisms that underlie the lobe-specific and age-dependent development of prostatic hyperplasia in the Brown Norway rat model. The current study addresses whether age-dependent and lobe-specific differences in the rates of prostate epithelial cell proliferation, specifically in response to androgen, play a central role in the development of hyperplasia.

It is well established that the androgens testosterone (T) and its metabolite 5-dihydrotestosterone are essential for the development, differentiation, and functional maintenance of the human and rat prostate glands (3). The effects of androgens on cell survival and proliferation have attracted considerable attention related to the pathogenesis of prostate cancer (4) and BPH (5). In aging men (6, 7, 8, 9), as well as aging rats (10, 11), serum T levels decrease, yet dramatic imbalances in androgen-responsive cell death and cell proliferation lead to age-dependent pathological overgrowth of the prostate in cancer and hyperplasia (12). The primary actions of androgens are mediated by the androgen receptor (AR), a nuclear hormone receptor and ligand-activated transcription factor (3). AR levels are hormonally regulated, and changes in the levels of AR expression and ligand availability are contributing factors in determining the androgen responsiveness of the prostate (13, 14, 15). In young, 4-month-old adult Brown Norway rats, AR levels are higher in the ventral lobe than in the dorsal and lateral lobes (15). Interestingly, by 24 months of age when histological epithelial cell hyperplasia is observed in the rat prostate, AR levels have increased in the dorsal and lateral lobes, but decreased in the ventral lobe, when compared with levels in young adult rats. We previously showed that androgen withdrawal by castration causes rapid and extensive apoptosis of epithelial cells in the ventral lobe, but not the dorsal or lateral lobes of young and aged Brown Norway rats (16, 17). Moreover, exogenous administration of T stimulated a dose-dependent increase in the magnitude of epithelial cell hyperplasia that was unique to the dorsal and lateral lobes of aged Brown Norway rats (18). Hyperplasia was not seen in the dorsal or lateral lobes of young rats, or in the ventral lobe of young or old rats. Together, these findings suggest that androgens may differentially affect both epithelial cell death/survival and cell proliferation in the prostate lobes of the Brown Norway rat as a function of age.

The majority of cells in the adult prostate are quiescent at the G₀ stage of the cell cycle. The critical step in the initiation of cell proliferation is the escape of cells from cell cycle arrest and their entry into the active phase of the cell cycle through the G₁/S restriction point (reviewed in Ref. 19). A burst of cyclin D1-cyclin-dependent kinase (cdk) 4/6 activity is essential for the G₁/S transition to occur in mammalian cells. Activation of the cyclin D1-cdk4/6 complex can initiate phosphorylation and inactivation of retinoblastoma (Rb) protein (reviewed in Refs. 20 and 21). The phosphorylation of Rb frees the E2F transcription factor to activate the transcription of S-phase genes, such as cyclin E. The complex of cyclin E-cdk2 activity further enhances Rb phosphorylation, forming a positive feedback loop. In addition, the activated cyclin E-cdk2 complex can phosphorylate p27^Kip1, a critical member of the Cip/Kip family of cdk inhibitors (22). Phosphorylation of p27^Kip1 signals its recognition and ubiquitination by the SCF (Skp1, Cullin, and F-box protein) ubiquitin E3 ligase (composed of Skp1, Cul1, and the F-box protein, Skp2), that targets p27^Kip1 to the proteasome for degradation (23). The resulting degradation of p27^Kip1 in turn leads to a burst in cyclin E-cdk2 activity.

In the study described here, we investigated the dose-dependent effects of T administration on cell proliferation in the lateral, dorsal, and ventral lobes of the prostate in young and old Brown Norway rats after tissue regression due to androgen withdrawal by castration. In previous studies we showed that regression of the prostate lobes occurred after androgen withdrawal but that apoptosis of epithelial cells was seen only in the ventral lobe, and not in the lateral or dorsal lobes (16, 17). These results suggest that increased survival of cells contributes to the lobe-specific development of age-dependent hyperplasia in the dorsal and lateral lobes. However, other studies showed dose-dependent increased DNA contents of the lateral and dorsal lobes specific to old rats after T administration (18). Based upon these latter studies, we hypothesized that age-dependent differences in the sensitivity of the dorsal and lateral lobes to androgen, related in part to the increased expression of AR in these lobes of aged rats (15), might also promote increased rates of cell proliferation leading to hyperplasia in these lobes of older animals, even in the face of declining serum T levels. In fact, increased numbers of Ki67-positive cells were detected in the lateral and dorsal lobes of aged rats compared with young rats, despite diminished endogenous serum levels of T in aged rats. Moreover, levels of cyclins D1 and E and cdks 4 and 6 were higher in the lateral and dorsal lobes of aged rats than in young rats. To test further the hypothesis of differential androgen sensitivity, T was administered to castrated rats to mimic either the higher or lower serum concentrations of T naturally present in the peripheral circulation of young and old rats, respectively. Interestingly, we observed both age-dependent and lobe-specific differences in the kinetics of cell proliferation consistent with changes in the levels of protein expression, posttranslational modification, and subcellular localization of the cell cycle regulatory proteins cyclin D1, cdk4, cdk6, p27^Kip1, cyclin E, cdk2, and Rb in response to androgen stimulation. Most significantly, our data support a mechanism by which differential responsiveness to androgens provokes higher rates of epithelial cell proliferation in the lateral and dorsal lobes of old rats than in young rats, consistent with the age-dependent development of hyperplasia, whereas no age-dependent differences were observed for the ventral lobe.

	Materials and Methods

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Experimental animals
Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal protocols were approved by the Johns Hopkins University Animal Care and Use Committee. Adult male Brown Norway rats of 4 (young) and 24 (aged) months of age were purchased from Harlan (Indianapolis, IN) by special arrangement with the National Institute on Aging (Bethesda, MD). The rats were housed under standard conditions with access to food and water ad libitum. Animals were castrated via the scrotal route under ether anesthesia with removal of both the testes and epididymides. Fourteen days after castration, animals (n = 6 per age per treatment group) were implanted sc with T-containing capsules that were pre-equilibrated overnight in sterile PBS (pH 7.4). Capsules were prepared by filling SILASTIC brand tubing (No. 602-305, 1.98-mm inside diameter, 3.18-mm outside diameter; Dow Corning, Midland, MI) with T (Steraloids, Wilton, NH), and sealing the ends with silicone type A medical adhesive (No. 891; Dow Corning).

Tubing of different lengths was calibrated in vivo to release T sufficient to maintain steady-state serum concentrations of T equivalent to that of young and aged rats. In 4-month-old castrated animals, the appropriate lengths of the capsules were determined to be 0.1 and 0.5 cm to mimic the endogenous serum T levels of aged and young rats, respectively. Relative to the increased body weight, capsules of 0.5 and 1.0 cm were implanted in 24-month-old castrated rats to mimic the serum T levels of intact aged and young rats, respectively. Additional 4 and 24-month-old castrated animals were implanted with capsules of 6.0 cm to establish superphysiological concentrations of serum T. One hour before euthanasia, animals were injected ip with 5-bromodeoxyuridine (BrdU) (Roche Applied Science, Indianapolis, IN) at a concentration of 10 mg/kg body weight in sterile PBS (pH 7.4). Young and aged rats were killed at 1–4 and 7 d after implantation of T-filled capsules.

Measurement of serum T concentration
Blood was collected and allowed to clot for 2 h on ice. The serum was separated by centrifugation and stored frozen (–80 C) until assayed. Serum aliquots of 100 µl were extracted twice with 4 ml anhydrous ethyl ether, and the combined extracts were taken to dryness under nitrogen. The T concentration (ng/ml) was determined by RIA as previously described (2). All determinations were in duplicate, and the assay was sensitive to 0.05 ng/ml.

Dissection of prostatic lobes
Harvest of the individual prostate lobes was performed as previously described (2). Briefly, the entire urogenital complex was cut from the abdominal cavity of animals immediately after euthanasia and immersed in ice-cold PBS (pH 7.4). The ventral, lateral, and dorsal lobes were separated under a dissecting microscope, blotted to remove excess PBS, and weighed. Each lobe was snap frozen in liquid nitrogen, and stored at –80 C or immersion fixed in 4% neutral buffered paraformaldehyde and embedded in paraffin.

Western blot analysis
Frozen prostate tissues were homogenized in cell lysis buffer [50 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, protease inhibitor cocktail (Roche Applied Science), 1 mM phenylmethylsulfonylfluoride, 10 mM NaF, and 1 mM Na₃VO₄]. Tissue lysates were sonicated to shear genomic DNA and cleared by centrifugation at 14,000 x g for 20 min at 4 C. The supernatants were retrieved and frozen at –80 C until used in immunoprecipitation and immunoblot assays. Protein concentrations were measured by the BCA assay (Bio-Rad Laboratories, Hercules, CA). Aliquots of tissue lysates (equivalent to 50 µg protein) were resolved by electrophoresis on 8 or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA), which were stained with Ponceau S to verify equal protein loading and transfer of the samples. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBS-T) and probed with the following antibodies: anti-cyclin D1(DCS6; Cell Signaling Technologies, Beverly, MA); anti-cdk4 (DCS-35; BD Biosciences PharMingen, San Diego, CA); anti-cdk6 (C-21; Santa Cruz Biotechnologies, Santa Cruz, CA); anti-Rb (G3-245; BD Biosciences PharMingen); anti-phospho-Rb Ser807/811 (no. 9308; Cell Signaling Technologies); anti-phospho-Rb Ser780 (no. 9307; Cell Signaling Technologies); anti-phospho-Rb Ser795 (no. 9301; Cell Signaling Technologies); anti-p27Kip1 (no. 2552; Cell Signaling Technologies); anti-cyclin E (M-20; Santa Cruz Biotechnologies); anti-cdk2 (no. 610145; BD Biosciences PharMingen); and anti-β-actin (A5441; Sigma-Aldrich, St. Louis, MO). After washing in PBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ), developed using West Pico enhanced chemiluminescent reagent kit (Pierce, Rockford, IL), and exposed to Hyperfilm ECL film (Amersham Biosciences). Signal intensities for individual proteins were quantified by scanning of films on a Typhoon 9200 imaging system using ImageQuant software (Amersham Biosciences) and normalized to β-actin protein signal intensity for the same sample.

Immunoprecipitation assays
Tissue lysates were precleared by incubating 1 ml lysate normalized to contain 500 µg total protein with 100 µl 50% vol/vol slurry of protein G agarose beads (Sigma-Aldrich) at 4 C for 1 h on a rotator. Protein G agarose beads were removed by centrifugation at 14,000 g at 4 C for 10 min, and the supernatant was transferred to a clean centrifuge tube. For immunoprecipitation, anti-Rb antibody was added to the supernatant and constantly rotated overnight at 4 C, followed by the addition of 50 µl 50% protein G agarose beads for 2 h at 4 C with mild agitation. After three washes with 1 ml ice-cold lysis buffer, the beads were resuspended in 3x Laemmli sample buffer and boiled immediately. Proteins were resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, and immunoblot analyses were performed using anti-Rb antibody and each of the three different site-specific anti-phospho Rb antibodies as described previously.

Hematoxylin and eosin staining
Tissue sections (6 µm) were cut from the paraffin-embedded ventral, dorsal, and lateral prostatic lobes from intact control, castrated, and castrated T-treated rats. Prostate tissue sections were stained with hematoxylin and eosin and viewed under a light microscope.

BrdU-labeling index (LI)
Prostate tissue sections were deparaffinized in xylene and rehydrated through an ethanol gradient to water. The DNA was partially denatured by treatment with 0.1 N HCl for 20 min. After a 5-min wash in PBS, antigen retrieval was performed by boiling in 10 mM citrate buffer (pH 6.0) for 2 min. Sections were allowed to cool to room temperature for 30 min and washed three times in PBS for 5 min each. Nonspecific binding was blocked by incubation of sections in PBS-T containing 20% normal goat serum (Sigma-Aldrich) and 1% BSA for 30 min at room temperature. Sections were washed briefly with PBS-T and incubated with BrdU antibody (1:1000 dilution, BU33; Sigma-Aldrich) in PBS-T with 1% BSA overnight at 4 C. After three washes in PBS-T for 5 min each, the sections were incubated with Alexa Fluor 488 conjugated goat antimouse secondary antibody (1:150 dilution; Invitrogen-Molecular Probes, Eugene, OR) in PBS-T containing 10% goat serum and 1% BSA for 1 h at room temperature. The sections were then washed three times in PBS-T for 5 min each. Mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Prolong Gold Anti-fade reagent with DAPI; Invitrogen) was added, and sections were coverslipped. Immunofluorescence was observed on a Nikon Eclipse E800 microscope (Nikon, Melville, NY), and images were captured using a CCD camera (Princeton Instruments, Trenton, NJ) and IPLab Spectrum Analysis Software (Scanalytics, Fairfax, VA). The BrdU-LI was calculated as the number of BrdU-positive nuclei in epithelial cells divided by the total number of epithelial cell nuclei counted expressed as a percentage. Nuclei were counted in four areas per tissue section for approximately 1000–2000 total epithelial cells, and this was repeated for three different tissue sections from each lobe of three different animals in each treatment group.

Immunocytochemistry
Processing of prostate tissue sections was as described previously for deparaffinization, rehydration, and antigen retrieval. Sections were incubated for 10 min in 2% H₂O₂ to block endogenous peroxidase activity. Nonspecific binding was blocked with PBS containing 10% species-specific serum and 1% BSA before incubation in PBS containing 1% BSA and specific antibodies. The primary antibodies were the same as those used previously for immunoblots with the addition of Ki67 (clone MM1; Novocastra Laboratories Ltd., Newcastle upon Tyne, UK). For immunofluorescence, antigen binding of primary antibody was visualized using Alexa Fluor 488-conjugated goat antimouse secondary antibody (Invitrogen-Molecular Probes) and/or Alexa Fluor 594 conjugated goat antirabbit secondary antibody (Invitrogen-Molecular Probes). Fluorescence microscopy for BrdU and Ki67 was performed using a Nikon Eclipse E800 microscope, and images were captured using a CCD camera and IPLab Spectrum Analysis Software. The confocal images of cyclin D1 and cdk4 subcellular localization were captured using a Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss, Inc., Thornwood, NY).

Statistical analyses
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 (4 months old) and aged (24 months old) rats were compared by t test (P 0.05).

	Results

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Spontaneous epithelial cell hyperplasia
Focal areas of epithelial cell hyperplasia were evident histologically in the dorsal (Fig. 1, D and G) and lateral (Fig. 1, E and H), but not the ventral (Fig. 1F), prostate lobes from aged (24 months old) compared with young (4 months old) (Fig. 1, A–C) Brown Norway rats. Ki67 is expressed across the active phases of the cell cycle and serves as a biomarker for cell proliferation. We detected expression of Ki67 in numerous epithelial cell nuclei in the dorsal and lateral lobes of 24-month-old rats; these Ki67-positive nuclei were scattered throughout the glandular epithelium (Fig. 2, D and E) but were particularly prominent in focal areas of hyperplasia (Fig. 2, G and H) within these lobes. By comparison, very few Ki67 positive nuclei were detected in epithelial cells of the ventral lobe from 24-month-old rats (Fig. 2F) or the three different prostate lobes from 4-month-old rats (Fig. 2, A–C). These observations suggested a higher rate of epithelial cell proliferation in the dorsal and lateral lobes of aged rats.

View larger version (117K): [in this window] [in a new window]   FIG. 1. Morphology of prostate lobes in 4- and 24-month-old Brown Norway rats. Representative tissue sections from the dorsal (DP), lateral (LP), and ventral (VP) prostate lobes were stained with hematoxylin and eosin to illustrate the histological changes in prostate morphology that occur in rats between 4 and 24 months of age. The dorsal, lateral, and ventral prostate lobes from 4-month-old rats are shown in panels A–C, respectively (magnification, x200). The dorsal, lateral, and ventral prostate lobes from 24-month-old rats are shown in panels D–F, respectively (magnification, x200). Higher magnification (x400) images from the dorsal and lateral prostate lobes of 24-month-old rats are shown in panels G and H, respectively, to highlight the obvious presence of hyperplasia of epithelial cells. Bar, 100 µm.

View larger version (53K): [in this window] [in a new window]   FIG. 2. Immunofluorescent detection of Ki67 in prostate lobes of 4- and 24-month-old Brown Norway rats. Expression of the biomarker, Ki67, of cell proliferation in tissue sections from the dorsal (DP), lateral (LP), and ventral (VP) prostate lobes was identified by immunofluorescent microscopy. Specific Ki67 (green) immunofluorescent staining was detected in the nuclei of cells as the cyan merged signal in the presence of DAPI (blue) nuclear counterstain. Few Ki67 positive nuclei were detected in the dorsal, lateral, and ventral prostate lobes from 4-month-old rats shown in panels A–C, respectively (magnification, x200). Focal hot spots of Ki67 positive nuclei consistent with areas of epithelial cell hyperplasia were detected in the dorsal in panels D (magnification, x200) and G (x400), and in the lateral prostate lobes in panels E (x200) and H (x400), but not in the ventral prostate lobes in panel F (x200) from 24-month-old rats. Bar, 100 µm.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Serum T levels in intact, castrated, and T-treated 4- and 24-month (Mo)-old Brown Norway rats. T concentrations in serum collected from 4- and 24-month-old Brown Norway rats were measured by RIA. Age-dependent serum T concentrations in intact control rats and 14 d after castration in rats of both ages are shown in panel A. Castrated 4- (panel B) and 24-month rats (panel C) were implanted with T-filled SILASTIC brand capsules of 0.1, 0.5, 1.0, or 6.0 cm in length for 1, 2, 3, 4, or 7 d, and serum T concentrations were determined at the time of prostate tissue collection. Mean serum T concentrations are expressed as ng/ml ± SEM (n = 6 rats per group).

View larger version (37K): [in this window] [in a new window]   FIG. 4. Tissue weights of each prostate lobe from intact, castrated, and T-treated 4- and 24-month (Mo)-old Brown Norway rats. Individual prostate lobe weights are shown for 4- (panels A–D) and 24-month-old rats (panels E–H). The weights of the ventral (VP), lateral (LP), and dorsal (DP) lobes are shown for intact and 14-d castrated rats of 4 and 24 months of age in panels A and E, respectively. The weights of the dorsal, lateral, and ventral lobes are shown in panels B–D, respectively, for 4-month-old castrated rats treated with 0.1, 0.5, or 6.0 cm T-filled capsules for 1, 2, 3, 4, or 7 d. The weights of the dorsal, lateral, and ventral lobes are shown in panels F–H, respectively, for 24-month-old castrated rats treated with 0.5, 1.0, or 6.0 cm T-filled capsules for 1, 2, 3, or 4 d. Tissue weights are expressed as mg wet weight/lobe ± SEM (n = 6 rats per group). *, Significantly different from intact control (P < 0.05). **, Significantly different all other T-treated groups (P < 0.05).

View larger version (36K): [in this window] [in a new window]   FIG. 5. Time course and rates of epithelial cell proliferation in the prostate lobes of 4- and 24-month-old castrated Brown Norway rats after T treatment. Castrated rats of 4 (solid bars) and 24 months of age (stippled bars) were implanted with T-filled capsules to establish serum T concentrations equivalent to the endogenous levels of T normally present in the blood of 4- (young-T; left column panels) and 24-month-old rats (aged-T; right column panels). Rats were injected with the thymidine analog, BrdU, 1 h before euthanasia to allow the incorporation of BrdU into newly synthesized DNA in replicating cells. BrdU-positive nuclei were detected by immunofluorescent detection of BrdU, and the total number of nuclei was quantified using DAPI staining. The rates of epithelial cell proliferation were determined from the BrdU-LI (number of BrdU-positive epithelial cell nuclei per total number of luminal epithelial cell nuclei x 100%) at 1, 2, 3, and 4 d during T treatment. Data are expressed as the percentage of BrdU-positive epithelial cells ± SEM (n = 3 animals per group). *, Significantly different for 4- and 24-month-old rats (P < 0.05). DP, Dorsal prostate; LP, lateral prostate; VP, ventral prostate.

View larger version (40K): [in this window] [in a new window]   FIG. 6. Immunoblots of cyclins, cdks, and p27 proteins in the prostate lobes from intact, castrated, and T-treated 4- and 24-month-old Brown Norway rats. Western blot analyses for levels of cyclin D1, cdk4, cdk6, cyclin E, cdk2, and p27 protein expression that regulate the G1/S cell cycle transition were conducted for the dorsal (DP) (upper-left panel), lateral (LP) (upper-right panel), and ventral (VP) (lower-left panel) prostate lobes from intact control, 14-d castrated, and castrated plus 3-d T-treated 4- and 24-month-old rats. Serum T levels were established to mimic the endogenous T levels in 24 (aged-T; 0.8 ng/ml) or 4-month-old rats (young-T; 1.5 ng/ml). β-Actin was used to show equal loading of protein in each lane. The numbers below individual lanes in each panel were calculated as the ratio of the intensity of each protein band to the intensity of the actin band; the ratio of the bands from the young intact control rats was set to 1.0, and values for all other lanes are represented relative to this value. Each sample was derived by pooling the prostate tissues from three animals within each experimental group. All results were replicated in two separate experiments.

Hypophosphorylated Rb is the active form of Rb, whereas (hyper)-phosphorylation by cdk4/6 and cdk2 on multiple serine residues leads to its inactivation and the release of E2F transcription factors that are involved in the active transcription of S-phase genes. Figure 7 shows Western blots of total Rb protein (anti-Rb) and its phosphorylation on specific serine residues (795, 780, and 807/811) in each prostate lobe from young and aged intact rats, in 14-d castrated rats, and in castrated rats treated with T for 3 d. Total levels of Rb were similar within each lobe of the prostate in young and aged animals and were not significantly affected by castration or subsequent T administration. By comparison, levels of Rb phosphorylated at serine residues 780, 795, and 807/811 were 1.6- to 3.8-fold higher in the dorsal (Fig. 7, upper-left panel) and lateral (Fig. 7, upper-right panel) lobes of intact aged animals than in the same lobes of intact young rats; age-dependent differences were not seen in the ventral lobe (Fig. 7, bottom panel). Castration of young and aged rats caused the levels of phosphorylated Rb to decrease by as much as 90% or more in all three lobes of young and aged rats. Administration of T to castrated young and aged rats increased the levels of phosphorylation at each of the serine-specific sites. The levels of these phosphorylated forms of Rb consistently exceeded the levels present in intact control animals only in the dorsal and lateral lobes of young rats.

View larger version (46K): [in this window] [in a new window]   FIG. 7. Immunoblots of Rb protein, Rb, and its phosphorylated isoforms in the prostate lobes from intact, castrated, and T-treated 4- and 24-month-old Brown Norway rats. Protein expression levels of Rb and its serine residue specific phosphorylated isoforms were determined by Western blots using the protein fraction obtained after immunoprecipitation of total Rb protein using a Rb-specific antibody from tissue lysates normalized to contain 500 µg total protein of the dorsal (DP) (upper-left panel), lateral (LP) (upper-right panel), and ventral (VP) (lower-left panel) prostate lobes from intact control, 14-d castrated, and castrated plus 3-d T-treated 4- and 24-month-old rats. Serum T levels were established to mimic the endogenous T levels in 24 (aged-T; 0.8 ng/ml) or 4-month-old (young-T; 1.5 ng/ml) rats. The numbers below individual lanes in each panel were calculated as the ratio of the intensity of each protein band to the intensity of the actin band; the ratio of the bands from the young intact control rats was set to 1.0, and values for all other lanes are represented relative to this value. Each sample was derived by pooling the prostate tissues from three animals within each experimental group. All results were replicated in two separate experiments.

Subcellular (co)localization of cyclin D1 and cdk4
The subcellular cytoplasmic or nuclear localization of cyclin D1 and cdk4 was determined by confocal immunofluorescent microscopy in tissue sections from each lobe of the prostates from intact, castrated, and castrated T-treated young and aged rats (Figs. 8–10). In intact rats, cyclin D1 was predominantly localized to the cytoplasm but was observed in the nuclei of a small number of epithelial cells in the dorsal (Fig. 8, G and M) and lateral (Fig. 9, G and M), but not the ventral (Fig. 10, G and M) lobes of young and aged rats. Notably, nuclear localization of cyclin D1 was evident in some epithelial cells within focal areas of hyperplasia in the dorsal (Fig. 8N) and lateral (Fig. 9N) lobes of intact aged rats. Cyclin D1 was retained in the cytoplasm of epithelial cells in all prostate lobes of young (Figs. 8A, 9A, and 10A) and aged (Figs. 8H, 9H, and 10H) castrated rats, and this predominantly cytoplasmic localization persisted during d 1 (Figs. 8, B and I, 9, B and I, and 10, B and I) and d 2 (Figs. 8, C and J, 9, C and J, and 10, C and J) after T treatment. By d-3 T treatment (Figs. 8, D and K, 9, D and K, and 10, D and K), the increased nuclear localization of cyclin D1 was evident in epithelial cells in all lobes of young and aged rats but began to wane by d 4 (Figs. 8, E and L, 9, E and L, and 10, E and L) and decreased further in young rats by d 7 (Figs. 8F, 9F, and 10F). Therefore, the nuclear localization of cyclin D1 closely correlated with the time course of cell proliferation shown in Fig. 5.

View larger version (89K): [in this window] [in a new window]   FIG. 8. Immunofluorescent localization of cyclin D1 and cdk4 in epithelial cells of the dorsal prostate lobes from intact, castrated, and T-treated 4- and 24-month-old Brown Norway rats. Rats of 4 (top portion) and 24 months of age (bottom portion) were castrated, and the dorsal prostate (DP) was allowed to regress for 14 d (panels A and H), and subsequently treated with 0.5 cm (4 months old) or 1.0 cm (24 months old) T-filled capsules for 1 (panels B and I), 2 (panels C and J), 3 (panels D and K), 4 (panels E and L), or 7 d (panel F). The dorsal prostate from intact 4 (panel G, normal) and 24-month-old rats (panels M, normal, and N, focal hyperplasia) are also shown. Within panels A–N, confocal immunofluorescent microscopy was used to detect the cytoplasmic and nuclear localization of cyclin D1 (green; top-left corner) and cdk4 (red; top-right corner) along with the DAPI-stained nuclei (blue; bottom-left corner). The merged images are shown in the bottom right corner of each panel. Bar, 10 µm.

View larger version (90K): [in this window] [in a new window]   FIG. 9. Immunofluorescent localization of cyclin D1 and cdk4 in epithelial cells of the lateral prostate lobes from intact, castrated, and T-treated 4- and 24-month-old Brown Norway rats. Rats of 4 (top portion) and 24 months of age (bottom portion) were castrated, and the lateral prostate (LP) was allowed to regress for 14 d (panels A and H), and subsequently treated with 0.5 cm (4 months old) or 1.0 cm (24 months old) T-filled capsules for 1 (panels B and I), 2 (panels C and J), 3 (panels D and K), 4 (panels E and L), or 7 d (panel F). The lateral prostate from intact 4 (panel G, normal) and 24-month-old rats (panel M, normal, and panel N, focal hyperplasia) are also shown. Within panels A–N, confocal immunofluorescent microscopy was used to detect the cytoplasmic and nuclear localization of cyclin D1 (green; top-left corner) and cdk4 (red; top-right corner), along with the DAPI-stained nuclei (blue; bottom-left corner). The merged images are shown in the bottom right corner of each panel. Bar, 10 µm.

View larger version (89K): [in this window] [in a new window]   FIG. 10. Immunofluorescent localization of cyclin D1 and cdk4 in epithelial cells of the ventral prostate lobes from intact, castrated, and T-treated 4- and 24-month-old Brown Norway rats. Rats of 4 (top portion) and 24 months of age (bottom portion) were castrated, and the ventral prostate (VP) was allowed to regress for 14 d (panels A and H), and subsequently treated with 0.5 cm (4 months old) or 1.0 cm (24 months old) T-filled capsules for 1 (panels B and I), 2 (panels C and J), 3 (panels D and K), 4 (panels E and L), or 7 d (panel F). The ventral prostate from intact 4- and 24-month-old rats are shown in panels G and M, respectively. Within panels A–M, confocal immunofluorescent microscopy was used to detect the cytoplasmic and nuclear localization of cyclin D1 (green; top-left corner) and cdk4 (red; top-right corner), along with the DAPI-stained nuclei (blue; bottom-left corner). The merged images are shown in the bottom right corner of each panel. Bar, 10 µm.

In summary, cdk4 is localized within nuclei of epithelial cells, including those within focal areas of hyperplasia in the dorsal and lateral lobes of aged rats in which it colocalized with cyclin D1 in a small subset of these cells. The predominant nuclear localization of cdk4 after T treatment contrasted with the relatively few cells in which cyclin D1 was localized to the nucleus. These observations suggest that the nuclear colocalization of cyclin D1 and cdk4 within nuclei of individual cells after T treatment is a transient event that is most closely associated with the time-dependent nuclear localization of cyclin D1. Formation of the nuclear cyclin D1/cdk4 complex triggers the G₁/S cell cycle transition and then rapidly reestablishes the cytoplasmic localization of cyclin D1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The appearance of cellular hyperplasia due to significantly increased numbers of epithelial cells in the dorsal and lateral lobes of aging Brown Norway rats challenges the notion that cell turnover remains balanced throughout the lifespan. The results from our studies establish a new paradigm in which epithelial cell proliferation, as well as cell survival (16), in the rat prostate is lobe specific and age dependent. We previously reported the absence of cell death in the dorsal and lateral lobes of Brown Norway rats after castration (16, 17). Therefore, increases in the BrdU-LIs for the dorsal and lateral lobes in response to physiologically relevant levels of T in our current experiments were somewhat unexpected because there seemingly was no requirement for cell renewal in these lobes after castration. However, T stimulates the proliferation of epithelial cells in these lobes without the prior loss of cells after castration, thereby adding to the total number of epithelial cells present in the tissue and generating the histological appearance of hyperplasia. With regard to the age dependency of hyperplasia, we observed higher BrdU-LIs for the dorsal and lateral lobes of castrated aged rats than for young rats in response to T, thus suggesting that the sensitivity and/or responsiveness of these lobes to androgen is greater in aged rats. The increased sensitivity of the dorsal and lateral lobes in aged rats may be related to the age-dependent increases in AR levels in these lobes (15). Moreover, the age-dependent differences in cell proliferation are accentuated by the T dose-dependent responses observed in the BrdU-LIs for the dorsal and lateral lobes. Epithelial cell proliferation was increased in these lobes of aged rats at levels of serum T that mimic the reduced endogenous levels present in rats at this age, whereas the same levels of T evoked only a minimal proliferative response in these lobes in young rats. These findings suggest that increases in cell proliferation provide a major contribution to epithelial cell hyperplasia in aged rats consistent with earlier observations of dose-dependent and age-related increases in DNA content in the dorsal and lateral lobes of aged but not young rats in response to T administration over a period of 3 months (18).

Results in the ventral lobe present an interesting contrast to those from the lateral and dorsal lobes. In previous studies we observed significant cell death in the ventral lobe of young rats but lesser levels of apoptosis in aged rats after castration (16), and in the current study, ventral lobe weights did not decrease as much in castrated aged rats as in castrated young rats. Although these observations predict that fewer cells die by apoptosis in the ventral lobe of aged rats compared with young rats, T did not evoke age-dependent differences in the number of proliferating cells in this lobe. The rates of cell proliferation in the ventral lobe of young and aged rats were similar as measured by the BrdU-LIs at each of the doses of T. Notably, our observations that the effects of T on cell proliferation in the ventral lobe were independent of the age of the animals are consistent with the absence of age-dependent epithelial hyperplasia in this lobe.

The critical step required for the proliferation of cells is their transition from cell cycle arrest to entry into the active phase of the cell cycle through the G₁/S restriction point. We were initially surprised by the novel finding that significant numbers of epithelial cells in the lateral and dorsal lobes, but not the ventral lobe, of aged than young rats expressed the biomarker of cell proliferation, Ki67. This finding suggested that cells in these lobes, even in intact aged rats with diminished levels of serum T, were in the active phases of the cell cycle. The higher levels of cyclins D1 and E protein expression in the dorsal and lateral lobes of intact aged rats compared with young rats are consistent with the critical role for these cyclins, particularly cyclin D1, in the cell cycle regulatory network that pushes cells into the active phase of the cell cycle (19), thus promoting higher rates of cell proliferation in aged rats. By contrast, an inverse age relationship for cyclins D1 and E expression was observed in the ventral lobe in which age-dependent hyperplasia is absent.

These findings established a basis for investigating the relationship between age-adjusted levels of T and stimulation of cell proliferation relative to the expression of cyclins D1 and E, cdks 2, 4, and 6, the cdk inhibitor p27, and phosphorylation status of Rb protein. The cyclin D1-cdk4/6 complex initiates and the cyclin E-cdk2 complex augments the (hyper)-phosphorylation of the Rb protein, Rb, leading to the release of E2F, which induces entry into S phase of the cell cycle (20, 21). Previous studies had primarily examined the effects of castration and T replacement on the expression of these cell cycle regulatory proteins in the ventral lobe from young adult rats (24, 25), whereas effects related to age or, more specifically, on the dorsal and lateral lobes were largely ignored. We observed that levels of cyclins D1 and E generally declined after castration and increased in response to T replacement. Interestingly, the magnitude of the increase of these cyclins after T treatment was greater in the dorsal and lateral lobes of castrated aged rats than in young rats, in agreement with the age-dependent differences in the BrdU-LIs in these lobes. cdks 2, 4, and 6 were also affected by androgen, their levels of expression being higher in intact rats and after androgen replacement than in castrated rats. The effects of castration and androgen treatment on levels of cdk4 and cdk6 were most striking in the dorsal and lateral lobes of young and aged rats, whereas the effects of androgen on cdk2 were more apparent in young rats. We showed that changes in the phosphorylation status of Rb, rather than absolute changes in the level of Rb protein, occur in response to androgen. The increased phosphorylation of Rb at multiple serine residues was evident in all three prostate lobes of intact and T-treated castrated rats.

The cdk inhibitor, p27, plays an important role in cell cycle regulation both as an inhibitor of cdks and as a protein involved in the assembly of cyclin-cdk complexes (19, 22). Previous studies showed that p27 null mice developed enlarged, hyperplastic prostate glands (26, 27), and tissue specimens from men with BPH had nearly undetectable levels of p27 mRNA and protein compared with normal prostate tissues (26). In our experiments, p27 levels in all three prostate lobes were increased in castrated rats and suppressed after T replacement.

Based upon our immunoblot data, levels of cyclin D1 most closely paralleled the age-dependent and lobe-specific differences in epithelial cell proliferation, consistent with Ki67 expression in intact rats and T-stimulated BrdU-LIs in castrated rats. Interestingly, cyclin D1 appeared in the nuclei of some epithelial cells in the dorsal and lateral lobes but not in the ventral lobe of intact rats, consistent with Ki67 positive immunostaining. In castrated rats, cyclin D1 was predominantly localized to the cytoplasm of epithelial cells in all lobes, whereas its binding partner, cdk4, was seen in both cytoplasm and nucleus. Moreover, nuclear localization of cyclin D1-cdk4 complexes appeared to be a transient event within individual cells based upon the limited numbers of cells in which both cyclin D1 and cdk4 proteins were colocalized within the nucleus at any given time after T stimulation. A greater number of cells exhibited sustained nuclear residence of cdk4 in comparison to cyclin D1, for which nuclear localization was highly dependent upon the time after T treatment. When cell turnover was minimal as in intact rats, cyclin D1 was predominantly localized to the cytoplasm in all three prostate lobes, however, nuclear colocalization of cyclin D1 and cdk4 was detected within nuclei of epithelial cells in the dorsal and lateral lobes of intact aged rats. After T treatment of castrated rats, the cytoplasmic to nuclear shift of cdk4 occurred more rapidly in the dorsal and lateral lobes of aged rats within the first day of treatment, compared with the dorsal and lateral lobes of young rats in which this happened at d 2. By d-3 and 4 T treatment, the vast majority of epithelial cells exhibited nuclear localization of cdk4. By contrast, the nuclear localization of cyclin D1 was transient and time dependent. On d 1 and 2 after T treatment, very few nuclei contained cyclin D1, but this increased dramatically by d 3 and then declined on subsequent days. Together, these data support the premise that nuclear colocalization of cyclin D1/cdk4 complexes, which is most dependent upon the transient nuclear localization of cyclin D1, correspond to the time frame of the proliferative response in the prostate lobes of young and aged rats.

In summary, our studies of cell proliferation in the prostate lobes of young and aged intact rats and castrated rats after administration of age-appropriate physiological levels of T clearly illustrate how age-dependent and lobe-specific proliferation of epithelial cells in the dorsal and lateral lobes contributes to the development of prostatic hyperplasia in the aging Brown Norway rat. Our initial observation of greater numbers of Ki67 positive cells in the lateral and dorsal lobes of intact aged rats than in young rats led to more definitive studies showing higher levels of T-stimulated proliferation of epithelial cells in these lobes of aged rats. These differences were confirmed by the BrdU-LIs, critical differences in the levels of cell cycle regulatory proteins that control the G₁/S restriction point and the time-dependent nuclear colocalization of cyclin D1-cdk4 protein complexes. Importantly, we discovered age-related differences in cell cycle regulatory proteins in T-treated castrated animals and intact animals that reflect responses to changes in the hormonal milieu. Other studies suggest that androgens may have indirect effects on prostate epithelial cell proliferation and survival via andromedins (growth factors, cytokines, and survival factors) that are secreted by stromal cells and act in a paracrine fashion within the stromal-epithelial microenvironment (28). Additional studies will define whether epithelial cell hyperplasia in the aging Brown Norway rat is the result of age-dependent intrinsic changes in epithelial cells, and/or extrinsic changes mediated by the heterogeneous prostatic microenvironment and hormonal milieu.

	Acknowledgments

 
We thank J. Folmer for expert advice in microscopic techniques and H. Chen for assistance with RIA. We appreciate the helpful discussions with Drs. W. Nelson, J. Isaacs, and A. De Marzo, and critical reading of the manuscript by Dr. B. Zirkin. Confocal fluorescent microscopy was conducted in the Integrated Imaging Center of Johns Hopkins University.

	Footnotes

 
This work was supported by National Institutes of Health Grant R01-AG020999 (to T.R.B.).

Submitted by J.Y. in partial fulfillment of the requirements for the Doctor of Philosophy degree to Johns Hopkins University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 25, 2007

Abbreviations: AR, Androgen receptor; BPH, benign prostatic hyperplasia; BrdU, 5-bromodeoxyuridine; cdk, cyclin-dependent kinase; DAPI, 4',6-diamidino-2-phenylindole; LI, labeling index; PBS-T, PBS containing 0.1% Tween 20; Rb, retinoblastoma; T, testosterone.

Received September 13, 2007.

Accepted for publication October 12, 2007.

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