Increased prostate cell proliferation and loss of cell differentiation in mice lacking prostate epithelial androgen receptor

Abstract

Developmental studies of the prostate have established that ductal morphogenesis, epithelial cytodifferentiation, and proliferation/apoptosis are regulated by androgens acting through stromal androgen receptor (AR). Here, we found mice lacking epithelial AR within the mature prostate (pes-ARKO) developed prostate tissue that was less differentiated and hyperproliferative relative to WT littermates. Epithelial AR protein was significantly decreased in 6-week-old mice and was nearly absent by ≥24 weeks of age. Circulating levels of testosterone, external genitalia, or fertility were not altered in pes-ARKO mice. A significant (P < 0.05) increase in bromo-deoxyuridine-positive epithelia was observed in ventral and dorsal-lateral prostates of pes-ARKO mice at 24 weeks of age. Less differentiation was observed as indicated by decreased epithelial height and glandular infolding through 24 weeks of age, differentiation markers probasin, PSP-94, and Nkx3.1 were sig nificantly decreased, and epithelial sloughing and luminal cell apoptosis increased from 6 to 32 weeks of age in pes-ARKO mice. Gain of function occurred by crossing pes-ARKO to the T857A transgenic mice containing constitutively activated AR. In T857A-pes-ARKO mice prostates were of normal size, contained glandular infoldings, and maintained high secretory epithelium, and the appropriate prostatic epithelial proliferation was restored. Collectively, these results suggest that prostatic epithelial AR plays an important role in the homeostasis of the prostate gland. These data support the hypothesis that epithelial AR controls prostate growth by suppressing epithelial proliferation in the mature gland.

Androgens and epithelial-mesenchymal interactions are necessary for prostate growth and development. Androgen signaling occurs through the androgen receptor (AR) (1, 2), which is found in both stroma and epithelium within the prostate. Mice lacking a functional androgen/AR signaling fail to develop normal prostate glands (3, 4). Pioneering studies on normal prostatic development showed that stromal, but not epithelial AR, is essential for specification of epithelial cell identity, bud formation, ductal branching, proliferation, and apoptosis (5, 6). In contrast, experimental evidence from study of anaplastic prostate cancer cell lines has led to the idea that epithelial AR, when activated by androgen, increases cellular proliferation (7–9). This notion is the central premise for androgen ablation therapy, a key treatment for advanced or metastatic prostate cancer. Although many studies demonstrate that stromal, but not epithelial, AR mediates key events during normal prostatic development (5, 6), these studies were typically evaluated over short periods of time, thus recapitulation of events that may take months to manifest a phenotype were not examined. To date, there are no models or techniques that effectively evaluate the role of epithelial AR in situ or in vivo within the mature adult and aging prostate. In addition, it is unclear as to why the mature prostate maintains homeostasis without active proliferation in an androgenic milieu.

In the adult prostate, androgen deprivation leads to luminal cell apoptosis and dedifferentiation, resulting in an increased proportion of basal cells to luminal cells (10, 11). In normal prostate, benign prostatic hyperplasia, and prostate cancer, androgen deprivation decreases growth, increases apoptosis, and reduces tumor volume and is thought to be acting on the epithelial or carcinoma cells. Often, however, the effect is temporary and after removal of androgens, abnormal epithelial cells persist and inevitably grow independent of hormonal stimulus. Every year, >30,000 men die and many more suffer from this enigmatic process. To better understand the role of androgens/AR signaling in normal and malignant prostate epithelial function, it is imperative to evaluate gain and loss of function of AR within the prostatic epithelia. Here, we report the generation of the conditional knockout AR (pes-ARKO) mice that lack AR only in prostate epithelia.

Results and Discussion

Generation and Characterization of pes-ARKO Mice.

Pes-ARKO mice contain a prostate epithelial specific promoter (12) driving cre-recombinase in floxed AR mice (3). Expression of the probasin promoter transgene within the epithelium has been reported to be increasingly expressed from 2 to 7 weeks and sustained expression within the luminal cells is observed throughout life (13). To verify AR gene deletion within the prostate epithelium of pes-ARKO mice, candidate mice were genotyped for probasin-cre transgene and conditional flox-AR allele (Fig. 1a). We also evaluated the specificity of recombination in several key organs by RT-PCR, using primers directed toward exons 1 and 3 of the AR gene. Deletion of AR exon 2 was confirmed by the detection of truncated transcripts via RT-PCR present within the ventral prostate (VP) and dorsal-lateral prostate (DLP) of pes-ARKO mice only (Fig. 1b). The lobe specific expression is consistent with the probasin promoter transgene driven expression in other models (14). No other tissues in WT or pes-ARKO mice contained truncated forms of AR DNA.

Fig. 1.

Characterization of pes-ARKO mice. (a) Genotypes of WT (Left) and pes-ARKO mice (Right, KO). The presence of transgenes cre (110 bp) and floxAR (540 bp) were observed only within pes-ARKO mice. Only WT mice have WT AR gene. IL-2 RNA is present in both mice and serves as PCR genotyping control. (b) Evaluation of AR and floxed AR using RT-PCR was performed by using primers that span exons 1–3 of the AR gene. This result shows one band for WT AR (305 bp) in WT mice. In pes-ARKO mice, which lack exon 2, both WT band (WT AR is present in stroma) and the truncated transcript band (153 bp; deletion of exon 2) appear within DLP and VP, but only the WT band appears in other tissues. (c and d) Evaluation of external (c) and internal (d) organs demonstrated no differences between strains, except for the larger VP size in pes-ARKO mice. (e) Assessment of protein expression of AR (Left) and probasin (Right) in VPs of WT vs. pes-ARKO mice demonstrate a decrease over time in pes-ARKO mice VPs but not in WT mice VPs. (f) To assess fertility, pups per litter from WT female × WT males (red bar) or × pes-ARKO (blue bar) males were compared and found to be not significantly different (Left). Serum testosterone levels (Right) were similar in WT (red bar) and pes-ARKO (blue bar) male mice at weeks 12 and 24, and thus the changes in prostatic phenotypes are not likely to be due to changes in circulating androgens. SV, seminal vesicle; Kid, kidney; U, ureter; AP, anterior prostate; Pr, all lobes of prostate; T, testis; Pe, glans penis. *, P < 0.05; ***, P < 0.001.

There were no differences in external characteristics, including anogenital distances between WT and pes-ARKO mice (Fig. 1c). The internal urogenital organs, excluding prostatic lobes, organs also showed no differences between WT and pes-ARKO mice (Fig. 1d Upper). In contrast, the VPs in pes-ARKO mice had substantially larger VPs at week 24 (Fig. 1d Lower Right). Neither the DLP nor anterior prostate within pes-ARKO mice significantly changed in size.

We confirmed the progressive loss of AR by immunohistochemistry. A labeling index for epithelial AR was determined and tabulated (Fig. 1e Left). AR protein localized in epithelial nuclei slowly decreased with age in pes-ARKO mice compared with WT littermates. By week 24, epithelial AR detection was rare. To indirectly evaluate epithelial AR signaling, we quantified staining intensity of probasin, an androgen-regulated protein in mature animals. Probasin intensity was similar in pes-ARKO and WT littermates until 12 weeks of age, when the reduced level in pes-ARKO samples compared with WT approached significance (P = 0.057). By week 24 and thereafter, this difference was significant (P = 0.027) (Fig. 1e Right). These data suggest that probasin expression is normal before significant loss of AR and epithelial AR decline precedes the loss of an AR-dependent secreted protein, thus confirming earlier studies by Donjacour and Cunha (15).

To determine whether pes-ARKO mice contain abnormalities other than enlarged VPs, we evaluated fertility. We found that there were no significant differences in litter size when either WT or pes-ARKO males were mated to WT females (Fig. 1f). To rule out the possibility that elevated VP size might be due to circulating androgen levels, we measured serum testosterone levels by ELISA. We observed little difference between the WT and pes-ARKO males at 12 or 24 weeks of age (Fig. 1f Right). Together, results shown in Fig. 1 demonstrate an effective time-dependent depletion of the AR that is confined to the prostatic epithelium and that consequences of the AR gene deletion appear to be restricted to the prostate and are without the influence of serum testosterone.

Loss of Differentiated Glandular Structure in pes-ARKO Mice.

The histomorphology of prostates was checked weekly from 2 through 6 weeks of age and then biweekly thereafter until 32 weeks for the anterior prostate, DLPs, and VPs. Early on, the pes-ARKO glands looked normal, showing considerable prostatic budding, and differentiation into tall, columnar glandular epithelium (Fig. 2). In pes-ARKO mice at 9 weeks of age, some ducts within the VP contained epithelial cells that were shorter in height or low cuboidal as compared with taller or columnar epithelia from WT littermates. Concurrent with loss of prostatic epithelial morphology was the loss of glandular infolding in the pes-ARKO mice (Fig. 2a). These changes in histomorphology increased over time, until, at 24 weeks (and later), nearly all ducts contained a dedifferentiated epithelium of low height and lack of glandular infolding (Fig. 2a).

Fig. 2.

Histomorphological changes in the VP of pes-ARKO mice. (a) H&E staining of VPs of WT mice shows glandular infoldings (arrowheads) and tall secretory epithelium through week 32. In pes-ARKO (KO) littermates, we saw these features only at weeks 3 and 6. In week-9 pes-ARKO mice, some VP ducts lose glandular infoldings (*) and have short, poorly differentiated epithelial cells compared with WT littermates. The change in epithelia is evident in ≈50% of ducts within the VP of week 14 pes-ARKO mice. At week 24 (and older), in pes-ARKO mice nearly all glandular infolding and high secretory cells are lost, and the enlarged ducts have many sloughed epithelial cells, fragmented nuclei, and immune cells. By week 32, pes-ARKO mice lack glandular infolding and have squat epithelial cells. (Magnification: ×100; Inset, ×400.) (b–e) At week 14, in pes-ARKO mice, the VP continues to lose glandular infolding. Layers of sloughed epithelial cells are abundant in the prostate lumen. (b) In week 14 pes-ARKO mice, some glandular infoldings (arrow) are present. (c–e) In pes-ARKO mice, infoldings (arrow) become smaller and shorter (c), and infoldings (arrow) constrict (red arrow) at their base (d). Ultimately (e), putative glandular infoldings (arrow) are lost, and sloughed luminal cells appear within the lumen.

In pes-ARKO mice at 14 weeks of age, an increased number of cells were found as detached layers within the prostatic lumen (Fig. 2e). In ducts where histological dedifferentiation appeared, normal glandular infolding could be observed (Fig. 2b) as well as a range of infoldings with constrictions at their base (Fig. 2 c and d). As the infoldings became narrower at their base, cells appeared to have lost polarity, as observed by nuclei moving from basal to apical location. Ultimately glandular infoldings were lost, and apparently sloughed into the prostatic lumen (Fig. 2e).

Loss of Epithelial AR Decreases Androgen-Regulated Gene Expression.

VPs from WT and pes-ARKO mice of different ages were stained for AR and androgen-regulated probasin [supporting information (SI) Fig. 5). At week 3, AR staining in both epithelial and stromal cells was evident in the two strains. At week 6, the pes-ARKO prostates start to have noticeably weaker epithelial AR staining and by week 24 AR staining in the epithelium was undetectable. There were also a small percentage of epithelial cells within the DLP of pes-ARKO mice that lacked AR protein; however, nearly all luminal cells of the anterior prostate were positive for AR. The decline of probasin staining in VP epithelium of the pes-ARKO mice lagged behind that of AR, but was also gone by week 24 (SI Fig. 5). Importantly, stromal AR was seen at all stages evaluated.

Probasin, Nkx3.1, and prostatic secretory protein-94 (PSP94) are three prostate-specific proteins known to be transcriptionally regulated by androgens (16, 17). However, it is unknown whether stromal AR or epithelial AR is responsible for their expression. To evaluate loss of epithelial AR signaling on downstream gene expression, we performed quantitative PCR on VP RNA from WT and pes-ARKO mice at weeks 6, 12, 18, and 32. In pes-ARKO VPs, transcriptional down-regulation of probasin and PSP94 were observed by week 18 and remained lower through the final time point at week 32 (Fig. 3a). Nkx3.1 is a transcription factor that governs prostate morphogenesis and patterning (18) and is a marker of tumor initiation and progression (19). Nkx3.1 gene expression is significantly (P < 0.05) decreased by 12 weeks and remained low through week 32 (Fig. 3a). The decreased expression of Nkx3.1 coincides with the marked loss of glandular infolding. Thus, AR signaling is significantly decreased within prostate epithelia by week 12, and epithelial AR is the major transcriptional factor to regulate these genes.

Fig. 3.

Loss of epithelial AR leads to loss of androgen-regulated protein and gene expression and increased proliferation. (a) Androgen-regulated gene transcription decreases as epithelial ARs are lost. Quantitative PCR was done on VPs from WT (red bar) and pes-ARKO (blue bar) mice. Androgen-regulated genes probasin, PSP94, and Nkx3.1 are all down-regulated in pes-ARKO prostates compared with WT prostates. (Magnification: ×100; Inset, ×400.) (b) VPs were collected from WT (red bar) and pes-ARKO (blue bar) mice during different stages and analyzed for proliferation. Proliferation, as determined by BrdU-positive nuclei primarily occurs in epithelial cells at all stages evaluated. (c) Epithelial cell proliferation is significantly (P < 0.01) higher in pes-ARKO than in WT littermates. Prepuberty, 2–3 weeks; puberty, 4–6 weeks; postpuberty, 7–8 weeks; early adult, 9–22 weeks; late adult, 24–32 weeks. *, P < 0.05.

Although epithelial AR is initially decreased by week 6, androgen-regulated factors (probasin, PSP94, Nkx3.1) have a delayed response in their reduction in gene expression. It is unclear what causes this delay, which might be due to an as-yet-undescribed mechanism that is epithelial AR-independent, for example, stromal AR dependency. These data agree with the report suggesting that epithelial AR governs secretory protein expression (15) and suggest that loss of epithelial AR signaling leads to both biochemical and structural dedifferentiation of the mature prostate.

Mature Prostate Epithelial Cell Proliferation Is Increased in pes-ARKO Mice.

Tissue growth occurs through hypertrophy and/or hyperplasia and is balanced by cell death. The normal adult prostate is growth-quiescent, whereas in prostatic disease organ size and epithelial proliferation increases. At 24 weeks of age, VPs of the pes-ARKO mice were larger than those of their WT littermates. As noted in our histological examination of VPs from pes-ARKO mice, epithelial cells shrunk in size, suggesting that VP enlargement was not due to hypertrophy (Fig. 2a, 14–32 wk). To check for VP proliferation, we evaluated BrdU incorporation. Up to week 14, in prepubescent and mature prostates regardless of WT or knockout genotype, BrdU incorporation was not different and was primarily localized to epithelial cell nuclei (Fig. 3b). However, by week 24, concurrent with nearly complete loss of epithelial AR, BrdU incorporation was significantly higher in prostatic epithelium of pes-ARKO mice than in WT littermates (Fig. 3 b–c and SI Fig. 6 c and d), which was confirmed to have elevated proliferating cell nuclear antigen (PCNA) within prostatic epithelium of pes-ARKO (data not shown). The observed significant increase (P < 0.05) in proliferation was evident in all prostatic lobes except for the anterior prostate. Although a significant increase was observed in DLP and VP, only the VP increased in size. The reason for the increased VP size is unclear. Because prostate epithelia of the VP had a substantially higher BrdU labeling-index relative to DLP, it may be that increased cellular proliferation of the VP led to its increased size, whereas a smaller proliferation rate observed in the DLP did not lead to a significant increase in size. It is possible that loss of AR might increase epithelial proliferation through an Nkx3.1 mediated pathway, because hyperplasia has been observed in Nkx3.1 deficient mice. Although in pes-ARKO mice, increased proliferation was observed through 56 and 80 weeks of age (n = 2; data not shown), no carcinoma in situ was observed. It has been widely interpreted that proliferation of normal prostatic epithelial cells (7, 8) and hence carcinoma cells (20, 21) are induced to proliferate by androgens acting directly through epithelial AR. However, others have demonstrated that androgens acting directly on epithelial AR might not directly regulate proliferation (15, 22, 23). Maintenance of epithelial proliferation in aged adult prostate lacking epithelial AR suggests that epithelial androgen/AR-signaling might induce production of proliferation suppressors within luminal epithelial cells. Such epithelial derived proliferation suppressors might act directly to suppress epithelial proliferation or may act indirectly to regulate epithelial or stromal production of growth factors, which in turn regulate epithelial proliferation. The lack of AR within the epithelium of pes-ARKO mice spatially recapitulates what is observed in fetal and neonatal prostate development in that AR is present only within the stroma and not in the epithelium (24, 25). Interestingly, during this time of development, epithelial proliferation is high (26, 27). The results presented here represent a concept in prostate biology in which epithelial AR is capable of controlling epithelial mitogenesis by acting as a suppressor of epithelial proliferation in the mature prostate.

Using immunocytochemical markers for basal and luminal epithelial cells, we then determined that, as the pes-ARKO animals matured, the p63-positive basal epithelial cell population increased during puberty and then remained elevated, while the cytokeratins (CK)8/18-positive luminal epithelial cell population declined. In contrast, in the WT animals, the basal cell number declined with age, whereas the luminal cell (CK8/18-positive cells) population remained stable (SI Fig. 7).

To evaluate cell death, we used histological and TUNEL staining in VPs from pes-ARKO mice (SI Fig. 7b). We saw little apoptosis or necrosis in the prostatic epithelium of pes-ARKO mice. In the lumen, however, we saw considerable cell death in CK8-positive cells, but not CK5-positive cells (SI Fig. 3). These data demonstrate that lack of epithelial androgen/AR-signaling leads to sloughing of epithelial cells into the lumen (Fig. 2 b–d and SI Fig. 6a) and ultimately epithelial cell death (SI Fig. 6b).

To evaluate which population of epithelial cells increased over time in pes-ARKO mice, we identified each cell type, using cell specific markers. Basal and luminal cells were identified histochemically, using antibodies directed toward CK5 or p63 (28, 29) and CK8/18 (30), respectively. Expression of p63 is critical for maintaining the progenitor-basal cell population that is necessary to sustain epithelial development and morphogenesis (31, 32). As expected, the number of basal cells decreased over time in WT mice, whereas p63 positive basal cell numbers remained elevated through 32 weeks of age in pes-ARKO (SI Fig. 7a). BrdU-positive cells were primarily colocalized with CK5-positive basal cells and to a lesser extent with CK8-positive cells (SI Fig. 6c). Localization of CK8/18 and pan-cytokeratin-positive luminal cells were similar in WT and pes-ARKO through puberty. However, as pes-ARKO mice aged and AR protein expression decreased, expression of CK8/18 and pan-cytokeratin were diminished (SI Fig. 7b).

Sloughing and Apoptosis of Epithelia in the Prostate of pes-ARKO Mice.

To evaluate cell death we used histological analysis and TUNEL staining in VPs from pes-ARKO mice (SI Fig. 6). However, within the lumen, layers of sloughed epithelium, immune cells, and fragmented DNA were observed. TUNEL analyses showed numerous TUNEL-positive cells or nuclear fragments within the prostatic lumen of pes-ARKO mice. The lack of apoptosis within the epithelial layer was not surprising, because lack of androgen/AR signaling during castration induced prostate apoptosis has been shown to be mediated through the stroma (23). Because there were few apoptotic cells within the intact epithelium, this suggested that TUNEL-positive epithelial cells within the lumen had undergone anoikis (33) by detaching from their basement membrane before the detection of DNA fragmentation, leading to an accumulation of TUNEL-positive DNA and scavenging immune cells within the lumen.

Restoring Functional AR Via Knockin of T857A-AR Restores pes-ARKO to a Normal Prostate Phenotype.

Because cellular proliferation and lack of differentiation were observed with removal of WT AR within the prostate epithelia, we wanted to determine whether growth and morphological attributes could be rescued after knockin of constitutively activated T857A-AR (mouse AR mutant equivalent to functional human mutant AR, T877A), found in LNCaP cells and human prostate tumors (34, 35) into prostate epithelia of pes-ARKO mice. Therefore, we created triple compound mutant mice containing T857A-AR, flox-AR, and ARRPB2-cre, resulting in the T857A/pes-ARKO mice, which have no WT AR within the prostate epithelium but express transgenic T857A-AR. Genomic DNA (PCR), mRNA (RT-PCR), and protein assays (immunohistochemistry) all demonstrated that deletion of WT AR and appropriate expression of T857A-AR in T857A/pes-ARKO mice occurred (data not shown). We saw no external differences between T857A/pes-ARKO and WT or pes-ARKO mice. Gross dissection of T857A/pes-ARKO mice revealed little differences in internal urogenital organs between WT and pes-ARKO mice at any stage. Importantly, VPs were similar in size compared with WT littermates. VPs were much smaller in T857A/pes-ARKO mice than in pes-ARKO mice. As anticipated, restoring AR signaling in pes-ARKO mice generated a normal glandular epithelial phenotype quite similar to that of WT mice at week 32 (Fig. 4a). This included the presence of glandular infolding and tall secretory columnar cells. In addition to restoring normal prostate architecture, the expression of functional AR within the epithelia of T857A/pes-ARKO mice stimulated biochemical changes and reexpression of differentiation markers within the epithelium. These changes included increased gene expression of secretory proteins PSP94 and probasin (Fig. 4b). To measure epithelial cell proliferation in T857A/pes-ARKO mice, we determined the BrdU labeling index as described above. Importantly, VPs in T857A/pes-ARKO mice were smaller than those in pes-ARKO mice, suggesting a lack of proliferation like that in prostate from WT mice. The restoration of androgen/AR action within pes-ARKO mice significantly reduced epithelial proliferation to levels not different from WT littermates (Fig. 4b Right). Collectively, these gain-of-function experiments show that AR can suppress prostate epithelial proliferation both in situ and in vivo, a role that has previously been ascribed to stromal factors. However, in those studies implicating stromal factors, tissues were evaluated at 1–2 months, whereas our studies implicating epithelial AR exhibited such effects at >6 months (5, 6).

Fig. 4.

Expression of T857A mutant AR transgene in pes-ARKO mice reverts VP phenotype to WT VPs. To determine whether reexpression of AR(T857A) could rescue the pes-ARKO phenotype, we created compound transgenic mice, and their prostates were evaluated. (a) H&E staining of 32 week-old VPs from WT, pes-ARKO, and pes-ARKO/T857A mice. Note that epithelium in pes-ARKO/T857A mice are very similar in morphology, cell height, architecture, and glandular infolding to WT mice. (Magnification: ×100; Inset, ×400.) (b) pes-ARKO/T857A mice have normal AR-regulated gene transcription levels and proliferation rates. Quantitative PCR for probasin (orange bars) expression in VPs at 32 weeks. In pes-ARKO/T857A mice probasin expression is significantly increased compared with pes-ARKO mice but not different compared with WT. Quantitative PCR for PSP-94 (green bars) expression in VPs at week 32. In pes-ARKO/T857A mice PSP-94 expression is significantly increased compared with pes-ARKO mice but not different compared with WT. BrdU-labeling index (blue bars) in week 32 VPs. In pes-ARKO/T857A mice epithelial cell proliferation is significantly decreased compared with pes-ARKO mice but not different compared with WT. *, P < 0.05.

Conclusion

A key signature of the adult normal prostate gland is the lack of proliferation even in the presence of growth stimulating androgens. This is in contrast to benign prostate hyperplasia and prostate cancer, in which epithelial cells acquire the ability to proliferate. Here, we show two seminal findings in the area of cell biology. First, we report in vivo and in situ that in mature prostatic epithelium, AR is critical for maintaining the differentiated phenotype and overall homeostasis of the gland. Moreover, selective removal of AR signaling in luminal and basal epithelial cells stimulates mitogenesis of the otherwise growth quiescent adult prostate. These data support the hypothesis that epithelial AR maintains homeostasis through induction of epithelial proliferation suppressors or through the decreased production of proliferation-stimulatory factors. These proliferation regulators may indirectly mediate stromal factors or act directly on the putative AR-negative progenitor (i.e., stem cell transit amplifying basal or intermediate) cells, thereby inhibiting epithelial proliferation. The mechanisms by which AR mediates these processes might be multifactorial, most likely involving paracrine factors. Normal prostate growth might require delicate temporal and spatial balance between the proliferative role of stromal AR and the proliferation-suppressive role of epithelial AR. Our findings recast the role of androgen/AR signaling within the prostate and suggest that future chemoprevention strategies might need to target stromal-AR-mediated-factors rather that epithelial-AR-mediated-factors to prevent early stages of carcinogenesis.

Methods

Generation of Transgenic Mice.

To generate pes-ARKO mice, we mated ARRPB2-Cre transgenic mice (13) (C57BL/6N; National Institutes of Health, Bethesda, MD) with mice (C57BL/6J) containing the conditional AR allele (floxed AR; SI Fig. 8) (3). To generate pes-ARKO/T857A AR mice, we interbred the three transgenic mice, ARRPB2-Cre mice (C57BL/6N), floxed AR mice (C57BL/6J), and T857A AR mice (FVB) (gift from N. Greenburg, Fred Hutchinson Cancer Research Center, Seattle, WA).

Immunohistochemistry.

Pes-ARKO specimens: All prostatic lobes were embedded in the same block and sections prepared at 5 μm. Immunodetection was performed as described in refs. 36 and 37. The antibodies used were anti-AR (C-19, 1:200), anti-probasin (1:300), anti-CK8/18 or anti-probasin R-15 (1:100), anti-PCNA (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-p63 (1:50) (Abcam, Cambridge, MA). The ratio of AR-positive to total nuclei was calculated in at least 500 cells examined in each of three randomly selected regions.

RNA Isolation and Analysis.

Total cellular RNA was isolated from each lobe and used to synthesize random primed first-strand complementary DNA for analysis by RT-PCR or real-time PCR (36). Amplification of AR exon 2, Nkx3.1, probasin, and PSP 94 were normalized to beta-actin in each sample. Sequences used were as follows: Probasin, 5′-ATC ATC CTT CTG CTC ACA CTG CAT G-3′ (forward), 5′-ACA GTT GTC CGT GTC CAT GAT ACG C-3′ (reverse); PSP-94, 5′-CCT GTA AGG AGT CCT GCT TTG TC-3′ (forward), 5′-ATG CTG GCT CTG CCT TCT GAG T-3′ (reverse); Nkx3.1, 5′-AGA CAC GCA CTG AAC CCG AGT CTG ATG CAC-3′ (forward), 5′-AGA CAG TAC AGG TAG GGG TAG TAG GGA TAG C-3′ (reverse).

Apoptosis Assay.

The in situ cell death detection kit (Roche Pharmaceuticals, Nutley, NJ) was used according to the manufacturer's instructions for detection of apoptotic cells.

BrdU Labeling Indices.

Mice were injected with BrdU (30 μg per g of body weight; Sigma–Aldrich, St. Louis, MO) i.p. 24 h before killing. The BrdU-labeled epithelial cells were detected employing a monoclonal anti-BrdU antibody (Zymed Laboratories, South San Francisco, CA) according to manufacturer's direction. The labeled cells were calculated from multiple fields of each slide. Several sections from each prostate were analyzed to obtain the mean of BrdU positive epithelial cells. The means of the proliferating cells from each lobe of prostate were reported.

Other Methods.

We performed testosterone quantification, PCR, and Western blot assays as described in ref. 36.

Statistics.

We presented the data as the mean ± SD. We made comparisons between groups, using a two-sided Student's t test. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were considered significant.

Abbreviations

AR

androgen receptor

DLP

dorsal-lateral prostates

pes-ARKO

conditional knockout AR

VP

ventral prostate.