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Carcinogenesis Advance Access originally published online on August 25, 2005
Carcinogenesis 2006 27(2):216-224; doi:10.1093/carcin/bgi219
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Carcinogenesis vol.27 no.2 © Oxford University Press 2005; all rights reserved.

Sex steroids have differential effects on growth and gene expression in primary human prostatic epithelial cell cultures derived from the peripheral versus transition zones

Alexander Kirschenbaum 3, Xin-Hua Liu 1, Shen Yao 1, Goutham Narla 2, Scott L. Friedman 2, 5, John A. Martignetti 4, 5 and Alice C. Levine 1, *

1 Divisions of Endocrinology and 2 Liver Diseases, Department of Medicine, and 3 Department of Urology, 4 Department of Human Genetics and 5 Department of Oncological Science, Mount Sinai School of Medicine, New York, NY 10029, USA

* To whom correspondence should be addressed at: Department of Medicine, Box 1055, Annenberg Building, Room 23-70, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. Tel: +1 212 241 7509; Fax: +1 212 423 0508; E-mail: alice.levine{at}mountsinai.org


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The majority of human prostate cancers arise from the peripheral zone (PZ). Prostate epithelial stem cells have been localized to the basal epithelial cell compartment. In addition, basal cells have been shown to maintain luminal epithelial cell differentiation and may mediate signals between the stromal and luminal cell compartments. Therefore, the study of adult prostate basal cells derived from different prostate zones may give insights into the mechanisms underlying normal and abnormal prostate growth. We herein compare the basal and sex steroid-stimulated expression and activity of several genes/proteins that are known to be critical in prostate cancer development in primary cultures of basal cells derived from the transition zone (TZ) and PZ of prostatectomy specimens. Our results demonstrate that prostate basal cells derived from the PZ versus TZ are more viable in culture, particularly in response to sex steroid addition. PZ cells exhibit higher telomerase activity and increased expression levels of androgen receptor, the anti-apoptotic protein bcl-2, and the dominant-negative splice variant of Kruppel-like Factor 6. PZ cells have lower basal expression levels of estrogen receptor-beta, the pro-apoptotic protein Bax, and cell-cycle inhibitor proteins (p53, p21waf1/Cip1). Finally, we demonstrate divergent responses to sex hormones in the two basal cell populations. The gene expression pattern in the PZ cells may partially explain the predominance of prostate cancer development in this region.

Abbreviations: AR, androgen receptor; BPH, benign prostatic hyperplasia; CK, cytokerotin; E2, 17ß-estadiol; ER, estrogen receptor; DHT, dihydrotestosterone; HGPIN, high grade prostatic intraepithelial neoplasia; hTERT, human telomerase reverse transcriptase subunit; KLF6, Kruppel-like zinc finger transcription factor; PSA, prostate specific antigen; PSMA, prostate specific membrane antigen; PZ, peripheral zone; SNP, single nucleotide polymorphism; TZ, transition zone


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
It is well known that prostate cancer and high grade prostatic intraepithelial neoplasia (HGPIN) arise predominantly in the peripheral zone (PZ), whereas benign prostatic hyperplasia (BPH) develops largely in the transition zone (TZ) (1–4). Histologically, the epithelial compartments of both zones are similar, consisting of basal, luminal and neuroendocrine cells. Basal cells are believed to play a pivotal role in normal and neoplastic prostate growth because of their strategic location (between stroma and luminal cells), the identification of a small population of stem cells in the basal cell layer and the demonstration that they are essential for the maintenance of normal luminal cell differentiation (5,6). Although the exact cell of origin of prostate cancer is presently unknown, most prostate cancer and HGPIN cells express prostate specific antigen (PSA), androgen receptors (AR) and other markers of luminal cell differentiation. Notably, the hallmark of prostate cancer histology is the absence of a basal cell layer, implying that basal cells may serve a tumor suppressor function. The precise roles of various tumor suppressor proteins [i.e. p53, Kruppel-like zinc finger transcription factor 6 (KLF6)], growth activators (i.e. telomerase), apoptotic and antiapoptotic molecules, and sex steroids in these complex cellular interactions that predispose to prostatic neoplasia are not well delineated.

Sex steroids are unequivocally linked to the development of prostate cancer. Androgens are essential for normal prostate differentiation, the maintenance of luminal epithelial cells and the development of prostatic diseases. Accumulating data, derived from both human and animal studies, indicate that estrogens are also important in these processes. Estrogens have been demonstrated to promote the development of prostate cancer (7–9). Neonatal estrogenization or long-term treatment of adult animals with androgens plus estrogens leads to dysplasia and invasive cancer in rodent models (7,10). It has been proposed that estrogenic influences lead to the accumulation of spontaneous genetic errors in progenitor cells (11). A recent report indicates that estradiol induces a functional inactivation of p53 by intracellular redistribution in a human breast cancer cell line (12). However, there is also data demonstrating that estrogens act as growth inhibitors and differentiating agents in the prostate (7,13). These reportedly divergent effects of estrogens likely reflect the presence of two distinct receptors, estrogen receptor alpha (ER{alpha}) and beta (ERß). ERß expression localizes predominantly to the prostatic basal epithelial cell compartment of the normal human prostate, whereas ER{alpha} is mainly expressed in the stroma of the gland (8). Studies with ER knockout mice conclude that the ER{alpha} subtype of estrogen receptor mediates the imprinting effects of neonatal estrogens on the future development of HGPIN (14). In contrast, ERß appears to mediate estrogenic influences on epithelial cell differentiation in the rodent prostate (15). Other studies confirm that ERß exerts a protective effect against aberrant cell proliferation and carcinogenesis in the prostate (7,8,16).

In the present study, we developed a technique for the isolation and growth in culture of prostate basal cells derived from PZ and TZ of radical prostatectomy specimens. The expression and activity of several genes/proteins that are known to be important in prostate carcinogenesis were examined. The effects of estrogen and androgen on the expression of those genes/proteins were also determined. Our results demonstrate that prostate basal cells derived from PZ and TZ differ in their p53 activity, the expression of hTERT, KLF6, p21waf1/Cip1, Bax and Bcl-2, as well as sex hormone receptors. Moreover, we show that the responses to sex hormones are different in the two basal cell populations. Basal and steroid hormone-induced gene expression in PZ cells favor a more proliferative phenotype.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Prostate tissue specimens and cell culture
Prostate specimens were obtained from five patients undergoing radical prostatectomy for clinically localized prostate carcinoma at the Mount Sinai Medical Center between February and December, 2004 under the guidelines of the Institutional Review Board. Prostate specimens were transected in the mid-portion and PZ and TZ were clearly identified. Frozen sections were obtained prior to fine needle aspiration (PZ and TZ) and were examined after H & E staining under light microscopy by a pathologist. Only areas that did not contain prostate cancer were subsequently aspirated. Utilizing a 20-gauge needle, cells were aspirated from PZ and TZ by a single examiner. The fine needle aspiration, via mechanical dissociation, yields only prostatic epithelial cells and avoids the problem of fibroblast contamination in the primary cultures. After centrifugation, cells were immediately plated on 100-mm culture dishes coated with collagen I, and cultured in a humidified 5% CO2 incubator in Keratinocyte medium (Gibco, Grand Island, NY) containing 20% fetal bovine serum (FBS), 2.5 ng/ml epidermal growth factor (EGF) at 37°C. After initial plating, the cultures contained a mixed population of cells, as assessed by immunohistochemical staining for basal cell-specific (anticytokeratin 903) and luminal cell-specific (PSA and cytokeratin 902) markers (~95% basal, 5% PSA-positive luminal type cells). However, after 48 h in culture and during subsequent passages, all cultures had a uniform cytokeratin expression pattern (basal cell-specific) and did not express PSA. The basal cell phenotype of cultures in subsequent passages was confirmed by both immunohistochemistry and western blot. Each pair of cells was passaged 4–5 times, and all experiments were performed using cells from either the second or third passages. For cell growth experiments, 5 x 104 cells were seeded in each well of 12-well plates and growth assessed by counting cell number in a hemocytometer. All growth experiments were performed in triplicate for each individual sample. The doses and time periods for estradiol and DHT treatments were chosen based on previous experiments from our laboratory and others (11,12,17,18).

Immunohistochemistry
Basal cells were cultured on coverslips for 3 days, washed three times with phosphate buffered saline (PBS) and fixed/permeabilized in prechilled 95% ethanol at –20°C for 30 min. After ethanol aspiration, the cultures were allowed to air dry at 4°C, and stained with targeted primary antibodies for 1 h at room temperature. The coverslips were then washed with PBS three times and incubated with secondary antibodies conjugated with avidin–biotin complex (Vector Labs, Burlingame, CA). The antibodies used were cytokeratin (CK)-903 (antibodies against cytokeratins 1, 5, 10 and 14, selectively stains prostate basal epithelial cells), CK-902 (antibodies against cytokeratin 8 and 18, selectively stains prostate luminal epithelial cells) (both were purchased from Enzo Diagnotics, Farmingdale, NY) and PSA (Santa Cruz, Santa Cruz, CA).

Total RNA extraction and real-time PCR
Cells were incubated in serum-free medium and treated with or without various steroid hormones. Total RNA was extracted with Trizol Reagent (Gibco BRL, Gaithersberg, MD). cDNA was prepared by incubating 1 µg of total RNA in 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT and RNase inhibitors with 250 U of reverse transcriptase, 1 µM of each dNTP and random primers (0.05 µM, Gibco BRL) for 60 min at 37°C. The fragment was PCR amplified by using specific primers. For human telomerase reverse transcriptase (hTERT), forward: 5'-CGGAAGAGTGTCTGGAGCAA-3' and reverse: 5'-GGATGAAGCGGAGTCTGGA-3'. Primers for ß-actin: forward: 5'-GAAGAG-CTACGAGCTGCC-3'; and reverse: 5'-TGATCC-ACATCTGCTGGA-3'. PCR was initiated in a thermal cycle programmed at 95°C for 5 min, 94°C for 30 s, 60°C for 30 s, 72°C for 45 s, and amplified with 28 cycles for ß-actin, and 35 cycles for hTERT. The amplified products were visualized on ethidium-stained 1.5% agarose gels.

Preparation of cell lysates, cytosolic and nuclear proteins
Basal cells cultured under the desired conditions were lysed as described previously (19). Briefly, cells were rinsed twice with ice-cold PBS and scraped with 1.5 ml of PBS containing 4 mM iodoacetate. After centrifugation, the pellets were resuspended in CHAPS extraction solution [10 mM CHAPS, 2 mM EDTA (pH 8.0) and 4 mM iodoacetate in PBS] with protease inhibitors. The samples were then incubated for 30 min on ice and centrifuged at 15 000 g for 10 min. The supernatants were collected and stored at –70°C. Proteins from the cytosolic and nuclear fractions were isolated using a commercial kit purchased from PIERCE (Rockford, IL), according to the manufacturer's instructions. Protein content was assayed using a kit from Bio-Rad (Hercules, CA).

Immunoblotting
Cell lysates were electrophoresed on SDS–PAGE transferred to a polyvinylidene difluoride membrane (DuPont, NEN), and incubated with targeting antibodies overnight at 4°C. Secondary horseradish peroxidase-linked donkey anti-mouse IgG (Amersham, Arlinton Heights, IL) was used. Filters were developed by the enhanced chemiluminescence system (Amersham). Antibodies against p53 were purchased from Cell Signaling Tech. (Beverly, MA). Antibodies against AR, KLF6, p21waf1/Cip1, and Bax were obtained from Santa Cruz. Antibodies against Bcl-2, ER{alpha} and ERß were purchased from Calbiochem (San Diego, CA). Antibodies against CK14 and CK18 were products from BD Biosciences (Palo Alto, CA). Actin was used as the internal control in all western blot analyses. Membrane stripping was performed using western blot stripping buffer (Pierce).

Luciferase assay
Luciferase assay was performed as described previously (20). The reporter plasmid, pGL3-Luc-E1bTATA, a gift from Dr J. Manfredi (Department of Oncological Sciences, Mount Sinai School of Medicine), was used to assay p53 transcriptional activity. The plasmid contains the p21 promoter sequences and the coding region for firefly luciferase upstream of the minimal adenovirus E1b promoter. Cells were cultured in 12-well cluster plates and cotransfected with 1 µg of either the reporter plasmid, empty pGL3-Luc vector or empty pcDNA plasmid. Cells were cotransfected with a ß-galactosidase expression vector driven by the CMV promoter (ClonTech) as an internal control. Total DNA transfected into each plate was 2 µg in each well of a 12-well plate. After 40 h, the transfected cells were lysed by scraping into reporter buffer (ClonTech), total protein concentration was determined and luciferase and ß-galactosidase activities were assayed and quantitated using a TD-20e Luminometer. The resulting activities were normalized to protein concentrations and ß-galactosidase activity.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Both PZ- and TZ-derived cultures express predominantly basal cell markers
Previous reports demonstrate that basal versus luminal prostate epithelial cells can be differentiated based upon their protein expression patterns. Basal cells express high molecular weight cytokeratins 5 and 14, whereas luminal cells express low molecular weight cytokeratins 8 and 18. In addition, there are other basal cell-specific proteins (i.e. p63, ER-beta, maspin, c-met) and luminal cell-specific markers [PSA and prostate specific membrane antigen (PSMA)] (5,6,21). Immunohistochemical staining with both anti-CK903 and CK902 antibodies, which recognize basal cell-specific markers and luminal cell-specific markers respectively, as well as PSA revealed that cultures derived from both the PZ and TZ, expressed only basal cell-specific markers after 48 h in culture and during all subsequent passages (Figure 1A). Immunoblotting was also performed with the CK14 (a basal cell marker) and CK18 (a luminal cell marker) to confirm the basal cell phenotype of these cultures (Figure 1B). The cytokeratin and PSA expression patterns did not change from passages 1 to 5 as determined by immunohistochemistry (data not shown) nor did estradiol or DHT addition alter the basal phenotype, as assessed by western blot (Figure 1B). The demonstration that adjacent frozen tissue sections contained only benign pathology (See Materials and methods) coupled with the basal cell phenotype of the cultured cells, indicates that both the PZ- and TZ-derived cultures are derived from non-cancerous tissue, as basal cells are absent in areas of prostate cancer.


Figure 1
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  		Fig. 1. Determination of basal versus luminal cell-specific markers in cultured prostate epithelial cells. (A) Immunohistochemical analysis of basal cell and luminal cell markers in basal cells. Basal cells were cultured on coverslips for 3 days, and stained with antibodies against CK 903 (basal cell-specific cytokeratins) or CK 902 (luminal cell-specific cytokeratins) or PSA (luminal cell-specific) for 1 h at room temperature. Samples were examined under a microscope (magnification x200). (B) Western blot analysis on the expression of basal cell and luminal cell markers in basal cells. Basal cells were cultured in serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total protein was isolated and subjected to western blot analysis using an antibody against CK14. After stripping, the membrane was sequentially re-probed with CK18 and actin. In each lane 20 µg of protein were loaded. Data shown are representative of paired samples from five individual patients.

 
Expression of AR, ER{alpha} and ERß in PZ- versus TZ-derived cultures
The effects of steroid hormones are mediated through their cognate receptors. In addition to androgens, estrogen signaling pathways have regained considerable attention in contemporary prostate cancer research. Divergent effects of estrogens may be due to differential expression of the ER{alpha} and ERß subtypes. We examined the expression of AR, ER{alpha} and ERß in the two cell populations. As shown in Figure 3, levels of ER{alpha} expression were similar in cultures derived from the two zones and was not modulated by steroid hormone addition. Both PZ and TZ cells expressed similar basal levels of AR, however, the levels were further increased by the treatment with E2, DHT or the combination of E2 plus DHT only in PZ cells. Finally, PZ cells had lower expression levels of ERß and no induction of its expression by steroid hormone addition. In contrast, TZ cells had higher basal expression levels of ERß, which were further increased by the addition of E2 (Figure 2).


Figure 2
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  		Fig. 2. Steroid hormone receptor expression in PZ and TZ basal cells. (A) Western blot analysis. Basal cells were cultured in serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total protein was isolated and subjected to western blot analysis using an antibody against AR. After stripping, the membrane was sequentially re-probed with ER{alpha}, ERß and actin antibodies. In each lane 20 µg of protein were loaded. Data shown are representative of paired samples from five individual patients. (B) Quantitative analysis of AR, ER{alpha} and ERß protein expression in PZ- and TZ-derived basal cells. The relative amount of specific protein was quantitated by densitometry. Data expressed as the ratios of receptor proteins to actin, and shown are mean ± SE of paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells.

 
Comparison of cell viability in TZ- versus PZ-derived basal cells
We initially compared the basal cell from PZ versus TZ, and examined the effects of estrogen and androgen on this parameter. As demonstrated in Figure 3, during a period of 7 days in culture in serum-free medium, PZ cells had increased cell viability compared to TZ cells. Treatment with E2, or DHT, or a combination of E2 and DHT, further increased the viability of PZ cells (90, 60 and 110%, respectively). In contrast, cells derived from the TZ exhibited a significantly lower response to these steroid hormones. Only DHT alone produced a marginally significant increase in TZ cell viability (35%). As previously noted, neither passage in culture nor steroid hormone treatment altered the cytokeratin or PSA expression pattern of the cultured cells, which exhibited a basal cell phenotype throughout the course of these experiments.


Figure 3
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  		Fig. 3. Comparison of cell viability in TZ- versus PZ-derived basal cells. (A) Basal cells were plated in 12-well cluster dishes with 1 ml serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 7 days. Medium was changed every other day. Cells were harvested, and cell viability was determined by counting the number of living cells (identified by trypan blue staining) in a hemacytometer. (B) Quantitative analysis of cell viability in TZ- versus PZ-derived basal cells. The data represent mean ± SEM in paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells.

 
Estrogen and androgen prevent p53 nuclear localization in PZ-derived basal cells
Although the effect of p53 in prostate cancer development is controversial, a recent report suggested that non-functional p53 protein may be one of the triggers for cellular proliferation in the diseased prostate (3). Girinski et al. (22) also demonstrated that inactivation of p53 protein (rather than gene mutation) might underlie the cancerous predisposition of PZ basal cells. In addition, estrogen has been reported to induce p53 functional inactivation via its intracellular redistribution (12). To determine the cellular mechanisms that might underlie the observed growth characteristics, we examined the expression, intracellular localization and activity of p53 in PZ versus TZ cells. As demonstrated in Figure 4A, TZ-derived basal cells expressed 2-fold higher levels of p53 protein than PZ cells. The addition of E2 and DHT, singly or in combination, had no significant effect on the expression of p53 protein sampled from total cell lysates. However, the same treatments increased p53 levels in the cytosolic fraction (where the protein is inactive) with a concomitant decrease in nuclear protein levels, only in PZ-derived cells (Figure 4A and B). This suggests that sex steroids can inactivate p53 protein in PZ cells by preventing its nuclear translocation.


Figure 4
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  		Fig. 4. Estrogenic and androgenic induction of intracellular redistribution of p53 in basal cells. (A) Western blot of total cell lysates. Basal cells were cultured in serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total protein and the protein in the cytoplasmic and nuclear fractions were isolated and subjected to western blot analysis using an antibody against endogenous p53. In each lane 20 µg of protein were loaded. Data shown are representative of paired samples from five individual patients. (B) Quantitative analysis of cytosolic and nuclear p53 protein expression in PZ- and TZ-derived basal cells. The relative amount of specific protein was quantitated by densitometry. Data expressed as ratio of the protein to actin, and shown are mean ± SE of paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells. (C) Estrogen-inhibited p53 transcriptional activity in PZ-derived basal cells. Basal cells were cultured in 12-well cluster plates and co-transfected with a reporter plasmid and a ß-galactosidase expression vector. After transfection, serum-free medium was replaced and cells were treated with either vehicle, or 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Cells were harvested and cell lysates were prepared for luciferase assays. Luciferase activities are normalized for protein concentrations and ß-gal activities, and expressed as the mean ± SE in paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells.

 
Estrogen inhibits p53 transcriptional activity in PZ-derived basal cells
We investigated the effects of estrogen and androgen on p53 transcriptional activity using a luciferase reporter transactivation assay. PZ and TZ basal cells were transfected with a plasmid containing the p21 promoter sequence and the coding region for firefly luciferase upstream of the minimal adenovirus E1b promoter. Basal and sex steroid-induced p53 activity was assayed and compared in the two cell populations. Figure 4C demonstrates that TZ cells exhibited higher basal p53 activity than PZ cells. The p53 activity in PZ cells was further reduced by the addition of E2, DHT or the combination of the two hormones. Only DHT alone had a modest downregulatory effect on p53 activity in TZ-derived cells (Figure 4C). These results are consistent with the data obtained on western blot analysis (Figure 4A and B), and imply that decreased p53 activity in PZ cells may be due to sequestration of the protein in the cytoplasm, particularly in response to steroid hormone addition.

Differential expression of full-length wild-type KLF6 and the SV1 splice variant (KLF6-SV1) in PZ and TZ basal cells
KLF6 has recently been identified as a tumor suppressor gene, which is inactivated by either allelic loss or somatic mutation in sporadic prostate cancers (23,24) colorectal cancer (25), malignant glioma (26) and nasopharyngeal cancer (27). In both prostate and lung cancers, decreased KLF6 expression is predictive of a poor clinical outcome (28,29). We identified a KLF6 single nucleotide polymorphism (SNP) that was associated with an increased lifetime risk of prostate cancer (30). The SNP increased production of the alternatively spliced KLF6 isoform, KLF6-SV1, which antagonizes wild-type (wt) KLF6 growth-suppressive functions and increases prostate cancer cell proliferation, colony formation and cellular invasion (31). We next examined the basal and steroid hormone-regulated expression of wt-KLF6 and KLF6-SV1 in the two basal cell populations. As shown in Figure 5, both PZ- and TZ-derived basal cells expressed low levels of wt-KLF6. E2 marginally increased wt-KLF6 in both zones. In addition, both PZ and TZ cells expressed KLF6-SV1, but the expression levels of this antagonist to wt-KLF6 were 2-fold higher in the PZ cells (Figure 5A and B). Thus, there is a marked difference between the KLF6-SV1/wt-KLF6 ratios present in PZ- and TZ-derived basal cells and PZ-derived cells more closely resemble the increased splicing ratios associated with a more tumorigenic phenotype (30).


Figure 5
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  		Fig. 5. KLF6 expression in PZ and TZ basal cells. (A) Western blot analysis. Basal cells were cultured in serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total protein was isolated and subjected to western blot analysis using an antibody against KLF6. In each lane 20 µg of protein were loaded. Data shown are representative of paired samples from five individual patients. (B) Quantitative analysis of wt KLF6 and KLF6 SV1 protein expression in PZ- and TZ-derived basal cells. The relative amount of specific protein was quantitated by densitometry. Data are expressed as a ratio of wt-KLF6 or KLF6 SV1 protein to actin expression, and shown are the Mean ± SE of paired samples from five individual patients. **P < 0.05, *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells.

 
Differential expression of p21waf1/Cip1, Bax, and bcl-2 in PZ- versus TZ-derived basal cells
Cell number is determined by the balance between cellular proliferation and apoptosis. Loss of cell cycle control is a common event in malignancies. p21waf1/Cip1 protein, a well-known downstream product of the p53 gene, is a key cyclin-dependent kinase inhibitor (32). A recent report indicates that the growth-suppressive activity of wt-KLF6 is linked to p53-independent transactivation of p21waf1/Cip1 (30). Bax and Bcl-2 are critical proteins that, respectively, positively and negatively regulate cell apoptosis. We next determined the expression of those proteins in the two basal cell populations. As shown in Figure 6, while the levels of both p21waf1/Cip1 and Bax proteins were undetectable in PZ cells, TZ cells expressed moderate levels of these proteins. While PZ cells expressed relatively higher basal levels of the anti-apoptotic protein Bcl-2, there was no significant difference in the pattern of Bcl-2 expression in response to steroid hormone addition in the two cell populations (Figure 6).


Figure 6
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  		Fig. 6. Differential expression of p21waf1/Cip1, Bax, and Bcl-2 in PZ versus TZ-derived basal cells. (A) Western blot analysis. Basal cells were cultured in serum-free medium in the presence or absence of either 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total protein was isolated and subjected to western blot analysis using antibodies against p21. After stripping, the membrane was sequentially re-probed with Bax, bcl-2 and actin antibodies. In each lane 20 µg of protein were loaded. Data shown are representative of paired samples from five individual patients. (B) Quantitative analysis of p21, Bax and bcl-2 protein expression in PZ- and TZ-derived basal cells. The relative amounts of specific proteins were quantitated by densitometry. Data are expressed as the ratio of specifc protein expression to actin expression, and shown are Mean ± SE of paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and *P < 0.01 versus the control of TZ cells.

 
Estrogen and androgen increase telomerase expression in PZ-derived basal cells
Telomerase is a cellular reverse transcriptase that catalyzes the synthesis and extension of telomeric DNA (33). This enzyme is specifically activated in most malignant tumors but is usually inactive in normal somatic cells. Telomerase activity is modulated by changes in the expression level as well as the extent of phosphorylation of its catalytic subunit, hTERT (33). It has been reported that both estrogens and androgens can modulate hTERT activity (34–36). We compared basal and sex steroid-stimulated hTERT expression levels in the PZ- and TZ-derived basal cells. RT–PCR analysis (Figure 7A and B) demonstrated that hTERT expression was 3-fold higher in PZ cells than in TZ cells. Treatment with E2 and DHT, alone or in combination, significantly upregulated hTERT expression in PZ cells only. E2 addition to TZ cultures, in contrast, had a modest inhibitory effect on hTERT expression (Figure 7).


Figure 7
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  		Fig. 7. Effects of estrogen and androgen on hTERT expression in basal cells. (A) RT–PCR. Basal cells were cultured in serum-free medium and treated with either vehicle or 0.1 µM E2 or 0.1 µM DHT, or a combination of E2 and DHT for 3 days. Total RNA was then isolated, and RT–PCR was performed using specific primers for hTERT. (B) Quantitative analysis of hTERT mRNA expression in PZ- and TZ-derived basal cells. The relative amount of specific mRNA was quantitated by densitometry. Data are expressed as a ratio of hTERT to ß-actin expression, and shown are mean ± SE of paired samples from five individual patients. *P < 0.01 versus the control of PZ cells, and **P < 0.05 versus the control of TZ cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
These studies demonstrate that PZ-versus TZ-derived prostate epithelial cell cultures have similar basal cell-specific cytokeratin expression patterns and do not express PSA. The phenotype of these cells, therefore, is similar to that previously reported with primary prostate epithelial cell cultures, and more representative of a basal cell population than a luminal cell population (21,37). Our demonstration that all cultures expressed AR, may indicate that these cells represent a particular subpopulation of human basal cells. Previous studies investigating the expression of AR in the human prostate have yielded conflicting results. Bonkhoff and Remberger (38) demonstrated nuclear AR expression in the basal cell layer of the normal and hyperplastic human prostate. A subsequent report (39) demonstrated that only a very small population of basal cells (<1%) had AR expression. A more recent study demonstrated variable AR immunostaining in basal cells, with strong basal cell ERß immunoreactivity, and ER{alpha} immunoreactivity localized to stromal cells (40). A previous report demonstrated AR immunoreactivity in primary cultures of both basal and luminal human prostate epithelial cells (41). We postulate that our cultured cells may represent a subpopulation of post-stem cells located in the basal layer. These cells have previously been designated as transit amplifying cells that are capable of more rapid growth during prostate regeneration (5).

Although the zone-specific cultures exhibited the similarities mentioned above, we found evidence of inherent differences in their in vitro growth characteristics, gene expression levels and response to steroid hormones. Specifically, the PZ cells are more viable in culture. This may be explained by a combination of lower basal expression of genes that induce cell cycle arrest and apoptosis (p53, p21waf1/Cip1 and Bax) and higher basal expression of growth-promoting and anti-apoptotic genes (i.e. telomerase, KLF6-SV1 and Bcl-2) in the PZ cultures.

The role of tumor suppressor proteins in prostate neoplasia is not well delineated. Although alterations in the p53 gene have been reported in more than half of all human cancers (32), p53 mutations have not been commonly reported in prostate cancer, and tend to be noted only in more advanced stages (42,43). The present study demonstrates higher p53 protein expression and transcriptional activity in basal cells derived from the TZ compared with that seen in the PZ. The higher p53 activity in the TZ-derived cells probably reflects both higher protein expression levels and increased nuclear accumulation. Lower p53 activity in the PZ-derived cultures may partially explain the loss of p21waf1/Cip1 and Bax protein expression, as well as the increased Bcl-2 expression. Our findings are consistent with previous reports that identify non-functional (silencing) p53 protein as a trigger for basal cell overgrowth and prostate cancer development in the peripheral zone (3,22).

KLF6 is a prostate cancer tumor suppressor gene that induces p21waf1/Cip1 expression in a p53-independent manner (23). We previously identified a common KLF6 germline single nucleotide polymorphism (SNP) that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms (30). While KLF6 acts as a classic tumor suppressor, we recently reported that the SNP-increased isoform KLF6-SV1 has the opposite effect on prostate cancer cells both in vitro and in vivo. Inhibition of KLF6-SV1 activity by siRNA technology reduced tumor growth by 50% and decreased the expression of a number of growth- and angiogenesis-related genes (31). In the present study, we found increased expression of KLF6-SV1 in cultures derived from the peripheral zone. These findings, coupled with decreased p53 expression, nuclear localization and activity, may coordinately explain the lack of p21waf1/Cip1 expression observed in the PZ-derived cultures.

PZ-derived cells had increased basal telomerase activity compared with TZ-derived cells. Activation of telomerase, which has been detected in >95% of tumor samples studied, has been identified as an essential requirement for unlimited cell proliferation (44). A prior report demonstrated that hTERT expression levels in human prostate cancer tissue samples positively correlates with advanced tumor grade and stage (45). The increased telomerase expression noted in the PZ cultures identifies them as having a more stem cell-like phenotype than the TZ-derived cells.

Our data gives some insight into the molecular mechanisms underlying the observed carcinogenic effects of sex steroids in the human prostate. Steroid hormone addition increased cell numbers in PZ cultures and had little effect on this parameter in TZ cultures. Enhancement of cell numbers by steroid hormones in PZ cells may be attributed to their modulation of a variety of factors. Androgen and estrogen addition decreased p53 nuclear localization and activity to a much greater extent in the PZ cells. Steroid hormone addition increased telomerase activity only in PZ-derived cells. With regard to telomerase, steroid hormone regulation of hTERT expression has previously been reported in a variety of cancer cell lines (34–36). Estrogens can reverse telomerase silencing in normal telomerase-negative ovary epithelial cells by transcriptional activation of hTERT (46). A study in normal human prostate epithelial cells demonstrated increases in hTERT mRNA and activity following estrogen addition. That report further showed estradiol-dependent hTERT promoter induction and demonstrated that both ER{alpha} and ERß bound this sequence (47).

The differential effects of sex steroids in the TZ- versus PZ-derived cells are likely due to differences in the relative expression levels of AR, ER{alpha} and ERß. Although ER{alpha} protein levels are similar in the two cell populations, PZ cultures exhibit higher inducible expression of AR and lower ERß expression. In the TZ cultures, ERß expression is further increased by treatment with androgens and estrogens. As ERß has been shown to have growth inhibitory, differentiating effects on non-neoplastic prostate epithelium (7,8,15), estrogen signaling via this pathway in TZ-derived cells may be responsible for estrogenic induction of p53 nuclear localization/activity and p21waf1/Cip1 expression. Although AR expression was nearly identical, AR signaling was suppressed in TZ-derived cells, possibly due to increased p53 activity. In support of this conclusion, Shenk et al. (48) reported that p53 represses androgen-induced transcriptional activity in a human prostate cancer cell line.

Although the cytokeratin expression pattern of the cultured cells is consistent with a basal cell phenotype, the expression of AR and telomerase suggests that they are a post-stem cell population that can undergo significant growth and may be the cells of origin for prostate cancer. Transition zone cells may be relatively protected from malignant transformation due to enhanced ERß and p21 expression and higher p53 activity. In contrast, higher expression levels of anti-apoptotic and growth-promoting genes, coupled with steroid hormone enhancement of some of these factors in PZ-derived cells, offers a plausible explanation for the increased incidence of prostate cancer in the peripheral zone.


   Acknowledgments
 
This work was supported by grants-in aid from the Department of Defense (DAMD 17-00-1-0090) of the U.S. government, and the T.J. Martell Foundation for Leukemia, Cancer and Aids Research. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 26, 2005; revised July 22, 2005; accepted August 19, 2005.


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