Mechanisms of acquired androgen independence during arsenic-induced malignant transformation of human prostate epithelial cells.

BACKGROUND: Prostate cancer progression often occurs with overexpression of growth factors and receptors, many of which engage the Ras/mitogen-activated protein MAP kinase (MAPK) pathway. OBJECTIVES: In this study we used arsenic-transformed human prostate epithelial cells, which also show androgen-independent growth, to study the possibility that chronic activation of Ras/MAPK signaling may contribute to arsenic-induced prostate cancer progression. METHODS: Control and chronic arsenic-transformed prostate epithelial cells (CAsE-PE) were compared for Ras/MAPK signaling capacities using reverse transcription-polymerase chain reaction and Western blot analyses. RESULTS: We found activation of HER-2/neu oncogene in transformed CAsE-PE cells, providing molecular evidence of androgen independence in the transformed cells. CAsE-PE cells displayed constitutively increased expression of unmutated K-Ras (6-fold), and the downstream MAP kinases A-Raf and B-Raf (2.2-fold and 3.2-fold, respectively). There was also increased expression of phosphorylated MEK1/2 and Elk1 in the transformant cells. The MEK1/2 inhibitor, U0126, blocked PSA overexpression in CAsE-PE cells. CONCLUSION: Thus, arsenic-induced malignant transformation and acquired androgen independence are linked to Ras signaling activation in human prostate epithelial cells. Chronic activation of this pathway can sensitize the androgen receptor to subphysiologic levels of androgen. This may be important in arsenic carcinogenesis and provide a mechanism that may be common for prostate cancer progression driven by diverse agents.

Acquisition of apoptotic resistance in cadmium-transformed human prostate epithelial cells: Bcl-2 overexpression blocks the activation of JNK signal transduction pathway.

BACKGROUND: We have recently shown that cadmium can induce malignant transformation of the human prostate epithelial cell line (RWPE-1) and that these cadmium-transformed prostate epithelial (CTPE) cells acquire apoptotic resistance concurrently with malignant phenotype. OBJECTIVE: The present study was designed to define the mechanism of acquired apoptotic resistance in CTPE cells. METHODS: Various molecular events associated with apoptosis were assessed in control and CTPE cells that were obtained after 8 weeks of continuous cadmium exposure. RESULTS: Compared with control, CTPE cells showed a generalized resistance to apoptosis induced by cadmium, cisplatin, or etoposide. Signal-regulated mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases (JNK1 and JNK2), and p38 were phosphorylated in a cadmium concentration-dependent fashion in CTPE and control cells. However, phosphorylated JNK1/2 levels and JNK kinase activity were much lower in CTPE cells. The pro-apoptotic gene Bax showed lower transcript and protein levels, whereas the anti-apoptotic gene Bcl-2 showed higher levels in CTPE cells. The ratio of Bcl-2/Bax, a key determinant in apoptotic commitment, increased more than 4-fold in CTPE cells. In Bcl-2-transfected PT-67 cells, phosphorylated JNK1/2 levels were much lower after apoptogenic stimulus, and apoptosis induced by cadmium or etoposide was reduced compared with control. Mutation of tyrosine to serine at the 21st amino acid of the Bcl-2 protein BH4 domain resulted in a loss both of suppression of JNK1/2 phosphorylation and its anti-apoptotic function. CONCLUSIONS: CTPE cells become resistant to apoptosis during malignant transformation, and disruption of the JNK pathway and Bcl-2 overexpression play important roles in this resistance. Bcl-2 BH4 domain is required for modulating JNK phosphorylation and anti-apoptotic function.

Tumor suppressor gene inactivation during cadmium-induced malignant transformation of human prostate cells correlates with overexpression of de novo DNA methyltransferase.

BACKGROUND: Aberrant DNA methylation is common in carcinogenesis. The typical pattern appears to involve reduced expression of maintenance DNA methyltransferase, DNMT1, inducing genomic hypomethylation, whereas increased expression of de novo DNMT3a or 3b causes gene-specific hypermethylation. OBJECTIVES: During cadmium-induced malignant transformation, an unusual pattern of genomic hypermethylation occurred that we studied to provide insight into the roles of specific DNMTs in oncogenesis. METHODS: Gene expression and DNA methylation were assessed in control and chronic cadmium-transformed prostate epithelial cells (CTPE) using reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, methylation-specific PCR, and methyl acceptance assay. RESULTS: During the 10-weeks of cadmium exposure that induced malignant transformation, progressive increases in generalized DNMT enzymatic activity occurred that were associated with over-expression of DNMT3b without changes in DNMT1 expression. Increased DNMT3b expression preceded increased DNMT enzymatic activity. Procainamide, a specific DNMT1 inhibitor, reversed cadmium-induced genomic DNA hypermethylation. Reduced expression of the tumor suppressor genes, RASSF1A and p16, began about the time DNMT3b overexpression first occurred and progressively decreased thereafter. RASSF1A and p16 promoter regions were heavily methylated in CTPE cells, indicating silencing by hypermethylation, while the DNA demethylating agent, 5-aza-2'-deoxycytidine, reversed this silencing. DNMT1 inhibition only modestly increased RASSF1A and p16 expression in CTPE cells and did not completely reverse silencing. CONCLUSIONS: These data indicate that DNMT3b overexpression can result in generalized DNA hypermethylation and gene silencing but that DNMT1 is required to maintain these effects. The pattern of genomic DNA hypermethylation together with up-regulation of DNMT3b may provide a unique set of biomarkers to specifically identify cadmium-induced human prostate cancers.

Cholecalciferol (vitamin D3) and the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) are synergistic for chemoprevention of prostate cancer.

Prostate cancer, the most commonly diagnosed cancer among American men, develops slowly over many years. The long latent period of 20 to 30 years, involved in the multistep process of carcinogenesis, provides an important opportunity to block or reverse progression to a malignant state. Vitamin A (retinoids) and vitamin D not only have the ability to block steps in the process of carcinogenesis but they can also modulate or reverse some malignant characteristics of cancer cells. However, at high levels, vitamins A and D have undesirable side effects, thus, limiting effective dose levels and efficacy. Therefore, combination treatment at low doses, to increase efficacy and avoid toxicity, is of special interest. This study examines the effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in combination with cholecalciferol (vitamin D3) on growth, and on the expression of vimentin, matrix metalloproteinase-2 (MMP-2), and retinoid and vitamin D receptor expression, using the non-tumorigenic, human prostate epithelial cell line RWPE-1. Treatment with 4-HPR and cholecalciferol resulted in synergistic growth inhibition when compared to that caused by each agent alone. A decrease in vimentin expression and MMP-2 activity, and up-regulation of vitamin D receptor (VDR) and some of the retinoid-X (RXRs) and retinoic acid receptor (RARs) subtypes, was observed. These results suggest that combined treatment with 4-HPR and cholecalciferol, at doses lower than what might be effective with single agents, increases their efficacy and suggest that this may serve as an effective strategy for chemoprevention and treatment of prostate cancer.

Inorganic arsenite-induced malignant transformation of human prostate epithelial cells.

Although several epidemiologic studies show an association between arsenic exposure and prostate cancer, it is still unknown whether human prostate epithelial cells are directly susceptible to arsenic-induced transformation. This study was designed to determine whether the nontumorigenic human prostate epithelial cell line RWPE-1 could be malignantly transformed in vitro by arsenite. RWPE-1 cells were continuously exposed to 5 micro M arsenite and monitored for signs of transformation, assessed as changes in matrix metalloproteinase-9 levels. After 29 weeks of exposure, the arsenite-exposed RWPE-1 cells (referred to as CAsE-PE) showed a marked increase in matrix metalloproteinase-9 secretion, a common finding in prostate malignancies. Malignant transformation was confirmed when CAsE-PE cells produced aggressive undifferentiated malignant epithelial tumors in nude mice. The tumors stained positive for human prostate-specific antigen, confirming their origin. These results are the first report of arsenite-induced malignant transformation of a human epithelial cell line and provide an important in vitro model for studying the mechanisms underlying arsenic-induced carcinogenesis in humans.

Chemoprevention of prostate cancer by cholecalciferol (vitamin D3): 25-hydroxylase (CYP27A1) in human prostate epithelial cells.

The 20-30 year latency period for prostate cancer provides an important opportunity to prevent the development of invasive cancer. A logical approach for chemoprevention to reduce incidence is to identify agents, such as, vitamin D, which can inhibit cell proliferation and induce differentiation, are safe, and readily available to the public at low cost. Epidemiological evidence suggests that vitamin D deficiency is associated with increased risk for prostate cancer. We examined the ability and mechanisms of action of cholecalciferol (vitamin D(3)), a precursor of the most biologically active hormone calcitriol, to block or reverse premalignant changes. The immortalized, non-tumorigenic, RWPE-1 human prostate epithelial cell line, was used. Results show that cholecalciferol, at physiological levels: (i) inhibits anchorage-dependent growth (ii) induces differentiation by increasing PSA expression and (iii) exerts its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptors (RXRs), and androgen receptor (AR). Furthermore, we discovered that human prostate epithelial cells constitutively express appreciable levels of 25-hydroxylase CYP27A1 protein, the enzyme which catalyzes the conversion of cholecalciferol to 25(OH)D(3), and that CYP27A1 is up-regulated by cholecalciferol. Recent studies show that human mitochondrial CYP27A1 can also catalyze 1alpha-hydroxylation of 25(OH)D(3) to calcitriol. The presence of 25-hydroxylase in human prostate epithelial cells has not previously been shown. Since human prostate epithelial cells have the necessary enzymes and the rare ability to locally convert cholecalciferol to the active hormone calcitriol, we propose that they are a prime target for chemoprevention of prostate cancer with cholecalciferol whose safety is well established as a supplement in vitamins and fortified foods.

Cholecalciferol (vitamin D3) inhibits growth and invasion by up-regulating nuclear receptors and 25-hydroxylase (CYP27A1) in human prostate cancer cells.

Epidemiological evidence suggests an inverse relationship between prostate cancer and serum vitamin D levels. We examined the ability of cholecalciferol (vitamin D(3)), a calcitriol precursor, to inhibit or reverse cellular changes associated with malignant transformation and invasion and explored its mechanisms of action. The RWPE2-W99 human prostate epithelial cell line, which forms slow-growing tumors in nude mice, was used because it mimics the behavior of the majority of primary human prostate cancers. Cholecalciferol, at physiological levels: (i) inhibited anchorage-dependent and -independent growth; (ii) induced differentiation by decreasing vimentin expression with a concomitant decrease in motility/chemotaxis; (iii) decreased MMP-9 and MMP-2 activity with concomitant decrease in invasion; and (iv) exerted its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptor-alpha (RXR-alpha), and androgen receptor (AR) in a dose-dependent manner. Furthermore, we found that RWPE2-W99 prostate cancer cells, similar to RWPE-1 cells (Tokar and Webber. Clin Exp Metast 2005; 22: 265-73), constitutively express the enzyme 25-hydroxylase CYP27A1 which is markedly up-regulated by cholecalciferol. Cholecalciferol has effects similar to those of calcitriol on growth, MMP activity, and VDR. The ability of CYP27A1 to catalyze the conversion of cholecalciferol to 25(OH)D(3) and of 25(OH)D(3) to calcitriol has been reported. RWPE2-W99 cells, similar to RWPE-1 cells, appear to have the rare ability to locally convert cholecalciferol to the active hormone calcitriol. Because it can inhibit cellular changes associated with malignant transformation and invasion, we propose that cholecalciferol may be an effective agent for the treatment of prostate cancer.

Acquisition of androgen independence by human prostate epithelial cells during arsenic-induced malignant transformation.

Lethal phenotypes of human prostate cancer are characterized by progression to androgen independence, although the mechanisms behind this progression remain unclear. Arsenic is a potential human prostate carcinogen that may affect tumor progression. In this study, we used a prostate cancer cell model in which an immortalized, nontumorigenic human prostate epithelial cell line (RWPE-1) had been malignantly transformed by chronic low-level arsenic to help determine whether arsenic affects prostate tumor progression. Control and CAsE-PE (chronic-arsenic-exposed human prostate epithelial) cells were continuously maintained in a complete medium [keratinocyte serum-free medium (K-SFM) with bovine pituitary extract and epidermal growth factor] or in a steroid-depleted medium (K-SFM alone). The arsenic-transformed cells showed a more rapid proliferation rate in complete medium than did control cells and also showed sustained proliferation in steroid-reduced medium. Although both control and CAsE-PE cells showed similar levels of androgen receptor (AR), androgens were less effective in stimulating cell proliferation and AR-related gene expression in CAsE-PE cells. For instance, dihydrotestosterone caused a 4.5-fold increase in prostate-specific antigen transcript in control cells but only a 1.5-fold increase in CAsE-PE cells. CAsE-PE cells also showed relatively low levels of growth stimulation by nonandrogen steroids, such as estradiol. Thus, arsenic-induced malignant transformation is associated with acquired androgen independence in human prostate cells. This acquired androgen independence was apparently not due to AR up-regulation, increased activity, or altered ligand specificity. The precise manner in which arsenic altered CAsE-PE growth and progression is undefined but may involve a bypass of AR involving direct stimulation of downstream signaling pathways.

Selection of cell lines with enhanced invasive phenotype from xenografts of the human prostate cancer cell line WPE1-NB26.

Prostate cancer is a leading cause of death from cancer in American men and metastasis the main cause of death. To better understand the disease and accelerate development of new therapies, in vivo models that reflect different disease stages are needed. A family of cell lines that mimics multiple steps in cancer development and tumor progression has been developed in our laboratory from the parent, non-tumorigenic, RWPE-1 cell line by transformation with N-methyl-N-nitrosourea (MNU). The MNU cell lines mimic multiple steps in tumor progression where WPE1-NB26 is the most malignant cell line. WPE1-NB26 cells form metastases in the lungs of athymic, male, nude mice after intravenous injection. Two new cell lines, WPE1-NB26-64 and WPE1-NB26-65, showing more malignant characteristics than the parent WPE1-NB26 cell line, were derived from tumors after subcutaneous injection of WPE1-NB26 cells into nude mice. The WPE1-NB26-64 and WPE1-NB26-65 cell lines show an increase in anchorage-dependent growth and invasive ability as compared to the parent WPE1-NB26 cells. While the parent WPE1-NB26 cells express barely detectable levels, the new cell lines produce high levels of matrix metalloproteinase MMP-2 and detectable levels of MMP-9. By immunostaining, all three cell lines were positive for cytokeratins CK18 and CK5/14. These cell lines, having the same lineage, represent additional steps in the multi-step process of tumor progression and provide novel and useful cell models for studies on tumor progression and for drug development for the treatment of prostate cancer.

Alteration of cellular phenotype and responses to oxidative stress by manganese superoxide dismutase and a superoxide dismutase mimic in RWPE-2 human prostate adenocarcinoma cells.

To study biologic effects of increased manganese superoxide dismutase (MnSOD) on cell behavior, we overexpressed MnSOD in a human prostate cancer cell line RWPE-2 by cDNA transfection. Stable transfectants of MnSOD showed a two- to threefold increase in MnSOD protein and enzymatic activity and a decrease in growth rate with prolonged cell population doubling times. Western blot analysis showed a 1.5- to twofold increase in the cyclin-dependent kinase inhibitor p21(Waf1) in MnSOD transfectants. Overexpression of MnSOD resulted in a seven- to eightfold increase in reduced glutathione (GSH), 18- to 26-fold increase in oxidized glutathione (GSSG), and a two- to threefold decrease in the ratio of GSH to GSSG. MnSOD-overexpressing cells showed an increase in sensitivity to the cytotoxicity of buthionine sulfoximine, a glutathione-depleting agent, and vitamin C, but a decrease in sensitivity to sodium selenite. Treatment with a superoxide dismutase (SOD) mimic MnTMPyP resulted in similar effects of MnSOD overexpression on cell responses to vitamin C and selenium. These data demonstrate that overexpression of MnSOD or treatment with SOD mimics can result in antioxidant or prooxidant effects in cells, depending on the presence of other antioxidants and prooxidants. MnSOD also has redox regulatory effects on cell growth and gene expression. These findings suggest that MnSOD and SOD mimics have the potential for cancer prevention or treatment.

Intercellular communication and human prostate carcinogenesis.

Gap-junction-mediated intercellular communication (GJIC) is required for completion of embryonic development, tissue homeostasis, and regulation of cell proliferation and death. Although, as emphasized in several reports, defects or disruption of GJIC may be important in carcinogenesis, the potential role of GJIC in the onset and progression of human prostate cancer remains ill-defined. The gap junction channel-forming connexins (Cx) comprise a multigene family of highly conserved proteins that are differentially expressed in a tissue- and development-specific manner; changes in connexin expression are also commonly seen during cellular differentiation. However, when multiple connexins are concurrently expressed, gap junction channels may consist of more than one connexin species. This is important, because only certain pairings give rise to functional channels. In our studies, we investigated GJIC in a panel of both nontumorigenic (RWPE-1) and malignant (RWPE-2, LNCaP, DU-145) human prostate epithelial cells, compared to a normal rat liver epithelial F344 (WB-1) cell line, as it was found to be junctionally proficient. In addition, expression and regulation of Cx43 and Cx32 were also inspected using western blot analysis. The ability of hormones, antihormones, and the antihypertensive drug forskolin to restore GJIC in nontumorigenic and malignant human prostate epithelial cells was examined by the scrape-loading/dye transfer (SL/DT) or fluorescence recovery after photobleaching (FRAP) methods using an Ultima laser cytometer. Results from both assays showed that neither nontumorigenic nor malignant prostate cells have functional GJIC. However, both estrone (E1) and forskolin (FK) induced a significant increase (4.4- and 2.8-fold, respectively) in cell-cell communication only in the RWPE-1 cells. Interestingly, the use of Matrigel, a solubilized basement membrane, as substrate for cell attachment and growth resulted in the rescue of GJIC activity in RWPE-1 cells, as revealed by the SL/DT method. Furthermore, E1 induced a twofold increase in connexin 43 (Cx43), whereas forskolin caused a 50% reduction in Cx32 expression in RWPE-1 cells. These data suggest that agents that increase Cx43:Cx32 ratio may restore GJIC in junctionally deficient cells, providing a basis for the development of new strategies for the prevention and treatment of human prostate cancer.

Connexin expression in nonneoplastic human prostate epithelial cells.

Expression of gap-junction proteins connexins (Cx), specifically Cx43, Cx32, and Cx26, in both nontumorigenic (RWPE-1) and tumorigenic (RWPE-2) human prostate epithelial cells as well as in two cell clones (WPEI-7 and WPEI-10) originating from the RWPE-1 cell line was investigated. The aim was to determine whether individual connexins are differentially expressed in cultured cells. Western blot analysis revealed striking differences in the expression of individual connexins in the cell lines studied. In particular, Cx43 is largely expressed in RWPE-1 and WPEI-10 cells, whereas Cx32 is expressed predominantly in RWPE-2 and WPEI-7 cells. In addition, both forskolin and estrone increase Cx43 expression levels in WPEI-10 cells, with no apparent effect on WPEI-7 cells. Conversely, forskolin and especially estrone induce a marked increase of Cx32 in WPEI-7 cells, whereas Cx32 expression is limitedly affected by both agents in WPEI-10 cells. Overall, expression levels of Cx43 and Cx32 appear to be inversely related, with RWPE-1 and WPEI-10 cells having a significantly higher Cx43 to Cx32 ratio than that observed in RWPE-2 and WPEI-7 cells. We recently reported that junctional communication could be rescued in RWPE-1 cells by either forskolin or estrone and that restoration of GJIC is associated with an increase of Cx43 or a decrease of Cx32, or both, eventually leading to a marked rise of the Cx43 to Cx32 ratio. Studies are currently ongoing in our laboratories to assess the potential effect of agents increasing the Cx43 to Cx32 ratio on GJIC activity in these systems.

Arsenic-specific stem cell selection during malignant transformation.

BACKGROUND: Arsenic is a carcinogen that targets the urogenital system, including the prostate. Although the mechanisms for arsenic-induced carcinogenesis are undefined, arsenic drives overaccumulation of stem cells and cancer stem cells (CSCs) in vivo and in vitro, indicating that these cells are a key target population. Disruption of stem cell population dynamics may be critical to acquisition of cancer phenotype. We tested the hypothesis that prostate stem cells have a survival selection advantage during arsenic exposure that favors their accumulation and facilitates their malignant transformation. METHODS: Innate and acquired resistance to acute (24-72 hours of exposure) and chronic (6 weeks of exposure) arsenite-induced cytolethality and apoptosis were assessed in a human prostate stem cell line (WPE-stem) and the mature parental cell line (RWPE-1). Real-time reverse transcription-polymerase chain reaction and/or Western blot analysis was used to measure the expression of apoptosis-, stress-, and arsenic-related genes. Arsenic-, cadmium-, and N-methyl-N-nitrosourea-induced isogenic malignant transformants of RWPE-1 cells were compared for acquisition of CSC-like qualities by holoclone and sphere formation assays, growth in soft agar, and expression of CSC biomarkers. All statistical tests were two-sided. RESULTS: WPE-stem cells showed innate resistance to arsenic-induced cytolethality (arsenite concentration lethal to 50% of the cells [LC(50)] = 32.4 microM, 95% confidence interval [CI] = 31.5 to 33.3 muM) and apoptosis compared with parental RWPE-1 cells (LC(50) = 10.4 muM, 95% CI = 7.4 to 13.4 microM). Compared with RWPE-1 cells, WPE-stem cells showed noticeably higher expression of antiapoptotic (ie, BCL2, MT), stress-related (ie, NFE2L2, SOD1, PRODH), and arsenic adaptation (ie, ABCC1, GSTP1) factors and noticeably lower expression of proapoptotic factors (ie, BAX, caspases 3, 7, 8, and 9). WPE-stem cells also showed hyper-adaptability to chronic arsenite exposure (5 microM, 6 weeks) compared with RWPE-1 cells (LC(50) = 94.7 vs 32.1 microM, difference = 62.6 muM, 95% CI = 53.3 to 71.9 muM) at levels that in previous work induced a malignant phenotype in RWPE-1 after 30 weeks of exposure. Quantification of CSC-like cells in isogenic RWPE-1 transformants showed that marked overproduction was unique to a malignant phenotype acquired in response to arsenic exposure but not in response to cadmium or N-methyl-N-nitrosourea exposure. CONCLUSIONS: An apparent stem cell survival advantage with regard to arsenic causes selection during malignant transformation that manifests itself as an overabundance of CSC-like cells specifically after arsenic-driven acquisition of malignant phenotype. The increased resistance to apoptosis and arsenite hyper-adaptability of WPE-stem cells suggests that arsenite transformation of RWPE-1 cells involves an increase in the number of CSC-like cells.

Estrogen signaling and disruption of androgen metabolism in acquired androgen-independence during cadmium carcinogenesis in human prostate epithelial cells.

BACKGROUND: Lethal prostate cancers often become androgen-independent due to androgen receptor (AR) overexpression. The role of cadmium in prostate tumor progression was determined. METHODS: Control and cadmium-transformed prostate epithelial cells (CTPE) were compared for steroid-induced proliferation, steroid receptor expression, and androgen metabolism. RESULTS: CTPE cells showed rapid proliferation in complete medium and sustained proliferation in steroid-reduced medium. Androgens stimulated significantly less cell proliferation and AR-related genes expression in CTPE cells. 5alpha-Dihydrotestosterone increased PSA expression more effectively in control cells. Flutamide reduced 5alpha-dihydrotestosterone-stimulated growth less effectively in CTPE cells compared to control. CTPE cells showed decreased p27 expression. Estrogen receptors were overexpressed and estradiol markedly stimulated proliferation in CTPE cells. In CTPE cells 5alpha-aromatase was markedly increased, while 5alpha-reductase was decreased. CONCLUSIONS: Cadmium-induced malignant transformation stimulates androgen independence, unrelated to AR expression or activity. Increased estrogen receptor and 5alpha-aromatase expression suggest estrogen signaling may be critical to this process.

Stem/progenitor and intermediate cell types and the origin of human prostate cancer.

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.

Molecular events associated with arsenic-induced malignant transformation of human prostatic epithelial cells: aberrant genomic DNA methylation and K-ras oncogene activation.

Numerous studies link arsenic exposure to human cancers in a variety of tissues, including the prostate. Our prior work showed that chronic arsenic exposure of the non-tumorigenic, human prostate epithelial cell line, RWPE-1, to low levels of (5 microM) sodium arsenite for 29 weeks resulted in malignant transformation and produced the tumorigenic CAsE-PE cell line. The present work focuses on the molecular events occurring during this arsenic-induced malignant transformation. Genomic DNA methylation was significantly reduced in CAsE-PE cells. A time course experiment showed that during malignant transformation DNA methyltransferase activity was markedly reduced by arsenic. However, DNA methyltransferase mRNA levels were not affected by arsenic exposure. Microarray screening showed that K-ras was highly overexpressed in CAsE-PE cells, a result further confirmed by Northern blot and Western blot analyses. Since ras activation is thought to be a critical event in prostate cancer progression, further detailed study was performed. Time course experiments also showed that increased K-ras expression preceded malignant transformation. Mutational analysis of codons 12, 13, and 61 indicated the absence of K-ras mutations. The K-ras gene can be activated by hypomethylation, but our study showed that CpG methylation in K-ras promoter region was not altered by arsenic exposure. Arsenic metabolism studies showed RWPE-1, CAsE-PE, and primary human prostate cells all had a very poor capacity for arsenic methylation. Thus, inorganic arsenic-induced transformation in human cells is associated with genomic DNA hypomethylation and K-ras overexpression. However, overexpression of K-ras occurred without mutations and through a mechanism other than promoter region hypomethylation.

Altered apoptotic gene expression and acquired apoptotic resistance in cadmium-transformed human prostate epithelial cells.

BACKGROUND: Cadmium is a suspected prostatic carcinogen, although the underlying mechanisms are unclear. To investigate these mechanisms, we performed molecular comparisons between the cadmium-transformed prostate epithelial cell line CTPE and the nontumorigenic parental line RWPE-1. METHODS: Gene expression patterns were compared by using cDNA arrays, RNase protection assays, and Western blots. Apoptosis was analyzed by using flow cytometry to quantify apoptotic nuclei and an enzyme-linked immunosorbent assay method to measure DNA fragmentation. Caspase-3 activity was measured colorimetrically. RESULTS: Among the genes down-regulated in CTPE cells were those encoding several members of the caspase family of apoptotic proteases as well as the apoptotic regulator Bax. Ribonuclease protection assays confirmed global down-regulation of caspase gene expression in CTPE. Decreased Bax expression in CTPE was confirmed by Western blots, which also revealed increased expression of anti-apoptotic Bcl-2. Consistent with these changes, CTPE cells exhibited increased resistance to apoptosis induced by cadmium, cisplatin, and etoposide. CTPE cells also exhibited lower caspase-3 activity vs. RWPE-1 after etoposide treatment. CONCLUSIONS: CTPE cells exhibited altered expression of important apoptotic regulators as well as resistance to several apoptotic stimuli. We hypothesize that acquired apoptotic resistance may be a key aspect of cadmium-induced malignant transformation of prostate epithelial cells and that this may contribute to both tumor initiation and the acquisition of aggressive characteristics subsequent to tumor formation.

Chronic arsenic-exposed human prostate epithelial cells exhibit stable arsenic tolerance: mechanistic implications of altered cellular glutathione and glutathione S-transferase.

Acquisition of stable arsenic tolerance in human cells following chronic arsenic exposure has not been previously reported. In the present work, we describe acquisition of stable arsenic tolerance in the human prostate epithelial cell line RWPE-1 following chronic arsenic exposure in vitro. RWPE-1 cells continuously exposed to 5 microM sodium arsenite for > or =18 weeks exhibited dramatic resistance to acute arsenite toxicity. The LC50 for acute arsenite exposure in these chronic arsenic-exposed prostate epithelial (CAsE-PE) cells was 43.8 microM versus 17.6 microM in control cells. Similar results were obtained using the antineoplastic agent arsenic trioxide. This tolerance was stable, as CAsE-PE cells grown in arsenic-free medium for 5 weeks retained their resistant phenotype. Compared to control cells, CAsE-PE cells showed a 90% reduction in arsenic accumulation over 24 h coupled with a 2.6-fold increase in the rate of arsenic efflux. CAsE-PE cells had increased basal GSH levels (4.9-fold) and increased GST activity (2.4-fold) and both GSH depletion and inhibition of GST activity abolished arsenic tolerance. Arsenic tolerance was also abolished by treatment with inhibitors of the Mdr1 and Mrp1 transporters, although no increases in mdr1 or mrp1 gene expression were observed. Our results indicate that this tolerance in human cells involves increases in GSH levels and GST activity that allow for more efficient arsenic efflux by MRP1 and MDR1. This study represents the first report of stable acquired arsenic tolerance in human cells, which could have important implications for both the toxicology and the pharmacology of arsenic.

Effects of cyclic stretch on prostatic cells in culture.

PURPOSE: The fundamental process in the development of benign prostatic hyperplasia (BPH) is a loss of homeostasis between cell proliferation and apoptosis. Prostatic smooth muscle cells contract under adrenergic control. The response of a cell to stretch may have a role in the pathogenesis of BPH. MATERIALS AND METHODS: Monolayer cultures of human prostatic stromal and epithelial cell lines were exposed to cyclic stretch for 48 hours. RESULTS: Cyclic stretch conferred resistance to etoposide induced apoptosis. Underlying this apoptotic resistance was increased expression of the anti-apoptotic Bcl-2 family of proteins. As measured by thymidine incorporation, the rate of proliferation also increased in benign epithelial cells under cyclic stretch conditions. Furthermore, an increase in the production of platelet-derived growth factor by stromal cells and transforming growth factor-beta by epithelial cells occurred under such conditions. CONCLUSIONS: The observed changes in proliferation and apoptosis may contribute to the understanding of BPH, ultimately leading to therapeutic and preventive applications.