ANTICANCER RESEARCH 25: 1-8 (2005)

Androgen and Androgen Receptor Antagonist Responsive Primary African-American Benign Prostate Epithelial Cell Line

YONGPENG GU1, KEE-HONG KIM1, DAEJIN KO1, SHIV SRIVASTAVA1, JUDD W. MOUL1, DAVID G. MCLEOD2 and JOHNG S. RHIM1

1Center for Prostate Disease Research (CPDR), Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; 2Urology Service, Department of Surgery, Walter Reed Army Medical Center, Washington D.C., U.S.A.

Abstract. The generation of suitable in vitro models is critical AR gene sequencing analysis indicated that this cell line for understanding the process associated with the development expresses wild-type AR. The cell growth is stimulated by and progression of prostate cancer in high-risk African- dihydrotestosterone (1.0nM DHT) and the stimulatory effect is American men. However, the generation of long-term human blocked by flutamide (50 nM). Chromosome analysis revealed prostate epithelial cell lines derived from primary human that this cell line is aneuploid human male (XO), with most prostate epithelium have been unsuccessful due to the absence chromosome counts in the 80’s-90’s range. There were losses of of in vitro immortalization. We have successfully established an chromosomes Y, 1q, 4q, 10q, 12p and 22 and gains of immortal human prostate epithelial cell line from primary chromosomes 15 and 20. This is the first documented case of benign tissues of African-American prostate cancer patients by the establishment of an androgen and androgen receptor using telomerase. The actively proliferating secondary African- antagonist responsive African-American epithelial cell line American prostate epithelial RC-165N cells, derived from derived from benign prostate tissue with telomerase. This benign prostate tissue of a radical prostatectomy specimen, were unique in vitro model may be useful for the study of prostate transduced through infection with a retrovirus vector expressing cancer biology and the molecular basis of prostate the human telomerase catalytic subunit (hTERT). A high level carcinogenesis, especially for high-risk African-American men. of telomerase activity was detected in RC-165N/hTERT cells but not in RC-165N cells. RC-165N/hTERT cells are currently African-American men have a significantly higher incidence growing well at passage 50 whereas RC-165N cells senesced and mortality rates from prostate cancer compared to within passage 3. RC-165N/hTERT cells exhibit epithelial Caucasian men in North America, indicating that prostate morphology. These immortalized cells showed no cell growth in cancer is a major public health problem in this population soft agar, and no tumor formation in SCID mice. The RC- (1). The etiology of these racial differences in the clinical 165N/hTERT cells express androgen-regulated prostate-specific manifestation of prostate cancer is unclear; hormonal, homobox gene. NKX 3.1 and epithelial cell specific cytokeratin genetic, behavioral and environmental factors have all been 8, androgen receptor (AR), prostate stem cell antigen and p16, implicated (2). To understand the many factors suspected but not PSA. AR protein was detected by Western blot analysis. of contributing to the development of this malignancy, there is a need for an in vitro culture system. However, no suitable in vitro models for the study of African-American prostate cancer are available. Abbreviations: HPE, human prostate epithelial; PSA, prostate- specific antigen; AR, androgen receptor; HPV, human papilloma Androgen regulation of prostate growth, as well as the virus; SV40, Simian papovavirus; K-SFM, keratinocyte serum-free widely-used androgen deprivation therapy for prostate medium; TRAP, telomeric repeat amplification protocol; CK, cancer treatment, necessitates a better understanding of the cytokeratin. role of androgen in prostate cancer biology. The well- characterized LNCaP prostate cancer cell line is known to Correspondence to: Johng S. Rhim, MD, Center for Prostate express AR, but it is a mutant receptor (3). Therefore, Disease Research, 4301 Jones Bridge Road, Bethesda, MD 20814, generation of primary prostate cell lines expressing AR will U.S.A. Tel: 301-295-3165, Fax: 301-295-1978, e-mail: [email protected] have significant impact in evaluating the role of the androgen signaling pathway. Key Words: Benign human prostate cells, African-American, Telomerase is an enzyme responsible for replicating hTERT, androgen, androgen receptor antagonist. telomeres and is composed of an RNA subunit containing

0250-7005/2005 $2.00+.40 1 ANTICANCER RESEARCH 25: 1-8 (2005) an integral catalytic subunit, hTERT (human telomerase Diego, CA, USA). The Protein concentration was determined with reverse transcriptase) (4). Telomerase is expressed low in BCA protein assay kit (PIERCE). Twenty mg of proteins from each most normal tissues in vivo, but is known to become lysate were electrophoresed in 4 – 12% SDS- polyacrylamide gels and then electro-transferred to PVDF membrane (Invitrogen, activated during tumorigenesis, including prostate Carlsbad, CA, USA). The PVDF membrane was blocked in 5% carcinogenesis (5). Recent findings have directly implicated nonfat milk in 1xPBS and 0.05% Tween-20 and then exposed to AR telomerase in the escape from senescence (4). Senescence specific monoclonal antibody (SC-7305, H-280, Santa Cruz, CA, can be prevented by introducing the hTERT into human USA) at a dilution of 1:100 for 1 hour at room temperature. The cells (6-8). Thus, an alternative approach to immortalizing antimouse secondary polyclonal antibody (SC-2005, Santa Cruz) various human cells has been developed recently by was then applied at a dilution of 1:500 for 1 hour at room transfecting human cells with a vector expressing the hTERT temperature. Antigen antibody complexes were detected with an enhanced chemilluminescence detection kit (ECL. PIERCE). gene. To our knowledge, no successful establishment of immortal primary African-American prostate epithelial cells Androgen and AR antagonist sensitivity assay. Both stock solution of with viral agents or telomerase has been reported. Our goal DHT (10 mM) and flutamide (10 mM) were prepared in absolute is to generate new, continuously proliferating human ethanol and subsequently diluted by corresponding medium. The prostate epithelial cell lines from primary prostate benign same passage of the cells was used for the DHT and flutamide tissues of African-American prostate cancer patients by sensitivity assays. The RC-165N/hTERT cells (1 x 105) in the KGM using telomerase. medium were plated into individual wells of sterile six-well plates. After 24 hours, the BPE and rEGF in the KGM were replaced by Materials and Methods 0.1% BSA solution (designated as KGM/BSA medium) and androgen was added at 0, 0.01, 0.1, 1.0 and 10.0 nM DHT (Sigma, St. Louis, MO, USA) in a final concentration of ethanol 0.01% Cell culture. Epithelial cells (RC-165N) were derived from benign (v/v), respectively. The plates were placed into a 37ÆC incubator prostate tissue of an African-American patient who underwent with 5% CO atmosphere for 4 days with a change of medium radical prostatectomy. Benign prostate tissue was confirmed under 2 including DHT doses after 2 days. The cells were counted by a light microscopy by an experienced pathologist. Primary cultures particle counter (Coulter Counter Z1, Beckman) after were established by the explant-outgrowth method as described (9). trypsinization. For AR blocking study in the presence of exogenous For serial passages, routine trypsinization was used. Keratinocyte DHT doses, cells (3 x 104) in the KGM/BSA medium were plated serum-free medium (Cat. 10724-011, Gibco, Gaithersburg, MD, into individual wells of sterile 24-well plates. After 24 hours USA) supplemented with bovine pituitary extract (BPE), settlement, a single dose of flutamide was then spiked into 24-well recombinant epidermal growth factor (rEGF), 1% PSN antibiotic plate at a final concentration of 50 nM. For the dose-dependent mixtures (Cat. 15640-055, Gibco), and 1% amphotericin B (Cat. antagonist sensitivity assay, cells (3 x 104) in the KGM medium 15290-018, Gibco) was used for growing and maintaining the cells were plated into individual wells of sterile 24-well plates. After and designated as KGM medium (9). settlement, flutamide (0, 0.1, 1, 10, 100 nM) was added in sterile 24-well plates without addition of DHT. After the cells had been Generation of the RC165N/hTERT cell line. The actively allowed to grow 4 days, a colorimetric assay kit (Cell Titer 96 One proliferating secondary RC165N cells were infected with a Solution Cell Proliferation Assay, G358/1, Promega, Madison, WI, recombinant retrovirus construct, LXSN-hTERT(generously USA) was used to determine the number of viable cells in provided by Vimla Band, Ph.D., New England Medical Center, proliferation assay following the manufacturer’s instruction. In Boston, MA, USA) containing the hTERT (7). brief, 120 ÌL of reagent solution was added into individual wells (600 ÌL medium) and the plates were incubated for 2 hours at Telomerase assay. Cellular extracts were assayed for telomerase 37ÆC in a humidified 5% CO2 atmosphere. A 96-well plate reader activity with TRAP assay (10). This assay amplifies telomeric recorded the absorbance at 490 nm after incubation and transfer repeats; the repeat length is longest in cells exhibiting telomerase of 200 Ìl of resulting reaction solution from 24-well plates. The activity and shortest in cells exhibiting little or no telomerase activity. dynamic linear range between absorbance at 490 nm and up to 2 x 105 cells/well (RC-165N/hTERT) was examined and used to keep RT-PCR assay. The RT-PCR assay was performed as described the measured absorbance within linear range. previously (9). Briefly, total RNAs from culture cells were extracted with RNAzol B (TEL-TEST Inc., Friendswood, TX, Statistical analysis. The differences in mean values of the relative USA). One Ìg of total RNA was reverse transcribed into cDNA intact cell ratio between untreated and treated groups were with RNA PCR-kit (Perkin-Elmer, Foster, CA, USA) and 1/10 of analyzed by one-way analysis of variance with Dennett’s pair-wise the reverse-transcribed product from each sample was used for multiple comparison test (n=3). p<0.05 was considered statistically PCR to amplify NKX 3.1, AR, PSA, PSCA, CK 8, GAPDH and significant. p16, respectively. The primer sequences and the expected size of the PCR products were the same as described previously (9). AR sequencing. Total RNA was extracted from RC165N/hTERT cells and mRNA was reverse transcribed into cDNA with the RNA Western blot analysis. Cell lysates were prepared from confluent cells PCR-kit (Perkin-Elmer). Six pairs of primers were used to amplify by T-PER tissue protein extraction reagent (PIERCE, Rockford, 6 fragments which cover the whole encoding sequence of AR; the IL, USA) containing protease inhibitor cocktail (Calbiochem, San detailed primer sequence information is:

2 Gu et al: Androgen and Androgen Receptor Antagonist Responsive Benign Prostate Cell Line

Table I. Properties of RC-165N cells and the telomerase-transduced RC- 165N/hTERT cell line.

RC-165N RC-165N/hTERT

Life-span >Passage 3

Growth (PD time) ND 35 hours

Telomerase activity Not present Present

Colony formation in soft agar ND Negative

Gene expression by RT-PCR NKX3.1 + + Cytokeratin 8 + + PSCA + + P16 + + PSA - - Androgen receptor + + GAPDH + +

Tumorigenicity in SCID mice ND Negative

ND=not done PD=population doubling

Figure 1. Telomerase activity in extracts from cultured RC-165N/hTERT cells. Cells were analyzed for telomerase activity by TRAP assay as described (10). (A) LNCaP cells, (B) RC-165N/hTERT (passage 2), (C) RC-165N/hTERT (passage 10).

Sense Anti-sense (Forward) (Reverse)

5’-GAAGTAGGTGGAAGATTCAGCCA-3’ 5’-GTTGTCA GAAATGGTCGAAGTG-3’ 5’-ATGGAAGTGCAGTTAGGGCTGGGA-3’ 5’-ACTTCG CGCACGCTCTGGAA-3’ 5’-AGGAAGCAGTATCCGAAGGCA-3 5’-GCCAGG GTACCACACATCAGGT-3’ 5’-ACCTGATGTGTGGTACCCTGGC-3’ 5’-ATCAGG GGCGAAGTAGAGCATC-3’ 5’-AGCCCCACTGAGGAGACAACC-3’ 5’-TCACTG GGTGTGGAAATAGATGGG-3’ 5’-GAAGCTGACAGTGTCACACAT-3’ 5’-TCACT GGGTGTGGAAATAGATGGG-3’

PfuTurbo DNA Polymerase (Stratagene, La Jolla, CA, USA) was used to increase the fidelity of PCR (11). Gel purification was performed to isolate the PCR products. Direct sequencing was carried out by 6 purified fragments. To confirm the repetition number of CAG and GGC, the PCR products containing CAG and GGC repeats were amplified by the following two pairs of primers:

Sense Anti-sense (Forward) (Reverse)

5’-TCCAGAGCGTCCGCGAAGT-3’ 5’-GTTGT CAGAAATGGTCGAAGTG-3’ 5’-GAGGTTACACCAAAGGGCTAGAA-3’ 5’-GCCAGG GTACCACACATCAGGT-3’

3 ANTICANCER RESEARCH 25: 1-8 (2005)

Figure 2. Morphology of the RC-165N/hTERT cell line. This culture shows typical epithelial cell morphology.

PRISM Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used following the manufacturer’s instruction.

Colony formation in soft agar. A cell suspension (5x105 cells/ml) in 5 ml of 0.35% Noble agar with KGM was overlaid into a 60-mm dish containing 0.5% agar base. Colonies of > 0.2 mm in diameter were counted on day 21.

Tumorigenicity in SCID mice. Tumorigenicity assay was carried out in SCID mice. Briefly, viable cells (1x107) in 0.2 ml of PBS were injected subcutaneously into the mid-dorsal intrascapular region of adult SCID mice to determine tumorigenicity. The SCID mice were observed for 6 months for the appearance of tumor developments.

Cytogenetic analysis. Chromosome counts, ploid distribution and Giemsa (G)-banded karyotypes were prepared by standard protocol, as described previously (12). Results Figure 3. Analysis of RT-PCR products generated from the RC-165N and the RC-165N/hTERT cell lines. Each cell line was also evaluated without Immortalization of RC-165N cells following hTERT transduction. reverse transcriptase activation, which resulted in an internal control. The To determine whether RC-165N cells were immortalized by figure shows the representative data through quadruple experiments. the overexpression of hTERT, we introduced a retrovirus construct expressing hTERT into secondary RC-165N cells through overnight infection in the presence of polybrene (10 These two amplified fragments were integrated into Zero Blunt Ìg/ml). Non-infected cells could not be propagated serially TOPO PCR Cloning plasmid (Invitrogen), and transformed into E. coli. Plasmids extracted from positive colonies were analyzed with beyond 3 subcultures (Table π). In contrast, the hTERT- EcoRI. Plasmids containing the correct size of fragments were infected RC-165N cells have apparently unlimited life-span sequenced with M13 forward and reverse primers. The 3100 ABI and have been successfully subcultivated for >100 population

4 Gu et al: Androgen and Androgen Receptor Antagonist Responsive Benign Prostate Cell Line

Figure 4. Androgen receptor (AR) protein detection. Lysate (20 Ìg protein) of each cell line were electrophoresed, electroblotted and then detected with the Anti-AR monoclonal antibody and a secondary antibody. Lanes 1: LNCaP cells, 2: LNCaP cells (5 Ìg proteins), 3: 267B1 cells, 4: MLCSV40 cells, 5: DU-145 cells, 6, RC-165N/hTERT cells. 267B1 and MLCSV40 cells are previously immortalized human prostate cell lines.

Figure 5. Androgen sensitivity assay in a box plot format. Sensitivity to androgen (DHT) was assessed for RC-165N/hTERT cells after growth in serum-free K-SFM media with 0.1% BSA in the presence of 0, 0.1, 1.0, 10.0 and 100.0 nM DHT for 4 days. Significant difference in cell growth was observed at 10.0 nM DHT (p<0.05) in stimulation and 10.0 nM DHT (p<0.05) in inhibition. The numbers of cell counts were expressed as the mean value of triplicate observations (n=3).

Figure 6. A. The effect of 50 ÌM flutamide on the proliferation of RC- doublings (>50 subcultures) over the course of one year with 165N/hTERT cells in the presence of DHT doses in a box plot format. The no evidence of decreased proliferation capacity. addition of 50 nM flutamide undifferentiated the cell growth despite the presence of DHT doses (Group p=0.069). Thus, a single dose of Telomerase activity in RC-165N/hTERT cells. To confirm that flutamide (50 nM) blocked the stimulatory effects of DHT on the proliferation of RC-165N/hTERT cells. At 1.0 nM DHT, that showed the the immortalized RC-165N/hTERT cells do contain the maximum stimulation effect (Figure 5), no difference from the control transduced hTERT, the telomerase repeat amplification sample was observed in the presence of 50 nM flutamide (p=0.771). The protocol (TRAP) (10) was carried out. A high level of values of relative proliferation (%), described by the ratio of the telomerase activity was detected in RC-165N/hTERT cells absorbance at 490 nm of untreated control sample to those of the treated (Figure 1). samples, were expressed as the mean value of triplicate observations. B. Inhibition of cell proliferation by flutamide in the absence of exogenous DHT in a box plot format. The proliferation of RC-165N/hTERT cells was Phenotype of RC-165N/hTERT cells. The RC-165N/hTERT cells significantly inhibited by the presence of flutamide in a dose-dependent have the typical epithelial cell morphology (Figure 2). RC- fashion (p<0.05). The dose with initial proliferation inhibition was 165N/hTERT cells, like parent RC-165N cells, expressed an observed at 10 nM flutamide (p<0.001). The inhibition doses (ID) with epithelial cell-specific cytokeratin 8, androgen-regulated 50% and 25% effectiveness were calculated as 45 and 290 nM for flutamide, respectively. The values of relative proliferation (%) were prostate-specific gene NKX 3.1, AR, PSCA, p16 and GAPDH, computed by the ratio of the absorbance at 490 nm of the untreated but no detectable PSA was expressed (Figure 3). The RC- control sample to those of the treated samples, and were expressed as the 165N/hTERT cells did not grow in soft agar. In addition, the mean value of triplicate observations.

5 ANTICANCER RESEARCH 25: 1-8 (2005)

Figure 7. Karyotype of the RC-165N/hTERT cell line. Loss of chromosome Y and various marker formation were observed.

RC-165N/hTERT cells were non-tumorigenic for up to 6 of DHT increased to 1.0 nM, the cell count increased, but cell months after subcutaneous inoculation in SCID mice (Table I). growth decreased as the DHT concentration further increased up to 10 nM. The cell growth showed modest biphasic profile, Androgen receptors. To determine whether RC-165N/hTERT which was similar to that of LNCaP cells with respect to DHT cells express detectable AR protein, Western immunoblot doses (14, 15). Statistical analysis indicates that the mean analysis was carried out. LNCaP and DU145 cells were used value difference between control and 1.0 nM or 100 nM DHT as a positive and a potential negative control, respectively. treated was significant (p<0.05). The RC-165N/hTERT cells The results (Figure 4) indicate production of androgen responded with maximum proliferation to 1.0 nM DHT, but receptor in RC-165N/hTERT cells. A strong-band for LNCaP cell proliferation returned to the control level at 10.0 and was and weaker band for the other cell lines, including the RC- significantly lower at 100.0 nM DHT (Figure 5). 165N/hTERT cells, were observed. No AR production was detected in DU-145. AR antigonist responsiveness of RC-165N/hTERT cells. The After careful investigation of cDNA sequences, no functionality of the cells with wild-type AR was further mutation was found in all 8 exons, including codon 877, which characterized by an AR antagonist (flutamide) in terms of mutated in LNCaP and some clinical samples (3,13). The cell proliferation inhibition. The summary of inhibition by number of CAG and GGC repetition is 26 and 17, flutamide is presented in Figure 6A,B. Fifty nM flutamide respectively. Based on our sequencing data, we concluded that was spiked to the same setup as the DHT dose dependence the RC-165N/hTERT cells express wild-type AR. test (Figure 6A). A single dose of flutamide (50 nM) successfully reversed the stimulation of cell proliferation by Androgen sensitivity of RC-165N/hTERT cells. To examine if stimulatory doses of DHT (p=0.069). This indicates that the cell growth was sensitive to androgen, the RC- molar excess of an antagonist competes with DHT to AR 165N/hTERT cells were grown in the KGM/BSA medium at proteins and inhibits the agonistic activity of 1.0 nM DHT in different doses of DHT for 4 days. The BPE and rEGF the RC-165N/hTERT cells. In contrast to the LNCaP cells supplements of K-SFM were replaced by HPLC purified 0.1% that express mutated AR and are ineffectively responsive to bovine serum albumin solution to minimize endogenous flutamide (16), the competition between DHT and flutamide androgen or AR-activating components. As the concentration to wild-type AR in the RC-165N/hTERT cells is evident.

6 Gu et al: Androgen and Androgen Receptor Antagonist Responsive Benign Prostate Cell Line

For further examination of the antagonistic effect on cell to locus 1q 42.2-43 (18, 19) and appeared to represent the most proliferation in the basal KGM medium with supplements frequent known locus predisposing to hereditary prostate (BPE and rEGF), the cells were treated by flutamide doses cancer in Western and Southern Europe (20). Possible evidence (Figure 6B). The proliferation of the cells in the KGM medium of a prostate cancer susceptibility locus on chromosomes 4q and was inhibited in a dose-dependent fashion. A significant 20 has been reported (21, 22). There are reports of loss of inhibition of proliferation was observed at 10 ÌM flutamide and heterozygosity for both 10p and 10q in prostate cancer greater (p<0.0001). The computed inhibition doses (ID) with described in the literature (23,24). 50% and 25% effectiveness were 45 and 290 nM for flutamide, We and others have previously developed reliable methods respectively. After linearization of the inhibitory doses of for generating and characterizing human prostate epithelial cell flutamide with logarithm transformation and linear regression lines from normal prostate tissue by viral DNA oncoproteins of the relative cell proliferation, ID values were calculated. such as SV40 and high-risk HPVs (25-28). However, no such Figure 6B also indicates the existence of either trace amounts of virally-induced African-American prostate epithelial cell line endogenous androgen or of AR-activating components in the has been established. These virally-induced cell models are not K-SFM medium with the BPE and EGF due to no prior ideal, because the immortalizing cells frequently contain viral addition of exogenous DHT. These data suggest that the cell oncogenic DNA and accompany major cytogenic alterations. line not only grows in a DHT-dependent fashion, but also Development of in vitro human cell models that mimic human flutamide can block the AR-signaling pathway (Figure 6A,B). prostate cancer progression would be ideal. As described above, Based on the functional characterization of both agonistic we have succeeded in immortalization by the introduction of (DHT) and antagonistic (flutamide) effects on cell telomerase into primary prostate epithelial cells derived from proliferation, we concluded that the RC-165N/hTERT cell line African-American benign prostate tissue. As shown by other responds to the presence of agonist (DHT) in stimulation, and human cells (4, 6-8), we anticipated that the hTERT- of AR antagonist in inhibition of cell proliferation mediated by immortalized cell would maintain the phenotypic and genetic functional and wild-type androgen receptors. characteristics of its primary cells. Conceptually, the successful establishment of spontaneous immortalized human prostate Chromosome analysis. Chromosome study was performed at epithelial cells derived from benign prostate tissue would be passage 24 on 30 metaphases. The cell line is aneuploid human ideal and a major breakthrough in prostate cancer research. male (XO), with most chromosome counts in the 80’s to 90’s However, the generation of such cell lines is an extremely rare range. The model number is 90. There are observed 2X’s in event and, to date, has not been reported. Establishment of a each karyotype. Looking at the normal chromosomes and spontaneously immortalized primary androgen-responsive markers, there is loss of Y, 1q (1q42>qter) in marker M8/M8A, African-American prostate cancer cell line has recently been 4q (q26>qter) in marker M5, 10q (q22>qter) in M9, 12p reported (29). However, this cell line does not express the full (12p12.1>pter) in marker M7, some loss of N22. There is 7p neoplastic phenotype. loss in marker M2 but most likely this is translocated to marker As described, this RC-165N/hTERT cell line expresses M7. There are further gains of chromosome 15 and 20 and several important biomarkers including wild-type androgen unknown material in M1 [(3q+)], M3[13p+], M4 [t(14q;?)], receptor. The most valuable feature of this cell line is its M6[16q+] and M8 [der (1)t(1q41;+hsr) (Figure 7). capability of androgen sensitivity and blockage of AR signaling pathway by an AR antagonist (flutamide). The novel cell line Discussion may provide a useful in vitro model for the study of prostate cancer biology, and for molecular characterization of prostate The present study appears to represent the first documented carcinogenesis, especially for the high-risk African-American case of the establishment of a human prostate epithelial cell population. We have, for the first time to our knowledge, line from high-risk African-American benign prostate tissue succeeded in the immortalization of primary African-American immortalized by telomerase. The immortalized RC- benign prostate epithelial cells in culture by transduction with a 165N/hTERT cells show epithelial morphology and are grown retrovirus vector expressing hTERT. The telomerase expression in serum-free medium. They showed no cell growth in soft agar in primary African-American benign prostate epithelial cells and no tumor formation in SCID mice. They expressed CK8, can itself induce immortalization and does not induce changes NKX3.1, PSCA, p16 and AR, but did not express PSA. The associated with a transformed phenotype. These immortalized cell line was aneuploid and showed losses of chromosomes Y, cells express prostate-specific markers including androgen and 1q, 4q, 10q, 12p, and 22 and gains of chromosomes 15 and 20. androgen receptor antagonist responsiveness. These cells Some of the alterations of chromosomes 1q, 4q, 10q, 20 and provide a useful tool to study the biology and the pathology of Y observed in the RC-165N/hTERT cell line have been already benign prostate epithelial cells, to understand the steps leading reported in the literature (17). It is interesting to note that a to malignant prostate transformation, especially for high-risk predisposing gene for early-onset prostate cancer was mapped African-American men.

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