Immortalization of Primary Human Prostate Epithelial Cells by C-Myc

Research Article

Immortalization of Primary Human Prostate Epithelial Cells by c-Myc

Jesu´s Gil,1 Preeti Kerai,3 Matilde Lleonart,4 David Bernard,5 Juan Cruz Cigudosa,6 Gordon Peters,1 Amancio Carnero,7 and David Beach2

1Molecular Oncology Laboratory, Cancer Research UK, London Research Institute; 2Center for Cutaneous Biology, Institute for Cell and Molecular Sciences, London, United Kingdom; 3University of Cambridge, Cancer Research UK, Department of Oncology and the Medical Research Council Cancer Cell Unit, Hutchinson/Medical Research Council Research Centre, Cambridge, United Kingdom; 4Department of Pathology, Hospital Vall d’Hebron, Universitat Autonoma de Barcelona, Barcelona, Spain; 5Free University of Brussels, Laboratory of Molecular Virology, Faculty of Medicine, Brussels, Belgium; and 6Cytogenetics Unit, Biotechnology Program and 7Experimental Therapeutics Program, Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain

Abstract keratinocytes grown on feeder layers could be immortalized by hTERT alone, others have found a requirement for inactivation of A significant percentage of prostate tumors have amplifica- INK4a tions of the c-Myc gene, but the precise role of c-Myc in the Rb/p16 pathway even in these conditions (9). prostate cancer is not fully understood. Immortalization of The establishment of improved models of human cancer human epithelial cells involves both inactivation of the Rb/ progression relies on the expression of defined combinations of p16INK4a pathway and telomere maintenance, and it has been genes that mirror those naturally arising in cancer. In this context, recapitulated in culture by expression of the catalytic subunit significant advances have been achieved recently (10–13). Transfor- of telomerase, hTERT, in combination with viral oncoproteins. mation of different human primary cells has been achieved by serial introduction of hTERT, SV40 large T (inactivating at least the Here, we show the immortalization of human prostate INK4a epithelial cells (HPrEC) by a single genetic event, the p16 /Rb and the p53 pathways), SV40 small t (affecting c-Myc expression of the c-Myc oncogene. Myc stabilizes telomere stability through PP2A regulation (14, 15)), and oncogenic Ras (16). length in HPrEC through up-regulation of hTERT expression However, these models rely on the use of viral oncoproteins. As and overrides the accumulation of cell cycle inhibitory viruses are implicated in a limited spectrum of cancer types, we proteins, such as p16INK4a. Overall, HPrECs expressing c-Myc reasoned that human epithelial cells immortalized by cellular genes retain many characteristics of normal cells, such as the that are amplified in naturally occurring cancers could represent a induction of a senescence-like growth arrest in response to first step toward the generation of more realistic models of human oncogenic Ras, an intact p53 response, and an absence of cancer. Such models have already been established in human gross karyotypic abnormalities. However, HPrECs expressing fibroblast by taking advantage of short hairpin RNA technology or c-Myc lack a Rb/p16INK4a checkpoint and can be transformed using cells derived from patients with germ line mutations in INK4a without the need for additional genetic lesions in that p16 (17, 18). pathway. These results give a partial explanation for the The oncogene c-Myc fulfils many of the expectations for a gene physiologic role of c-Myc overexpression in prostate cancer. involved in immortalization of human epithelial cells. Deregulated (Cancer Res 2005; 65(6): 2179-85) expression of c-Myc caused by gene amplification, chromosomal translocation, or proviral insertion is widely associated with Introduction tumorigenesis in both human cancers and rodent models (19). Ectopic expression of c-Myc has been shown to increase the life Normal human somatic cells undergo a limited number of span of IMR-90 fibroblasts and HMECs, an effect that is in part divisions before entering an irreversible growth arrest state defined attributed by the activation of hTERT transcription (20, 21). In this as replicative senescence (1). This life span varies between different context, although 85% to 90% of human tumor cells maintain their cell types and is also dependent on culture conditions (2). The telomeres by up-regulation of telomerase (22), fewer than 30% have acquisition of unlimited proliferative ability is probably a key step amplifications of the hTERT gene (23), and deregulation of Myc during tumorigenic progression (3). It has been proposed that the may represent an alternative mechanism. In addition, c-Myc has restoration of telomerase activity is sufficient to immortalize human been shown to promote cell proliferation in the presence of diploid fibroblasts, endothelial cells, T lymphocytes, and some other p16INK4a or hypophosphorylated pocket proteins (24). cell types (4–6). However, different studies have identified two Prostate cancer is the most common noncutaneous malignancy requirements for immortalization of human epithelial cells: diagnosed in American men (25), but there is only a limited maintenance of telomere length and inactivation of the Rb/p16INK4a understanding of its genetic and molecular basis (26). Different pathway (7, 8). Thus, expression of hTERT in combination with viral studies have shown that c-Myc mRNA is commonly overexpressed in oncoproteins, such as the E7 product of human papillomavirus 16 hyperplasic and malignant prostate (27), and the c-Myc gene is or the SV40 large T antigen, is sufficient to immortalize human amplified in between 11% and 40% of prostate cancers in different keratinocytes and human mammary epithelial cells (HMEC; ref. 8). studies (28–30). Although c-Myc amplification has been mainly Although Ramirez et al. (2) suggest that both HMEC and associated with metastatic progression, c-Myc is also amplified at lower levels in initial stages of prostate carcinogenesis (30). Interestingly, high levels of hTERT mRNA in prostate tumors Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). correlate with high c-Myc mRNA expression (31). Recently, we have Requests for reprints: Jesu´s Gil, Molecular Oncology Laboratory, Cancer Research uncovered a role for c-Myc in androgen-independent prostate UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom. Phone: 44-2072693594; Fax: 44-2072693094; E-mail: [email protected]. cancer cell growth (32) coherent with the observed c-Myc I2005 American Association for Cancer Research. amplification in late stages of prostate cancer. In addition, c-Myc www.aacrjournals.org 2179 Cancer Res 2005; 65: (6). March 15, 2005

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Cancer Research overexpression has been used for modeling human-like prostate Biotechnology), and anti-p27 (sc-1641, Santa Cruz Biotechnology). The cancer in a murine model (33). However, a molecular explanation for corresponding peroxidase-labeled secondary antibody (Santa Cruz Biotech- the role of c-Myc amplification during early stages of human prostate nology) was detected using enhanced chemiluminescence Western blotting cancer is still needed. reagents (Amersham Biosciences). Analysis of Retroviral Integration Sites. Genomic DNA (15 Ag) from With the aim of reconstructing prostate cancer progression, we HPrEC c-Myc cells was digested with EcoRI and hybridized to a 32P-labeled derived primary human prostate epithelial cells (HPrEC) and asked c-Myc probe. whether they could be immortalized by several viral and cellular Reverse Transcription-PCR Analysis. Cells were homogenized in Trizol genes. Among the genes tested, only c-Myc was able to immortalize (Invitrogen, San Diego, CA) and total RNA was isolated according to the HPrEC as a single agent. In addition, HPrEC expressing c-Myc manufacturer’s recommendations. Complementary DNAs were synthesized lacked Rb/p16INK4a checkpoint control and became transformed using the first-strand cDNA synthesis kit (Roche, Indianapolis, IN) and without the need for additional alterations in that pathway. These amplified in a final volume of 50 AL containing 150 Amol/L deoxynucleotide observations clarify the role of observed c-Myc amplifications in triphosphate, 2 mmol/L MgCl2, 1 unit Taq gold polymerase (Applied A ARF prostate cancer. Biosystems, Foster City, CA), and each primer at 1 mol/L. For p14 , DMSO was added at a final concentration of 1%. Primers used were as follows: p14ARF forward GAGTGGCGCTGCTCACCTC, p14ARF reverse TACCGTGC- Materials and Methods GACATCGCGAT, and h-actin as described elsewhere (36). Twenty-five to 30 amplification cycles were done at 94jC for 1 minute, 55jC for 1 minute, and Establishment of Primary HPrEC Cultures. A primary culture of 72jC for 1 minute, with an initial denaturation step of 5 minutes at 95jC. HPrEC was derived from the histologically normal prostate (data not shown) of an adult male undergoing a cystectomy for bladder carcinoma. Briefly, the tissue was minced and digested in RPMI 1640 (Life Technologies, Results Gaithersburg, MD) containing 5% FCS and 400 units/mL collagenase. The digested tissue was plated in collagen IV–coated plates and maintained Expression of c-Myc Bypasses Senescence and Extends Life with PrEGM (BioWhittaker, Walkersville, MD) serum-free medium accord- Span of HPrEC. To study the requirements for immortalization of ing to the manufacturer’s instructions. HPrEC, we derived a primary culture of HPrEC from normal prostate Cell Culture and Retroviral-Mediated Gene Transfer. The ampho- tissue from an adult donor. These cells showed typical epithelial tropic packaging cell line LinXA was maintained in DMEM (Life Tech- morphology, stained positive for a pan-cytokeratin antibody, and nologies) and 10% FCS (Sigma, St. Louis, MO). HPrEC were established as expressed multiple keratins as well as several prostate markers (such explained above or acquired from Clonetics (East Rutherford, NJ) and were as prostate-specific phosphatase, several kallikreins, and the maintained in PrEGM. PC-3 cells were maintained in RPMI 1640 and 10% transcription factor Nkx3.1) as assessed by microarray analyses FCS. Retrovirus production and infection of target cells was done according (data not shown). HPrECs had a finite life span in culture of f20 PDs to ref. 20. HPrEC were selected using 250 ng/mL puromycin, 100 Ag/mL f G418, 500 ng/mL blasticidin, or 20 Ag/mL hygromycin as required. (Fig. 1A). HPrEC at PD 10 were infected with retroviral vectors Telomerase Assays. Telomeric repeat amplification protocol assay was expressing either green fluorescent protein, the catalytic subunit of done as described before (20). PCR products were electrophoresed on 12% telomerase (hTERT), c-Myc, Mdm2, human papillomavirus 16 E7, or polyacrylamide gels. Gels were dried and autoradiographed. Telomeric both human papillomavirus 16 E6 and E7. As expected, green restriction fragment length was measured as described before (34). At least fluorescent protein expression did not increase the life span of three independent experiments were done with identical results. HPrECs. Infection with either Mdm2- or E7-expressing retroviruses Growth Curves. Cumulative population doublings per passage were resulted in a modest increase of life span, but eventually the cells calculated as log2 (number of cells at time of subculture divided by number stopped proliferating (Fig. 1A). Introduction of hTERT resulted in an of cells plated) and plotted against total time in culture to assess replicative extension of proliferative potential up to f60 PD, but cells senesced life span. Three independent experiments were done using cells from two different donors. For 12-day growth curves, 20,000 cells per well were plated thereafter (Fig. 1B). Only HPrECs expressing the E6 and E7 into 24-well plates. At the indicated times, cells were washed with PBS, fixed oncoproteins of human papillomavirus 16 or c-Myc maintained in 5% glutaraldehyde, and rinsed with distilled water. Cells were stained proliferation beyond 110 PDs. Similar results were obtained using with 0.1% crystal violet (Sigma) and processed as described before (35). All commercially available HPrEC from an independent donor (Sup- experiments were done in triplicate. plementary Fig. 1) and in independent infections (data not shown). 4 Soft Agar Assays. Cells (10 ) were plated in RPMI 1640 plus 10% FCS The efficiencies of retroviral infection (generally >80%) and the in 0.35% agar above a layer of 0.7% agar. After 3 weeks, colonies were continuity of the growth curves suggested that the majority of the visualized and counted. PC-3 cells were used as positive control. All experi- cells transduced with c-Myc had become immortal and argued ments were done in triplicate. against selection for clonal variants. To confirm this impression, we Karyotypic and Fluorescence In situ Hybridization Analysis. Exponentially growing cultures were incubated with 100 nmol/L colcemid compared the colony forming ability of the parental cells infected (Sigma) for 1 hour, resuspended in hypotonic buffer (75 mmol/L KCl), with empty vector with HPrEC expressing c-Myc or E6 + E7 at f incubated for 20 minutes at 37jC, fixed by addition of cold ethanol/acetic various times after infection. At passage 4, 12 F 3% of the vector acid, and applied to chilled microscope slides. Chromosome preparations of only control cells formed colonies under these conditions and the cultured cells were counted for the number of metaphase spreads. Forty Myc- and E6 + E7–expressing pools showed very similar properties at metaphase spreads were analyzed for each cell line. this and subsequent passages (data not shown). As a more direct test Immunoblotting. For immunoblot analysis, total cell extracts were of clonality, we also compared the patterns of provirus integration in fractionated by gel electrophoresis (SDS-PAGE) and proteins were transferred the Myc-transduced HPrECs at early and late passages. In EcoRI- to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). digested genomic DNA, each fragment hybridized to the Myc probe Membranes were incubated with the following primary antibodies: anti-c- Myc (sc-764, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p16INK4a (sc- corresponds to a virus-host junction and equates to a single 468, Santa Cruz Biotechnology), anti-phospho-Rb (554164, PharMingen, San integrated provirus. The hybridization patterns were indicative of a Diego, CA), anti-h-actin (A5316, Sigma), anti-p53 (sc-126, Santa Cruz polyclonal population of cells; importantly, there was no evidence for Biotechnology), anti-p21 (sc-397, Santa Cruz Biotechnology), anti-hTERT emergence of a clonal population on prolonged passaging (Fig. 1C). (sc-7212, Santa Cruz Biotechnology), anti–cyclin A (sc-596, Santa Cruz Finally, karyotypic analyses of the HPrEC expressing c-Myc indicated

Cancer Res 2005; 65: (6). March 15, 2005 2180 www.aacrjournals.org

Figure 1. c-Myc immortalizes HPrEC. A, growth curve of HPrEC derived from a healthy donor. Cumulative population doublings were plotted against time (days). B, senescence-associated h-gal staining of HPrEC infected with indicated retroviruses at PD f20 [green fluorescent protein (GFP)], PD f30 (E7 and Mdm2), PD f60 (hTERT), and PD >100 [E6/E7 and c-Myc (cmyc)]. C, analysis of retroviral integration sites in HPrEC expressing c-Myc showing that such cells are polyclonal. D, karyotypic analysis of HPrEC expressing c-Myc, showing the genetic alteration observed in 2.5% of the cultures.

that they were generally diploid. Recent reports suggest that Myc rendered HPrEC resistant to arrest by cyclin-dependent kinase overexpression can induce genomic instability in primary human inhibitors. To explore this possibility, the immortalized HPrEC fibroblasts (37), but we found only one example of a chromosomal cultures were infected with retroviruses encoding LacZ (as control) alteration, der(7)t(5;7), that was present in 2.5% of the Myc- or murine versions of p16INK4a or p27KIP1, and pools of cells were immortalized cells at very late passage (Fig. 1D). Taken together, selected for drug resistance. Expression of p16INK4a and p27KIP1 was the data are consistent with the notion that c-Myc can immortalize monitored by immunoblotting with antibodies specific for the HPrECs without the need for additional mutations. murine proteins (Fig. 3A; data not shown). On infection with a Myc Expression Activates Telomerase in HPrEC. As telomere p16INK4a retrovirus, the hTERT-expressing cells arrested with a maintenance is a prerequisite for immortalization of human cells, we senescence-like phenotype (Fig. 3A; data not shown), whereas c-Myc- tested the effect of c-Myc expression on HPrEC telomeres. To this expressing HPrEC continued to proliferate. Thus, c-Myc allowed end, we first analyzed the average lengths of the telomeric end HPrEC to tolerate elevated levels of p16INK4a expression as shown fragments by Southern blotting. Expression of either hTERTor c-Myc in other cell types (24). In contrast, p27KIP1 expression provoked an resulted in the maintenance of telomere length relative to the vector arrest in both c-Myc- and hTERT-expressing HPrEC. only control cells, whereas introduction of E7 or Mdm2 had no effect To further analyze the impact of c-Myc on cell cycle regulation, we (Fig. 2A). This correlated with the presence of telomerase activity as examined the ability of HPrEC to grow in chemically defined measured by the telomeric repeat amplification protocol assay medium with or without growth factors. As shown in Fig. 3B, hTERT- (Fig. 2B). Finally, we analyzed hTERT protein levels by immunoblot- expressing cells stop growing in the absence of growth factors, ting. Consistent with previous studies in other human cell types correlating with a decrease in the number of cells in S phase as (20, 21), we found that c-Myc up-regulates the expression of hTERT measured by BrdUrd incorporation (Fig. 3C) and a decline in the to levels comparable with that achieved with hTERT retroviral phosphorylation of pRb as monitored with a phosphospecific infection (Fig. 2C). Thus, c-Myc allows maintenance of the telomeres antibody (Fig. 3D). In contrast, c-Myc-expressing cells grew in HPrECs by increasing hTERT expression and telomerase activity. regardless of the presence of growth factors, and pRb phosphory- Effect of c-Myc on Cell Cycle Progression. Senescence is lation was maintained (Fig. 3B-D). accompanied by an increase in the levels of several cyclin-dependent Interestingly, expression of c-Myc results in increased levels of kinase inhibitors, notably p16INK4a (35). However, it has been cyclin A when compared with both parental cells and hTERT- reported previously that, in model systems at least, ectopic c-Myc expressing HPrEC (Supplementary Fig. 2). We also noted a slight can override the effects of p16INK4a, p21CIP1, and p27KIP1 (24, 38). It increase in cyclin D1 levels but no significant changes in cyclin D2 or was therefore interesting to consider whether hTERT or c-Myc had p27KIP1 (data not shown). www.aacrjournals.org 2181 Cancer Res 2005; 65: (6). March 15, 2005

Figure 2. c-Myc expression activates telomerase in HPrEC. A, telomere length was analyzed by hybridization of genomic DNA with a telomere-specific oligonucleotide after eight passages in culture (PD f20-30) in parental HPrEC (lane 1) or HPrEC expressing hTERT (lane 2), E7 (lane 3), Mdm2 (lane 4), or c-Myc (lane 5). B, cellular extracts of parental and retrovirally transduced HPrEC were tested for telomerase activity using the telomeric repeat amplification protocol assay. C, expression of hTERT and c-Myc and h-actin was analyzed by Western blot using specific antibodies. Numbers, passage numbers.

HPrECs Expressing c-Myc Have a Normal Response to stresses (39, 40). We therefore assessed these variables in c-Myc Genotoxic Agents. Human somatic cells immortalized by hTERT immortalized HPrEC. As illustrated in Fig. 4A, both hTERT and c- retain many characteristics of primary cells, such as cell cycle Myc transduced HPrEC showed the expected accumulation of p53 checkpoints and p53 activation in response to diverse genotoxic when treated with etoposide accompanied by induction of its target

Figure 3. Effect of c-Myc expression on the p16INK4a/Rb pathway. A, HPrEC expressing hTERT or c-Myc were infected with LacZ (as a control), p16INK4a, or p27KIP1 retroviruses, drug selected, and cell growth was analyzed in triplicate as explained in Materials and Methods. B, growth curves of HPrEC maintained in chemically defined medium supplemented with (com.) or without (min.) growth factors. HPrECs expressing c-Myc or hTERT were grown for the indicated times in medium depleted of growth factors. C, percentage of cells incorporating BrdUrd after a 1-hour pulse. D, extracts from cells treated as in C were analyzed by Western blotting using antibodies directed against pRb.

Cancer Res 2005; 65: (6). March 15, 2005 2182 www.aacrjournals.org

that whereas E7 alone was unable to overcome Ras-induced growth arrest in either context, E6 allowed continued proliferation of the c- Myc-expressing but not the hTERT-expressing HPrEC cells in the presence of Ras (Fig. 5). This implied that Ras was inducing a p53- dependent arrest that could not be overridden by Myc. Introduction of RasV12 or c-Myc into primary cells has been shown to result in up-regulation of both p16INK4a and p14ARF (17, 43– 45) and this also holds true for HPrEC. Analysis of p16INK4a and p14ARF expression by immunoblotting and reverse transcription- PCR, respectively, showed that infection of HPrEC with retroviruses encoding c-Myc or H-RasV12 caused increased expression of both genes relative to empty vector controls (Fig. 6A and B). For p16INK4a, the effects were comparable with the up-regulation that occurs Figure 4. HPrEC immortalized by c-Myc retain a normal p53 response. A, cells were treated with 25 Amol/L etoposide for 24 hours, and p53, p21, and naturally on continued passaging of HPrEC (cf. passages 3 and 10). ARF h-actin levels were analyzed by Western blot. B, cells were treated with different Although the p14 levels were significantly elevated by Myc and concentrations of Adriamycin for 24 hours and viability was analyzed. Ras, this had no discernible effect on the viability of the cells following treatment with Adriamycin (Fig. 6B). Introduction of E6 further enhanced the levels of p14ARF because of loss of gene p21CIP1. Induction of p21CIP1 was not observed in HPrEC suppression by p53 (46), but in the absence of p53 the cells were expressing E6 and E7 due to ablation of p53 by E6. Treatment of more resistant to the effects of Adriamycin. Importantly, c-Myc or HPrEC cells expressing hTERT or c-Myc with 1 Amol/L etoposide the combination of Myc, Ras, and SV40 small t antigen (see below) also caused a marked reduction in the number of cells in S phase did not seem to alter the viability of the HPrEC cells under these with a corresponding increase of cells in G (data not shown), an 2 conditions. effect that was abolished by expression of E6. Similarly, treatment of To evaluate the impact of c-Myc on transformation of HPrEC the hTERT- or Myc-transduced cells with increasing concentrations rather than immortalization, we compared the ability of the HPrEC of Adriamycin caused a dose-dependent loss of cell viability, cells expressing c-Myc or hTERT together with different combina- consistent with a p53-dependent apoptotic response (Fig. 4B). tions of oncogenes to form anchorage-independent colonies. As Impairment of the p53 pathway by expression of E6 reduced the shown previously in other human cell types (12), we found that in sensitivity to Adriamycin. Taken together, these results indicate that HPrEC immortalized by hTERT, addition of oncogenic Ras, E6 + E7, the p53 pathway is operational in c-Myc-expressing HPrEC. and SV40 small t enabled them to grow in soft agar (Fig. 6D). Transformation of HPrEC Expressing c-Myc. Expression of Omission of any of these genes compromised this phenotype. In oncogenic Ras in human somatic cells results in a growth arrest contrast, with c-Myc immortalized HPrEC, expression of E7 was not termed premature senescence (35), and this occurs irrespective of necessary for acquisition of anchorage independence, consistent the expression of hTERT (39, 41). In line with these observations, with the ability of c-Myc to override the p16INK4a/pRb checkpoint. expression of Val12 variant of H-Ras or the downstream effector Raf (42) caused a senescence-like growth arrest in both hTERT- and c- Myc-expressing HPrEC (Fig. 5, left). In both cases, Ras-induced Discussion growth arrest was negated by introduction of the human The ultimate aims of this investigation are to establish a model papillomavirus 16 E6 and E7 genes. Further investigation indicated that allows the in vitro reconstruction of events leading to

Figure 5. Requirements for bypassing oncogenic Ras-induced senescence in HPrEC. HPrEC expressing the indicated genes were infected with control retroviruses (o) or retroviruses expressing RasV12 (.). Cells were drug selected for 5 days, and cell growth was analyzed in triplicate as described in Materials and Methods for 12 days. www.aacrjournals.org 2183 Cancer Res 2005; 65: (6). March 15, 2005

DNA.8 To our knowledge, the work presented here is the first evidence that overexpression of c-Myc alone can result in complete immortalization of normal human epithelial cells. There are several reasons to believe that immortalization does not require an additional genetic event. In the first place, we did not see any evidence for extensive apoptosis on infection of the HPrEC cultures with the c-Myc retrovirus; other studies have made similar observations (17). Moreover, infection efficiencies were >80%, suggesting that the resultant cell pools were likely to be polyclonal, and our analyses of proviral insertion sites support this contention. Expression of c-Myc in HPrEC did not abrogate the DNA damage checkpoint, and both p53 and its target p21CIP1 were induced by DNA damage as effectively as in cells expressing hTERT (Fig. 4A). Although c-Myc can cause DNA damage through the production of reactive oxygen species (37), we did not observe gross karyotypic abnormalities in the great majority of c-Myc-expressing HPrEC, suggesting that genomic instability is not a facet of c-Myc-mediated immortalization. The ability of c-Myc to immortalize HPrEC relates primarily to two functions: its ability to up-regulate telomerase activity (Fig. 2) and its ability to override the p16INK4a/Rb checkpoint (Fig. 3A). As shown in other epithelial cell types (8), expression of E7, which is known to target pRb and related pocket proteins (50), enables hTERT to immortalize HPrEC (data not shown), consistent with the view that disruption of the p16INK4a/Rb pathway is needed to negate replicative senescence of HPrEC (7, 51). The ability of c-Myc to bypass this checkpoint is additionally important because c-Myc, as with other oncogenic agents, causes up-regulation of p16INK4a (Fig. 6A). Our finding that c-Myc-expressing HPrEC cells have elevated levels of cyclin A may account for resistance to p16INK4a- mediated arrest (Supplementary Fig. 2), but there are potentially Figure 6. Genetic events involved in transformation of HPrEC. A, effect of many other ways in which c-Myc can influence the expression and c-Myc (M) or RasV12 (R) compared with vector-infected HPrEC cells (V) over p16INK4a expression was analyzed by Western blot. B, effect of c-Myc or function of cell cycle regulators (38, 52–55). For example, c-Myc seems RasV12, E6, c-Myc + E6 (E6 + M), or c-Myc + E6 + RasV12 + small t (E6 + MRt) to reduce growth factor dependence in HPrEC as also noted in human expression in HPrEC over p14ARF expression was analyzed by reverse diploid fibroblasts (17). However, in our hands, c-Myc did not overcome transcription-PCR. C, viability of the corresponding cells after treatment with KIP1 0.2 Ag/mL Adriamycin for 24 hours. HPrECs expressing hTERT or c-Myc were ap27 -mediated arrest in HPrEC in contrast to previous studies in serially infected with retroviruses expressing the indicated genes with (+)or rodent fibroblasts. In this context, it is interesting to note that p27KIP1 without (À) small t and subsequently infected with retroviruses expressing levels have prognostic significance in prostate cancer (56–58). RasV12. Growth in soft agar was monitored and colonies were scored after 3 weeks. Cell type differences may also apply to the apparent links between c-Myc and hTERT expression. We and others have shown that late- prostate cancer progression and to clarify the role of c-Myc in this passage HMECs expressing hTERT can show amplification of c-Myc process. The introduction of a defined combination of genes in (11, 59), although this is presumably a stochastic event rather than a primary human cells allows the generation of tailor-made models direct effect. In the present work, HPrEC expressing hTERT did not of cancer progression. However, several limitations reduce the express elevated levels of c-Myc or acquire growth factor utility of the currently developed models. One is the use of viral independence (Figs. 2C and 4B; data not shown). oncoproteins as pivotal components of the transformation Identification of the genetic events that cooperate with c-Myc in protocol. In addition, most use ectopic expression of hTERT to the bypass of Ras-induced senescence and transformation of HPrEC maintain telomere length in these cells. Although telomerase constitute a first step for reconstructing prostate cancer progression activity is present in most of human tumors, the hTERT gene itself in vitro. Interestingly, in accordance with the ability of c-Myc to is amplified in a limited number of cases (47). Therefore, we override the p16INK4a/Rb checkpoint, additional alterations in this believe that the immortalization of HPrEC by c-Myc may provide pathway are not required for transformation, whereas the hTERT- an alternative and more physiologic start point for studying expressing counterparts require inactivation of both pRb and p53 prostate cancer progression. (Fig. 6). Thus, the amplification of the c-Myc gene that is observed in Although c-Myc has been shown previously to extend the life span a high percentage of prostate tumors may have multiple explan- of fibroblasts and HMECs (20), the consequences of Myc expression ations. At early stages, c-Myc can confer a proliferative advantage by may be context dependent. For example, it has been reported that immortalizing prostate cells and allowing them to grow under Myc causes terminal differentiation in primary keratinocytes (48) limited growth factor conditions. At later stages, c-Myc may and premature senescence in some strains of fibroblast (17), an contribute to androgen-independent growth of the prostate cancers outcome that may depend on the basal levels of p16INK4a and the as reported previously (32). extent to which the cells have been exposed to culture stress (49). In the published study on HMECs (20), the cells used had already INK4a shutdown p16 expression due to methylation of the genomic 8 Unpublished observations.

Cancer Res 2005; 65: (6). March 15, 2005 2184 www.aacrjournals.org

Acknowledgments 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 Received 12/23/2003; revised 12/20/2004; accepted 1/11/2005. with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: Cancer Research UK, Hugh and Catherine Stevens Fund, Long- We thank G. Muir for donation of prostate tissue; D. Hudson for help in initial term Fellowships from EMBO and Human Frontiers Science Program (J. Gil), Brussels establishment of prostate primary cultures; L. Martı´nez for technical assistance; Capital Fellowship (D. Bernard), and Long-term Intra-European Marie Curie R. Weinberg, W.C. Hahn, and M. McMahon for the gift of retroviruses; J. Masters for Fellowship. helpful suggestions and discussions; and M. Collado for review of the article.

References 20. Wang J, Xie LY, Allan S, Beach D, Hannon GJ. Myc changes associated with a transformed phenotype. Nat activates telomerase. Genes Dev 1998;12:1769–74. Genet 1999;21:111–4. 1. Hayflick L. The limited in vitro lifetime of human 21. Wu KJ, Grandori C, Amacker M, et al. Direct 41. Wei W, Jobling WA, Chen W, Hahn WC, Sedivy JM. diploid cell strains. Exp Cell Res 1965;37:614–36. activation of TERT transcription by c-MYC. Nat Genet Abolition of cyclin-dependent kinase inhibitor 2. Ramirez RD, Morales CP, Herbert BS, et al. Putative 1999;21:220–4. p16INK4a and p21Cip1/Waf1 functions permits Ras- telomere-independent mechanisms of replicative aging 22. Shay JW, Bacchetti S. A survey of telomerase activity induced anchorage-independent growth in telomerase- reflect inadequate growth conditions. Genes Dev 2001; in human cancer. Eur J Cancer 1997;33:787–91. immortalized human fibroblasts. Mol Cell Biol 2003;23: 15:398–403. 23. Zhang A, Zheng C, Lindvall C, et al. Frequent 2859–70. 3. Greider CW. Telomerase activation. One step on the amplification of the telomerase reverse transcriptase 42. Zhu J, Woods D, McMahon M, Bishop JM. Senescence road to cancer? Trends Genet 1999;15:109–12. gene in human tumors. Cancer Res 2000;60:6230–5. of human fibroblasts induced by oncogenic Raf. Genes 4. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of 24. Alevizopoulos K, Vlach J, Hennecke S, Amati B. Dev 1998;12:2997–3007. life-span by introduction of telomerase into normal Cyclin E and c-Myc promote cell proliferation in the 43. Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, human cells. Science 1998;279:349–52. presence of p16INK4a and of hypophosphorylated DePinho RA. Role of the INK4a locus in tumor 5. Rufer N, Migliaccio M, Antonchuk J, Humphries RK, retinoblastoma family proteins. EMBO J 1997;16: suppression and cell mortality. Cell 1996;85:27–37. Roosnek E, Lansdorp PM. Transfer of the human 5322–33. 44. Zindy F, Eischen CM, Randle DH, et al. Myc signaling telomerase reverse transcriptase (TERT) gene into T 25. Isaacs W, De Marzo A, Nelson WG. Focus on prostate via the ARF tumor suppressor regulates p53-dependent lymphocytes results in extension of replicative poten- cancer. Cancer Cell 2002;2:113–6. apoptosis and immortalization. Genes Dev 1998; tial. Blood 2001;98:597–603. 26. Abate-Shen C, Shen MM. Molecular genetics of 12:2424–33. 6. Yang J, Chang E, Cherry AM, et al. Human endothelial prostate cancer. Genes Dev 2000;14:2410–34. 45. Palmero I, Pantoja C, Serrano M. p19ARF links the cell life extension by telomerase expression. J Biol Chem 27. Mirchandani D, Zheng J, Miller GJ, et al. tumour suppressor p53 to Ras. Nature 1998;395:125–6. 1999;274:26141–8. Heterogeneity in intratumor distribution of p53 46. Stott FJ, Bates S, James MC, et al. The alternative 7. Jarrard DF, Sarkar S, Shi Y, et al. p16/pRb pathway mutations in human prostate cancer. Am J Pathol product from the human CDKN2A locus, p14(ARF), alterations are required for bypassing senescence in 1995;147:92–101. participates in a regulatory feedback loop with p53 and human prostate epithelial cells. Cancer Res 1999;59: 28. Kaltz-Wittmer C, Klenk U, Glaessgen A, et al. FISH MDM2. EMBO J 1998;17:5001–14. 2957–64. analysis of gene aberrations (MYC, CCND1, ERBB2, RB, 47. Zhang W, Kapusta LR, Slingerland JM, Klotz LH. 8. Kiyono T, Foster SA, Koop JI, McDougall JK, Galloway and AR) in advanced prostatic carcinomas before and Telomerase activity in prostate cancer, prostatic intra- DA, Klingelhutz AJ. Both Rb/p16INK4a inactivation and after androgen deprivation therapy. Lab Invest epithelial neoplasia, and benign prostatic epithelium. telomerase activity are required to immortalize human 2000;80:1455–64. Cancer Res 1998;58:619–21. epithelial cells. Nature 1998;396:84–8. 29. Nupponen NN, Kakkola L, Koivisto P, Visakorpi T. 48. Gandarillas A, Watt FM. c-Myc promotes differenti- 9. Dickson MA, Hahn WC, Ino Y, et al. Human keratino- Genetic alterations in hormone-refractory recurrent ation of human epidermal stem cells. Genes Dev cytes that express hTERT and also bypass a p16(INK4a)- prostate carcinomas. Am J Pathol 1998;153:141–8. 1997;11:2869–82. enforced mechanism that limits life span become 30. Visakorpi T, Kallioniemi AH, Syvanen AC, et al. 49. Benanti JA, Galloway DA. Normal human fibroblasts immortal yet retain normal growth and differentiation Genetic changes in primary and recurrent prostate are resistant to RAS-induced senescence. Mol Cell Biol characteristics. Mol Cell Biol 2000;20:1436–47. cancer by comparative genomic hybridization. Cancer 2004;24:2842–52. 10. Sonoda Y, Ozawa T, Hirose Y, et al. Formation of Res 1995;55:342–7. 50. Munger K, Basile JR, Duensing S, et al. Biological intracranial tumors by genetically modified human 31. Latil A, Bieche I, Pesche S, et al. VEGF overexpression activities and molecular targets of the human astrocytes defines four pathways critical in the in clinically localized prostate tumors and neuropilin-1 papillomavirus E7 oncoprotein. Oncogene 2001;20: development of human anaplastic astrocytoma. Cancer overexpression in metastatic forms. Int J Cancer 7888–98. Res 2001;61:4956–60. 2000;89:167–71. 51. Schwarze SR, Shi Y, Fu VX, Watson PA, Jarrard DF. 11. Elenbaas B, Spirio L, Koerner F, et al. Human breast 32. Bernard D, Pourtier-Manzanedo A, Gil J, Beach DH. Role of cyclin-dependent kinase inhibitors in the cancer cells generated by oncogenic transformation of Myc confers androgen-independent prostate cancer cell growth arrest at senescence in human prostate primary mammary epithelial cells. Genes Dev 2001; growth. J Clin Invest 2003;112:1724–31. epithelial and uroepithelial cells. Oncogene 2001;20: 15:50–65. 33. Ellwood-Yen K, Graeber TG, Wongvipat J, et al. Myc- 8184–92. 12. Hahn WC, Dessain SK, Brooks MW, et al. Enumer- driven murine prostate cancer shares molecular 52. Mateyak MK, Obaya AJ, Sedivy JM. c-Myc regulates ation of the simian virus 40 early region elements features with human prostate tumors. Cancer Cell cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle necessary for human cell transformation. Mol Cell Biol 2003;4:223–38. progression at multiple independent points. Mol Cell 2002;22:2111–23. 34. Strahl C, Blackburn EH. Effects of reverse transcrip- Biol 1999;19:4672–83. 13. Hahn WC, Counter CM, Lundberg AS, tase inhibitors on telomere length and telomerase 53. Hermeking H, Rago C, Schuhmacher M, et al. Beijersbergen RL, Brooks MW, Weinberg RA. Creation activity in two immortalized human cell lines. Mol Cell Identification of CDK4 as a target of c-MYC. Proc Natl of human tumour cells with defined genetic elements. Biol 1996;16:53–65. Acad Sci U S A 2000;97:2229–34. Nature 1999;400:464–8. 35. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe 54. Carrano AC, Eytan E, Hershko A, Pagano M. 14. Yeh E, Cunningham M, Arnold H, et al. A signalling SW. Oncogenic ras provokes premature cell senescence SKP2 is required for ubiquitin-mediated degradation pathway controlling c-Myc degradation that impacts associated with accumulation of p53 and p16INK4a. Cell of the CDK inhibitor p27. Nat Cell Biol 1999;1: oncogenic transformation of human cells. Nat Cell Biol 1997;88:593–602. 193–9. 2004;6:308–18. 36. Gil J, Bernard D, Martinez D, Beach D. Polycomb 55. Goodrich DW, Lee WH. Abrogation by c-myc of G1 15. Chen W, Possemato R, Campbell KT, Plattner CA, CBX7 has a unifying role in cellular lifespan. Nat Cell phase arrest induced by RB protein but not by p53. Pallas DC, Hahn WC. Identification of specific PP2A Biol 2004;6:67–72. Nature 1992;360:177–9. complexes involved in human cell transformation. 37. Vafa O, Wade M, Kern S, et al. c-Myc can induce DNA 56. Cheville JC, Lloyd RV, Sebo TJ, et al. Expression of Cancer Cell 2004;5:127–36. damage, increase reactive oxygen species, and mitigate p27KIP1 in prostatic adenocarcinoma. Mod Pathol 16. Hahn WC, Weinberg RA. Modelling the molecular p53 function: a mechanism for oncogene-induced 1998;11: 324–8. circuitry of cancer. Nat Rev Cancer 2002;2:331–41. genetic instability. Mol Cell 2002;9:1031–44. 57. Cote RJ, Shi Y, Groshen S, et al. Association of p27KIP1 17. Drayton S, Rowe J, Jones R, et al. Tumor suppressor 38. Vlach J, Hennecke S, Alevizopoulos K, Conti D, Amati levels with recurrence and survival in patients with p16INK4a determines sensitivity of human cells to B. Growth arrest by the cyclin-dependent kinase stage C prostate carcinoma. J Natl Cancer Inst transformation by cooperating cellular oncogenes. inhibitor p27KIP1 is abrogated by c-Myc. EMBO J 1996; 1998;90:916–20. Cancer Cell 2003;4:301–10. 15:6595–604. 58. Tsihlias J, Kapusta LR, DeBoer G, et al. Loss of cyclin- 18. Voorhoeve PM, Agami R. The tumor-suppressive 39. Morales CP, Holt SE, Ouellette M, et al. Absence of dependent kinase inhibitor p27KIP1 is a novel prognostic functions of the human INK4A locus. Cancer Cell cancer-associated changes in human fibroblasts im- factor in localized human prostate adenocarcinoma. 2003;4:311–9. mortalized with telomerase. Nat Genet 1999;21:115–8. Cancer Res 1998;58:542–8. 19. Bouchard C, Staller P, Eilers M. Control of cell 40. Jiang XR, Jimenez G, Chang E, et al. Telomerase 59. Wang J, Hannon GJ, Beach DH. Risky immortaliza- proliferation by Myc. Trends Cell Biol 1998;8:202–6. expression in human somatic cells does not induce tion by telomerase. Nature 2000;405:755–6.

www.aacrjournals.org 2185 Cancer Res 2005; 65: (6). March 15, 2005

Jesús Gil, Preeti Kerai, Matilde Lleonart, et al.

Cancer Res 2005;65:2179-2185.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/6/2179

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2005/04/01/65.6.2179.DC1

Cited articles This article cites 58 articles, 25 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/6/2179.full#ref-list-1

Citing articles This article has been cited by 16 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/6/2179.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/6/2179. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.