Immortalization of Primary Human Prostate Epithelial Cells by c-Myc

A significant percentage of prostate tumors have amplifications of the c-Myc gene, but the precise role of c-Myc in prostate cancer is not fully understood. Immortalization of human epithelial cells involves both inactivation of the Rb/ p16 pathway and telomere maintenance, and it has been recapitulated in culture by expression of the catalytic subunit of telomerase, hTERT, in combination with viral oncoproteins. Here, we show the immortalization of human prostate epithelial cells (HPrEC) by a single genetic event, the expression of the c-Myc oncogene. Myc stabilizes telomere length in HPrEC through up-regulation of hTERT expression and overrides the accumulation of cell cycle inhibitory proteins, such as p16. Overall, HPrECs expressing c-Myc retain many characteristics of normal cells, such as the induction of a senescence-like growth arrest in response to oncogenic Ras, an intact p53 response, and an absence of gross karyotypic abnormalities. However, HPrECs expressing c-Myc lack a Rb/p16 checkpoint and can be transformed without the need for additional genetic lesions in that pathway. These results give a partial explanation for the physiologic role of c-Myc overexpression in prostate cancer. (Cancer Res 2005; 65(6): 2179-85)


Introduction
Normal human somatic cells undergo a limited number of divisions before entering an irreversible growth arrest state defined as replicative senescence (1).This life span varies between different cell types and is also dependent on culture conditions (2).The acquisition of unlimited proliferative ability is probably a key step during tumorigenic progression (3).It has been proposed that the restoration of telomerase activity is sufficient to immortalize human diploid fibroblasts, endothelial cells, T lymphocytes, and some other cell types (4)(5)(6).However, different studies have identified two requirements for immortalization of human epithelial cells: maintenance of telomere length and inactivation of the Rb/p16 INK4a pathway (7,8).Thus, expression of hTERT in combination with viral oncoproteins, such as the E7 product of human papillomavirus 16 or the SV40 large T antigen, is sufficient to immortalize human keratinocytes and human mammary epithelial cells (HMEC; ref. 8).Although Ramirez et al. (2) suggest that both HMEC and keratinocytes grown on feeder layers could be immortalized by hTERT alone, others have found a requirement for inactivation of the Rb/p16 INK4a pathway even in these conditions (9).
The establishment of improved models of human cancer progression relies on the expression of defined combinations of genes that mirror those naturally arising in cancer.In this context, significant advances have been achieved recently (10)(11)(12)(13).Transformation of different human primary cells has been achieved by serial introduction of hTERT, SV40 large T (inactivating at least the p16 INK4a /Rb and the p53 pathways), SV40 small t (affecting c-Myc stability through PP2A regulation (14,15)), and oncogenic Ras (16).However, these models rely on the use of viral oncoproteins.As viruses are implicated in a limited spectrum of cancer types, we reasoned that human epithelial cells immortalized by cellular genes that are amplified in naturally occurring cancers could represent a first step toward the generation of more realistic models of human cancer.Such models have already been established in human fibroblast by taking advantage of short hairpin RNA technology or using cells derived from patients with germ line mutations in p16 INK4a  (17,18).
The oncogene c-Myc fulfils many of the expectations for a gene involved in immortalization of human epithelial cells.Deregulated expression of c-Myc caused by gene amplification, chromosomal translocation, or proviral insertion is widely associated with tumorigenesis in both human cancers and rodent models (19).Ectopic expression of c-Myc has been shown to increase the life span of IMR-90 fibroblasts and HMECs, an effect that is in part attributed by the activation of hTERT transcription (20,21).In this context, although 85% to 90% of human tumor cells maintain their telomeres by up-regulation of telomerase (22), fewer than 30% have amplifications of the hTERT gene (23), and deregulation of Myc may represent an alternative mechanism.In addition, c-Myc has been shown to promote cell proliferation in the presence of p16 INK4a or hypophosphorylated pocket proteins (24).
Prostate cancer is the most common noncutaneous malignancy diagnosed in American men (25), but there is only a limited understanding of its genetic and molecular basis (26).Different studies have shown that c-Myc mRNA is commonly overexpressed in hyperplasic and malignant prostate (27), and the c-Myc gene is amplified in between 11% and 40% of prostate cancers in different studies (28)(29)(30).Although c-Myc amplification has been mainly 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 correlate with high c-Myc mRNA expression (31).Recently, we have uncovered a role for c-Myc in androgen-independent prostate cancer cell growth (32) coherent with the observed c-Myc amplification in late stages of prostate cancer.In addition, c-Myc overexpression has been used for modeling human-like prostate cancer in a murine model (33).However, a molecular explanation for the role of c-Myc amplification during early stages of human prostate cancer is still needed.
With the aim of reconstructing prostate cancer progression, we derived primary human prostate epithelial cells (HPrEC) and asked whether they could be immortalized by several viral and cellular genes.Among the genes tested, only c-Myc was able to immortalize HPrEC as a single agent.In addition, HPrEC expressing c-Myc lacked Rb/p16 INK4a checkpoint control and became transformed without the need for additional alterations in that pathway.These observations clarify the role of observed c-Myc amplifications in prostate cancer.

Materials and Methods
Establishment of Primary HPrEC Cultures.A primary culture of 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, Gaithersburg, MD) containing 5% FCS and 400 units/mL collagenase.The digested tissue was plated in collagen IV-coated plates and maintained with PrEGM (BioWhittaker, Walkersville, MD) serum-free medium according to the manufacturer's instructions.
Cell Culture and Retroviral-Mediated Gene Transfer.The amphotropic packaging cell line LinXA was maintained in DMEM (Life Technologies) and 10% FCS (Sigma, St. Louis, MO).HPrEC were established as explained above or acquired from Clonetics (East Rutherford, NJ) and were maintained in PrEGM.PC-3 cells were maintained in RPMI 1640 and 10% FCS.Retrovirus production and infection of target cells was done according to ref. 20.HPrEC were selected using 250 ng/mL puromycin, 100 Ag/mL G418, 500 ng/mL blasticidin, or 20 Ag/mL hygromycin as required.
Telomerase Assays.Telomeric repeat amplification protocol assay was done as described before (20).PCR products were electrophoresed on 12% polyacrylamide gels.Gels were dried and autoradiographed.Telomeric restriction fragment length was measured as described before (34).At least three independent experiments were done with identical results.
Growth Curves.Cumulative population doublings per passage were calculated as log 2 (number of cells at time of subculture divided by number of cells plated) and plotted against total time in culture to assess replicative 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 into 24-well plates.At the indicated times, cells were washed with PBS, fixed in 5% glutaraldehyde, and rinsed with distilled water.Cells were stained with 0.1% crystal violet (Sigma) and processed as described before (35).All experiments were done in triplicate.
Soft Agar Assays.Cells (10 4 ) were plated in RPMI 1640 plus 10% FCS in 0.35% agar above a layer of 0.7% agar.After 3 weeks, colonies were visualized and counted.PC-3 cells were used as positive control.All experiments were done in triplicate.
Karyotypic and Fluorescence In situ Hybridization Analysis.Exponentially growing cultures were incubated with 100 nmol/L colcemid (Sigma) for 1 hour, resuspended in hypotonic buffer (75 mmol/L KCl), incubated for 20 minutes at 37jC, fixed by addition of cold ethanol/acetic acid, and applied to chilled microscope slides.Chromosome preparations of cultured cells were counted for the number of metaphase spreads.Forty metaphase spreads were analyzed for each cell line.
Analysis of Retroviral Integration Sites.Genomic DNA (15 Ag) from HPrEC c-Myc cells was digested with EcoRI and hybridized to a 32 P-labeled c-Myc probe.
Reverse Transcription-PCR Analysis.Cells were homogenized in Trizol (Invitrogen, San Diego, CA) and total RNA was isolated according to the manufacturer's recommendations.Complementary DNAs were synthesized using the first-strand cDNA synthesis kit (Roche, Indianapolis, IN) and amplified in a final volume of 50 AL containing 150 Amol/L deoxynucleotide triphosphate, 2 mmol/L MgCl 2 , 1 unit Taq gold polymerase (Applied Biosystems, Foster City, CA), and each primer at 1 Amol/L.For p14 ARF , DMSO was added at a final concentration of 1%.Primers used were as follows: p14 ARF forward GAGTGGCGCTGCTCACCTC, p14 ARF reverse TACCGTGC-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 72jC for 1 minute, with an initial denaturation step of 5 minutes at 95jC.

Results
Expression of c-Myc Bypasses Senescence and Extends Life Span of HPrEC.To study the requirements for immortalization of HPrEC, we derived a primary culture of HPrEC from normal prostate tissue from an adult donor.These cells showed typical epithelial morphology, stained positive for a pan-cytokeratin antibody, and expressed multiple keratins as well as several prostate markers (such as prostate-specific phosphatase, several kallikreins, and the transcription factor Nkx3.1) as assessed by microarray analyses (data not shown).HPrECs had a finite life span in culture of f20 PDs (Fig. 1A).HPrEC at PD f10 were infected with retroviral vectors expressing either green fluorescent protein, the catalytic subunit of telomerase (hTERT), c-Myc, Mdm2, human papillomavirus 16 E7, or both human papillomavirus 16 E6 and E7.As expected, green fluorescent protein expression did not increase the life span of HPrECs.Infection with either Mdm2-or E7-expressing retroviruses resulted in a modest increase of life span, but eventually the cells stopped proliferating (Fig. 1A).Introduction of hTERT resulted in an extension of proliferative potential up to f60 PD, but cells senesced thereafter (Fig. 1B).Only HPrECs expressing the E6 and E7 oncoproteins of human papillomavirus 16 or c-Myc maintained proliferation beyond 110 PDs.Similar results were obtained using commercially available HPrEC from an independent donor (Supplementary Fig. 1) and in independent infections (data not shown).
The efficiencies of retroviral infection (generally >80%) and the continuity of the growth curves suggested that the majority of the cells transduced with c-Myc had become immortal and argued against selection for clonal variants.To confirm this impression, we compared the colony forming ability of the parental cells infected with empty vector with HPrEC expressing c-Myc or E6 + E7 at various times after infection.At passage 4, f12 F 3% of the vector only control cells formed colonies under these conditions and the Myc-and E6 + E7-expressing pools showed very similar properties at this and subsequent passages (data not shown).As a more direct test of clonality, we also compared the patterns of provirus integration in the Myc-transduced HPrECs at early and late passages.In EcoRIdigested genomic DNA, each fragment hybridized to the Myc probe corresponds to a virus-host junction and equates to a single integrated provirus.The hybridization patterns were indicative of a polyclonal population of cells; importantly, there was no evidence for emergence of a clonal population on prolonged passaging (Fig. 1C).Finally, karyotypic analyses of the HPrEC expressing c-Myc indicated that they were generally diploid.Recent reports suggest that Myc overexpression can induce genomic instability in primary human fibroblasts (37), but we found only one example of a chromosomal alteration, der(7)t(5;7), that was present in 2.5% of the Mycimmortalized cells at very late passage (Fig. 1D).Taken together, the data are consistent with the notion that c-Myc can immortalize HPrECs without the need for additional mutations.
Myc Expression Activates Telomerase in HPrEC.As telomere maintenance is a prerequisite for immortalization of human cells, we tested the effect of c-Myc expression on HPrEC telomeres.To this end, we first analyzed the average lengths of the telomeric end fragments by Southern blotting.Expression of either hTERT or c-Myc resulted in the maintenance of telomere length relative to the vector only control cells, whereas introduction of E7 or Mdm2 had no effect (Fig. 2A).This correlated with the presence of telomerase activity as measured by the telomeric repeat amplification protocol assay (Fig. 2B).Finally, we analyzed hTERT protein levels by immunoblotting.Consistent with previous studies in other human cell types (20,21), we found that c-Myc up-regulates the expression of hTERT to levels comparable with that achieved with hTERT retroviral infection (Fig. 2C).Thus, c-Myc allows maintenance of the telomeres in HPrECs by increasing hTERT expression and telomerase activity.
Effect of c-Myc on Cell Cycle Progression.Senescence is accompanied by an increase in the levels of several cyclin-dependent kinase inhibitors, notably p16 INK4a (35).However, it has been reported previously that, in model systems at least, ectopic c-Myc can override the effects of p16 INK4a , p21 CIP1 , and p27 KIP1 (24,38).It was therefore interesting to consider whether hTERT or c-Myc had rendered HPrEC resistant to arrest by cyclin-dependent kinase inhibitors.To explore this possibility, the immortalized HPrEC cultures were infected with retroviruses encoding LacZ (as control) or murine versions of p16 INK4a or p27 KIP1 , and pools of cells were selected for drug resistance.Expression of p16 INK4a and p27 KIP1 was monitored by immunoblotting with antibodies specific for the murine proteins (Fig. 3A; data not shown).On infection with a p16 INK4a retrovirus, the hTERT-expressing cells arrested with a senescence-like phenotype (Fig. 3A; data not shown), whereas c-Mycexpressing HPrEC continued to proliferate.Thus, c-Myc allowed HPrEC to tolerate elevated levels of p16 INK4a expression as shown in other cell types (24).In contrast, p27 KIP1 expression provoked an arrest in both c-Myc-and hTERT-expressing HPrEC.
To further analyze the impact of c-Myc on cell cycle regulation, we examined the ability of HPrEC to grow in chemically defined medium with or without growth factors.As shown in Fig. 3B, hTERTexpressing cells stop growing in the absence of growth factors, correlating with a decrease in the number of cells in S phase as measured by BrdUrd incorporation (Fig. 3C) and a decline in the phosphorylation of pRb as monitored with a phosphospecific antibody (Fig. 3D).In contrast, c-Myc-expressing cells grew regardless of the presence of growth factors, and pRb phosphorylation was maintained (Fig. 3B-D).
Interestingly, expression of c-Myc results in increased levels of cyclin A when compared with both parental cells and hTERTexpressing HPrEC (Supplementary Fig. 2).We also noted a slight increase in cyclin D1 levels but no significant changes in cyclin D2 or p27 KIP1 (data not shown).HPrECs Expressing c-Myc Have a Normal Response to Genotoxic Agents.Human somatic cells immortalized by hTERT retain many characteristics of primary cells, such as cell cycle checkpoints and p53 activation in response to diverse genotoxic stresses (39,40).We therefore assessed these variables in c-Myc immortalized HPrEC.As illustrated in Fig. 4A, both hTERT and c-Myc transduced HPrEC showed the expected accumulation of p53 when treated with etoposide accompanied by induction of its target  gene p21 CIP1 .Induction of p21 CIP1 was not observed in HPrEC expressing E6 and E7 due to ablation of p53 by E6.Treatment of HPrEC cells expressing hTERT or c-Myc with 1 Amol/L etoposide also caused a marked reduction in the number of cells in S phase with a corresponding increase of cells in G 2 (data not shown), an effect that was abolished by expression of E6.Similarly, treatment of the hTERT-or Myc-transduced cells with increasing concentrations of Adriamycin caused a dose-dependent loss of cell viability, consistent with a p53-dependent apoptotic response (Fig. 4B).Impairment of the p53 pathway by expression of E6 reduced the sensitivity to Adriamycin.Taken together, these results indicate that the p53 pathway is operational in c-Myc-expressing HPrEC.
Transformation of HPrEC Expressing c-Myc.Expression of oncogenic Ras in human somatic cells results in a growth arrest termed premature senescence (35), and this occurs irrespective of the expression of hTERT (39,41).In line with these observations, expression of Val 12 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 growth arrest was negated by introduction of the human papillomavirus 16 E6 and E7 genes.Further investigation indicated 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 p53dependent 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 p16 INK4a and p14 ARF (17,(43)(44)(45) and this also holds true for HPrEC.Analysis of p16 INK4a and p14 ARF 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 p16 INK4a , the effects were comparable with the up-regulation that occurs naturally on continued passaging of HPrEC (cf.passages 3 and 10).Although the p14 ARF levels were significantly elevated by Myc and 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 p14 ARF because of loss of suppression by p53 (46), but in the absence of p53 the cells were more resistant to the effects of Adriamycin.Importantly, c-Myc or the combination of Myc, Ras, and SV40 small t antigen (see below) did not seem to alter the viability of the HPrEC cells under these conditions.
To evaluate the impact of c-Myc on transformation of HPrEC rather than immortalization, we compared the ability of the HPrEC cells expressing c-Myc or hTERT together with different combinations of oncogenes to form anchorage-independent colonies.As shown previously in other human cell types (12), we found that in HPrEC immortalized by hTERT, addition of oncogenic Ras, E6 + E7, and SV40 small t enabled them to grow in soft agar (Fig. 6D).Omission of any of these genes compromised this phenotype.In contrast, with c-Myc immortalized HPrEC, expression of E7 was not necessary for acquisition of anchorage independence, consistent with the ability of c-Myc to override the p16 INK4a /pRb checkpoint.

Discussion
The ultimate aims of this investigation are to establish a model that allows the in vitro reconstruction of events leading to  prostate cancer progression and to clarify the role of c-Myc in this process.The introduction of a defined combination of genes in primary human cells allows the generation of tailor-made models of cancer progression.However, several limitations reduce the utility of the currently developed models.One is the use of viral oncoproteins as pivotal components of the transformation protocol.In addition, most use ectopic expression of hTERT to maintain telomere length in these cells.Although telomerase activity is present in most of human tumors, the hTERT gene itself is amplified in a limited number of cases (47).Therefore, we believe that the immortalization of HPrEC by c-Myc may provide an alternative and more physiologic start point for studying prostate cancer progression.
Although c-Myc has been shown previously to extend the life span of fibroblasts and HMECs (20), the consequences of Myc expression may be context dependent.For example, it has been reported that Myc causes terminal differentiation in primary keratinocytes (48) and premature senescence in some strains of fibroblast (17), an outcome that may depend on the basal levels of p16 INK4a and the extent to which the cells have been exposed to culture stress (49).In the published study on HMECs (20), the cells used had already shutdown p16 INK4a expression due to methylation of the genomic DNA. 8To 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 p21 CIP1 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 p16 INK4a /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 p16 INK4a /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 p16 INK4a (Fig. 6A).Our finding that c-Myc-expressing HPrEC cells have elevated levels of cyclin A may account for resistance to p16 INK4amediated arrest (Supplementary Fig. 2), but there are potentially many other ways in which c-Myc can influence the expression and function of cell cycle regulators (38,(52)(53)(54)(55).For example, c-Myc seems to reduce growth factor dependence in HPrEC as also noted in human diploid fibroblasts (17).However, in our hands, c-Myc did not overcome a p27 KIP1 -mediated arrest in HPrEC in contrast to previous studies in rodent fibroblasts.In this context, it is interesting to note that p27 KIP1 levels have prognostic significance in prostate cancer (56)(57)(58).
Cell type differences may also apply to the apparent links between c-Myc and hTERT expression.We and others have shown that latepassage HMECs expressing hTERT can show amplification of c-Myc (11,59), although this is presumably a stochastic event rather than a direct effect.In the present work, HPrEC expressing hTERT did not express elevated levels of c-Myc or acquire growth factor independence (Figs.2C and 4B; data not shown).
Identification of the genetic events that cooperate with c-Myc in the bypass of Ras-induced senescence and transformation of HPrEC constitute a first step for reconstructing prostate cancer progression in vitro.Interestingly, in accordance with the ability of c-Myc to override the p16 INK4a /Rb checkpoint, additional alterations in this pathway are not required for transformation, whereas the hTERTexpressing counterparts require inactivation of both pRb and p53 (Fig. 6).Thus, the amplification of the c-Myc gene that is observed in a high percentage of prostate tumors may have multiple explanations.At early stages, c-Myc can confer a proliferative advantage by immortalizing prostate cells and allowing them to grow under limited growth factor conditions.At later stages, c-Myc may contribute to androgen-independent growth of the prostate cancers as reported previously (32). .Genetic events involved in transformation of HPrEC.A, effect of c-Myc (M ) or RasV12 (R) compared with vector-infected HPrEC cells (V) over p16 INK4a expression was analyzed by Western blot.B, effect of c-Myc or RasV12, E6, c-Myc + E6 (E6 + M ), or c-Myc + E6 + RasV12 + small t (E6 + MRt ) expression in HPrEC over p14 ARF expression was analyzed by reverse transcription-PCR.C, viability of the corresponding cells after treatment with 0.2 Ag/mL Adriamycin for 24 hours.HPrECs expressing hTERT or c-Myc were serially infected with retroviruses expressing the indicated genes with (+) or without (À ) small t and subsequently infected with retroviruses expressing RasV12.Growth in soft agar was monitored and colonies were scored after 3 weeks.

Figure 2 .
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.

Figure 3 .
Figure 3.Effect of c-Myc expression on the p16 INK4a /Rb pathway.A, HPrEC expressing hTERT or c-Myc were infected with LacZ (as a control), p16 INK4a , or p27 KIP1 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.

Figure 4 .
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 h-actin levels were analyzed by Western blot.B, cells were treated with different concentrations of Adriamycin for 24 hours and viability was analyzed.

Figure 5 .
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.

Figure 6
Figure 6.Genetic events involved in transformation of HPrEC.A, effect of c-Myc (M ) or RasV12 (R) compared with vector-infected HPrEC cells (V) over p16 INK4a expression was analyzed by Western blot.B, effect of c-Myc or RasV12, E6, c-Myc + E6 (E6 + M ), or c-Myc + E6 + RasV12 + small t (E6 + MRt ) expression in HPrEC over p14 ARF expression was analyzed by reverse transcription-PCR.C, viability of the corresponding cells after treatment with 0.2 Ag/mL Adriamycin for 24 hours.HPrECs expressing hTERT or c-Myc were serially infected with retroviruses expressing the indicated genes with (+) or without (À ) small t and subsequently infected with retroviruses expressing RasV12.Growth in soft agar was monitored and colonies were scored after 3 weeks.