[CANCER RESEARCH 59, 2957–2964, June 15, 1999]

p16/pRb Pathway Alterations Are Required for Bypassing Senescence in HumanProstate Epithelial Cells1

David F. Jarrard, 2 Somdatta Sarkar, Yan Shi, Thomas R. Yeager, Gregg Magrane, Hidefumi Kinoshita,Nadine Nassif, Lorraine Meisner, Michael A. Newton, Frederic M. Waldman, and Catherine A. ReznikoffDepartments of Human Oncology [S. S., M. A. N., C. A. R.], Surgery [D. F. J., Y. S., H. K., N. N.], and Toxicology [D. F. J., T. R. Y., L. M., C. A. R.], University of WisconsinComprehensive Cancer Center and Medical School, Madison, Wisconsin 53792; and Division of Molecular Cytometry and Department of Laboratory Medicine, University ofCalifornia at San Francisco, San Francisco, California 94143 [G. M., F. M. W.]

ABSTRACT

The cell cycle regulatory genesp16/CDKN2 and RB are frequentlydeleted in prostate cancers. In this study, we examined the role of alter-ations in p16 and pRb during growth, senescence, and immortalizationinvitro of human prostate epithelial cells (HPECs). HPECs are establishedfrom normal prostate tissues and cultured on collagen-coated dishes. Ourresults show that p16 is reproducibly elevated at senescence in HPECs.HPECs are immortalized using human papilloma virus 16 E6 and/or E7 asmolecular tools to inactivate p53 and/or pRb, respectively. Immortaliza-tion occurs infrequently in this system and only after a latent periodduring which additional genetic/epigenetic changes are thought to occur.Notably, all of the E6-immortalized HPEC lines but none of the E7 linesshow inactivation ofp16/CDKN2(by deletion, methylation, or mutation) inassociation with immortalization. In contrast, E7 lines, in which pRbfunction is abrogated by E7 binding, retain the high levels of p16 observedat senescence. Thus, all lines show either a p16 or pRb inactivation.Analysis of six independent lines from metastatic prostate cancers revealsa similar loss of either p16 or pRb. Comparative genomic hybridization ofHPECs shows that gains of chromosomes 5q, 8q, and 20 are nonrandomlyassociated with bypassing senescence (probability5 0.95). These resultssuggest that high levels of the cyclin-dependent kinase inhibitor p16mediate senescence G1 arrest in HPECs and that bypassing this block bya p16/pRb pathway alteration is required for immortalization in vitro andpossibly tumorigenesisin vivo. Our results further indicate that inactiva-tion of the p16/pRb pathway alone is not sufficient to immortalize HPECsand that additional genetic alterations are required for this process.

INTRODUCTION

Normal human cells have a finite proliferative potential (usually50–100 doublings)in vitro, after which cell cycle arrest occurs (1).This process is termed replicative senescence, and in contrast toapoptotic or programmed cell death, it represents a stable, protractedstate in which cells remain viable and metabolically active. Theprocess of immortalization requires overcoming or escaping replica-tive cellular senescence. Bypassing senescence is thought to be im-portant in the development of human cancer for several reasons (2, 3):(a) immortalization increases cell susceptibility to malignant progres-sion by permitting the extensive cell divisions that are necessary forcells to acquire multiple genetic alterations; (b) tumors, both naturallyoccurring and experimentally induced, often contain cells that areimmortal or have an extended replicative life span (4, 5); and (c)genetic regions associated with the immortalization of cell typesinvitro are found to be altered in clinical cancers of the same cell type(6). Therefore, an understanding of the genes controlling normalprostate epithelial cell mortality may provide relevant information on

the mechanisms underlying abnormal cell growth and tumorigenesisin prostate epithelial cells.

A series of classical observations have shown that replicative lifespan can be extended by the expression of DNA tumor virus onco-proteins, including SV40 and HPV163 E6 and E7 (3, 7). These tumorviruses are known to inactivate p53 and pRb (8), thus implicatingthese antiproliferative proteins in the process of overcoming senes-cence. This initial period of extended life span requires overcomingthe M1 (mortality) block. However, overcoming M2, which results inan infinite life span, clearly requires additional genetic events (9, 10).Immortalization occurs infrequently, which indicates that the inacti-vation of several pathways is required. Alterations in other cell cycleregulatory genes, includingp21WAF1 and p16/CDKN2,have beenimplicated in mediating senescence (11–13). Notably, all of thesegenes encode putative tumor suppressor proteins, the functions ofwhich are lost in many human cancers, including prostate cancers(14–19).

Increases in wild-type p53 induce antiproliferation signals, includ-ing the downstream cyclin-dependent kinase p21, that are distinctfrom apoptosis (20). Levels of p53 have been found to increase at latepassage in human fibroblasts, putatively contributing to senescencegrowth arrest (11, 21). Inhibiting the expression of p53, either byantisense RNA methods (22) or by use of p53 transdominant mutants(23), leads to a delay in senescence. Elevation of p21 levels has alsobeen found in aging fibroblasts (13). However, because levels de-crease at senescence, the significance of the role of p21 in senescencegrowth arrest is unclear. Its role in tumorigenesis is also uncertain: arecent study showed that mice with a homozygous deletion ofp21/WAF1 failed to form tumors (24).

pRb and p16 function in a common pathway that appears to beimportant in the growth arrest of cells after a finite number ofpopulation doublings. Phosphorylation regulates pRb function byinducing E2F release and the subsequent expression of E2F-depend-ent proteins, such as cdc2 and cyclin A. These genes are not expressedin senescent fibroblasts consistent with a block in pRb phosphoryla-tion at senescence (25). Furthermore, pRb is found in a hypophos-phorylated state in senescent fibroblasts (26). Overcoming this senes-cence block is possible by fusion with cells containing the viraloncogeneE7, which binds unphosphorylated pRb (26). p16 blockspRb phosphorylation by binding cdk4 and cdk6 and inhibiting theirassociation with cyclin D (27). This results in a failure of pRbphosphorylation and E2F release and culminates in G1 cell cyclearrest. Several studies have shown that p16 levels increase in humanand rodent fibroblasts as cells are passed to terminal senescence (11,28). However, only one study to date has demonstrated increased p16at senescence in a human epithelial cell type, namely urothelial cells(12).

p16 and pRb inactivation are generally not found in the samecancer, consistent with the observation that they function within the

Received 11/30/98; accepted 4/15/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant CA76184 and a Howard Hughes FacultyDevelopment award (to D. F. J.).

2 To whom requests for reprints should be addressed, at G5/347 Clinical ScienceCenter, University of Wisconsin School of Medicine, 600 Highland Avenue, Madison, WI53792. E-mail: jarrard@surgery.wisc.edu.

3 The abbreviations used are: HPV16, human papilloma virus 16; HPEC, humanprostate epithelial cell; FBS, fetal bovine serum; RT-PCR, reverse transcriptase-PCR;CGH, comparative genomic hybridization; SA, senescence-associated.

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same pathway (29, 30). In support of this model, the cell cycle arrestinduced by the introduction of p16 occurs only in cells that retainfunctional pRb (31). The importance of p16 as a tumor suppressor isemphasized by its frequent inactivation in different types of humanmalignancies (32). We recently reported thatp16/CDKN2,located at9p21, was deleted or hypermethylated in.50% of advanced prostatecancers and that this finding was associated with transcriptionalinactivation (19).RB loss of heterozygosity is also frequently found inprostate cancers (16, 17), although whether homozygous alterationsoccur is not known. The loss of pRb expression does correlate with apoor clinical prognosis (33).

In summary, an alteration in the p16/pRb pathway is commonlyseen in prostate cancers. Several lines of evidence discussed abovesuggest that these alterations may play a role in bypassing the tumorsuppressor checkpoint of cellular senescence. Here, we test this hy-pothesis using an HPECin vitro transformation system that usesHPV16 E6 and E7 to inactivate p53 and pRb, respectively. We reporthere for the first time with HPECs that p16 levels are reproduciblyelevated at senescence and that bypassing senescence in HPV16 E6-or E7- transformed cells is always accompanied by an alteration in thep16/pRb pathway. Furthermore, we identify additional genetic alter-ations, including15q, 18q, and120, that are nonrandomly associ-ated with HPEC immortalization. We conclude that alteration of thep16/pRb pathway is necessary but not sufficient for overcomingsenescence in HPECs.

MATERIALS AND METHODS

Tissue Acquisition/Prostate Epithelial Cell Culture. Biopsies of histo-logically confirmed normal prostate tissue from men (ages 47–65 years)without diagnosed prostate cancer were obtained from cystoprostatectomyspecimens or from cadavers. The culture system used is based on the work ofPeehlet al. (34, 35) and Reznikoffet al. (36). To establish HPECs, we mincedfresh prostate tissues and plated them on 100-mm collagen dishes in F-121medium with penicillin/streptomycin (9). F-121 is a supplemented Ham’smedium (Life Technologies, Inc., Gaithersburg, MD; intermediate Ca21, 0.3mM) that also contains 1% FBS. Bovine pituitary extract (25mg/ml; LifeTechnologies, Inc.) and cholera toxin (100 ng/ml; Life Technologies, Inc.)were added to this medium. HPECs were passaged routinely when confluentafter dispersion with trypsin-EDTA (Life Technologies, Inc.). Prostate cancercell lines were grown in RPMI with 10% FBS.

Immunohistochemistry. Immunohistochemistry was performed on early-passage HPECs and HPEC E6- and E7- immortalized cell lines after harvestingand 10% formalin fixation (37). Primary antibodies and dilutions includedcytokeratins 5 (1:500; Enzo) and 18 (1:100; Sigma Chemical Co., St. Louis,MO) and prostate-specific antigen (1: 100; Sigma). Biotinylated antimouse orrabbit IgG was used as a secondary antibody (Sigma). Staining was accom-plished with the ABC reagent (Vector Laboratories, Burlingame, CA) and thesubstrate diaminobenzidine. Immunohistochemistry was duplicated on each ofthree independent HPEC cultures. Controls included tissues with no primaryantibody and uncultured normal prostate tissues.

SA b-Galactosidase Staining.HPECs were transferred to chamberedslides (Nunc, Naperville, IL) and grown for 2 days. Cells were then rinsed andfixed in a b-galactosidase stain solution as described (38). Positiveb-galac-tosidase staining, as defined by blue cytoplasmic and nuclear staining, was amarker for senescence and was not present in differentiated or apoptotic cells(38).

Transformation of HPECs with HPV16 E6 and/or E7. Transformationof HPECs with HPV16 E6 and/or E7 and selection for immortal clones wasperformed as described for human urothelial cells (9). Briefly, retrovirusescarrying either the HPV16 E6 and/or E7 gene(s) (received from Dr. D.Galloway, Seattle, WA) were prepared (39). Subconfluent proliferating HPECswere infected with 103–105 infectious viral units at early-passage (;5 3 105

cells per 100-mm dish) in 3 ml of 1% FBS-F-121 containing 4 mg/mlpolybrene (Sigma). The virus was then removed after 6 h, and selection with50 mg/ml G418 (Life Technologies, Inc.) was performed for a minimum of 7

days. Two uninfected control dishes were monitored for senescence. Clonesinfected with E6, E7, or E6/E7 were checked for expression of E6 and E7transcripts using RT-PCR as described previously (9). Southern blot analysiswas also performed as described previously (9) using a 0.8-kbBamHI-HindIIIfragment from p1321 that contains the HPV16 E6/E7 genes. Fifteenmg ofDNA were loaded in each lane.

Western Blot Analysis. Western blots were performed as described pre-viously (12) Briefly, cells were lysed in buffer containing protease inhibitorsand resolved (50mg/lane) on 12.5% SDS-polyacrylamide gels. After transferto a nylon blot (Immobilon P; Millipore, Bedford, MA), primary antibodieswere applied. These included specific antibodies to p53 (AB2; OncogeneScience), phosphorylated and unphosphorylated forms of pRb (14001A;PharMingen), p16 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), andp21(Oncogene Science). Immunoreactive proteins were visualized using en-hanced chemiluminescence. Urothelial cells immortalized with HPV16 E7 (12)were used as positive controls for p16, p53, and p21. Immunoblotting wasperformed in duplicate.

Sequencing ofp16/CDKN2and Methylation Analysis. Sample DNA wasamplified by PCR using primers spanning exons 1 and 2 of thep16/CDKN2gene (40, 41). The amplified products were cloned into pCR 2.1-TOPO usingthe TOPO TA cloning kit from Invitrogen according to manufacturer’s in-structions. The cloned fragments were sequenced using an automated DNAsequencer. Methylation analysis was performed as described previously usinga 340-bpp16/CDKN2exon 1 probe (19). Serial concentrations of HPEC E6-15DNA was used in Southern blotting to confirm deletion ofp16/CDKN2.

CGH of HPV16 HPEC Immortal Lines and Statistical Analysis. Hy-bridization of differentially labeled immortalized cell lines to metaphase chro-mosomes from normal peripheral blood was performed exactly as describedpreviously (42). We have described how we define changes in the relative copynumber of DNA sequences (i.e., gains and losses) in an earlier manuscript (43).CGH loss and gain data were analyzed using a simple statistical model, asdescribed previously (44). Briefly, the null hypothesis asserts that changes aresporadic and thus present randomly according to a background rate that isconstant among chromosome arms. The alternative hypothesis allows thatsome arms exhibit an elevated rate of change. The model also allows thepossibility that changes on the p and q arms are linked. Gain data exhibit apattern significantly different from one would expect under the null hypothesis,with P 5 0.001, whereas loss data are consistent with sporadic change(P 5 0.31).

RESULTS

Growth and Senescence of HPECsin Vitro . The growth ofHPEC explants on collagen-coated dishes in F-121 medium results incultures of prostate epithelial cells that are epithelial in morphology(i.e., tightly adherent and polygonal) and apparently uncontaminatedwith other cell types (Fig. 1A). After four to five passages, HPECproliferation slows, and cells enter a nonproliferative state. TheseHPECs adopt a morphological phenotype characteristic of senescence(Fig. 1B) and acquire SAb-galactosidase activity (Ref. 38; Fig. 1C).

Characterization of Cultured HPECs. To assess the phenotypeof cultured HPECs using our conditions, we performed immunohis-tochemical staining for cytokeratin markers of luminal or basal pros-tate epithelial cells (45, 46) in both uncultured tissue sections andcultured HPECs. Cytokeratin 5, a basal cell marker, and cytokeratin18, a luminal protein, are both expressed (data not shown). Anothermarker of differentiated luminal epithelial cells, prostate-specific an-tigen, is not found at detectable levels in HPECs using immunohis-tochemistry. It is, however, detectable using RT-PCR (data notshown). These results demonstrate that HPECs coexpress both basaland luminal keratins, thus representing the amplifying or stem cellpopulation that is hypothesized to be the cellular precursor to prostatecancer (46). Additionally, expression of these prostate epithelialmarkers essentially excludes contamination by cells of stromal origin.

Elevation of p16 but not p21 or p53 at Senescence in NormalHPECs. To determine whether alterations of pRb, p53, p21, and p16might play a role in mediating prostate epithelial senescence, we

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compared protein levels in proliferating (presenescent) and senescentHPECs. In contrast to previous studies in fibroblasts (11), resultsshowed no alterations in p53 or p21 levels at senescence in HPECs.However, HPECs at senescence reproducibly show at least a 10-foldelevation of p16 (Fig. 2), when compared to early-passage prolifer-ating HPECs. We also note a consistently decreased level of phos-phorylated pRb in senescent cells consistent with a G1 cell cycleblock. pRb is detectable on longer exposure. The antibody used forpRb detection recognizes primarily phosphorylated forms of pRb.

Generation of Isogeneic HPV16 E6 and/or E7 HPEC Lines.HPV16 E6 selectively abrogates p53, whereas HPV16 E7 binds andinactivates pRb. These viral genes were used as molecular tools totest, in HPECs, the functional significance of losing pRb and p53 inthe process of overcoming senescence. Results showed that immor-

talization of human epithelial cell lines by HPV16 genes occurs in twostages (M1 and M2), as was initially described in mammary epithelialcells by Shayet al. (47). After infection of HPECs at passage 2 (P2)and selection with G418, all HPV16-transformed cultures (E6/E7, E6,and E7) showed an extended life span of two to three additionalpassages (eightversusfive passages) before undergoing senescence.E6- and/or E7-transformed HPECs then entered an extended crisisperiod lasting 2–3 months in which no net gain in cell numbers wasevident. After 2–3 months, small, single colonies of HPV16-trans-formed HPECs began to proliferate and could eventually be dispersedand passaged. These colonies represent the emergence of immortalclones from the so-called M2 stage, and they occurred at a lowfrequency of;1 3 1025. E6/E7 immortal colonies typically emergesooner than E6 or E7 colonies (;6 weeksversus12 weeks). Usingthis approach, we established three sets of HPV16 E6, E7, and E6/E7HPEC immortal cell lines. Each set of cell lines was generated froma single initial prostate epithelial culture taken from one of threedifferent individuals, thus the lines can be defined as isogeneic inorigin.

Phenotypic differences are noted between the E6- and E7-trans-formed prostate epithelial cell lines. E6 HPEC and E6/E7 HPEC linesare characterized by a pleomorphic cell size, irregular shape, andloose adhesion (Fig. 3A). In contrast, E7 HPEC cells have flat, tightlyadherent cells more typical of normal epithelial morphology (Fig. 3B).All HPV16-transformed HPEC lines are now at P40 or more (400doublings), consistent with the immortal phenotype. Cytokeratinstaining for 5 and 18 confirmed their epithelial origin.

Independence and Clonality of HPV16 E6 and/or E7 HPECs.Southern blot analysis for HPV16 insertion was performed on theeight HPV cell lines generated at an early-passage (P183P24). Lineswere not considered immortal until approximately P15. Results showa single integration site in 5 cell lines, as indicated by a single bandon Southern blot (Fig. 4A). Two bands are demonstrated in HPEClines E6-9, E6/E7-9, and E7–14. Given their apparent clonal origin(see above) and equal band intensity at different passages, these two

Fig. 1. Phase contrast microscopy and SA-b-galactosidase staining of proliferating and senescent normal human prostate epithelial cells in culture.A, early-passage HPECsdemonstrate tightly juxtaposed cells that grow in large islands (phase microscopy,3200).B, nonproliferating passage five HPECs demonstrate morphological changes consistent withsenescence including flattened, enlarged cells, and multiple nuclei.C, SA-b-galactosidase staining (38) of nonproliferating prostate epithelial cells demonstrates blue nuclear/cytoplasmic staining indicating senescence (3400). Early-passage cultures (data not shown) were negative for SA-b-galactosidase staining.

Fig. 2. Western analysis of p53, pRb, p16, and p21 in early- and late-passage, senescentnormal prostate epithelial cells. Protein expression from three separate proliferatinghuman prostate epithelial cell cultures is shown (Pre-Senescent,Lanes 1–3). These cellswere continuously passaged and analyzed at passage 5 after growth arrest (Senescent,Lanes 1–3). Senescence was confirmed by positive SA-b-galactosidase staining (data notshown). p16 is strongly expressed in senescent cells compared to early-passage cells. Incontrast, no alterations in expression are found in p53 or p21. Phosphorylated pRb levelsare decreased at senescence. HPEC-E7 is used as a positive control because it stronglyexpresses p53, p21, and p16.

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bands probably represent two integration sites in each of these celllines. RT-PCR confirms the correct expression of E6 and/or E7 RNAwithin the E6, E7, or E6/E7 HPEC lines (Fig. 4B).

p16 or pRb Inactivation in HPV16 Immortal HPECs and inMetastatic Prostate Cancer Lines.The sufficiency of pRb or p53inactivation in overcoming senescence during immortalization wasevaluated in HPECs. Consistent with a functioning HPV16 E6, West-ern blot analysis of HPEC E6 and E6/E7 immortal lines (P20) showsthat p53 levels are undetectable (Fig. 5). In contrast, HPEC E7 celllines show slightly elevated p53 levels compared to normal HPECs(Fig. 2). p21 expression was positively regulated by p53, and consist-ent with this, p21 was strongly expressed in HPEC E7. In contrast,even with prolonged Western exposure times, p21 protein was notdetectable in p53 negative HPEC E6 lines. Therefore, p21 levelscorrelate with p53 expression and p53 function in E7 cell lines wasconfirmed.

pRb is nondetectable in the E7 cell lines and present at lowlevels in E6/E7 HPEC when compared to proliferating normalHPECs. The low level of pRb detected in HPEC E6/E7 lines mayrepresent incomplete ubiquitin-induced degradation of pRb (48).pRb was strongly detected in HPEC E6 lines, as expected. Theselevels of pRb protein were comparable to those seen in normalprostate epithelium. However, in all three HPEC E6 lines, immor-talization is accompanied by p16 loss. In contrast, p16 is abun-dantly expressed in the HPEC E7 and E6/E7 immortal lines (thathave lost pRb function). These elevated levels of p16 are compa-rable to the levels seen in HPECs at senescence (Fig. 2). Highlevels of p16 do not inhibit proliferation in E7-transformed HPECsbecause of the inactivation of downstream regulatory protein pRb.Therefore, all HPV16 E6- and/or E7-transformed HPECs showedeither p16 or pRb inactivation.

To further test the association between inactivation of pRb expres-sion or p16 in bypassing senescence in HPECs, we evaluated a seriesof immortal prostate cancer cell lines (Fig. 5). These lines derive fromindependent biopsies of metastatic prostate cancer. Two cell lines,Du145 and LnCaP, showed no pRb expression. The Du145 prostatecancer cell line contains a knownRB mutation (49). In both of these

lines, p16 was present and detectable by Western analysis. All othermetastatic immortal prostate cancer lines expressed apparently wild-type pRb, but no p16 protein was detectable. These results demon-strate a loss of p16 or pRb in all immortalized prostate cancer cells.

Abnormal p53 expression was detected in the majority of metastaticcell lines, including PC3, TSU-PR1, DuPro, and PPC-1. Inactivatingmutations within the coding exons (exons 5–8) have been describedpreviously in PC3 and TSU-PR1 (50). Du145 contains a stabilizedmutant p53. The mutant p53 was undetectable in PC3 and TSU-PR1.In LnCaP, p53 is wild type, and low levels were detectable only byimmunoprecipitation (50). Therefore, p53 inactivation was frequentbut not necessary for overcoming senescence and acquisition of themetastatic phenotype in prostate cancer.

CDKN2/p16 Is Inactivated by Hypermethylation, Deletion, orMutation in HPEC E6 and Metastatic Prostate Cancer Lines.Toidentify mechanisms underlying the loss of p16 expression inimmortal HPEC E6 lines and in representative metastatic prostatecancer lines described above, we performed methylation analysisusing Southern blot (Fig. 6). Loss of p16 expression in two of threeHPEC E6 lines (E6-9 and E6-14) was due to biallelic DNAhypermethylation, as evidenced by the presence of a 6-kb fragmentafter restriction with the methylation-sensitive enzymeSmaI.Methylation of this region has been previously correlated with lossof transcription (40). The radiolabeled probe used for Southernanalysis demonstrates a reproducibly decreased signal (.50%) incell line HPEC E6-15, indicating a heterozygous deletion ofp16/CDKN2. This is consistent with CGH results (Table 1). Sequencingof exons 1 and 2 ofp16/CDKN2was performed in all HPEC E6lines. E6-15 contains a mutation in exon 1 in codon 33(GAG3TAG) that generates a stop codon and has been describedpreviously (51). A second mutation was detected downstream incodon 34 (GCG3ACG; Glu3Thr). A complete loss of p16 ex-pression from E6-15 resulted from a deletion of the other allele ofCDKN2/p16(Fig. 6).

An analysis of thep16/CDKN2locus in prostate cancer cell lines bySouthern demonstrated no methylation in LnCaP and Du145 (19),both of which express p16 by Western blot. However, a missense

Fig. 3. Phase-contrast microscopy of HPV16 E6and E7 HPEC lines. The morphology of HPV16immortal cells was recorded at passage 20 in sub-confluent dishes (3200 magnification).A, the mor-phology of E6 human prostate epithelial cell linesdemonstrates irregular shaped, loosely packed cellswith poor adherence. E6/E7 cell lines have a sim-ilar morphology (data not shown) to E6 lines.B, incontrast, E7 lines show flat, adherent cells withmorphology similar to early-passage HPECs.

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mutation in exon 2 on codon 84 (GAC3TAC; Arg3Leu) is presentin Du145. This was identified in all sequencing reactions performedand confirmed an earlier report of mutation in this line (19). It did notalter expression of the protein. Hypermethylation was detected inPC3, TSU-PR1 (data not shown), DuPro, and PPC-1. Therefore, theloss of p16 expression in HPEC E6 and metastatic prostate cancerlines can, in all cases, be explained by hypermethylation, mutation, ordeletion.

Additional Genomic Losses and Gains Accompany HPEC Im-mortalization. CGH on early-passage HPV16 immortalized HPECswas used to identify genetic regions that were gained or lost inassociation with immortalization (Table 1). A gain of chromosome 20was present in seven of eight HPEC-immortalized lines. 8q wasgained in four cell lines. Gain of 5q was seen in all HPEC E7. Lossof 8p, a commonly deleted region (;80%) in clinical prostate cancer(52), was present in one HPEC E6. Notably, CGH is relativelyinsensitive for the detection of genomic losses smaller than 10 Mb.Further mapping may detect smaller deletions. Nevertheless, thesefindings suggest that genetic alterations, including120, 18q, and15q may contribute to overcoming senescence.

To test this, we analyzed CGH loss and gain data using a simplestatistical model as described earlier (5, 44). The null hypothesisasserts that changes are sporadic and thus present randomly accordingto a background rate that is constant among chromosome arms. Thealternative hypothesis allows that some arms exhibit an elevated rateof change. The model allows the possibility that changes on the p andq arms are linked. Gain data exhibited a pattern that was significantlydifferent from that which one would expect under the null hypothesiswith P 5 0.001, whereas loss data were consistent with sporadicchange (P5 0.31). Among gains, 5q, 8q, and 20 were significant inthe sense that the probability exceeded 0.95 (in each case) that gain ofeach arm exhibited an elevated rate. No significant losses were de-tected in our sample group.

DISCUSSION

Replicative senescence is a mechanism of tumor suppression andrepresents a safeguard against the development of neoplasia (2, 4).Therefore, the identification of genes that function in normal replica-tive senescence in prostate epithelial cells is important for our under-standing of tumorigenesis in the prostate. In this study, we report forthe first time that replicative senescence in normal prostate epithelialcell cultures is reproducibly associated with an elevation of p16. We

Fig. 4. Demonstration of HPV16 E6 and E7 DNA insertion and gene expression.A,Southern analysis for HPV16 insertions. DNA from the eight cell lines was harvestedbetween passages 18 and 25, digested withHindIII, and probed with a radiolabeled 0.8-kbHPV16 fragment. Results show single bands in some lanes, demonstrating single sites forretroviral insertion and single clonal origin. Multiple bands seen inLanes 1,3, and5 (fromleft to right) represent multiple viral insertion sites.B, RT-PCR for HPV16 E6 or E7 geneexpression in human prostate epithelial cell lines. The HPEC cell lines immortalized withHPV16 E6, E7, or E6/E7 were assessed at passage 18–25 for expression of E6 and E7.Expression of the E6 (347 and 194 bp) and E7 gene products (165 bp) in immortalizedHPEC cell sets (HPEC 9, 14, and 15) is shown. Each set is derived from separate singleprostate epithelial tissue specimen.

Fig. 5. Western analysis of p53, pRb, p16, and p21 in HPV16 E6-, E7-, or E6/E7-immortalized prostate epithelial cells and metastatic prostate cancer cell lines.Lanes 1–8(from left to right), immortalized prostate epithelial cell lines generated by retroviraltransfection of HPV16 E7 and/or E6 that inactivate pRb and p53, respectively. p16expression is uniformly lost in HPV16 E6 cell lines in contrast to E7 cell lines. p16 is notdetectable by increasing the length of exposure or protein levels. Immortalized prostatecancer cell lines (Lanes 9–14) from metastatic prostate cancers demonstrate an inversecorrelation between p16 and pRb expression. p53 is wild type in cancer cell line LNCaPand shows detectable levels upon longer exposure.

Fig. 6. Southern blot of p16/CDKN2 in HPV16-immortalized prostate epithelial cellsand metastatic prostate cancer cell lines. The methylation-sensitive enzymeSmaI andflanking enzymeHindIII demonstrate hypermethylation (6.0 kb) in HPEC E6-9, HPECE6-14, DuPro, and PPC-1 cell lines. Digestion fragments of 2.1 and 3.9 kb are shown inunmethylated cell lines HPEC E6-15 and E7-9. HPEC E6-15 contains a heterozygousdeletion, as evidenced by the reproducible decrease in signal intensity. The other allele ofHPEC E6–15 is unmethylated but mutated (see text).

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also demonstrate for the first time using prostate epithelial cells thatbypassing senescence requires an alteration in the p16/pRb pathway.However, this latter alteration is apparently insufficient for bypassingsenescence, as nonrandom gains of 5q, 8q, and 20q also accompanyimmortalization.

Our data showing that p16 is elevated at senescence in prostateepithelial cells supports a model for replicative senescence that hasbeen previously proposed for human fibroblast and urothelial cells(11, 12). p16 specifically binds to and inhibits CDK4 blocking pro-gression of the cell cycle beyond G1. Thus, in this model, elevatedlevels of p16 play a critical role in senescence G1 growth arrest. Themechanisms underlying the dramatic increase in p16 levels at terminalsenescence have not been defined but may result from an accumula-tion of p16, possibly due to an increased stabilization of p16 mRNAand/or a loss of p16 repression by decreased levels of pRb (53). Wefind a consistent down-regulation of phosphorylated forms of pRb insenescent human prostate epithelial cells, which may, in turn, con-tribute to a failure of cell cycle progression. Functional pRb isrequired for normal G1 growth arrest, as studies using transfected andexpressed p16 have shown (31, 54). Our findings, therefore, implicatep16 as important in the G1 cell cycle arrest characteristic of senes-cence in normal prostate epithelial cells.

Notably, increases in expression of p21 and p53 do not occur duringreplicative cellular senescence in prostate epithelial cells. This resultis similar to findings in cultured human urothelial cells (12) but differsfrom findings in human and rodent fibroblasts (11, 28). Some studiessuggest that p21, which functions to arrest cells in cycle by binding tocyclins D, A, and E, is overexpressed in and is a mediator of repli-cative senescence in fibroblasts (13). However, other data question thefinding that p21 is elevated at terminal senescence in fibroblasts (11).It is also uncertain whether p53 up-regulation is critical to replicativesenescence. For example, p53-null fibroblasts from patients withLi-Fraumeni syndrome undergo normal senescence in the absence ofexpression changes in either p53 or p21 (55). Therefore, p21 and p53elevation at senescence may represent a characteristic of selectedfibroblast cell strains, but it does not appear to be important forsenescence G1 cell cycle arrest in human prostate (or urothelial)epithelial cells.

To test our hypothesis that p16 elevation plays a critical role inreplicative senescence, we examined the status of p16 and pRb inthree isogeneic sets of HPV16 E6 and/or E7 immortal prostate epi-thelial cells that bypassed senescence. Each cell set arises from aninitial single epithelial culture generated from one of three independ-ent normal prostate specimens. Our second important finding is thatovercoming the cell cycle block associated with senescence requiresan alteration in the p16/pRb pathway. In the cells lines containing E7,pRb function is lost by the binding of E7 oncoprotein to underphos-

phorylated pRb and by an enhancement of ubiquitin-induced degra-dation of pRb (48). HPV16 E7 immortal lines show elevated levels ofp16, similar to those seen at senescence. This finding supports theobservation that the cell cycle arrest imposed by p16 is only apparentin cells that retain functional pRb (31, 54). The high levels of p16 inE7 pRb deficient cells provide further evidence for a feedback regu-latory loop involving pRb and p16. We also tested spontaneouslyimmortalized cell lines derived from biopsies of metastatic prostatecancer and demonstrate that pRb expression is lost and p16 is elevatedin two of six lines.

An alternate pathway for reentry into the cell cycle from senescencein the presence of wild-type pRb involves a loss of p16 expression.Because pRb is a downstream component of the p16 pathway, loss ofp16 function would, in theory, function equivalently to pRb loss. Thisscenario is found in all HPEC E6-immortalized cell lines that havelost functional p53 and retained pRb. In the E6 cell lines tested, p16loss is mediated most commonly by DNA hypermethylation. We alsofind that one line, E6-15, contains an allelic deletion and a mutationon the remaining allele. Therefore, several common mechanisms forinactivating p16 are demonstrated in our experimental model usingnormal prostate epithelial cells. The requirement for a p16/pRb path-way alteration is also met in the spontaneously immortalized meta-static prostate cell lines. Loss of p16 expression due to hypermethy-lation of p16/CDKN2,along with wild-type pRb, is found in four ofsix of these cell lines. Hypermethylation ofp16/CDKN2is a selectivemechanism for inactivating p16 expression, and does not appear toalter the expression of the alternative reading frame splice variantp14/ARFin bladder cancer cell lines (56). The finding of methylationinactivation ofp16/CDKN2in prostate cancer and HPEC E6-immor-talized lines (six of nine) emphasizes a unique epigenetic feature ofthe tumorigenic process in prostate cells. Mutations ofp16/CDKN2occur rarely in prostate cancer (19) in contrast to other tumors (57,58). The Du145 cell line, which contains a pRb mutation, we havefound on sequencing to contain ap16/CDKN2missense mutation (19,59). However, wild-type p16 is apparently encoded from the secondallele in this pRb-negative line.

The results above document that an alteration in the p16/pRbpathway is critical for immortalization of HPEC. However, we haveidentified a number of additional genetic alterations that are nonran-domly associated with overcoming the senescence block in HPECs.Both HPEC E7- and E6-infected cells undergo a crisis period of lowto undetectable proliferation (M2 block) for several months beforeproliferative clones that give rise to immortal lines are detected. Themost significant genetic change, identified in seven of eight immortallines, is a gain of chromosome 20. Gain of chromosome 20 has beenseen in many human cancer types, including bladder, breast, ovarian,and prostate (9, 43, 60). By CGH, centromeric regions of 20 areamplified in almost half ofin vivo prostate cancer metastases (15).This amplification is infrequently found in primary prostate tumors.Finally, gain of chromosome 20 has been identified in human urothe-lial cells transformedin vitro by HPV16 (9). These observationsindicate the presence on chromosome 20 of one or several oncogenes.Several candidate genes have been identified, includingZNF217,NABC1, andCAS(61). Other regions of gain in HPEC E6 and E7 linesinclude 5q and 8q. 8q gains are noted infrequently in primary prostatecancers, but they occur commonly in metastatic and recurrent (80%)prostate cancers (15). c-Myc is located at 8q24, and Myc protein levelsare increased in E6 cells by an undefined posttranscriptional mecha-nism. Recently, it was found that telomerase induction is required forE6 epithelial immortalization, in addition to alterations in the p16/pRbpathway (10). Notably, we have demonstrated telomerase activity incultured normal prostate epithelium, as well as normal bladder, ureter,

Table 1 Genetic alteration in HPV16 E-6, E-7, and E6/E7-immortalized HPECsby CGH

Cell line Passagea Losses Gainsb

HPEC E7-9 26 4, 5, 8, 9, 14, 20c

HPEC E7-14 21 10p 5q, 8, 11, 20HPEC E7-15 16 5, 7, 20HPEC E6-9 25 5, 8q22–qter, 16, 20, YHPEC E6-14 14 22,Y 8q21.1–qter, 15q21–qter, 20HPEC E6-15 16 8p, 9p 9q31-qterHPEC E6/E7-9 24 7, 20,c YpHPEC E6/E7-14 17 Y 9, 20c

a Denotes total passage number since the initial culture was generated. Immortalizedcell lines generally arose between passages 8 and 12; therefore, these cultures representearly passages.

b Gains at 5q, 8q, and 20 are significant. The probability exceeds 0.95 (in each case)that that region exhibits an elevated rate of gain (44).

c In these cases, gains of chromosome 20 occurred to a greater degree than in otherlines, suggesting possible amplification.

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and mammary epithelium (62). It has also been noted that Myc proteinincreases telomerase mRNA through an undefined mechanism (63).

In the current model of prostate carcinogenesis, inactivation of thep16-pRb pathway appears to play an important role in overcoming thecell cycle block imposed by p16 at senescence.RB, on 13q14, is aregion of intermediate frequency (;30%) deletion in prostate cancersamples (16, 64) We have previously demonstrated thatp16/CDKN2alterations occur frequently (;50%) in prostate cancer (19). Given thepresent data supporting the mutual exclusion of p16 and pRb alter-ations within the same cell line, inactivation of this pathway mayrepresent an extremely common alteration in prostate cancer. Al-though this correlation has not been tested in prostate cancersin vivo,it has been demonstrated in several other tumors (29, 65). Inactivationof the p16/pRb pathway is necessary but not sufficient for immortal-ization of HPEC. Thus, ourin vitro model using E6 and E7 containsgenetic gains and losses also seen inin vivo tumors. Therefore, it maybe useful in identifying genes altered in prostate cancer and definingpathways of cancer progression via different combinations of geneticand epigenetic alterations.

ACKNOWLEDGMENTS

Our special thanks to Robert Huffman (Department of Surgery) and Dr.David Uehling (Division of Urology) of the University of Wisconsin for theirsupport.

REFERENCES

1. Hayflick, L. The limitedin vitro lifespan of human diploid cell strains. Exp. Cell Res.,25: 585–621, 1965.

2. Newbold, R. F., Overell, R. W., and Connell, J. R. Induction of immortality is an earlyevent in malignant transformation of mammalian cells by carcinogens. Nature(Lond.), 299: 633–635, 1982.

3. Smith, J. R., and Pereira-Smith, O. M. Replicative senescence: implications forinvivo aging and tumor suppression. Science (Washington DC),273: 63–67, 1996.

4. Dimri, G. P., and Campisi, J. Molecular and cell biology of replicative senescence.Cold Spring Harbor Symp. Quant. Biol.,59: 67–73, 1994.

5. Yeager, T. R., DeVries, S., Jarrard, D. F., Kao, C., Nakada, S. Y., Moon, T. D.,Bruskewitz, R., Stadler, W. M., Meisner, L. F., Gilchrist, K. W., Newton, M. A.,Waldman, F. M., and Reznikoff, C. A. Overcoming cellular senescence in humancancer pathogenesis. Genes Dev.,12: 163–174, 1998.

6. Reznikoff, C. A., Belair, C. D., Yeager, T. R., Savelieva, E., Blelloch, R. H.,Puthenveettil, J. A., and Cuthill, S. A Molecular genetic model of human bladdercancer pathogenesis. Semin. Oncol.,23: 571–584, 1996.

7. Shay, J. W., Pereira-Smith, O. M., and Wright, W. E. A role for both RB and p53 inthe regulation of human cellular senescence. Exp. Cell Res.,196: 33–39, 1991.

8. Kao, C., Huang, J., Wu, S. Q., Hauser, P., and Reznikoff, C. A. Role of SV40 Tantigen binding to pRB and p53 in multistep transformationin vitro of humanuroepithelial cells. Carcinogenesis (Lond.),14: 2297–2302, 1993.

9. Reznikoff, C. A., Belair, C., Savelieva, E., Zhai, Y., Pfeifer, K., Yeager, T., Thomp-son, K. J., DeVries, S., Bindley, C., and Newton, M. A. Long-term genome stabilityand minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-,immortalized human uroepithelial cells. Genes Dev.,8: 2227–2240, 1994.

10. Kiyono, T., Foster, S. A., Koop, J. L., McDougall, J. K., Galloway, D. A., andKlingelhutz, A. J. Both Rb/p16INK4A inactivation and telomerase activity are requiredto immortalize human epithelial cells. Nature (Lond.),396: 84–88, 1998.

11. Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D., and Barrett, J. C.Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicativesenescence of normal human fibroblasts. Proc. Natl. Acad. Sci. USA,93: 13742–13747, 1996.

12. Reznikoff, C. A., Yeager, T. R., Belair, C. D., Savelieva, E., Puthenveettil, J. A., andStadler, W. M. Elevated p16 at senescence and loss of p16 at immortalization inhuman papillomavirus 16 E6, but not E7, transformed human uroepithelial cells.Cancer Res.,56: 2886–2890, 1996.

13. Tahara, H., Sato, E., Noda, A., and Ide, T. Increase in expression level of p21/cip1/waf1 with increasing division age in both normal and SV40-transformed humanfibroblasts. Oncogene,10: 835–840, 1995.

14. Brooks, J. D., Bova, G. S., Ewing, C. M., Piantadosi, S., Carter, B. S., Robinson, J. C.,Epstein, J. I., and Isaacs, W. B. An uncertain role forp53gene alterations in humanprostate cancers. Cancer Res.,56: 3814–3822, 1996.

15. Cher, M. L., Bova, G. S., Moore, D. H., Small, E. J., Carroll, P. R., Pin, S. S., Epstein,J. I., Isaacs, W. B., and Jensen, R. H. Genetic alterations in untreated metastases andandrogen-independent prostate cancer detected by comparative genomic hybridiza-tion and allelotyping. Cancer Res.,56: 3091–3102, 1996.

16. Brooks, J. D., Bova, G. S., and Isaacs, W. B. Allelic loss of the retinoblastoma genein primary human prostatic adenocarcinomas. Prostate,26: 35–39, 1995.

17. Cunningham, J. M., Shan, A. L., Wick, M. J., McDonnell, S. K., Schaid, D. J., Tester,D. J., Qian, J. Q., Takahashi, S., Jenkins, R. B., Bostwick, D. G., and Thibodeau, S. N.Allelic imbalance and microsatellite instability in prostatic adenocarcinoma. CancerRes.,56: 4475–4482, 1996.

18. Aprikian, A. G., Sarkis, A. S., Fair, W. R., Zhang, Z. F., Fuks, Z., and Cordon-Cardo,C. Immunohistochemical determination of p53 protein nuclear accumulation in pros-tatic adenocarcinoma. J. Urol.,151: 1276–1280, 1994.

19. Jarrard, D. F., Bova, G. S., Ewing, C. M., Pin, S. S., Nguyen, S. H., Herman, J. G.,Cairns, P., Sidransky, D., Baylin, S. B., and Isaacs, W. B. Deletional, mutational andmethylation analyses of CDKN2 in primary and metastatic prostate cancer. GenesChromosomes Cancer,19: 90–96, 1997.

20. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell,88: 323–331,1997.

21. Atadja, P., Wong, H., Garkavtsev, I., Veillette, C., and Riabowol, K. Increasedactivity of p53 in senescing fibroblasts. Proc. Natl. Acad. Sci. USA,92: 8348–8352,1995.

22. Hara, E., Tsurui, H., Shinozaki, A., Nakada, S., and Oda, K. Cooperative effect ofantisense-Rb and antisense-p53 oligomers on the extension of life span in humandiploid fibroblasts, TIG-1. Biochem. Biophys. Res. Commun.,179: 528–534, 1991.

23. Bond, J. A., Blaydes, J. P., Rowson, J., Haughton, M. F., Smith, J. R., Wynford-Thomas, D., and Wyllie, F. S. Mutant p53 rescues human diploid cells from senes-cence without inhibiting the induction of SKI1/WAF1. Cancer Res.,55: 2404–2409,1995.

24. Brugarolas, J., Chandrasekaran, C., Gordon, J. I., Beach, D., Jacks, T., and Hannon,G. J. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature(Lond.), 377: 552–557, 1995.

25. Cristofalo, V. J., Pignolo, R. J., Cianciarulo, F. L., DiPaolo, B. R., and Rotenberg,M. O. Changes in gene expression during senescence in culture. Exp. Gerontol.,27:429–432, 1992.

26. Stein, G. H., Beeson, M., and Gordon, L. Failure to phosphorylate the retinoblastomagene product in senescent human fibroblasts. Science (Washington DC),249: 666–669, 1990.

27. Serrano, M., Hannon, G. J., and Beach, D. A new regulatory motif in cell-cyclecontrol causing specific inhibition of cyclin D/CDK4. Nature (Lond.),366: 704–707,1993.

28. Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S., and Peters, G. Regulation ofp16CDKN2 expression and its implications for cell immortalization and senescence.Mol. Cell. Biol., 16: 859–867, 1996.

29. Ueki, K., Ono, Y., Henson, J. W., Efird, J. T., von Deimling, A., and Louis, D. N.CDKN2/p16 or RB alterations occur in the majority of glioblastomas and areinversely correlated. Cancer Res.,56: 150–153, 1996.

30. Otterson, G. A., Kratzke, R. A., Coxon, A., Kim, Y. W., and Kaye, F. J. Absence ofp16INK4 protein is restricted to the subset of lung cancer lines that retains wildtypeRB. Oncogene,9: 3375–3378, 1994.

31. Lukas, J., Parry, D., Aagaard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G.,and Bartek, J. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumoursuppressor p16. Nature (Lond.),375: 503–506, 1995.

32. Ruas, M., and Peters, G. Thep16/CDKN2tumor suppressor gene and its relatives.Biochim. Biophys. Acta,1378: F115–F117, 1998.

33. Theodorescu, D., Broder, S. R., Boyd, J. C., Mills, S. E., and Frierson, H. F., Jr. p53,bcl-2 and retinoblastoma proteins as long-term prognostic markers in localizedcarcinoma of the prostate. J. Urol.,158: 131–137, 1997.

34. Peehl, D. M., and Stamey, T. A. Serum-free growth of adult human prostaticepithelial cells. In Vitro Cell. Dev. Biol.,22: 82–90, 1986.

35. Peehl, D. M., Wong, S. T., and Stamey, T. A. Clonal growth characteristics of adulthuman prostatic epithelial cells. In Vitro Cell. Dev. Biol.,24: 530–536, 1988.

36. Reznikoff, C. A., Johnson, M. D., Norback, D. H., and Bryan, G. T. Growth andcharacterization of normal human urotheliumin vitro. In Vitro, 19: 326–343, 1983.

37. Umbas, R., Schalken, J. A., Aalders, T. W., Carter, B. S., Karthaus, H. F., Schaafsma,H. E., Debruyne, F. M., and Isaacs, W. B. Expression of the cellular adhesionmolecule E-cadherin is reduced or absent in high-grade prostate cancer. Cancer Res.,52: 5104–5109, 1992.

38. Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E.,Linskens, M., Rubelj, I., and Pereira-Smith, O. A biomarker that identifies senescenthuman cells in culture and in aging skinin vivo. Proc. Natl. Acad. Sci. USA,92:9363–9367, 1995.

39. Halbert, C. L., Demers, G. W., and Galloway, D. A. The E7 gene of humanpapillomavirus type 16 is sufficient for immortalization of human epithelial cells.J. Virol., 65: 473–478, 1991.

40. Merlo, A., Herman, J. G., Mao, L., Lee, D. J., Gabrielson, E., Burger, P. C., Baylin,S. B., and Sidransky, D. 59 CpG island methylation is associated with transcriptionalsilencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat. Med.,1: 686–692, 1995.

41. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, Q., Harshman, K., Tavtigian, S. V.,Stockert, E., Day, R. S., III, Johnson, B. E., and Skolnick, M. H. A cell cycle regulatorpotentially involved in genesis of many tumor types. Science (Washington DC),264:436–440, 1994.

42. Kallioniemi, A., Kallioniemi, O. P., Piper, J., Tanner, M., Stokke, T., Chen, L., Smith,H. S., Pinkel, D., Gray, J. W., and Waldman, F. M. Detection and mapping ofamplified DNA sequences in breast cancer by comparative genomic hybridization.Proc. Natl. Acad. Sci. USA,91: 2156–2160, 1994.

43. Hovey, R. M., Chu, L., Balazs, M., DeVries, S., Moore, D., Sauter, G., Carroll, P. R.,and Waldman, F. M. Genetic alterations in primary bladder cancers and their metas-tases. Cancer Res.,58: 3555–3560, 1998.

44. Newton, M. A., Gould, M. N., Reznikoff, C. A., and Haag, J. D. On the statisticalanalysis of allelic-loss data. Stat. Med.,17: 1425–1445, 1998.

2963

p16/pRb PATHWAY ALTERATIONS IN HPEC IMMORTALIZATION

on May 31, 2020. © 1999 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

45. Bonkhoff, H., Stein, U., and Remberger, K. Multidirectional differentiation in thenormal, hyperplastic, and neoplastic human prostate: simultaneous demonstration ofcell-specific epithelial markers. Hum. Pathol.,25: 42–46, 1994.

46. Verhagen, A. P., Ramaekers, F. C., Aalders, T. W., Schaafsma, H. E., Debruyne,F. M., and Schalken, J. A. Colocalization of basal and luminal cell-type cytokeratinsin human prostate cancer. Cancer Res.,52: 6182–6187, 1992.

47. Shay, J. W., Wright, W. E., Brasiskyte, D., and Van der Haegen, B. A. E6 of humanpapillomavirus type 16 can overcome the M1 stage of immortalization in humanmammary epithelial cells but not in human fibroblasts. Oncogene,8: 1407–1413,1993.

48. Vousden, K. H. Regulation of the cell cycle by viral oncoproteins. Semin. CancerBiol., 6: 109–116, 1995.

49. Bookstein, R., Rio, P., Madreperla, S. A., Hong, F., Allred, C., Grizzle, W. E., andLee, W. H. Promoter deletion and loss of retinoblastoma gene expression in humanprostate carcinoma. Proc. Natl. Acad. Sci. USA,87: 7762–7766, 1990.

50. Isaacs, W. B., Carter, B. S., and Ewing, C. M. Wild-type p53 suppresses growth ofhuman prostate cancer cells containing mutantp53 alleles. Cancer Res.,51: 4716–4720, 1991.

51. Cairns, P., Mao, L., Merlo, A., Lee, D. J., Schwab, D., Eby, Y., Tokino, K., van derRiet, P., Blaugrund, J. E., and Sidransky, D. Rates of p16 (MTS1) mutations inprimary tumors with 9p loss. Science (Washington DC),265: 415–417, 1994.

52. Vocke, C. D., Pozzatti, R. O., Bostwick, D. G., Florence, C. D., Jennings, S. B., Strup,S. E., Duray, P. H., Liotta, L. A., Emmert-Buck, M. R., and Linehan, W. M. Analysisof 99 microdissected prostate carcinomas reveals a high frequency of allelic loss onchromosome 8p12–21. Cancer Res.,56: 2411–2416, 1996.

53. Boring, C. C., Squires, T. S., Tong, T., and Montgomery, S. Cancer statistics, 1994.CA Cancer J. Clin.,44: 7–26, 1994.

54. Okamoto, A., Demetrick, D. J., Spillare, E. A., Hagiwara, K., Hussain, S. P., Bennett,W. P., Forrester, K., Gerwin, B., Serrano, M., and Beach, D. H. Mutations and alteredexpression of p16INK4 in human cancer. Proc. Natl. Acad. Sci. USA,91: 11045–11049, 1994.

55. Medcalf, A. S., Klein-Szanto, A. J., and Cristofalo, V. J. Expression of p21 is notrequired for senescence of human fibroblasts. Cancer Res.,56: 4582–4585, 1996.

56. Gonzalgo, M. L., Hayashida, T., Bender, C. M., Pao, M. M., Tsai, Y. C., Gonzales,F. A., Nguyen, H. D., Nguyen, T. T., and Jones, P. A. The role of DNA methylation

in expression of thep19/p16locus in human bladder cancer cell lines. Cancer Res.,58: 1245–1252, 1998.

57. Caldas, C., Hahn, S. A., da Costa, L. T., Redston, M. S., Schutte, M., Seymour, A. B.,Weinstein, C. L., Hruban, R. H., Yeo, C. J., and Kern, S. E. Frequent somaticmutations and homozygous deletions of thep16 (MTS1) gene in pancreatic adeno-carcinoma. Nat. Genet.,8: 27–32, 1994.

58. Hussussian, C. J., Struewing, J. P., Goldstein, A. M., Higgins, P. A., Ally, D. S.,Sheahan, M. D., Clark, W. H., Jr., Tucker, M. A., and Dracopoli, N. C. Germline p16mutations in familial melanoma. Nat. Genet.,8: 15–21, 1994.

59. Liu, Q., Neuhausen, S., McClure, M., Frye, C., Weaver-Feldhaus, J., Gruis, N. A.,Eddington, K., Allalunis-Turner, M. J., Skolnick, M. H., Fujimura, F. K., and Kamb,A. CDKN2 (MTS1) tumor suppressor gene mutations in human tumor cell lines.Oncogene,11: 2455–2461, 1995.

60. Nishizaki, T., DeVries, S., Chew, K., Goodson, W. H., III, Ljung, B. M., Thor, A.,and Waldman, F. M. Genetic alterations in primary breast cancers and their metas-tases: direct comparison using modified comparative genomic hybridization. GenesChromosomes Cancer,19: 267–272, 1997.

61. Collins, C., Rommens, J. M., Kowbel, D., Godfrey, T., Tanner, M., Hwang, S. I.,Polikoff, D., Nonet, G., Cochran, J., Myambo, K., Jay, K. E., Froula, J., Cloutier, T.,Kuo, W. L., Yaswen, P., Dairkee, S., Giovanola, J., Hutchinson, G. B., Isola, J.,Kallioniemi, O. P., Palazzolo, M., Martin, C., Ericsson, C., Pinkel, D., and Gray J. W.Positional cloning ofZNF217andNABC1: genes amplified at 20q13.2 and overex-pressed in breast carcinoma. Proc. Natl. Acad. Sci. USA,95: 8703–8708, 1998.

62. Belair, C. D., Yeager, T. R., Lopez, P. M., and Reznikoff, C. A. Telomerase activity:a biomarker of cell proliferation, not malignant transformation. Proc. Natl. Acad. Sci.USA, 94: 13677–13682, 1997.

63. Wang, J., Xie, L. Y., Allan, S., Beach, D., and Hannon, G. J. Myc activatestelomerase. Genes Dev.,12: 1769–1774, 1998.

64. Ittmann, M. M., and Wieczorek, R. Alterations of the retinoblastoma gene in clini-cally localized, stage B prostate adenocarcinomas. Hum. Pathol.,27: 28–34, 1996.

65. Parry, D., Bates, S., Mann, D. J., and Peters, G. Lack of cyclin D-Cdk complexes inRb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressorgene product. EMBO J.,14: 503–511, 1995.

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