Proc. Nati. Acad. Sci. USA Vol. 91, pp. 11874-11878, December 1994 Medical Sciences Stepwise immortalization and transformation of adult human prostate epithelial cells by a combination of HPV-18 and v-Ki-ras (prostate /malignant transformation) JOHNG S. RHIM*, MUKTA M. WEBBERt, DIANA BELLOt, MYEONG SEON LEE*, PAUL ARNSTEIN*, LIAN-SHENG CHENt, AND GILBERT JAYt *Laboratory of Cellular and Molecular Biology, National Institute, Bethesda, MD 20892; tDepartments of Medicine and Zoology, Michigan State University, East Lansing, MI 48824; and tDepartment of Virology, Jerome H. Holland Laboratory, Rockville, MD 20855 Communicated by Gerald N. Wogan, August 4, 1994

ABSTRACT Recent investigations have shown the pres- (PCR), observed that 52% of the prostate carcinomas con- ence of ras gene and human papillomavirus (HPV) tained HPV-16 and/or HPV-18 sequences. Sarkar et al. (16) DNA in prostate carcinomas. In the present study, secondary also detected HPV DNA in human prostatic carcinomas, but adult human prostatic epithelial cells, upon transfection with a at a lower frequency, 13%. In recent study of Japanese plasmid containing the entire HPV-18 genome, acquired an patients with prostatic carcinomas, 41% of tissue samples indefinite life-span in culture but did not undergo malignant were positive for at least one type of the high-risk HPVs conversion. Subsequent infection of these immortalized cells (HPV-16, -18, and -33), the most common being HPV-18 (25% with the Kirsten murine sarcoma virus, which contains an of cases). In the same group of patients, cooccurrence of ras activated Ki-ras oncogene, induced morphological transforma- mutations and HPV infection was more frequent in stage C tion that led to the acquisition of neoplastic properties. These and stage D than in stage A and stage B carcinomas. findings demonstrate the malignant transformation of adult Therefore, at least among Japanese men with prostate can- human prostate epithelial cells in culture by a combination of cer, ras mutations and the presence of high-risk HPV DNA viral oncogenes and the successive roles of HPV infection and sequences have been linked at a relatively high frequency (9). Ki-ras activation in a multistep process responsible for prostate To obtain further insights into the mechanism of prostatic carcinogenesis. carcinogenesis we used newly developed tissue culture meth- ods to ascertain whether HPV-18 or Kirsten murine sarcoma is the most commonly diagnosed virus (Ki-MuSV) containing an activated Ki-ras oncogene in American men and the second leading cause ofmale cancer could confer a malignant phenotype on early passage human death in the United States (1). Compared with all other prostate epithelial (HPE) cells. We report the immortaliza- , the incidence of prostate carcinoma increases most tion of normal HPE cells by Polybrene-induced DNA trans- rapidly with age (2, 3). Despite the increasing prevalence of fection of the HPV-18 genome and subsequent conversion of the disease (4), the basic mechanisms underlying prostate such nontumorigenic but immortalized cells into tumorigenic cancer growth are largely unknown. To understand the many cells by the introduction of an activated Ki-ras oncogene. factors suspected to contribute to the development of this Our study demonstrates malignant transformation of HPE malignancy, there is a need for an in vitro culture system. cells in culture by a combination of HPV-18 and v-Ki-ras and Recent molecular studies have suggested roles for both the offers a unique in vitro model system for further analysis of activated ras oncogenes and the human papillomavirus molecular events underlying prostate carcinogenesis. (HPV) in prostate carcinogenesis. Previous reports have shown that increased expression of the ras p21 protein is AND associated with increasing histological grade in human pros- MATERIALS METHODS tatic cancer (5-7). Several prostate cancer cell lines have also Cell Cultures and Media. Epithelial cells were derived from been shown to express high levels of the Ha-ras gene (8). In the histologically normal prostate ofa 54-year-old white male addition, ras mutations have been reported in human prostate undergoing a cystectomy for bladder carcinoma. The tissue but not in normal or benign prostate sam- was minced and digested in RPMI 1640 medium containing ples (9-12); while a relatively high frequency (24%) of ras 5% fetal bovine serum and 400 units of collagenase per ml mutations has recently been detected in Japanese prostate (17). The digested tissue was inoculated into fibronectin and cancer patients (9), previous studies from the United States type IV collagen-coated plates in serum-free keratinocyte could detect ras only in <5% of samples analyzed, medium (GIBCO) containing 5 ng of epidermal growth factor although the number of cases examined was limited (10, 12). per ml. Secondary cultures grown in serum-free keratinocyte HPVs are associated with various anogenital carcinomas medium were used for transformation. and the presence ofHPV-16 and/or HPV-18 DNA sequences Transformation Assay. Secondary culture of prostate epi- has been detected in 90% of cervical carcinomas (13). Male thelial cells was infected at a multiplicity ofinfection of 100 sexual partners of women with cervical neoplasia were with the Ki-MuSV [baboon endogenous virus (BaEV)] (18). examined for the presence of infection; 59% demonstrated The infected cultures were subcultured at a 1:2 ratio every visible lesions and, of these, 49% contained HPV DNA (14). 7-10 days and were observed for the appearance of morpho- Because HPVs can be sexually transmitted, the presence of logical alterations. highly oncogenic HPV-16 and -18 in prostate cancer has been Plasmid. The plasmid pSHPV-18m (a gift from J. A. Di- examined. In a group of Canadian patients, McNicol and Paolo, National Cancer Institute) contains a single copy of Dodd (15), using the sensitive polymerase chain reaction the HPV-18 genome inserted into the EcoRI site ofpSV2 neo.

The publication costs ofthis article were defrayed in part by page charge Abbreviations: HPV, human papillomavirus; HPE, human prostate payment. This article must therefore be hereby marked "advertisement" epithelial; MuSV, murine sarcoma virus; SV40, simian virus 40; in accordance with 18 U.S.C. §1734 solely to indicate this fact. BaEV, baboon endogenous virus. 11874 Downloaded by guest on September 25, 2021 Medical Sciences: Rhim et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11875

Transfection. Polybrene-induced DNA transfection was (19) led to the appearance of actively growing colonies by carried out as described (19). Cells were transfected with 10 weeks 6-7. pg of DNA using Polybrene at a concentration of 10 pg/ml A number of cell lines were isolated from individual and incubated at 370C overnight. The cells were then shocked colonies using cloning cylinders and stored frozen in liquid with 30% dimethyl sulfoxide for 4 min. After 5 days, the cells nitrogen. We selected two clones, designated A-2 and C-1, were subcultured at a 1:2 ratio once a week. The cell culture for further characterization. A-2 and C-1 cells had the typical medium was changed twice weekly. polygonal arrangement of epithelial cells (Fig. 1A) but were Colony Formation in Soft Agar. A cell suspension (1 x 104 less polygonal than the parental HPE cells. Both cell lines cells per ml) in 5 ml of 0.35% Noble agar with serum-free have apparently an unlimited life-span; each has been suc- keratinocyte medium was overlaid into a 60-mm dish con- cessfully subcultured for >30 passages over the course of 1 taining a 0.6% agar base. Colonies >0.125 mm in diameter year with no evidence of decreased proliferative capacity were counted at 21 days. (Table 1). Tumorigenidty in Nude Mice. Cells (1 x 107) in 0.2 ml of To confirm that the two clones do contain the transfected phosphate-buffered saline (PBS) were injected subcutane- HPV-18 DNA, Southern blot hybridization analysis (20) was ously into the mid-dorsal interscapular region of adult 129J nude mice to determine tumorigenicity. Southern Blot Hybridization Analysis. DNA blot analysis was performed as described (20). High molecular mass DNA was digested with the appropriate restriction endonuclease and applied to a 1.0% agarose gel. After electrophoresis, the DNA fragments were transferred from the gel to a nitrocel- lulose membrane and hybridized to a random-primed 32p- labeled DNA probe. To confirm the presence of integrated, HPV-18 sequences, a 1.5-kb Xba I fiagment derived from HPV-18 and spanning the 3' end ofE6, the entire E7, and the 5' end of E9 coding regions was used. Western Immunoblot Analysis. Subconfluent cultures of the approprate cell clones were rinsed with PBS (pH 7.2) and lysed with a buffer containing 50 mM Tris HCl, 150 mM NaCl, 0.1% SDS, 1.0%1 Triton X-100, and 1.0%6 sodium deoxycholate. The protein concentration of the lysates was determined and equivalent amounts ofprotein from each cell line were immunoprecipitated with the appropriate antibody. The resulting immunoprecipitates were fractionated on a 12.5% polyacrylamide/SDS gel. After electrophoresis, pro- teins on the gel were electrocuted onto a nitrocellulose membrane, which was then allowed to react against the relevant antibody and 'm'I-labeled protein A (21). The anti- body against HPV-18 E7 was obtained from Louis Chow (University of Rochester) and used at 1:100 dilution. The antibody against p53 was from Stephen Ullrich (25) and was diluted at 1:200.

RESULTS In attempts to alter the growth properties of normal prostate epithelial cells, we used Ki-MuSV, whose activated Ki-ras oncogene has been detected in human prostate carcinomas (6, 7, 9, 11), and HPV-18, which has also been detected in human prostate carcinomas (9, 15, 16). Neither normal con- trol HPE nor Ki-MuSV-infected cells could be propagated serially beyond five subcultures (Table 1). In contrast, cells transfected with a plasmid (pHPV-18) containing the entire HPV-18 genome by Polybrene-induced DNA gene transfer Table 1. Biological properties of adult HPE cells exposed to Ki-MuSV and/or HPV-18 DNA No. of Activated Nude mice with passages Ki-ras tumors/mice Cells in culture expression inoculated* Primary HPE <5 ND ND + Ki-MuSV <5 ND ND + HPV-18 >30 - 0/5 + HPV-18 + Ki-MuSV >30 + 4/5t FIG. 1. Comparison of the morphology of HPV-18 DNA trans- Ki-MuSV (BaEV) was produced in human nonproducer cells by fected adult HPE clonal line C-1 (A) with Ki-MuSV-transformed superinfection with the BaEV (18). ND, not done. HPV-18 DNA transfected C-1 clonal line (B). (x23.) (C) Hematox- *Nude mice were inoculated with 107 cells. ylin/eosin-stained section of poorly differentiated carcinoma pro- tTumors were reestablished in tissue culture and confirmed to duced by inoculation of the Ki-MuSV-transformed HPV-18 DNA resemble the cells of origin by karyological analysis. transfected C-1 clonal line. (x9o.) Downloaded by guest on September 25, 2021 11876 Medical Sciences: Rhim et al. Proc. Natl. Acad. Sci. USA 91 (1994)

carried out using genomic DNA that had been digested with A B BamHI and a 1.5-kb Xba I DNA probe that spans the 3' end ofE6, the entire E7, and the 5' end ofEl coding regions. The detection of an z'4.2-kb fragment in clones A-2 and C-1, but not in the early passage parental HPE cells, demonstrates that HPV-18 sequences have been integrated into both cell clones (Fig. 2). As evidence for the acquisition of the morphological change being the consequence of expression of HPV-18 functions, we determined by Western immunoblot analysis

(21) the presence of the HPV-18 E7 gene product using a ! e rabbit antibody. Identification of an -15-kDa component in M 2 .+ M i3e3 clones A-2 and C-1, which is also present in HeLa cells but FIG. 3. Western immunoblot analysis of HPV-18 E7 protein and not in a simian virus 40 (SV40) ori- transfected HPE clone, cellular p53 protein in the HPV-18 transfected HPE clones. The is consistent with transformation requiring at least the ex- appropriate cell extracts were analyzed by using rabbit antisera pression of the E7 protein (Fig. 3A). Interestingly, although against either the HPV-18 E7 protein (A) or the cellular p53 protein the tumor suppressor protein p53 is detected in abundance in (B). The molecular mass markers (M) used were 97.4, 69, 46, 30, and 14.3 kDa. The arrowheads show the 15-kDa E7 protein (A) and the the SV40 orih transfected clone, presumably because of its 53-kDa p53 protein (B). Lanes 1, HPV-18 transfected HPE clone A-2. stabilization by binding to the SV40 T antigen, it is not Lanes 2, HPV-18 transfected HPE clone C-1. Lanes 3, SV40 ori- detectable in either of the two HPV-18 transfected clones transfected HPE clone. Lanes 4, HeLa cells. (The -30-kDa com- (Fig. 3B). This latter observation is consistent with E7 ponent detected with an excess of antiserum against p53 in all ofthe destabilizing p53, a process that has been proposed to result cell extracts is nonspecific and represents either cross-reactivity with in the loss of tumor suppressor activity. another cellular protein or the presence of an autoantibody in the Evidence for the human origin of the two lines was antiserum.) obtained by isoenzyme analysis and cell membrane species- specific immunofluorescence. Moreover, both cell lines further altered by the introduction of an activated Ki-ras showed human karyotype with a Y chromosome. When clone oncogene. Infection of the HPV-18 transfected C-1 line at C-1 was analyzed at passage 11 and clone A-2 at passage 15, passage 12 with Ki-MuSV, pseudotyped with BaEV to facil- both were aneuploid and had five and nine marker chromo- itate entry into human cells, resulted in a marked alteration somes, respectively. Immunocytochemical analysis showed in cell morphology. Two to 3 weeks after infection, the cells that both lines expressed cytokeratins 8 and 18, specific began to pile up in focal areas (Fig. 1B). The absence of any epithelial cell markers. Prostate-specific antigen was strongly detectable alteration induced by the helper virus alone im- present in early passages of both clones as well as parental plied that Ki-MuSV was responsible for inducing the trans- HPE cells, but only weakly present in later passages of both formed morphology. clones. The presence and expression ofthe Ki-ras oncogene in the When we analyzed for biological properties, we observed transformant were confimed by Western immunoblot anal- that neither cell lines grew in soft agar or produced tumors in ysis using a pan-reactive mouse antibody against the ras p21 129J nude mice even when 107 cells were injected (Table 1). protein. Using this analysis, the C-1 clone was found to Thus, HPV-18 infection was associated with the continued express a low level ofthe endogenous p21 protein (Fig. 4, lane proliferative capacity of adult HPE cells in culture without 2), as indicated on a SDS/polyacrylamide gel by a charac- the concomitant acquisition of a neoplastic phenotype. teristic doublet that represents the phosphorylated and non- The lack oftumorigenicity ofthe HPV-18 transfected lines phosphorylated forms of the gene product (18, 22). In addi- led us to inquire whether its growth properties might be tion to the endogenous p21, the Ki-MuSV transformed C-1 line also expressed a high level of the activated Ki-ras gene product (Fig. 4, lane 3), which is distinguishable by its slower kb migration on SDS/polyacrylamide gels. The expression of 9. the activated ras gene in the transformant is further sup- 7 ported by the presence of the transfected v-Ki-ras gene, as 5 shown by Southern blot hybridization analysis (data not 4 _ shown). HPV-18 DNA analysis indicated that its copy num- ber in the Ki-MuSV-transformed C-1 cells has remained 3. unchanged. When adult 129J nude mice were inoculated subcutane- 2 ously with 107 Ki-MuSV-transformed C-1 cells, tumors de- veloped within 3-4 weeks at the site ofinoculation (Table 1). Microscopic examination of sections of these tumors re- vealed poorly differentiated carcinomas consistent with neo- plastic transformation ofthe HPE cells (Fig. 1C). The tumor consisted of large, poorly differentiated epithelial cells with M 1 2 3 hyperchromatic, irregularly shaped nuclei. The presence ofa high mitotic index was consistent with rapid cell division. FIG. 2. Southern blot hybridization analysis of BamHI-digested Epithelial pearls were evident, suggesting squamous cell- genomic DNA from HPV-18 transfected HPE cell clones using a type differentiation. Small nondescript cells displaying little 1.5-kb Xba I DNA probe that spans the 3' end of E6, the entire E7, and the 5' end ofEl coding regions ofHPV-18. The arrowhead shows cytoplasm are mixed among the tumor cells. an -4.2-kb fragment that is indicative of the presence of the HPV One of the tumors was reestablished in culture and con- genome in the appropriate clones. Locations of the molecular mass firmed as human epithelial cells by isoenzyme and cytoker- markers (M) used are shown on the left. Lane 1, HPV-18 transfected atin analyses. Their deviation from the cells of origin was HPE clone A-2. Lane 2, HPV-18 transfected HPE clone C-1. Lane determined by karyological analysis. The cells cultured from 3, Early passage 91-1 HPE cells (negative control). a tumor in nude mice were aneuploid human male derivative Downloaded by guest on September 25, 2021 Medical Sciences: Rhim et al. Proc. Natl. Acad. Sci. USA 91 (1994) 11877 Additional evidence, however, points to frequent, in- creased expression of ras p21 proteins in prostate cancer. Increased expression of Ki-ras and Ha-ras was observed in grade I to grade III carcinomas, with p21 levels increasing with increasing grade, whereas noncancerous cells and most cells from early stage cancers did not express ras p21 (6, 7). Fifty-nine percent of moderately differentiated and 78% of poorly differentiated prostatic carcinomas showed increased ras expression (7). Further, Viola et al. (5), using monoclonal antibodies and immunohistochemical analysis, showed that while 19 tissue samples from patients with benign prostatic hyperplasia (BPH) were negative for p21 antigen, 2/6 pros- tates with grade I carcinoma, 4/6 with grade II, and 17/17 with high grades were positive for p21. The expression of large amounts ofras and myc mRNA in four human prostatic carcinoma cell lines (8) provides further evidence for the possible involvement of the ras oncogene in prostate car- M 1 2 3 4 M cinogenesis and tumor progression. The possible involvement of HPV in prostate cancer re- FIG. 4. Expression ofp21 ras protein in Ki-MuSV transformants. Subconfluent cultures of a positive control Ki-MuSV transformed quires equal attention. The frequency for the presence of human keratinocyte line (lane 1), HPV-18 transfected HPE clone C-1 high-risk HPV DNA (HPV-16 and -18) was studied in 68 line (lane 2), Ki-MuSV infected HPV-18 HPE clone C-1 line (lane 3), Japanese patients with prostate carcinomas using PCR am- and tumor line 129 Nu 3120-3 (lane 4) were rinsed with PBS and lysed plification (9). Forty-one percent were positive for at least with a buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, one type of high-risk HPV, the most common (25%) being 0.1% SDS, L.0o Triton X-100, and l.0o sodium deoxycholate. The HPV-18. ras mutations and HPV infections were more fre- protein concentration of the lysates was determined and equivalent queht in patients with advanced stages of the tumor. Fur- amounts ofprotein from each cell line were immunoprecipitated with thermore, there was the predominant presence of Ha-ras a pan-reactive mouse antibody against p21 (Cetus). The resulting immunoprecipitates were fractionated on a 12.5% polyacrylamide/ mutation and HPV-18 DNA in prostatic carcinoma metasta- SDS gel. After electrophoresis, proteins on the gel were electro- sizing to the bone. A similar prevalence ofthe HPV infection transferred onto a nitrocellulose membrane, which was then allowed that was described in the Japanese prostate carcinoma study to react against the same antibody against p21. The immunocomplex (9) has also been reported in Canadian prostate patients (15). on the nitrocellulose membrane was visualized by a subsequent Employing PCR, 14/15 BPH and 4/4 carcinomas showed the reaction with 125I-labeled protein A. Positions of the endogenous presence of HPV-16 and/or HPV-18. Four of 5 normal wild-type p21 doublet (arrowhead) and the exogenous activated p21 prostates did not show any HPV DNA and only 1/5 contained doublet (arrows) are indicated. The molecular mass (M) markers HPV-16 sequences (14). However, Sarkar et al. (16) recently used were 97.4, 69, 46, 30, and 14.3 kDa. demonstrated the presence of HPV-16 DNA in only three with chromosome counts in the hyperdiploid range. Four carcinomas (13%) from United States patients and concluded as marker chromosomes were detected, three and that HPV infection of the prostate in general is not (Ml, M2, common as had been previously reported (9, 15). Because Mll) of which corresponded to similar markers detected in oncogenic HPVs can be sexually transmitted, and high-risk the Ki-MuSV transformed C-1 cells. By isoenzyme pheno- HPV DNA was detected in prostate carcinomas, the possible type and distribution of normal and marker chromosomes, involvement of HPVs in prostate cancer needs to be further the tumor-derived line was clearly a derivative of the inoc- investigated. ulated Ki-MuSV transformed C-1 cells. The cultured tumor Our results demonstrate malignant transformation of adult cells continued to express the activated Ki-ras gene product, HPE cells in culture and support previous epidemiological as confirmed by Western immunoblot analysis (Fig. 4, lane studies that have implicated the K-ras oncogene and HPV 4). The prostate tumor cell line, upon reinjection into athymic infection in prostatic carcinogenesis. We have demonstrated nude mice, resulted in rapid tumor formation at a frequency that expression of the v-Ki-ras gene in an HPV-18 immor- of 100%1 (5/5). In contrast, subcutaneous inoculation of 107 talized adult HPE cell line facilitated malignant transforma- parental C-1 cells produced no tumors during the observation tion. Two or more alterations in cellular growth properties period of 6 months. seem to be required. The process was initiated by the acquisition of an unlimited growth potential as a result of DISCUSSION HPV-18 . HPV-18 has been shown to immor- talize human epithelial cells such as keratinocytes and cer- There is increasing evidence to support the idea that ras vical cells (23, 24). In addition to acquiring a continuous oncogene activation and HPV infection may be involved in proliferation capacity, the HPV-18 transfected HPE cells human prostatic carcinogenesis. Anwar et al. (9) recently showed an abnormal karyotype at the earliest measurable used PCR and DNA hybridization to examine the presence of time. Nonetheless, they lacked the ability to induce progres- ras mutations in paraffin-embedded specimens from 68 pros- sively growing tumors in nude mice. Thus, the alterations tatic carcinoma patients: 10 specimens each of normal and induced by HPV-18 DNA transfection appear to be necessary benign prostates were used as controls. Overall, 24% of all but by themselves are insufficient to induce progression to carcinomas showed ras mutations, which were more com- the neoplastic state. mon in advanced disease. Further, while 88% of the tumors Superinfection of early passage HPV-18 immortalized with bone metastasis had ras mutations, none of the normal adult HPE cells with Ki-MuSV, a virus with an activated or benign prostate tissues were found to contain any ras Ki-ras oncogene, resulted in further changes in their growth mutations. They concluded that ras mutations are relatively properties. Expression of the activated ras 21 protein, mor- frequent at least in prostate cancer specimens from Japanese phological alteration, increase in genetic instability, and patients, although two previous studies from the United ability to induce squamous cell carcinomas in athymic nude States showed that the frequency of ras mutations was much mice appeared to be concomitantly acquired properties as- lower (10, 12). sociated with the addition of the v-Ki-ras oncogene. The Downloaded by guest on September 25, 2021 11878 Medical Sciences: Rhim et al. Proc. Natl. Acad. Sci. USA 91 (1994) significance of the combined action of HPV-18 and the brugge, G. J., Romijn, J. C. & Trapman, J. (1985) Biochem. v-Ki-ras oncogene in the induction of malignant transforma- Biophys. Res. Commun. 132, 548-554. 9. Anwar, K., Nakakuki, K., Shiraishi, T., Naiki, H., Yatani, R. tion of HPE cells is emphasized by our finding that the & Inuzuka, M. (1992) Cancer Res. 52, 5991-5996. v-Ki-ras gene clone cannot induce neoplastic transformation 10. Carter, B. S., Epstein, J. E. & Isaacs, W. B. (1990) Cancer unless the cells have been immortalized by HPV-18. Thus, Res. 50, 6830-6832. the v-Ki-ras oncogene has the ability to complement with 11. Konishi, N., Enomoto, T., Buzard, G., Ohshima, M., Ward, HPV to induce full transformation. J. M. & Rice, J. M. (1992) Cancer 69, 2293-2299. Our results that to 12. Gumerlook, P. H., Poonamallee, U. R., Meyers, F. J. & show successive changes are required White, R. W. (1991) Cancer Res. 51, 1632-1637. induce malignant transformation of HPE cells of adult origin, 13. Zur Hausen, H. (1991) Virology 184, 9-13. the natural target of prostate carcinogenesis. Our unique 14. Barrasso, R., DeBrux, J., Croissant, 0. & Orth, G. (1987) N. multistep in vitro model should prove invaluable for defining Engl. J. Med. 317, 916-923. the molecular events underlying the development of human 15. McNicol, J. P. & Dodd, J. G. (1991) J. Urol. 145, 850-853. prostate cancer. 16. Sarkar, F. H., Sakv, N. A., Sreepathi, P. & Crissman, J. D. (1993) Prostate 22, 171-180. We thank J. A. DiPaolo, C. D. Woodworth, and H. zur Hausen for 17. Webber, M. M. (1988) in In Vitro Modelsfor CancerResearch, the plasmid containing the HPV-18 genome and the neomycin- eds. Webber, M. M. & Sekeley, L. I. (CRC, Boca Raton, FL), resistance gene pp. 3-24. and thank Dr. Wartinger for providing the prostate 18. Rhim, J. S., Jay, G., Price, F. M., Sanford, K. K. & Aaronson, tissue. S. A. (1985) Science 227, 1250-1252. 19. Rhim, J. S., Park, J. B. & Jay, G. (1989) Oncogene 4, 1403- 1. Boring, C. C., Squires, T. S. & Tong, T. (1993) Ca CancerJ. 1409. Clin. 43, 7-21. 20. Tanaka, K., Appella, E. & Jay, G. (1983) Cell 35, 457-465. 2. Coffey, D. S. (1993) Cancer 71, 880-886. 21. Maloy, W. L., Coligan, J. E., Barra, Y. & Jay, G. (1984) Proc. 3. Isaac, J. T. (1993) Cancer Metastasis Rev. 12, 1-2. Natl. Acad. Sci. 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