Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3874-3878, May 1992 Biochemistry Frequent amplification of c-mnc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus (hepatoceflular carcinoma/N-myc/) CATHERINE TRANSY*, GENEVIEVE FOUREL*, WILLIAM S. ROBINSONt, PIERRE TIOLLAIS*, PATRICIA L. MARIONt, AND MARIE-ANNICK BUENDIA*t *Unit6 de Recombinaison et Expression Gdndtique, Institut National de la Santd et de la Recherche Mddicale U163, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris, Cedex 15, France; and tDivision of Infectious Diseases, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305 Communicated by Andre Lwoff, January 23, 1992 (received for review November 5, 1991)

ABSTRACT Persistent infection with hepatitis B virus HCC through distinct and perhaps cooperative mechanisms. (HBV) is a major cause of hepatoceliular carcinoma (HCC) in However, the cellular factors involved in virally induced humans. HCC has also been observed in animals chronically oncogenesis remain largely unknown. infected with two other hepadnaviruses: ground squirrel hep- In this regard, hepadnaviruses infecting lower animals, atitis virus (GSHV) and woodchuck hepatitis virus (WHV). A such as the woodchuck hepatitis virus (WHV) and the ground distinctive feature of WHV is the early onset of woodchuck squirrel hepatitis virus (GSHV), represent interesting mod- tumors, which may be correlated with a direct role of the virus els. Chronic infection with WHV has been found to be as an insertional mutagen of myc : c-myc, N-myc, and associated with a high incidence and a rapid onset of HCCs predominantly the woodchuck N-myc2 retroposon. In the in naturally infected woodchucks (11), and the oncogenic present study, we searched for integrated GSHV DNA and capacity of the virus has been further demonstrated in genetic alterations ofmyc genes in ground squirrel HCCs. Viral experimentally infected hosts (12). Furthermore, molecular integration into host DNA was detected in only 3/14 squirrel studies of WHV-associated HCCs have shown a frequent tumors and did not result in insertional activation ofmyc genes, activation of myc genes: c-myc, N-mycl, the homolog of the despite the presence of a squirrel locus homologous to the known N-myc genes, and N-myc2, a functional N-myc ret- woodchuck N-myc2 . This suggests that GSHV may differ roposon identified in the woodchuck genome. Direct cisac- from WHV in its reduced ability to induce mutagenic integra- tivation of these genes by integrated WHV sequences has tion events. However, the high frequency of c-myc amplifica- been observed in about 50% oftumors (refs. 13 and 14 and our tion (6/14) observed in ground squirrel HCCs indicates that unpublished results), showing that WHV acts mainly as an myc genes might be preferential effectors in the tumorigenic insertional mutagen as described for several murine retrovi- processes associated with rodent hepadnaviruses, a feature not ruses (15, 16). reported so far in HBV-induced carcinogenesis. Together with In contrast, evidence for an association between persistent previous observations, our results suggest that hepadnaviruses, GSHV infection and HCC development in ground squirrels despite dose genetic and biological properties, may use differ- has been obtained only after a long-term study of captive ent pathways in the genesis of liver cancer. animals (17, 18), showing that HCCs occur at a lower rate and after a longer latency period in this model. Direct comparison Epidemiological studies have established that chronic infec- of WHV and GSHV for oncogenic potential in a common tion with human hepatitis B virus (HBV) is causally related host, the experimentally infected newborn woodchuck, has to the development ofhepatocellular carcinoma (HCC) (1, 2), shown that the two viruses differ significantly in their effi- placing HBV among the rare viruses involved in human ciency in inducing HCC (19). However, the host chromoso- cancer. Clues to the contribution of viral proteins in tumor- mal sites ofGSHV integrations in ground squirrel tumors and igenesis have been recently obtained using transgenic mice; effects on myc genes have not been investigated. thus, long-term overexpression of the HBV large surface In an attempt to address these questions, we have searched glycoprotein causes repeated liver necrosis/regeneration for potential alterations of myc genes in a panel of 14 ground processes that eventually lead to hepatocarcinogenesis (3). squirrel HCCs, including tumors from persistently infected, Constitutive expression of the viral X transcriptional activa- convalescent, and serologically negative animals. We report tor in the liver induces in here that, in contrast with the WHV/woodchuck system, hepatomas transgenic recipients, integrated GSHV DNA was detected in only a few tumors possibly through illegitimate transactivation ofcellular genes and did not result in insertional activation of myc genes. (4). Integration of viral sequences into the host genome, Interestingly, a high frequency of c-myc amplification in detected in almost all HBV-associated HCCs, might also these tumors was trigger hepatocyte transformation by cis or trans mecha- (6/14) observed. nisms. Insertional mutagenesis of cellular genes by HBV has been reported in two independent tumors, the target gene MATERIAL AND METHODS coding either for retinoic acid receptor (5) or for cyclin A (6), Animals and Tissue Samples. Beechey ground squirrels proteins involved in cell differentiation and in the cell cycle, (Spermophilus beecheyi) were trapped live at various loca- respectively. More frequently, rearrangement of HBV se- in quences upon integration may alter expression of the viral X tions northern California from 1980 and tested for markers protein (7) or generate a truncated preS2/S protein with of GSHV as described (20). Animals with ground squirrel transcriptional transactivator function (8-10). All together, hepatitis surface antigen (GSHsAg) and virion-associated these observations raise the possibility that HBV may induce Abbreviations: HBV, hepatitis B virus; WHV, woodchuck hepatitis virus; GSHV, ground squirrel hepatitis virus; HCC, hepatocellular The publication costs of this article were defrayed in part by page charge carcinoma; GSHsAg, ground squirrel hepatitis surface antigen; anti- payment. This article must therefore be hereby marked "advertisement" GSHs, antibody to GSHsAg. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

3874 Biochemistry: Transy et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3875

DNA polymerase activity in their sera were housed sepa- RESULTS rately from those with antibody to GSHsAg (anti-GSHs) or with no serological signs of past infection. The ages were Tumorous and nontumorous liver tissues were recovered a estimated as described (20). All animals listed as GSHV- from panel of 14 Beechey ground squirrels. The sex, age at positive in Table 1 were positive when trapped. Animals necropsy, and GSHV serology of the animals are listed in GL50, GL52, and CU03 seroconverted after a brief acute Table 1. Animals could be classified into three groups ac- infection resulting from experimental inoculation. Animals cording to the status of GSHV infection at the time of RV39 and RV50 were antibody-positive when trapped. Three necropsy: five animals with detectable GSHsAg in the serum were squirrels, PC65, PC72, and B30, each received a total of 4.64 persistently infected, five others with humoral antibod- mg of aflatoxin B1 per kg of body weight over a period of 16 ies to GSHsAg had recovered from acute infection, and the weeks as part of an experiment to study possible synergy of four remaining showed no serological signs of infection. the mycotoxin with the virus to cause an earlier appearance Replicative and Integrated Forms of GSHV DNA in Ground of HCC. Hepatoma tissues and adjacent nontumor liver Squirrel Tumors. Genomic DNA was extracted from coded tissues were resected from animals at necropsy and kept samples of HCCs and adjacent normal livers and digested frozen at -70°C until used. All animals had histologically with HindIII, a restriction enzyme with no recognition site in confirmed HCC. GL22 had both HCC and biliary carcinoma the GSHV genome (23). As shown in Fig. 1, Southern throughout most of the liver, and GL50 had extensive biliary analysis performed with a genomic viral probe revealed hyperplasia, biliary carcinoma, and HCC. abundant viral DNA replicative intermediates in the nontu- Southern Blot Analysis. Genomic DNA was extracted from morous liver and to a lesser extent in the tumors of all five coded tissue samples as described (14). After digestion with persistently infected animals. In addition, the tumorous liver the indicated restriction enzyme and electrophoresis, DNA from one animal (RV53) showed a discrete 10-kb band, was transferred onto reusable nylon membranes according to indicating the insertion of viral sequences into the host the manufacturer's recommendations (Zetaprobe; Bio-Rad). genome. Abundant viral transcripts were detected in these Membranes were hybridized with 32P-labeled probes as de- tissues (data not shown). scribed in ref. 13. Consistent with the serological data, no viral DNA repli- Northern Blot Analysis. Total RNA was extracted as de- cation was detected in the five anti-GSHs-positive animals scribed in ref. 13 and subjected to denaturing electrophoresis (Fig. 1). Viral DNA was, however, present as single inte- grated sequences in the tumors of two in agarose gels containing formaldehyde (21). Nylon mem- animals (RV39 and branes RV50), giving rise to 4.3- and 10.5-kb HindIII fragments, used for RNA transfer and hybridization procedures respectively. were as described above. No viral DNA was detected in the GSHV marker-free Probes. Detection of viral sequences was per- initially animals except in VP4, in which traces of viral DNA could be formed with a cloned WHV genome (22) and subsequently detected in the nontumorous liver. This result raised the with a cloned GSHV genome (23), kindly provided by C. possibility that GSHV could be present at a level below the Seeger. threshold of detection by most conventional methods in the The woodchuck N-myc2-derived probe spans 750 base seronegative animals. We therefore used PCR to reinvesti- pairs (bp) of the N-myc2 coding sequence and detects both gate the GSHV status ofVP4 and CU18, the two seronegative N-mycl and N-myc2 genes (13). The woodchuck N-mycl- animals for which tumor tissue was still available for DNA specific probe covers a region that is deleted in the N-myc2 preparation in conditions suitable for PCR analysis. As retroposon (13). shown in Fig. 2, a fragment of the size expected for the PCR The exon-specific probes from the cloned woodchuck amplification of GSHV sequences was obtained with the c-myc gene have been previously described (24). The probe tumor of the two seronegative animals tested. This 25 kilobases (kb) distal to the human c-myc gene (25) was fragment hybridized with a viral probe, confirming the spec- kindly provided by B. Henglein. The murine f-actin probe ificity of the amplification reaction (data not shown). was a generous gift of M. Buckingham (Paris). Detection of GSHV Sequences by Polymerase Chain Reac- Table 1. Status of GSHV and c-myc in ground squirrel HCCs tion (PCR). To avoid false-positive results due to possible GSHV cross-contamination between tissue samples upon DNA ex- DNAt c-myc traction, new samples of the tumorous livers from seroneg- Age, GSHV ampli- ative animals were used for genomic DNA isolation. Material Animal Sex* yr serologyt Rep Int fication was available only for CU18 and VP4. The liver DNA from GL59 M 5.0 + + - - a chronically infected animal (RV53) was prepared indepen- RV53 F 5.9 + + + + dently and used as a positive control. The primers used were TRO4 M 7.5 + + - + as follows: R1 (5'-GGCATGCGTCAGCAAACAC-3') is PC65§ F 6.5 + + - - complementary to positions 1327-1309 of the GSHV se- PC72§ M 7.5 + + - - quence as numbered in ref. 26. Li (5'-CCTATG- RV50 M 9.5 Ab+ - + + GAGTGGGCC-3') is homologous to positions 766-781. L2 GL52 M 6.5 Ab+ - - - (5'-TTGCTCATATGGATGATTT-3') is homologous to po- CU03 F 9.0 Ab+ - - + sitions 860-878. RV39 F 7.0 Ab+ - + - R1 and Li primers were used for the first step of PCR, GL50 M 5.5 Ab+ - - + performed with 1 jig of genomic DNA (1 ng in the case of the VP4 F 8.5 - - - - positive control) in the presence of 1.5 mM Mg2' and 2.5 units B30§ M 8.5 - - - - of Taq DNA polymerase (Perkin-Elmer/Cetus). Forty cycles GL22 M 9.1 - - - - ofPCR were performed with incubations of 1 min at 55°C, 1.5 CU18 F 9 - - - + min at 72°C, and 1 min at 94°C. One-hundredth ofthe reaction *M, male; F, female. was then to the product subjected second step of PCR (15 t+, GSHs-Ag-positive; Ab+, anti-GSHs-positive; -, no GSHV cycles), using R1 and L2 primers. One-tenth of this reaction serological marker. product was electrophoresed on a 1.5% agarose gel and WAs determined by Southern blot analysis: Rep, replicative forms of transferred onto nylon membrane for hybridization with the GSHV DNA; Int, integrated sequence of GSHV. GSHV probe. §These animals received aflatoxin B1 as described in the text. 3876 Biochemistry: Transy et al. Proc. Natl. Acad. Sci. USA 89 (1992) GL59 VP4 B30 RV50 GL52 GU02); RV39 RV5;9 GL2-. -L;

N- T SINT T TT N NT T NT T T NT NT NT T7 TV NT NT i F

Kt

')6- i& 6.6-

4-- w1

2, 2-

1 2 3 4 5 6 7 8 .-3 10 11 1? 1I 16 17 ?" : be?9) FIG. 1. Southern blot analysis of GSHV DNA in ground squirrel HCCs. Genomic DNAs (20 ,ug) were digested with HindI11 and hybridized with a probe covering the entire viral genome. Tumors (lanes T) and adjacent nontumorous tissues (lanes NT) were obtained from the indicated animals. Arrowheads indicate the integrated GSHV sequences.

Status of myc Loci in Ground Squirrel Tumors. The previ- was expressed at a detectable level in the tumorous livers ous finding of frequent insertional activation of c-myc and (data not shown). N-myc loci by WHV in woodchuck HCCs (13, 14) prompted In contrast, hybridization with a woodchuck c-myc exon 2 us to investigate the state of the myc family genes in squirrel probe revealed that c-myc gene amplification had occurred in tumors. As shown in Fig. 3 Upper, two major HindIll 6 ofthe 14 HCCs examined (Fig. 3 Lower and Table 1). In the fragments (3.5 and 9 kb in size) were detected in ground case of CU03, c-myc amplification was observed in both the squirrel genomic DNA upon hybridization with a woodchuck tumor and the nontumor tissue. Densitometric analysis ofthe N-myc2 probe. The 3.5-kb fragment was identical in size to autoradiograms, using hybridization intensity of the N-myc2 the woodchuck N-myc2 Hind1II fragment (13). It did not gene as baseline, indicated that copy number of the c-myc hybridize with a woodchuck probe specific for the classical gene in the amplified samples ranged between 5- and 25-fold N-myc gene, which revealed only the 9-kb fragment (data not higher than in the control nontumor samples. As shown in shown). Hybridization with N-myc2 probe after digestion Table 1, there was no apparent correlation between c-myc with several other restriction enzymes showed comigration amplification and the sex, age, or GSHV serology of the between bands detected in ground squirrel genomic DNA and animals. It may be noted, however, that c-myc amplification fragments generated by the woodchuck N-myc2 locus (data was not observed in the tumors from the three animals treated not shown). Taken together, these results strongly suggest with aflatoxin B1, a chemical inducer of hepatocarcinogen- to the esis. No alteration in the restriction pattern of the amplified the existence of a ground squirrel locus homologous c-myc gene was observed except for one tumor DNA (RV50; woodchuck N-myc2 retroposon. Fig. 3, lane 8) for which a wild-type and a rearranged form Inspection of the Southern blots shown in Fig. 3 Upper were coamplified. The novel band did not comigrate with the failed to reveal any gross alteration in the structure of the virus-specific band (compare lanes 8 in Figs. 1 and 3), N-myc loci in the squirrel HCCs. In addition, neither gene showing that rearrangement of the c-myc locus did not arise from nearby GSHV DNA integration. Further hybridization 0c CY) experiments with subregion-specific c-myc probes revealed a deletion encompassing intron 2 and exon 3 in the rearranged c-myc allele (not shown). Southern hybridization with a c) > m probe located 25 kb downstream from the human c-myc locus b p (25) revealed amplification of the homologous region in all c-myc-amplified squirrel tumors but not in the nontumorous liver of CU03, excluding that the amplification detected in 1353- this tissue reflected contamination by tumor cells (data not 603- shown). Finally, c-myc gene amplification correlated with significant increase of c-myc mRNA steady-state level as 310 demonstrated by Northern blot analysis (Fig. 4). DISCUSSION We have characterized a panel of 14 ground squirrel HCCs with respect to the presence of GSHV DNA and potential FIG. 2. PCR detection of GSHV sequences in tumor DNAs from alterations in the myc family protooncogenes, known to be seronegative animals. The indicated DNAs were used as templates for the PCR reactions. PCR products were analyzed in a 1.5% frequently involved in WHV-induced HCCs. agarose gel stained by ethidium bromide. Liver DNA from a chronic The serologically defined chronic carrier state correlated carrier (RV53) was used as a positive control and mouse liver DNA with active viral DNA replication as observed in earlier (ML), as a negative control. studies of GSHV-infected animals (17-19). The detection of Biochemistry: Transy et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3877

GL59 VP4 B30 RV50 GL52 CU03 RV39 RV53 GL22 GL50 TRO4 PC65 PC72 CU18 1m m m m m miI Ir-- I I m m I NT T NT T T NT NT T T NT T NT T NT NT Ti T2 T NT NT Ti T2 NT T T NT NT T T NT

9kb AW

_ _ N-myc2.W. low .- w w NWPi S w V - _ 3.5 kb _w _w _w W - .

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

c-myc 5.4 kb-. eS .* 4.5kb._.

FIG. 3. Southern blot analysis of N-myc and c-myc genes in ground squirrel HCCs. The blot shown in Fig. 1 was rehybridized with the woodchuck N-myc2 probe (Upper) and subsequently with a woodchuck c-myc exon 2 probe (Lower). Size and gene assignment of the bands are indicated on the left. In addition to the N-mycl- and -2-specific fragments, the woodchuck N-myc2 probe weakly detected a HindIII fragment that might be homologous to a woodchuck locus distantly related to N-myc (G.F., unpublished results). low levels of GSHV DNA in the tumors frorn seronegative account for the low number ofviral integrations in the ground squirrels is reminiscent of the situation descriibed in several squirrel HCCs. In this hypothesis, low overall chances of cases ofHBV-infected humans (27-29) and may reinforce the insertional mutagenesis by GSHV as compared with WHV established association between HCC developiment in ground might explain why insertional activation of myc loci, a squirrels and infection with GSHV (17, 18). frequent event in WHV-induced hepatocarcinogenesis, has Detection ofintegrated viral DNA in virtually all HBV- and not been observed in our study. We have shown here the WHV-associated HCCs has been widely docurnented and has existence of a squirrel locus homologous to the woodchuck suggested a contribution of viral integration to the oncogenic N-myc2 retroposon, the highly preferred target of insertional process by cis (5, 6, 13, 14) or trans (7, 8, 10) ffmechanisms. In mutagenesis by WHV (ref. 13 and Y. Wei and M.-A.B., this respect, it is striking that only three ofthe gground squirrel unpublished results). Since no GSHV integration at this locus HCCs analyzed here displayed integrated G'SHV DNA, in has been observed in our study, the possibility should also be each case as a single integration. This observeation, in agree- considered that squirrel N-myc2 is devoid of oncogenic ment with earlier ones (17, 18), implies that viiral integration capacity. However, preliminary sequencing data as well as is dispensable for malignant conversion andI/or tumorous restriction mapping of the ground squirrel N-myc2 locus growth of hepatocytes infected by GSHV. Giiven the strong (unpublished observations) suggest that it is highly homolo- selection operated by the tumorigenic proces,s, the average gous to its woodchuck counterpart. Furthermore, studies of frequency of viral integration may be differentt in tumor cells HCC development in GSHV-infected woodchucks (19) indi- as compared with normal hepatocytes. Ho)wever, a low cate differences in intrinsic oncogenic capacity of WHV and capacity of GSHV to integrate into the host genome might GSHV rather than in host/virus relationships. c-myc gene amplification was observed in a remarkably CU03 RV39 RV53 TR04 PC65 C,U18 RV50 high percentage ofthe ground squirrel tumors analyzed in the NT'T7 T present study (6/14). All 6 tumors showed increased steady- NT T NT state level ofc-myc message, suggesting that c-myc activation 28S- was involved in the neoplastic growth of the corresponding A tumors. In contrast, c-myc amplification seems to be only f -MVCR w occasional in HBV-associated HCCs (30) and has never been is- 18S- U,0 reported in WHV-induced HCCs. Since in vitro culture or in vivo transplantation of tumors * B often selects tumor cells showing c-myc amplification, the amplification is considered as a step in tumor progression rather FIG. 4. Northern blot analysis of c-myc mRNA irn squirrel HCCs. than an event initiating tumorigenesis (reviewed in ref. 31). Total (20 ,Ag per lane) from tumors (lanes T) and adjacent However, c-myc amplification as an initial event in the onco- nontumorous livers (lanes NT) were hybridized wiith a woodchuck genic process has been documented in the hepatocarcinogen- c-myc exon 2 probe (A) and a murine P-actin probe (B). Positions of esis induced in rats by a choline-free diet, since it occurs prior 28S and 18S ribosomal RNAs are indicated. to tumorigenesis and has been observed in 100%o oftumors (32). 3878 Biochemistry: Transy et al. Proc. Natl. Acad. Sci. USA 89 (1992) The observation of c-myc amplification in the nontumorous 10. Kekuld, A. S., Lauer, U., Meyer, M., Caselmann, W. H., Hof- liver of CU03 suggests that this genetic alteration might repre- schneider, P. H. & Koshy, R. (1990) Nature (London) 343,457-461. 11. Snyder, R. L. & Summers, J. (1980) in Viruses in Naturally Occur- sent an early step in GSHV-linked carcinogenesis. ring Cancers, Cold Spring Harbor Conferences on Cell Proliferation Association between c-myc amplification and viral infec- VII, eds. Essex, M., Todaro, G. & zur Hausen, H. (Cold Spring tion has been reported for other oncogenic viruses such as Harbor Lab., Cold Spring Harbor, NY), pp. 447-457. Epstein-Barr virus, Abelson murine leukemia virus, and 12. Popper, H., Roth, L., Purcell, R. H., Tennant, B. C. & Gerin, J. L. human papilloma virus (33-36). Molecular mechanisms lead- (1987) Proc. Natl. Acad. Sci. USA 84, 866-870. 13. Fourel, G., Trdpo, C., Bougueleret, L., Henglein, B., Ponzetto, A., ing to gene amplification are still poorly understood. How- Tiollais, P. & Buendia, M. A. (1990) Nature (London) 347, 294-298. ever, the existence of gene products that stimulate DNA 14. Hsu, T. Y., Moroy, T., Etiemble, J., Louise, A., Trepo, C., amplification in trans has been inferred from the dominant Tiollais, P. & Buendia, M. A. (1988) Cell 55, 627-635. phenotype of cell lines selected for their high rate of ampli- 15. Corcoran, L. M., Adams, J. M., Dunn, A. R. & Cory, S. (1984) Cell fication (reviewed in ref. 37). Whether GSHV could induce 37, 113-122. 16. Van Lohuinzen, M., Breuer, M. & Berns, A. (1989) EMBO J. 8, such stimulatory proteins is a matter of speculation. DNA 133-136. elements that stimulate DNA amplification in cis have also 17. Marion, P. L., Van Davelaar, M. J., Knight, S. S., Salazar, F. H., been isolated (37), and it has been reported that such se- Garcia, G., Popper, H. & Robinson, W. S. (1986) Proc. Natl. Acad. quences must be in a transcriptionally active region to exert Sci. USA 83, 4543-4546. their stimulatory effect (38). Stimulation of the transcription 18. Marion, P. L., Popper, H., Azcarraga, R. R., Steevens, C., Van Davelaar, M. J., Garcia, G. & Robinson, W. S. (1987) in Hepad- of c-myc might therefore increase its susceptibility to ampli- naviruses, UCLA Symposia on Molecular and Cellular Biology, fication processes. Noticeably, the HBV-encoded X protein New Series, eds. Robinson, W. S., Koike, K. & Will, H. (Liss, New has been shown to transactivate the c-myc promoter in York), pp. 337-348. transient transfection assays (39). Whether the GSHV X 19. Seeger, C., Baldwin, B., Hornbuckle, W. E., Yeager, A. E., Ten- nant, B. C., Cote, P., Ferrell, L., Ganem, D. & Varmus, H. E. protein shares this property with its HBV counterpart is not (1991) J. Virol. 65, 1673-1679. known, but it might be predicted on the basis of functional 20. Marion, P. L., Knight, S., Salazar, F. H., Popper, H. & Robinson, homology between the hepadnaviral X proteins (40). W. S. (1983) Hepatology 5, 435-439. The genetically related hepadnaviruses show similar biolog- 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular ical and pathological properties and might be expected to Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), pp. 202-203. develop common strategies in tumor induction. However, our 22. Ogston, C. W., Jonak, G. J., Rogler, C. E., Astrin, S. M. & Sum- present study ofGSHV-associated tumors, togetherwith earlier mers, J. (1982) Cell 29, 385-394. studies of HBV- and WHV-induced HCCs, reveals differences 23. Seeger, C., Ganem, D. & Varmus, H. E. (1984) J. Virol. 51, in at least some steps in the tumorigenic processes. Thus, 367-375. although our results indicate that myc family genes are prefer- 24. Moroy, T., Marchio, A., Etiemble, J., Trepo, C., Tiollais, P. & Buendia, M. A. (1986) Nature (London) 324, 276-279. ential effectors in both GSHV- and WHV-associated oncogen- 25. Henglein, B., Synovzik, H., Groitl, P. H., Bornkamm, G. W., esis, distinct mechanisms lead to activation ofmyc genes in the Hartl, P. & Lipp, M. (1989) Mol. Cell. Biol. 9, 2105-2113. two models, and this event has not been frequently observed in 26. Kodama, K., Ogasawara, N., Yoshikawa, H. & Murakami, S. HBV-induced HCCs. Pathways of progression toward the (1985) J. Virol. 56, 978-986. malignant state might depend on both specific virus properties 27. Liang, T. J., Baruch, Y., Ben-Porath, E., Enat, R., Bassan, L., Brown, N. V., Rimon, N., Blum, H. E. & Wands, J. R. (1991) (e.g., the capacity to integrate into the host genome) and Hepatology 13, 1044-1051. specific host factors (the woodchuck N-myc2 oncogene has no 28. Brdchot, C., Degos, F., Lugassy, C., Thiers, V., Zafrani, S., human homolog). It is, however, possible that initial steps in Franco, D., Bismuth, H., Trdpo, C., Benhamou, J. P., Wands, J., hepatocarcinogenesis are achieved in the same way by the three Isselbacher, K., Tiollais, P. & Berthelot, P. (1985) N. Engl. J. Med. viruses by still unknown mechanisms. 312, 270-276. 29. 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