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Associated with Past Or Ongoing Infection with a Hepadnavirus (Hepatoceflular Carcinoma/N-Myc/Retroposon) CATHERINE TRANSY*, GENEVIEVE FOUREL*, WILLIAM S

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Associated with Past Or Ongoing Infection with a Hepadnavirus (Hepatoceflular Carcinoma/N-Myc/Retroposon) CATHERINE TRANSY*, GENEVIEVE FOUREL*, WILLIAM S 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/retroposon) 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 genes: 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 gene. 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.
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