Alteration of Gene Expression in Human Hepatocellular Carcinoma with Integrated Hepatitis B Virus DNA AkihiroTamori, 1 YoshihiroYamanishi, 4 ShuichiKawashima, 5 MinoruKanehisa, 4 MasaruEnomoto, 1 HiromuTanaka, 2 ShojiKubo, 2 SusumuShiomi, 3 andShuheiNishiguchi 1 Abstract Purpose: IntegrationofhepatitisBvirus(HBV)DNAintothehumangenomeisoneofthemost importantstepsinHBV-relatedcarcinogenesis.ThisstudyattemptedtofindthelinkbetweenHBV DNA,theadjoiningcellularsequence,andalteredgeneexpressioninhepatocellularcarcinoma (HCC)withintegratedHBVDNA. Experimental Design: Weexamined15casesofHCCinfectedwithHBVbycassetteligation^ mediatedPCR.ThehumanDNAadjacenttotheintegratedHBVDNAwassequenced.Protein codingsequencesweresearchedforinthehumansequence.InfivecaseswithHBVDNAintegra- tion,fromwhichgoodqualityRNAwasextracted,geneexpressionwasexaminedbycDNA microarrayanalysis. Results: ThehumanDNAsequencesuccessivetointegratedHBVDNAwasdeterminedinthe15 HCCs.Eightprotein-codingregionswereinvolved:ras-responsiveelementbindingprotein1, calmodulin1,mixedlineageleukemia2(MLL2),FLJ333655,LOC220272,LOC255345, LOC220220,andLOC168991.The MLL2 genewasexpressedinthreecaseswithHBVDNA integratedintoexon3of MLL2 andinonecasewithHBVDNAintegratedintointron3of MLL2 . GeneexpressionanalysissuggestedthattwoHCCswithHBVintegratedinto MLL2 hadsimilar patternsofgeneexpressioncomparedwiththreeHCCswithHBVintegratedintootherlociof humanchromosomes. Conclusions: HBVDNAwasintegratedatrandomsitesofhumanDNA,andthe MLL2 genewas oneofthetargetsforintegration.OurresultssuggestthatHBVDNAmightmodulatehumangenes nearintegrationsites,followedbyintegrationsite^specificexpressionofsuchgenesduring hepatocarcinogenesis. Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world (1). Epidemiologic data suggest that hepatitis B virus (HBV) is closely related to hepatocarcino- genesis (2). Southern blot analysis has shown that HBV DNA is frequently integrated into the human genome in HCC (3). Such insertional mutagenesis is one of the most important mecha- nisms for the development of HBV-related HCC. Critical genes adjacent to integrated HBV DNA have been identified by molecular techniques. HBV DNA integration has occurred in an exon of the retinoic acid receptor B gene (4), and the cyclin A2 gene was identified at an early stage of HCC (5). However, these findings have not been reproduced. Woodchuck hepatitis virus DNA, which induces liver tumors in woodchucks, was found to be integrated in c-myc or N-myc in 30% of liver tumors, most of them exhibiting increased expression of these genes (6). It thus remains unclear which genes serve as targets for HBV DNA integration in human liver. Recent studies have reported that some modified PCR techniques could effectively detect the cellular DNA sequences adjacent to an integrated retroviral provirus (7, 8). One of these techniques, cassette ligation – mediated PCR, is used to selec- tively amplify utilized DNA when sequence information on a portion of the gene is available (9, 10). In this study, we used cassette ligation – mediated PCR to identify human genome sequences adjoining integrated HBV DNA in HCC. In three cases, HBV DNA was integrated into the mixed lineage leukemia 2 (MLL2 ) gene. To investigate changes in gene expression patterns caused by HBV DNA integration, we conducted cDNA microarray expression experiments. We confirmed that HCCs with HBV integrated into MLL2 had characteristic patterns of gene expression, compared with HCCs with HBV integrated into other loci of human chromosomes. Finally, we identified candidate genes whose expression was associated with MLL2. Materials and Methods Cell culture. PLC/PRF/5 cells were grown in DMEM supplemented with 10% fetal bovine serum. This cell line was used as a positive control of HBV DNA integration into human genome (11). Human Cancer Biology Authors’ Affiliations: Departments of 1 Hepatology, 2 Surgery, and 3 Nuclear Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan; 4 Bioinformatics Center, InstituteforChemicalResearch,KyotoUniversity,Kyoto, Japan;and 5 HumanGenomeCenter,InstituteofMedicalScience,Universityof Tokyo,Tokyo,Japan Received10/14/04;revised3/29/05;accepted5/20/05. Grant support: MinistryofEducation,Science,Sports,andCulture,Japan. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.Thisarticlemustthereforebeherebymarked advertisement inaccordance with18U.S.C.Section1734solelytoindicatethisfact. Requests for reprints: AkihiroTamori, Department of Hepatology, Osaka City University Graduate School of Medicine,1-4-3 Asahimachi, Abeno-ku, 545- 8585 Osaka, Japan. Phone: 81-6-6645-3811; Fax: 81-6-6646-1433; E-mail: atamori@med.osaka-cu.ac.jp. F 2005AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-04-2055 www.aacrjournals.org Clin Cancer Res 2005;11(16) August 15, 2005 5821 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/11/16/5821/1958592/5821-5826.pdf by guest on 21 February 2023