DNA double-strand breaks, potential targets for HBV integration

Abstract

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is one of the most frequently occurring cancers. Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis. More and more researches were designed to find the relationship of the two. In this study, we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks (DSBs), one of the most detrimental DNA damage. An 18-bp I-SceI homing endonuclease recognition site was introduced into the DNA of HepG2 cell line by stable DNA transfection, then cells were incubated in patients’ serum with high HBV DNA copies and at the same time, DSBs were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. By using nest PCR, the viral DNA was detected at the sites of the break. It appeared that integration occurred between part of HBV x gene and the I-SceI induced breaks. The results suggested that DSBs, as the DNA damages, may serve as potential targets for hepadnaviral DNA insertion and the integrants would lead to widespread host genome changes necessarily. It provided a new site to investigate the integration.

Keywords

DNA double-strand breaks / hepatitis B virus / integration / non-homologous end joining

References

[1] TakahashiS., ChayamaK.. Integration of hepatitis B virus DNA and hepatocellular carcinoma. J Gastroenterol Hepatol, 2005, 20(8): 1141-1142

[2] KimbiG.C., KramvisA., KewM.C.. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B. World J Gastroenterol, 2005, 11(41): 6416-6421

[3] MinamiM., DaimonY., MoriK., et al.. Hepatitis B virus-related insertional mutagenesis in chronic hepatitis B patients as an early drastic genetic change leading to hepatocarcinogenesis. Oncogene, 2005, 24(27): 4340-4348

[4] PeterM., RostyC., CouturierJ., et al.. MYC activation associated with the integration of HPV DNA at the MYC locus in genital tumors. Oncogene, 2006, 25(44): 5985-5993

[5] MiyagawaK.. Double-strand break repair and chromosomal instability. Rinsho Ketsueki, 2009, 50(6): 474-480

[6] BillC.A., SummersJ.. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc Natl Acad Sci USA, 2004, 101(30): 11 135-11 140

[7] GozuacikD., MurakamiY., SaigoK., et al.. Identification of human cancer-related genes by naturally occurring hepatitis B virus DNA tagging. Oncogene, 2001, 20(43): 6233-6240

[8] NickoloffJ.A., BrennemanM.A.. Analysis of recombinational repair of DNA double-strand breaks in mammalian cells with I-SceI nuclease. Methods Mol Biol, 2004, 262: 35-52

[9] ParanN., GeigerB., ShaulY., et al.. HBV infection and cell culture: evidence for multivalent and cooperate attachment. EMBO J, 2001, 20(16): 4443-4453

[10] KinnerA., WuW., StaudtC., et al.. γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Res, 2008, 36(17): 5678-5694

[11] SohnJ.A., LitwinS., SeegerC., et al.. Mechanism for cccDNA synthesis in hepadnaviruses. PLoS One, 2009, 4(11): e8093

[12] Mazet-WagnerA.A., BacletM.C., Loustaud-RattiV., et al.. Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients. J Virol Methods, 2006, 138(1–2): 70-79

[13] TakkenbergR.B., ZaaijerH.L., MolenkampR., et al.. Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus ovalently closed circular DNA in plasma of chronic hepatitis B patients. J Med Virol, 2009, 81(6): 988-995

[14] LöbrichM., ShibataA., BeucherA., et al.. γ-H2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization. Cell Cycle, 2010, 9(4): 662-669

[15] NassalM.. Hepatitis B viruses: reverse transcription a different way. Virus Res, 2008, 134(1–2): 235-249

[16] Alexandru CaruntuF., MolagicV.. cccDNA persistence during natural evolution of chronic HBV infection. Romanian J Gastroenterol, 2005, 14(4): 373-377

[17] SakuraiY., KomatsuK., AgematsuK., et al.. DNA double strand break repair enzymes function at multiple steps in retroviral infection. Retrovirology, 2009, 15(6): 114-127

[18] FeitelsonM.A., LeeJ.. Hepatitis B virus integration, fragile sites, and hepatocarcinogenesis. Cancer Lett, 2007, 252(2): 157-170

[19] CheminI., ZoulimF.. Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett, 2009, 286(1): 52-59

[20] PetersenJ., DandriM., BurkleA., et al.. Increase in the frequency of hepadnavirus DNA integration by oxidative DNA damage and inhibition of DNA repair. J Virol, 1997, 71(7): 5455-5463

[21] ShrivastavM., De HaroL.P., NickoloffJ.A., et al.. Regulation of DNA double-strand break repair pathway choice. Cell Res, 2008, 18(1): 134-147

[22] DandriM., BurdaM.R., BurkleA., et al.. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly (ADP-ribosyl)ation. Hepa tology, 2002, 35(1): 217-223

[23] AbelS., De KockM., van SchalkwykD.J., et al.. Altered lipid profile, oxidative status and hepatitis B virus interactions in human hepatocellular carcinoma. Prostaglandins Leukot Essent Fatty Acids, 2009, 81(5–6): 391-399

[24] BarashH., GrossE.R., EdreiY., et al.. Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks. Proc Natl Acad Sci U S A, 2010, 107(5): 2207-2212

[25] FeitelsonM.A., ReisH.M., TufanN.L., et al.. Putative roles of hepatitis B x antigen in the pathogenesis of chronic liver disease. Cancer Lett, 2009, 286(1): 69-79