1887

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Volume 96, Issue 8

Hepatitis B virus X protein activates the ATM–Chk2 pathway and delays cell cycle progression Free

Abstract

Genetic instability is intimately associated with tumour development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that γ-H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighbouring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx plasmids into cells induced accumulation of γ-H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser) and CDK1 (Tyr) was elevated; consequently, cell-cycle progression was delayed at G/M phase, suggesting that activation of the ATM–Chk2 pathway by the HBV X protein (HBx) induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced reactive oxygen species (ROS) production and led to the accumulation of γ-H2AX foci and the increased p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM–Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.

  • Received:
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© 2015 The Authors
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2015-08-01
2024-04-20
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References

  1. Amaddeo G., Cao Q., Ladeiro Y., Imbeaud S., Nault J.C., Jaoui D., Gaston Mathe Y., Laurent C., Laurent A., other authors. 2015; Integration of tumour and viral genomic characterisations in HBV-related hepatocellular carcinomas. Gut 64:820–829[PubMed] [CrossRef]
     [Google Scholar]
  2. Banáth J.P., Macphail S.H., Olive P.L. 2004; Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. Cancer Res 64:7144–7149 [View Article][PubMed]
     [Google Scholar]
  3. Bartek J., Lukas J. 2003; Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3:421–429 [View Article][PubMed]
     [Google Scholar]
  4. Bouchard M., Giannakopoulos S., Wang E.H., Tanese N., Schneider R.J. 2001; Hepatitis B virus HBx protein activation of cyclin A-cyclin-dependent kinase 2 complexes and G1 transit via a Src kinase pathway. J Virol 75:4247–4257 [View Article][PubMed]
     [Google Scholar]
  5. Bréchot C. 2004; Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 127:(Suppl 1)S56–S61 [View Article][PubMed]
     [Google Scholar]
  6. Chae S., Ji J.H., Kwon S.H., Lee H.S., Lim J.M., Kang D., Lee C.W., Cho H. 2013; HBxAPα/Rsf-1-mediated HBx-hBubR1 interactions regulate the mitotic spindle checkpoint and chromosome instability. Carcinogenesis 34:1680–1688 [View Article][PubMed]
     [Google Scholar]
  7. Chen Y., Poon R.Y. 2008; The multiple checkpoint functions of CHK1 and CHK2 in maintenance of genome stability. Front Biosci 13:5016–5029[PubMed]
     [Google Scholar]
  8. Cho M.Y., Cheong J.Y., Lim W., Jo S., Lee Y., Wang H.J., Han K.H., Cho H. 2014; Prognostic significance of catalase expression and its regulatory effects on hepatitis B virus X protein (HBx) in HBV-related advanced hepatocellular carcinomas. Oncotarget 5:12233–12246[PubMed] [CrossRef]
     [Google Scholar]
  9. Chung Y.L. 2013; Defective DNA damage response and repair in liver cells expressing hepatitis B virus surface antigen. FASEB J 27:2316–2327 [View Article][PubMed]
     [Google Scholar]
  10. Darbon J.M., Penary M., Escalas N., Casagrande F., Goubin-Gramatica F., Baudouin C., Ducommun B. 2000; Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells. J Biol Chem 275:15363–15369 [View Article][PubMed]
     [Google Scholar]
  11. Dejean A., Bougueleret L., Grzeschik K.H., Tiollais P. 1986; Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. Nature 322:70–72 [View Article][PubMed]
     [Google Scholar]
  12. Forgues M., Difilippantonio M.J., Linke S.P., Ried T., Nagashima K., Feden J., Valerie K., Fukasawa K., Wang X.W. 2003; Involvement of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles. Mol Cell Biol 23:5282–5292 [View Article][PubMed]
     [Google Scholar]
  13. Goldstone S., Pavey S., Forrest A., Sinnamon J., Gabrielli B. 2001; Cdc25-dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR. Oncogene 20:921–932 [View Article][PubMed]
     [Google Scholar]
  14. Haoudi A., Daniels R.C., Wong E., Kupfer G., Semmes O.J. 2003; Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response. J Biol Chem 278:37736–37744 [View Article][PubMed]
     [Google Scholar]
  15. Henkler F., Hoare J., Waseem N., Goldin R.D., McGarvey M.J., Koshy R., King I.A. 2001; Intracellular localization of the hepatitis B virus HBx protein. J Gen Virol 82:871–882[PubMed] [CrossRef]
     [Google Scholar]
  16. Kim S., Park S.Y., Yong H., Famulski J.K., Chae S., Lee J.H., Kang C.M., Saya H., Chan G.K., Cho H. 2008; HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint. Oncogene 27:3457–3464 [View Article][PubMed]
     [Google Scholar]
  17. Klein N.P., Schneider R.J. 1997; Activation of Src family kinases by hepatitis B virus HBx protein and coupled signaling to Ras. Mol Cell Biol 17:6427–6436[PubMed]
     [Google Scholar]
  18. Laurent-Puig P., Legoix P., Bluteau O., Belghiti J., Franco D., Binot F., Monges G., Thomas G., Bioulac-Sage P., Zucman-Rossi J. 2001; Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 120:1763–1773 [View Article][PubMed]
     [Google Scholar]
  19. Lim W., Kwon S.H., Cho H., Kim S., Lee S., Ryu W.S., Cho H. 2010; HBx targeting to mitochondria and ROS generation are necessary but insufficient for HBV-induced cyclooxygenase-2 expression. J Mol Med (Berl) 88:359–369 [View Article][PubMed]
     [Google Scholar]
  20. Ma J., Sun T., Park S., Shen G., Liu J. 2011; The role of hepatitis B virus X protein is related to its differential intracellular localization. Acta Biochim Biophys Sin (Shanghai) 43:583–588 [View Article][PubMed]
     [Google Scholar]
  21. Martin-Lluesma S., Schaeffer C., Robert E.I., van Breugel P.C., Leupin O., Hantz O., Strubin M. 2008; Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1. Hepatology 48:1467–1476 [View Article][PubMed]
     [Google Scholar]
  22. Miao J., Chen G.G., Chun S.Y., Lai P.P. 2006; Hepatitis B virus X protein induces apoptosis in hepatoma cells through inhibiting Bcl-xL expression. Cancer Lett 236:115–124 [View Article][PubMed]
     [Google Scholar]
  23. Mikhailov A., Shinohara M., Rieder C.L. 2004; Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J Cell Biol 166:517–526 [View Article][PubMed]
     [Google Scholar]
  24. Park J.H., Lee M.K., Kim H.S., Kim K.L., Cho E.W. 2003; Targeted destruction of the polymerized human serum albumin binding site within the preS2 region of the HBV surface antigen while retaining full immunogenicity for this epitope. J Viral Hepat 10:70–79 [View Article][PubMed]
     [Google Scholar]
  25. Pinsky B.A., Biggins S. 2005; The spindle checkpoint: tension versus attachment. Trends Cell Biol 15:486–493 [View Article][PubMed]
     [Google Scholar]
  26. Rahbari R., Sheahan T., Modes V., Collier P., Macfarlane C., Badge R.M. 2009; A novel L1 retrotransposon marker for HeLa cell line identification. Biotechniques 46:277–284[PubMed]
     [Google Scholar]
  27. Shih W.L., Kuo M.L., Chuang S.E., Cheng A.L., Doong S.L. 2003; Hepatitis B virus X protein activates a survival signaling by linking SRC to phosphatidylinositol 3-kinase. J Biol Chem 278:31807–31813 [View Article][PubMed]
     [Google Scholar]
  28. Shiloh Y. 2001; ATM and ATR: networking cellular responses to DNA damage. Curr Opin Genet Dev 11:71–77 [View Article][PubMed]
     [Google Scholar]
  29. Tarn C., Zou L., Hullinger R.L., Andrisani O.M. 2002; Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes. J Virol 76:9763–9772 [View Article][PubMed]
     [Google Scholar]
  30. Taylor W.R., Stark G.R. 2001; Regulation of the G2/M transition by p53. Oncogene 20:1803–1815 [View Article][PubMed]
     [Google Scholar]
  31. Wang J., Chenivesse X., Henglein B., Bréchot C. 1990; Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature 343:555–557 [View Article][PubMed]
     [Google Scholar]
  32. Wang J.H., Yun C., Kim S., Lee J.H., Yoon G., Lee M.O., Cho H. 2003; Reactive oxygen species modulates the intracellular level of HBx viral oncoprotein. Biochem Biophys Res Commun 310:32–39 [View Article][PubMed]
     [Google Scholar]
  33. Wang Y., Hong Y., Li M., Long J., Zhao Y.P., Zhang J.X., Li Q., You H., Tong W.M., other authors. 2013; Mutation inactivation of Nijmegen breakage syndrome gene (NBS1) in hepatocellular carcinoma and intrahepatic cholangiocarcinoma. PLoS One 8:e82426 [View Article][PubMed]
     [Google Scholar]
  34. Waris G., Huh K.W., Siddiqui A. 2001; Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via oxidative stress. Mol Cell Biol 21:7721–7730 [View Article][PubMed]
     [Google Scholar]
  35. Wen Y., Golubkov V.S., Strongin A.Y., Jiang W., Reed J.C. 2008; Interaction of hepatitis B viral oncoprotein with cellular target HBXIP dysregulates centrosome dynamics and mitotic spindle formation. J Biol Chem 283:2793–2803 [View Article][PubMed]
     [Google Scholar]
  36. Yun C., Lee J.H., Park H., Jin Y.M., Park S., Park K., Cho H. 2000; Chemotherapeutic drug, adriamycin, restores the function of p53 protein in hepatitis B virus X (HBx) protein-expressing liver cells. Oncogene 19:5163–5172 [View Article][PubMed]
     [Google Scholar]
  37. Yun C., Cho H., Kim S.J., Lee J.H., Park S.Y., Chan G.K., Cho H. 2004; Mitotic aberration coupled with centrosome amplification is induced by hepatitis B virus X oncoprotein via the Ras-mitogen-activated protein/extracellular signal-regulated kinase-mitogen-activated protein pathway. Mol Cancer Res 2:159–169[PubMed]
     [Google Scholar]
  38. Zhang X., Hou J., Lu M. 2013; Regulation of hepatitis B virus replication by epigenetic mechanisms and microRNAs. Front Genet 4:202 [View Article][PubMed]
     [Google Scholar]
  39. Zimmerman E.S., Chen J., Andersen J.L., Ardon O., Dehart J.L., Blackett J., Choudhary S.K., Camerini D., Nghiem P., Planelles V. 2004; Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation. Mol Cell Biol 24:9286–9294 [View Article][PubMed]
     [Google Scholar]
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Hepatitis B virus X protein activates the ATM–Chk2 pathway and delays cell cycle progression
J. Gen. Virol. 96, 2242 (2015); https://doi.org/10.1099/vir.0.000150
/content/journal/jgv/10.1099/vir.0.000150
/content/journal/jgv/10.1099/vir.0.000150
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