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BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells

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Abstract

Epstein-Barr virus (EBV)-encoded BARF1 (BamH1-A Rightward Frame-1) is expressed in EBV-positive malignancies such as nasopharyngeal carcinoma, EBV-associated gastric cancer, B-cell lymphoma and nasal NK/T-cell lymphoma, and has been shown to have an important role in oncogenesis. However, the mechanism by which BARF1 elicits its biological effects is unclear. We investigated the effects of BARF1 silencing on cell proliferation and apoptosis in EBV-positive malignant cells. We observed that BARF1 silencing significantly inhibits cell proliferation and induces apoptosis-mediated cell death by collapsing the mitochondrial membrane potential in AG876 and Hone-Akata cells. BARF1 knockdown up-regulates the expression of pro-apoptotic proteins and down-regulates the expression of anti-apoptotic proteins. In BARF1-down-regulated cells, the Bcl-2/BAX ratio is decreased. The caspase inhibitor z-VAD-fmk was found to rescue siBARF1-induced apoptosis in these cells. Immunoblot analysis showed significant increased levels of cleaved caspase 3 and caspase 9. We observed a significant increase in cytochrome c level as well as the formation of apoptosome complex in BARF1-silenced cells. In conclusion, siRNA-mediated BARF1 down-regulation induces caspase-dependent apoptosis via the mitochondrial pathway through modulation of Bcl-2/BAX ratio in AG876 and Hone-Akata cells. Targeting BARF1 using siRNA has the potential to be developed as a novel therapeutic strategy in the treatment of EBV-associated malignancies.

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References

  • Brunelle JK and Letai A 2009 Control of mitochondrial apoptosis by the Bcl-2 family. J Cell. Sci. 122 437–441

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chaitanya GV, Alexander JS and Babu PP 2010 PARP-1 cleavage fragments: Signatures of cell-death proteases in neurodegeneration. Cell Commun. Signal 8 31

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chang MS, Kim DH, Roh JK, Middeldorp JM, Kim YS, Kim S, Han S, Kim CW, et al. 2013 Epstein-Barr virus-encoded BARF1 promotes proliferation of gastric carcinoma cells through regulation of NF-κB. J. Virol. 87 10515–10523

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Danve C, Decaussin G, Busson P and Ooka T 2001 Growth transformation of primary epithelial cells with a NPC-derived Epstein–Barr virus strain. Virology 288 223–235

    Article  CAS  PubMed  Google Scholar 

  • De Turenne-Tessier M, Jolicoeur P, Middeldorp JM and Ooka T 2005 Expression and analysis of the Epstein-Barr virus BARF1-encoded protein from a tetracycline-regulatable adenovirus system. Virus res. 109 9–18

    Article  PubMed  Google Scholar 

  • Decaussin G, Sbih-Lammali F, de Turenne-Tessier M, Bouguermouh A and Ooka T 2000 Expression of BARF1 gene encoded by Epstein-Barr virus in nasopharyngeal carcinoma biopsies. Cancer Res. 60 5584–5588

  • Fiorini S and Ooka T 2008 Secretion of Epstein-Barr virus-encoded BARF1 oncoprotein from latently infected B cells. Virol. J. 5 70

    Article  PubMed Central  PubMed  Google Scholar 

  • Fulda S and Debatin KM 2006 Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25 4798–4811

    Article  CAS  PubMed  Google Scholar 

  • Gitlin L, Karelsky S and Andino R 2002 Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418 430–434

    Article  CAS  PubMed  Google Scholar 

  • Glaser R, Zhang HY, Yao KT, Zhu HC and Wang FX 1989 Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas. Proc. Natl. Acad. of Sci. USA 86 9524–9528

    Article  CAS  Google Scholar 

  • Gottlieb E, Armour SM, Harris MH and Thompson CB 2003 Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ. 10 709–717

    Article  CAS  PubMed  Google Scholar 

  • Gross A, McDonnell JM and Korsmeyer SJ 1999 BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13 1899–1911

    Article  CAS  PubMed  Google Scholar 

  • Hajra KM and Liu JR 2004 Apoptosome dysfunction in human cancer. Apoptosis Int. J. Program. Cell Death 9 691–704

    Article  CAS  Google Scholar 

  • Hoebe EK, Hutajulu SH, van Beek J, Stevens SJ, Paramita DK, et al. 2011 Purified hexameric Epstein-Barr virus-encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients. Clin. Vaccine Immunol. CVI 18 298–304

    Article  CAS  Google Scholar 

  • Hoebe EK, Le Large TYS, Tarbouriech N, Oosterhoff D, De Gruijl TD, et al. 2012a Epstein-Barr Virus-encoded BARF1 protein is a decoy receptor for macrophage colony stimulating factor and interferes with macrophage differentiation and activation. Viral Immunol. 25 461–470

    Article  CAS  PubMed  Google Scholar 

  • Hoebe EK, Wille C, Hopmans ES, Robinson AR, Middeldorp JM, et al. 2012b Epstein-Barr virus transcription activator R up-regulates BARF1 expression by direct binding to its promoter, independent of methylation. J. Virol. 86 11322–11332

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Houali K, Wang X, Shimizu Y, Djennaoui D, Nicholls J, et al. 2007 A new diagnostic marker for secreted Epstein-Barr virus–encoded LMP1 and BARF1 oncoproteins in the serum and saliva of patients with nasopharyngeal carcinoma. Clin. Cancer Res. 13 4993–5000

    Article  CAS  PubMed  Google Scholar 

  • Jiang R, Cabras G, Sheng W, Zeng Y and Ooka T 2009 Synergism of BARF1 with Ras induces malignant transformation in primary primate epithelial cells and human nasopharyngeal epithelial cells. Neoplasia N Y N 11 964–973

    CAS  Google Scholar 

  • Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE and Poirier GG 1993 Specific proteolytic cleavage of Poly(ADP-Ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 53 3976–3985

    CAS  PubMed  Google Scholar 

  • Korsmeyer SJ 1999 BCL-2 gene family and the regulation of programmed cell death. Cancer Res. 59 1693s–1700s

    CAS  PubMed  Google Scholar 

  • Ledgerwood EC and Morison IM 2009 Targeting the apoptosome for cancer therapy. Clin. Cancer Res. 15 420–424

    Article  CAS  PubMed  Google Scholar 

  • Ly JD, Grubb DR and Lawen A 2003 The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis Int. J. Program. Cell Death 8 115–128

    Article  CAS  Google Scholar 

  • Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, Schie RC, et al. 1995 Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182 1545–1556

    Article  CAS  PubMed  Google Scholar 

  • Oltvai ZN, Milliman CL and Korsmeyer SJ 1993 Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74 609–619

    Article  CAS  PubMed  Google Scholar 

  • Reed JC 1996 Balancing cell life and death: bax, apoptosis, and breast cancer. J. Clin. Invest. 97 2403–2404

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sakka E, Zur Hausen A, Houali K, Liu H, Fiorini S, et al. 2013 Cellular localization of BARF1 oncoprotein and its cell stimulating activity in human epithelial cell. Virus Res. 174 8–17

    Article  CAS  PubMed  Google Scholar 

  • Sall A, Caserta S, Jolicoeur P, Franqueville L, de Turenne-Tessier M, et al. 2004 Mitogenic activity of Epstein-Barr virus-encoded BARF1 protein. Oncogene 23 4938–4944

    Article  CAS  PubMed  Google Scholar 

  • Seto E, Yang L, Middeldorp J, Sheen TS, Chen JY, et al. 2005 Epstein–Barr Virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression. J Med. Virol. 76 82–88

    Article  CAS  PubMed  Google Scholar 

  • Seto E, Ooka T, Middeldorp J and Takada K 2008 Reconstitution of nasopharyngeal carcinoma-type EBV infection induces tumorigenicity. Cancer Res. 68 1030–1036

    Article  CAS  PubMed  Google Scholar 

  • Sheng W, Decaussin G, Sumner S and Ooka T 2001 N-terminal domain of BARF1 gene encoded by Epstein-Barr virus is essential for malignant transformation of rodent fibroblasts and activation of BCL-2. Oncogene 20 1176–1185

    Article  CAS  PubMed  Google Scholar 

  • Sheng W, Decaussin G, Ligout A, Takada K and Ooka T 2003 Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus. J. Virol. 77 3859–3865

    Article  PubMed Central  PubMed  Google Scholar 

  • Tanner JE, Wei MX, Alfieri C, Ahmad A, Taylor P, et al. 1997 Antibody and antibody-dependent cellular cytotoxicity responses against the BamHI A Rightward Open-Reading Frame-1 Protein of Epstein-Barr virus (EBV) in EBV-associated disorders. J. Infect. Dis. 175 38–46

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Tsao SW, Ooka T, Nicholls JM, Cheung HW, et al. 2006 Anti-apoptotic role of BARF1 in gastric cancer cells. Cancer Lett. 238 90–103

    Article  CAS  PubMed  Google Scholar 

  • Wei MX, Turenne-Tessier M, Decaussin G, Benet G and Ooka T 1997 Establishment of a monkey kidney epithelial cell line with the BARF1 open reading frame from Epstein-Barr virus. Oncogene 14 3073–3081

    Article  CAS  PubMed  Google Scholar 

  • Wei MX and Ooka T 1989 A transforming function of the BARF1 gene encoded by Epstein-Barr virus. EMBO J. 8 2897–2903

    PubMed Central  CAS  PubMed  Google Scholar 

  • Wei MX, Moulin JC, Decaussin G, Berger F and Ooka T 1994 Expression and tumorigenicity of the Epstein-Barr virus BARF1 gene in human Louckes B-lymphocyte cell line. Cancer Res. 54 1843–1848

    CAS  PubMed  Google Scholar 

  • Xue S, Labrecque LG, Lu QL, Ong SK, Lampert IA, et al. 2002 Promiscuous expression of Epstein-Barr virus genes in Burkitt’s lymphoma from the central African country Malawi. Int. J. Cancer 99 635–643

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Ohyashiki JH, Takaku T, Shimizu N and Ohyashiki K 2006 Transcriptional profiling of Epstein–Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection. Br. J. Cancer 94 599–608

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zur Hausen A, Brink AATP, Craanen ME, Middeldorp JM, Meijer CJLM, et al. 2000 Unique transcription pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric adenocarcinomas: Expression of the transforming BARF1 gene. Cancer Res. 60 2745–2748

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Middeldorp from the Department of Pathology, VU University Medical Center, for kindly providing the BARF1 antibody. We also thank Dr Ea Chee Kwee from the Institute of Biological Sciences, University of Malaya, for providing reagents in immunoprecipitation experiment, and Mr Lim Chun Shen for figure preparation for this manuscript.

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Authors and Affiliations

  1. Institute of Biological Sciences (Genetics and Molecular Biology), Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

    Taznim Begam Mohd Mohidin & Ching Ching Ng

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  1. Taznim Begam Mohd Mohidin
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  2. Ching Ching Ng
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Correspondence to Ching Ching Ng.

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Corresponding editor: Seyed E Hasnain

[Mohidin TBM and Ng CC 2015 BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells. J. Biosci. 40 1–11] DOI 10.1007/s12038-015-9502-z

Supplementary materials pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/mar2015/supp/Mohidin.pdf

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Mohidin, T.B.M., Ng, C.C. BARF1 gene silencing triggers caspase-dependent mitochondrial apoptosis in Epstein-Barr virus-positive malignant cells. J Biosci 40, 41–51 (2015). https://doi.org/10.1007/s12038-015-9502-z

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  • DOI: https://doi.org/10.1007/s12038-015-9502-z

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