The Epstein—Barr Virus Gene BHRF1, a Homologue of the Cellular Oncogene Bcl-2, Inhibits Apoptosis Induced by Gamma Radiation and Chemotherapeutic Drugs

  • N. J. McCarthy
  • S. A. Hazlewood
  • D. S. Huen
  • A. B. Rickinson
  • G. T. Williams
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 406)


Analysis of apoptosis, active and controllable cell death, has demonstrated that the size of a cell population can be regulated by changes in the cell death rate as well as in the rates of proliferation and differentiation. Factors which alter the rate of cell death, such as expression of the proto-oncogene bc1-2, can therefore directly affect the number of cells within a population. Bc1-2 has been. shown to suppress apoptosis in response to a variety of stimuli and to act as a complementary survival signal for the random acquisition of other oncogenic mutations, such as deregulated c-myc.

The Epstein Barr virus (EBV) gene BHRF1 was the first of a family of bc1-2 homologues now being identified. BHRF1 and bc1-2 share 25% primary amino acid sequence homology. Here we show that γ radiation and several cytotoxic anticancer agents induce apoptosis in Burkitt’s lymphoma (BL) cell lines, as has been found in several other systems. Using gene transfection studies we have also shown that expression of either BHRF 1 or bc1-2 in BL cell lines significantly suppresses apoptosis in response to a variety of anticancer treatments. This has confirmed that BHRF 1 is functionally homologous to bcl-2 in B-cells and suggests that BHRF1 may act to prevent apoptosis during EBV infection, maximising virus particle production, as has been suggested for other human and insect viral genes. Suppression of chemotherapeutic drug induced cell death by bcl-2 and BHRF1, as demonstrated in this cell system, results in resistance to a variety of different agents and may represent an alternative mechanism by which multidrug resistance arises during chemotherapy.


Programme Cell Death Epstein Barr Virus African Swine Fever African Swine Fever Virus Apoptotic Morphology 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    M.J. Arends and Wyllie, A., Apoptosis: mechanisms and roles in pathology. Int. Rev. Exp. Pathol., 32, 223–254 (1991).PubMedGoogle Scholar
  2. 2.
    G.T.Williams, Smith, C.A., McCarthy, N.J. and Grimes, E.A., Apoptosis: Final control point in cell biology. Trends Cell. Biol., 2: 263–267 (1992).PubMedCrossRefGoogle Scholar
  3. 3.
    A.J. Hale, Smith, C.A., Sutherland, L.C., Stoneman, V.E.A., Longthorne, V.L., Culhane, A.C. and Williams, G.T., Apoptosis: molecular regulation of cell death. Eur. J. Biochem. 1996 (in the press).Google Scholar
  4. 4.
    C.A.Smith, Gimes, E.A., McCarthy, N.J. and Williams, G.T., Multiple gene regulation of apoptosis: Significance in immunology and oncology. In:L.D. Tomei and F.O. Cope (eds.), Apoptosis. The molecular basis of cell death II,Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. (in press) (1994).Google Scholar
  5. 5.
    P. Golstein, Ojcius, D.M. and Young, J.D-E., Cell death mechanisms and the immune system. Immunol. Rev., 121: 29–65 (1991).PubMedCrossRefGoogle Scholar
  6. 6.
    G.T.Williams. Apoptosis in the immune system. J. Pathol., 173: 1–4 (1994).PubMedCrossRefGoogle Scholar
  7. 7.
    C.A. Smith, Williams, G.T., Kingston, R, Jenkinson, E.J. and Owen, J.J.T., Antibodies to the CD3/T-cell receptor complex induce death by apoptosis in immature T-cells in thymic cultures. Nature, 337: 181–184 (1989).PubMedCrossRefGoogle Scholar
  8. 8.
    Y. Shi, Sahai, B.M. and Green, D.R., Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymoccytes. Nature, 339: 625–626 (1989).PubMedCrossRefGoogle Scholar
  9. 9.
    Y.J.Liu, Cairns, J.A, Holder, M.J., Abbot, S.J., Jansen, K.U., Bonnefoy, J.Y., Gordon, J. and MacLennan, I.C.M.. Recombinant 25 kDa CD23 and interleukin la promote the survival of germinal centre B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis. Eur. J. Immunol. 21, 1107–1114 (1991).PubMedCrossRefGoogle Scholar
  10. 10.
    G.J.V.Nossal. The molecular and cellular basis of affinity maturation in the antibody response. Cell, 68: 1–2 (1992).Google Scholar
  11. 11.
    G.T.Williams, Smith, C. A., Spooncer, E., Dexter, T.M and Taylor, D. R., Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature, 343: 76–79 (1990).PubMedCrossRefGoogle Scholar
  12. 12.
    M.J.Koury and Bondurant, M.C., Control of red cell prodcution: the roles of programmed cell death (apoptosis) and erythropoietin. Transfusion, 30: 673–674 (1990).PubMedCrossRefGoogle Scholar
  13. 13.
    D.L.Vaux, Cory, S. and Adams, J.M.. Bc1–2 gene promotes haemopoietic cell survival and co-operates with c-myc to immortalize pre-B cells. Nature, 335: 440–42 (1988).PubMedCrossRefGoogle Scholar
  14. 14.
    D.Hockenbery, Nunez, G., Milliman, C., Schreiber, R.D. and Korsmeyer, S.J., Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature, 348: 334–336 (1990).PubMedCrossRefGoogle Scholar
  15. 15.
    Y.Tsujimoto, Stress resistance conferred by high level of Bcl-2a protein in human B-lymphoblastoid cell. Oncogene, 4: 1331–1336 (1989).PubMedGoogle Scholar
  16. 16.
    N.J.McCarthy, Smith, C.A. and Williams, G.T., Apoptosis in the development of the immune system: Growth factors, clonal selection and bd-2. Cancer Metastasis Rev., 11: 157–178 (1992).CrossRefGoogle Scholar
  17. 17.
    M.L.Cleary, Smith, S.D. and Sklar J., Cloning and structural analysis of cDNAs for bc1–2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell, 47: 19–28 (1986).CrossRefGoogle Scholar
  18. 18.
    G.T.Williams, Programmed cell death: Apoptosis and oncogenesis. Cell, 65: 1097–1098 (1991).CrossRefGoogle Scholar
  19. 19.
    S.A.Henderson, Huen, D., Rowe, M., Dawson, C., Johnson, G. and Rickinson, A., Epstein-Barr virus coded BHRF 1 protein, a viral homologue of bc1–2, protects human B cells from programmed cell death. Proc. Natl. Acad. Sci. U.S.A., 90: 8479–8483 (1993).CrossRefGoogle Scholar
  20. 20. T.Hickish, Robertson, D., Clarke, P., Hill, M., di Stefano, F., Clarke, C. and Cunnigham, D., Ultrastructural localisation of BHRF I: an Epstein-Barr virus gene product which has homology with bc1–2. Cancer Res. 54, 2808–2811. Google Scholar
  21. 21.
    J.W.Gratama, Oosterveer, M.A.P., Zwann, F.E., Lepoutre, J., Klein, G. and Ernberg, I., Eradication of Epstein Barr virus by allogenic bone marrow transplantation: implcations for sites of vital latency. Proc. Natl. Acad. Sci. USA, 85: 8693–8696 (1988).CrossRefGoogle Scholar
  22. 22.
    Q.Y.Yao, Ogan, P., Rowe, M., Wood, M. and Rickinson, A.B., Epstein Barr virus infected B cells persist in the cirulation of acyclovir-treated virus carriers. Int. J. Cancer, 43: 67–71 (1989).CrossRefGoogle Scholar
  23. 23.
    C.D.Gregory, Dive, C., Henderson, S.A., Smith, C.A., Williams, G.T., Gordon, J and Rickinson, A. B., Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature, 349: 612–614 (1991).CrossRefGoogle Scholar
  24. 24.
    S.A.Henderson, Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E. and Rickinson, A., Induction of bc1–2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell, 65: 1107–1115 (1991).CrossRefGoogle Scholar
  25. 25.
    E.White, Sabbatini, P. Debbas, M., Wold, W.S.M., Kusher, D. I. and Gooding L.R., The 19-Kilodalton Adenovirus Elb transforming protein inhibits programmed cell death and prevents cytolysis by tumour necrosis factor a. Mol. Cell. Biol., 12: 2570–2580.Google Scholar
  26. 26.
    J.L.Cleveland, Dean, M., Rosenberg, N., Wang, J.Y.J. and Rapp, U.R., Tyrosine kinase oncogenes abrogate interleukin 3 dependence of murine myeloid cells through signalling pathways involving c-myc: conditional regulation ofc-myc transcription by temperature sensitive v-abl. Mol. Cell Biol., 9:5685–5695 (1989).Google Scholar
  27. 27.
    R.J.Clem, Fechheimer, M. and Miller L. K., Prevention of apoptosis by a Baculovirus gene during infection of insect cells. Science, 254: 1388–1390 (1991).CrossRefGoogle Scholar
  28. 28.
    N.E.Crook, Clem, R.J. and Miller, L.K., An apoptosis inhibiting Baculovirus gene with a zinc finger like motif. J. Virol., 67: 2168–2174 (1993).Google Scholar
  29. 29.
    M.Hummel, and Kieff, E., Epstein Barr virus RNA VIII. Viral RNA in permissively infected B95–8 cells. J. Virol., 43: 262–272 (1982).Google Scholar
  30. 30.
    M.Hummel, and Kieff, E., Mapping of polypeptides encoded by the Epstein Barr virus genome in productive infection. Proc. Natl. Acad. Sci. U.S.A., 79: 5698–5702 (1982).CrossRefGoogle Scholar
  31. 31.
    T.J.McDonnell, Deane, N., Platt, F.M, Nunez, G., Jaeger, U., McKearn, J.P. and Korsmeyer S. J., Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell: 57: 79–88 (1989).CrossRefGoogle Scholar
  32. 32.
    T.J.McDonnell and Korsmeyer, S. J., Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14;18). Nature, 349: 254–256 (1991).CrossRefGoogle Scholar
  33. 33.
    J.J.Oudejans, van den Brule, A.J., Jiwa, N.M., de Bruin, P.C., Ossenkoppele, G.J., van der Valk, P., Walboomers, J.M. and Meijer, C.J., BHRF1, the Epstein-Barr virus (EBV) homologue of the Bc1–2 protooncogene, is transcribed in EBV-associated B-cell lymphomas and in reactive lymphocytes. Blood, 86: 1893–1902 (1995).Google Scholar
  34. 34.
    T.Yamada, and Ohyama, H. Radiation-induced interphase death of rat thymocytes is internally programmed. Int. J. Radiat. Biol.,53: 65–75 (1988).Google Scholar
  35. 35.
    M.A.Barry, Behnke, C.A. and Eastman, A., Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol., 40: 2353–2362 (1990).CrossRefGoogle Scholar
  36. 36.
    C.A.Evans, Owen-Lynch, P.J., Whetton, A.O. and Dive, C., Activation of the ableson tyrosine kinase activity is associated with suppression of apoptosis in haemopoietic cells. Cancer Res., 53: 1735–1738 (1993).Google Scholar
  37. 37.
    C.A.Dive and Hickman, J.A., Drug-target interactions: only the first step in a commitment to a programmed cell death. Br. J. Cancer, 64: 192–196 (1991).CrossRefGoogle Scholar
  38. 38.
    M.K.L.Collins, Marvel, J., Malde, P. and Lopez-Rivas, A., Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents. J. Exp. Med., 176: 1043–1051 (1992).CrossRefGoogle Scholar
  39. 39.
    T.Miyashita, and Reed, J.C., Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a leukaemia cell line. Blood, 81: 151–157 (1993).Google Scholar
  40. 40.
    A.H.Wyllie. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 284: 555–556 (1980).CrossRefGoogle Scholar
  41. 41.
    D.J.McConkey, Hartzell, P., Nicotera, P. and Orrenius, S., Calcium-activated DNA fragmentation kills immature thymocytes. FASEB J., 3: 1843–1849 (1989).Google Scholar
  42. 42.
    K.S.Sellins, and Cohen, J.J., Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J.Immunol., 139: 3199–3206 (1987).Google Scholar
  43. 43.
    A.J.Levine, Momand J. and Finlay, C.A., The p53 tumour suppressor gene. Nature, 351: 453–456 (1991).CrossRefGoogle Scholar
  44. 44.
    S.J.Baker, Fearon, E.R., Nigro, J.M., Hamilton, S.R., Preisinger, A.C., Jessup, J.M., vanTuinen, P., Ledbetter, D.H., Barker, D.F., Nakamura, Y., White, R. and Vogelstein, B., Chromosome 17 deletions and p53 gene mutations in colosectal carcinoma. Science, 244: 217–221 (1989).CrossRefGoogle Scholar
  45. 45.
    S.W.Lowe, Schmitt, E.M., Smith, S.W., Osborne, B.A. and Jacks, T., p53 is required for radiation induced apoptosis in mouse thymocytes. Nature, 362, 847–849 (1993).CrossRefGoogle Scholar
  46. 46.
    A.R.Clarke, Purdie, C.A., Harrison, D.J., Morris, R.G., Bird, C.C., Hooper, M.L. and Wyllie, A.H., Thymocyte apoptosis induced by p53 dependent and independent pathways. Nature, 362: 849–852 (1993).CrossRefGoogle Scholar
  47. 48.
    G.I.Evan, Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M, Waters, C.M. and Hancock, D. C., Induction of apoptosis in fibroblasts by c-myc protein. Cell, 63: 119–128 (1992).Google Scholar
  48. 49.
    L.H.Boise, Gonzalez-Garcia, M., Postema, C.E., Ding, L., Lindsten, T., Turka, L.A., Mao, X., Nunez, G. and Thompson, C.B., bcl-x, a bc1–2 related gene that functions as a dominant regulator of apoptotíc cell death. Cell, 74: 597–609 (1993).CrossRefGoogle Scholar
  49. 50.
    Z.N.Oltvai, Milliman, C.L. & Korsmeyer, S.J., Bc1–2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609–619 (1993).CrossRefGoogle Scholar
  50. 51.
    D.L.Vaux, Weissman, I.L. and Kim, S.K., Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science, 258: 1955–1957 (1992).Google Scholar
  51. 52.
    M.O.Hengartner, and Horvitz, H.R. C.elegans cell survival gene ced-9 encodes a functional homologue of the mammalian proto-oncogene bc1–2. Cell, 76: 665–676 (1994).Google Scholar
  52. 53.
    G.T. Williams, and Smith, C.A., Molecular regulation of apoptosis: Genetic controls on cell death. Cell, 74: 777–779 (1993).PubMedCrossRefGoogle Scholar
  53. 54.
    J.G.Neilan, Lu, Z., Afonso, C.L., Kutish, G.F., Sussman, M.D. and Rock, D.L., An African swine fever virus gene with similarity to the proto-oncogene bc1–2 and the Epstein-Barr virus gene BHRF1. J Viral., 67: 4391–4394 (1993).Google Scholar
  54. 55.
    G.R.Pearson, Luka, J., Petti, L., Sample, J., Birkenbach, M., Braun, D. and Keiff, E., Identification of an Epstein Barr virus early gene encoding a second comonent of the restricted early antigen complex. Virology, 160: 151–161 (1987).CrossRefGoogle Scholar
  55. 56.
    B.Tarodi, Subramanian, T. and Chinnadurai, G., Epstein-Barr virus BHRF1 protein protects against cell death induced by DNA-damaging agents and heterologous viral infection. Virology, 201: 404–407 (1994).CrossRefGoogle Scholar
  56. 57.
    C.W.Dawson, Eliopoulos, A.G., Dawson, J. and Young, L.S., BHRF1, a viral homologue of the BCL-2 oncogene, disturbs epithelial cell differentiation. Oncogene, 10: 69–77 (1995).Google Scholar
  57. 58.
    R.Dalla-Favera, Martinotti, S., Gallo, R.C., Erikson, J. and Croce, C.M., Science, 219: 963–967 (1983).CrossRefGoogle Scholar
  58. 59.
    F.Cavalli, Chemothreapy of non-Hodgkin’s lymphoma. Bailliere’s Clinical Haematology, 4: 157–179 (1991).CrossRefGoogle Scholar
  59. 60.
    R.Juliano, and Ling, V., J. Supramol. Strut., 4: 521–526 (1976).CrossRefGoogle Scholar
  60. 61.
    V.Ling. P-glycoprotein and resistance to anticancer drugs. Cancer, 69: 2603–2609 (1992).CrossRefGoogle Scholar
  61. 62.
    P.F.Juranka, Zastawny, R.L. and Ling, V., P-glycoprotein multidrug-resistance and a super family of membrane-associated transport proteins. FASEB J., 3: 2583–2592 (1989).Google Scholar
  62. 63.
    G.H.Mickisch, Merlino, G.T., Galski, H., Gottesman, M.M. and Pastan, I. Transgenic mice that express the human multidrug resistance gene in bone marrow enable a rapid identification of agents that reverse drug resistance. Proc. Natl. Acad. Sci. USA., 88: 547–551 (1991).CrossRefGoogle Scholar
  63. 64.
    T.C.Fisher, Milner, A.E., Gregory, C.D., Jackman, A., Aherne, G.W., Hartley, J.A., Dive, C. and Hickman, J.A., bc1–2 modulation of apoptosis induced by anticancer drugs: Resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res., 53: 3321–3326 (1993).Google Scholar
  64. 65.
    M.I.Walton, Whysong, D., O’Connor, P.M., Hockenbery, D., Korsmeyer, S.J. and Kohn, K.W., Constitutive expression of human bc1–2 modulates nitrogen mustard and camptothecin induced apoptosis. Cancer Res., 53: 1853–1861 (1993).PubMedGoogle Scholar
  65. 66.
    J.Lotem and Sachs, L. Regulation by bc12-, c-myc and p53 of susceptibility to induction of apoptosis by heat shock and cancer chemotherapy compounds in differentiation competent and defective myeloid leukaemic cells. Cell Growth Diff., 4: 41–47 (1993).PubMedGoogle Scholar
  66. 67.
    C.M.Rooney, Gregory, C.D., Rowe, M., Finerty, S., Edwards, C., Rupani, H. and Rickinson, A.B., Endemic Burkitt’s lymphoma: phenotypic analysis of Burkitt’s lymphoma biopsy cell and of the derived tumour cell lines. J.Natl. Cancer Inst., 77: 681–687 (1986).PubMedGoogle Scholar
  67. 68.
    K.Takada and Ono. Y., Synchronous and sequential activation of latently infected Epstein-Barr virus genomes. J.Virol., 63: 445–449 (1989).PubMedGoogle Scholar
  68. 69.
    L.Rymo, Lindahl, T., Povey, S. and Klien, G., Anaylsis of restriction endonuclease fragments of intracellular Epstein-Barr virus type A (EBNA 2A) and type B (EBNA 2B) isolates extends to the EBNA 3 family of proteins. Virology, 115: 115–124 (1981).PubMedCrossRefGoogle Scholar
  69. 70.
    A.H.Wyllie, Morris, R.G., Smith, A.L. and Dunlop, D., Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol., 142: 67–77 (1984).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • N. J. McCarthy
    • 1
    • 2
  • S. A. Hazlewood
    • 1
    • 3
  • D. S. Huen
    • 3
  • A. B. Rickinson
    • 3
  • G. T. Williams
    • 1
    • 2
  1. 1.Department of Biological SciencesKeele UniversityKeele, StaffordshireUK
  2. 2.Department of AnatomyUK
  3. 3.CRC Laboratories, Department of Cancer StudiesUniversity of Birmingham Medical SchoolBirminghamUK

Personalised recommendations