Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Manuscript
  • Published:

Lymphoma

Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line

Leukemia volume 14, pages 2149–2158 (2000)Cite this article

Abstract

CD95 (Fas/Apo-1) is a transmembrane molecule that induces apoptosis and plays a central role in the regulation of the immune response. The present study describes two new B lymphoid cell lines, B593 and BR97, derived from non-Hodgkin's lymphoma, which differ in susceptibility to CD95-mediated apoptosis. While B593 cells are sensitive to CD95-mediated apoptosis, BR97 cells are completely resistant. Activation of caspase-8 and caspase-3 proteases plays an important role in the CD95 signalling pathway. CD95 stimulation induced caspase-8 and caspase-3 activation in B593, but not in BR97 cells. However, activation of both caspase-8 and caspase-3 was achieved in BR97 cells treated with staurosporine. Furthermore, protein synthesis inhibition by cycloheximide restored sensitivity to CD95-mediated apoptosis and allowed activation of both caspase-8 and caspase-3 in BR97 cells. These results indicate that, in BR97 cells, both caspases are functional and suggest that CD95-apoptosis resistance may result from the presence of inhibitory factor(s). Constitutive high level expression of the apoptotic inhibitor c-FLIP was observed in the CD95-resistant BR97 cell line compared to B593. Moreover, downregulation of c-FLIP expression level by protein synthesis inhibition strictly correlated with restored sensitivity to CD95-mediated apoptosis in BR97 cells. Furthermore, we demonstrate that c-FLIP is recruited to the CD95 DISC in BR97 cells together with caspase-8 and FADD. The data presented in this study strongly suggests that, in a B-NHL-derived cell line, resistance to CD95-mediated apoptosis results from endogenous high level expression of apoptotic inhibitor c-FLIP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  1. Nagata S, Golstein P . The Fas death factor Science 1995 267: 1449–1455

    Article  CAS  PubMed  Google Scholar 

  2. Steller H . Mechanisms and genes of cellular suicide Science 1995 267: 1445–1449

    Article  CAS  PubMed  Google Scholar 

  3. Trauth BC, Klas C, Peters AMJ, Matzku S, Möller P, Falk W, Debatin KM, Krammer PH . Monoclonal antibody-mediated tumor regression by induction of apoptosis Science 1989 245: 301–305

    Article  CAS  PubMed  Google Scholar 

  4. Yonehara S, Ishii A, Yonehara M . A cell killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor J Exp Med 1989 169: 1747–1756

    Article  CAS  PubMed  Google Scholar 

  5. Matiba B, Mariani SM, Krammer PH . The CD95 system and the death of a lymphocyte Semin Immunol 1997 9: 59–68

    Article  CAS  PubMed  Google Scholar 

  6. Lowin B, Hahne M, Mattmann C, Tschopp J . Cytolytic T cell cytotoxicity is mediated through perforin and Fas lytic pathways Nature 1994 370: 650–652

    Article  CAS  PubMed  Google Scholar 

  7. Kägi D, Vignaux F, Ledermann B, Bürki K, Depraetere V, Nagata S, Hengartner H, Golstein P . Fas and perforin pathways as major mechanisms of T cell mediated cytotoxicity Science 1994 265: 528–530

    Article  PubMed  Google Scholar 

  8. Scott DW, Grdina T, Shi Y . T cell commit suicide, but B cells are murdered J Immunol 1996 156: 2352–2356

    CAS  PubMed  Google Scholar 

  9. Rieux Laucat F, Le Deist F, Hivroz C, Roberts IAG, Debatin KM, Fischer A, De Villartay JP . Mutation in Fas associated with human lymphoproliferative syndrome and autoimmunity Science 1995 268: 1347–1349

    Article  CAS  PubMed  Google Scholar 

  10. Fischer GH, Rosenberg FJ, Straus SE, Dale JK, Middelton LA, Lin AY, Strober W, Lenardo MJ, Puck JM . Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproloferative syndrome Cell 1995 81: 935–946

    Article  Google Scholar 

  11. Rathmell JC, Townsend SE, Xu JC, Flavell RA, Goodnow CC . Expansion or elimination of B cells in vivo: dual roles for CD40 and Fas (CD95) ligands modulated by the B cell antigen receptor Cell 1996 87: 319–329

    Article  CAS  PubMed  Google Scholar 

  12. Xerri L, Carbuccia N, Parc P, Hassoun J, Birg F . Frequent expression of Fas/APO-1 in Hodgkin's disease and anaplastic large cell lymphomas Histopathology 1995 27: 235–241

    Article  CAS  PubMed  Google Scholar 

  13. Nguyen PL, Harris NL, Ritz J, Robertson MJ . Expression of CD95 antigen and Bcl-2 protein in non-Hodgkin's lymphomas and Hodgkin's disease Am J Pathol 1996 148: 847–853

    CAS  PubMed Central  PubMed  Google Scholar 

  14. Debatin KM, Fahrig-Faissner A, Enenkel-Stoodt S, Kreuz W, Benner A, Krammer PH . High expression of APO-1 (CD95) on T-lymphocytes from HIV infected children Blood 1994 83: 3101–3103

    Article  CAS  PubMed  Google Scholar 

  15. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary ML, Delsol G, Dewolfpeeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, Mullerhermelink HK, Pileri SA, Piris MA, Ralfkiaer E, Warnke RA . A revised European–American classification of lymphoid neoplasmes: a proposal from the International Lymphoma Study Group Blood 1994 84: 1361–1392

    Article  CAS  PubMed  Google Scholar 

  16. Plumas J, Jacob MC, Chaperot L, Molens JP, Sotto JJ, Bensa JC . Tumor B cells from non-Hodgkin's lymphoma are resistant to CD95 (Fas/APO-1)-mediated apoptosis Blood 1998 91: 1–12

    Article  Google Scholar 

  17. Peter ME, Heufelder AE, Hengartner MO . Advances in apoptosis research Proc Natl Acad Sci USA 1997 94: 12736–12737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nicholson DW, Thornberry NA . Caspases killer proteases Trends Biochem Sci 1997 22: 299–306

    Article  CAS  PubMed  Google Scholar 

  19. Ashkenazi A, Dixit VM . Death receptor: signaling and modulation Science 1998 281: 1305–1308

    Article  CAS  PubMed  Google Scholar 

  20. Peter ME, Kischkel FC, Hellbardt S, Chinnaiyan AM, Krammer PH, Dixit VM . CD95 (APO1/Fas)-associating signalling proteins Cell Death Diff 1996 3: 161–170

    CAS  Google Scholar 

  21. Muzio M, Stockwell BR, Stennicke HR, Salvesen GS, Dixit VM . An induced proximity model for caspase-8 activation J Biol Chem 1998 273: 2926–2930

    Article  CAS  PubMed  Google Scholar 

  22. Fernandes-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L, Tomaselli KJ, Wang L, Yu Z, Croce CM, Salveson G, Earnshaw WC, Litwack G, Alnemri ES . Mch3, a novel human apoptotic cysteine protease highly related to CPP32 Cancer Res 1995 55: 6045–6052

    CAS  PubMed  Google Scholar 

  23. Enari M, Talanian RV, Wong WW, Nagata S . Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis Nature 1996 380: 723–726

    Article  CAS  PubMed  Google Scholar 

  24. Takahashi A, Hirata H, Yonehara S, Imai Y, Lee KK, Moyer RW, Turner PC, Mesner PW, Okazaki T, Sawai H, Kishi S, Yamamoto K, Okuma M, Sasada M . Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8 Oncogene 1997 14: 2741–2752

    Article  CAS  PubMed  Google Scholar 

  25. Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM . Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase Cell 1995 81: 801–809

    Article  CAS  PubMed  Google Scholar 

  26. Jacobson MD, Weil M, Raff MC . Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death J Cell Biol 1996 133: 1041–1051

    Article  CAS  Google Scholar 

  27. Faleiro L, Kobayashi R, Fearnhead H, Lazebnik Y . Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells EMBO J 1997 16: 2271–2281

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Thompson CB . Apoptosis in the pathogenesis and treatement of disease Science 1995 267: 1456–1462

    Article  CAS  PubMed  Google Scholar 

  29. Yang E, Korsmeyer SJ . Molecular thanatopsis: a discourse on the Bcl-2 family and cell death Blood 1996 88: 386–401

    Article  CAS  PubMed  Google Scholar 

  30. Kroemer G, Zamzami N, Susin SA . Mitochondrial control of apoptosis Immunol Today 1997 18: 44–51

    Article  CAS  PubMed  Google Scholar 

  31. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, Peter ME . Two CD95 (APO-1/Fas) signaling pathways EMBO J 1998 17: 1675–1687

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Peter ME, Kischkel FC, Scheuerpflug CG, Medema JP, Debatin KM, Krammer PH . Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex Eur J Immunol 1997 27: 1207–1212

    Article  CAS  PubMed  Google Scholar 

  33. Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schröter M, Burns K, Mattmann C, Rimoldi D, French LE, Tschopp J . Inhibition of death receptor signals by cellular FLIP Nature 1997 388: 190–195

    Article  CAS  PubMed  Google Scholar 

  34. Goltsev YV, Kovalenko AV, Arnold E, Varfolomeev EE, Brodianskii VM, Wallach D . CASH, a novel caspase homologue with death effector domains J Biol Chem 1997 272: 19641–19644

    Article  CAS  PubMed  Google Scholar 

  35. Inohara N, Koseki T, Hu Y, Chen S, Nunez G . CLARP, a death effector domain-containing protein interacts with caspase-8 and regulates apoptosis Proc Natl Acad Sci USA 1997 94: 10717–10722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kataoka T, Schröter M, Hahne M, Schneider P, Irmler M, Thome M, Froelich CJ, Tschopp J . FLIP prevents apoptosis induced by death receptors but non by perforin/granzyme B, chemotherapeutic drugs, and gamma irradiation J Immunol 1998 161: 3936–3942

    CAS  PubMed  Google Scholar 

  37. Callanan M, Le Baccon P, Mossuz P, Duley S, Bastard C, Hamoudi R, Dyer MJ, Klobeck G, Rimokh R, Sotto JJ, Leroux D . The IgG Fc receptor, Fc γ RIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma Proc Natl Acad Sci USA 2000 97: 309–314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C . A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V J Immunol Methods 1995 184: 39–51

    Article  CAS  PubMed  Google Scholar 

  39. Scaffidi C, Kischkel FC, Krammer PH, Peter ME . Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signalling complex Methods 1999 17: 287–291

    Article  CAS  PubMed  Google Scholar 

  40. Dirks W, Schöne S, Uphoff C, Quentmeier H, Pradella S, Drexler HG . Expression and function of CD95 (Fas/APO-1) in leukaemia–lymphoma tumor lines Br J Haematol 1997 96: 584–593

    Article  CAS  PubMed  Google Scholar 

  41. Xerri L, Bouabdallah R, Devilard E, Hassoun J, Stoppa AM, Birg F . Sensitivity to Fas-mediated apoptosis is null or weak in B-cell non-Hodgkin's lymphomas and is moderately increased by CD40 ligation Br J Cancer 1998 78: 225–232

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Yoshino T, Kondo E, Cao L, Takahashi K, Hayashi K, Nomura S, Akagi T . Inverse expression of Bcl-2 protein and Fas antigen in lymphoblasts in peripheral lymph nodes and activated peripheral blood T and B lymphocytes Blood 1994 83: 1856–1861

    Article  CAS  PubMed  Google Scholar 

  43. Miyawaki T, Uehara T, Nibu R, Tsuji T, Yachie A, Yonehara S, Taniguchi N . Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood J Immunol 1992 149: 3753–3758

    CAS  PubMed  Google Scholar 

  44. Garrone P, Neidhardt EM, Garcia E, Galibert L, van-Kooten C, Banchereau J . Fas ligation induces apoptosis of CD40-activated human B lymphocytes J Exp Med 1995 182: 1265–1273

    Article  CAS  PubMed  Google Scholar 

  45. Schattner EJ, Elkon KB, Yoo DH, Tumang J, Krammer PH, Crow MK, Friedman SM . CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and facilitates apoptosis through the Apo-1/Fas pathway J Exp Med 1995 182: 1557–1565

    Article  CAS  PubMed  Google Scholar 

  46. Scaffidi C, Schmitz I, Krammer PH, Peter ME . The role of c-FLIP in modulation of CD95-induces apoptosis J Biol Chem 1999 274: 1541–1548

    Article  CAS  PubMed  Google Scholar 

  47. Strasser A, Harris AW, Huang DCS, Krammer PH, Cory S . Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis EMBO J 1995 14: 6136–6147

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Chinnaiyan AM, Orth K, O'Rourke K, Duan H, Poirier GG, Dixit VM . Molecular ordering of the cell death pathway: Bcl-2 and Bcl-xL function upstream of the ced-3 like apoptotic proteases J Biol Chem 1996 271: 4573–4576

    Article  CAS  PubMed  Google Scholar 

  49. Huang DCS, Cory S, Strasser A . Bcl-2, Bcl-xL and adenovirus protein E1B19 kD are functionally equivalent in their ability to inhibit cell death Oncogene 1997 14: 405–414

    Article  CAS  PubMed  Google Scholar 

  50. Armstrong RC, Aja T, Xiang J, Gaur S, Krebs JF, Hoang K, Bai X, Korsmeyer SJ, Karanewsky DS, Fritz LC, Tomaselli KJ . Fas-induced activation of cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors J Biol Chem 1996 271: 16850–16855

    Article  CAS  PubMed  Google Scholar 

  51. Boise LH, Thompson CB . Bcl-xL can inhibit apoptosis in cells that have undergone Fas-induced proteases activation Proc Natl Acad Sci USA 1997 94: 3759–3764

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Medema JP, Scaffidi C, Krammer PH, Peter ME . Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex J Biol Chem 1998 273: 3388–3393

    Article  CAS  PubMed  Google Scholar 

  53. Schneider TJ, Grillot D, Foote LC, Nunez GE, Rothstein TL . Bcl-x protects primary B cells against Fas-mediated apoptosis J Immunol 1997 159: 4834–4839

    CAS  PubMed  Google Scholar 

  54. Vassilev A, Ozer Z, Navara C, Mahajan S, Uckun FM . Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex J Biol Chem 1999 274: 1646–1656

    Article  CAS  PubMed  Google Scholar 

  55. Ambrosini G, Adida C, Altieri DC . A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma Nature Med 1997 3: 917–921

    Article  CAS  PubMed  Google Scholar 

  56. Duckett CS, Nava VE, Gedrich RW, Clem RJ, Van Dongen JL, Gilfillan MC, Shiels H, Hardwick JM, Thompson CB . A conserved family of cellular genes related to the baculovirus iap gene and encoding apoptosis inhibitors EMBO J 1996 15: 2685–2689

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  57. Cascino I, Papoff G, De Maria R, Testi R, Ruberti G . CD95 receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas mediated apoptosis J Immunol 1996 156: 13–17

    CAS  PubMed  Google Scholar 

  58. Tamiya S, Etoh KI, Suzushima H, Takatsuki K, Matsuoka M . Mutation of CD95 (Fas/Apo-1) gene in adult T-cell leukemia cells Blood 1998 91: 3935–3942

    Article  CAS  PubMed  Google Scholar 

  59. Xerri L, Carbuccia N, Birg F . Search for rearrangements and/or allelic loss of the Fas/APO-1 gene in 101 human lymphomas Am J Pathol 1995 104: 424–430

    CAS  Google Scholar 

  60. Eberstadt M, Huang B, Chen Z, Meadows RP, Ng SC, Zheng L, Lenardo MJ, Fesik SW . NMR structure and mutagenesis of the FADD (Mort 1) death-effector domain Nature 1998 392: 941–945

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the French National League Against Cancer (Haute-Savoie), from the SIVOM, Region of Cluses (Haute Savoie), and from the ARAMIS association, France.

Author information

Authors and Affiliations

  1. Molecular Basis of Tumoral Progression, Research Group on Lymphoma, Albert Bonniot Institute, University Joseph Fourier, La Tronche, France

    M Irisarri, J Plumas, T Bonnefoix, M-C Jacob, C Roucard, M-A Pasquier, J-J Sotto & A Lajmanovich

Authors
  1. M Irisarri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Plumas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T Bonnefoix
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M-C Jacob
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C Roucard
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M-A Pasquier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J-J Sotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A Lajmanovich
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Irisarri, M., Plumas, J., Bonnefoix, T. et al. Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line. Leukemia 14, 2149–2158 (2000). https://doi.org/10.1038/sj.leu.2401954

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2401954

Keywords

This article is cited by

Search

Advanced search

Quick links