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Chronic Lymphocytic Leukemia

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism

Leukemia volume 20, pages 272–279 (2006)Cite this article

Abstract

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

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References

  1. Hale C, Bartholomew M, Taylor V, Stables J, Topley P, Tite J . Recognition of CD52 allelic gene products by CAMPATH-1H antibodies. Immunology 1996; 88: 183–190.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hale G . The CD52 antigen and development of the CAMPATH antibodies. Cytotherapy 2001; 3 (3): 137–143.

    Article  CAS  PubMed  Google Scholar 

  3. Hale GXM, Tighe HP, Dyer MJ, Waldmann H . The CAMPATH-1 antigen (CDw52). Tissue Antigens 1990; 35: 118–127.

    Article  CAS  PubMed  Google Scholar 

  4. Ratzinger G, Reagan JL, Heller G, Busam KJ, Young JW . Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood 2003; 101: 1422–1429.

    Article  CAS  PubMed  Google Scholar 

  5. Buggins AG, Mufti GJ, Salisbury J, Codd J, Westwood N, Arno M et al. Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab. Blood 2002; 100: 1715–1720.

    CAS  PubMed  Google Scholar 

  6. Hale G, Rye PD, Warford A, Lauder I, Brito-Babapulle A . The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa. J Reprod Immunol 1993; 23: 189–205.

    Article  CAS  PubMed  Google Scholar 

  7. Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Dyer MJ et al. Levels of expression of CD52 in normal and leukemic B and T cells: correlation with in vivo therapeutic responses to Campath-1H. Leuk Res 1998; 22: 185–191.

    Article  CAS  PubMed  Google Scholar 

  8. Hale G, Dyer MJ, Clark MR, Phillips JM, Marcus R, Riechmann L et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988; 2 (8625): 1394–1399.

    Article  CAS  PubMed  Google Scholar 

  9. Pawson R, Dyer MJ, Barge R, Matutes E, Thornton PD, Emmett E et al. Treatment of T-cell prolymphocytic leukemia with human CD52 antibody. J Clin Oncol 1997; 15: 2667–2672.

    Article  CAS  PubMed  Google Scholar 

  10. Osterborg A, Dyer MJ, Bunjes D, Pangalis GA, Bastion Y, Catovsky D et al. Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia* European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia. J Clin Oncol 1997; 15: 1567–1574.

    Article  CAS  PubMed  Google Scholar 

  11. Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002; 99: 3554–3561.

    Article  CAS  PubMed  Google Scholar 

  12. Crowe JS, Hall VS, Smith MA, Cooper HJ, Tite JP . Humanized monoclonal antibody CAMPATH-1H: myeloma cell expression of genomic constructs, nucleotide sequence of cDNA constructs and comparison of effector mechanisms of myeloma and Chinese hamster ovary cell-derived material. Clin Exp Immunol 1992; 87: 105–110.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hale G, Bright S, Chumbley G, Hoang T, Metcalf D, Munro AJ et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62: 873–882.

    CAS  PubMed  Google Scholar 

  14. Xia MQ, Hale G, Waldmann H . Efficient complement-mediated lysis of cells containing the CAMPATH-1 (CDw52) antigen. Mol Immunol 1993; 30: 1089–1096.

    Article  CAS  PubMed  Google Scholar 

  15. Hale G, Clark M, Waldmann H . Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes. J Immunol 1985; 134: 3056–3061.

    CAS  PubMed  Google Scholar 

  16. Rowan WC, Hale G, Tite JP, Brett SJ . Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes. Int Immunol 1995; 7: 69–77.

    Article  CAS  PubMed  Google Scholar 

  17. Rowan W, Tite J, Topley P, Brett SJ . Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells. Immunology 1998; 95: 427–436.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zent CS, Chen JB, Kurten RC, Kaushal GP, Marie Lacy H, Schichman SA . Alemtuzumab (CAMPATH 1H) does not kill chronic lymphocytic leukemia cells in serum free medium. Leuk Res 2004; 28: 495–507.

    Article  CAS  PubMed  Google Scholar 

  19. Grdisa M . Sensitivity of B-Cell Chronic Lymphocytic Leukemia to Rituximab and Campath-1H and Correlation with the Expression of Cell Cycle Regulatory Proteins. Croat Med J 2004; 45: 136–141.

    PubMed  Google Scholar 

  20. Shan D, Ledbetter JA, Press OW . Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 1998; 91: 1644–1652.

    CAS  PubMed  Google Scholar 

  21. Hofmeister JK, Cooney D, Coggeshall KM . Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. Blood Cells Mol Dis 2000; 26: 133–143.

    Article  CAS  PubMed  Google Scholar 

  22. Pedersen IM, Buhl AM, Klausen P, Geisler CH, Jurlander J . The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism. Blood 2002; 99: 1314–1319.

    Article  CAS  PubMed  Google Scholar 

  23. Byrd JC, Kitada S, Flinn IW, Aron JL, Pearson M, Lucas D et al. The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction. Blood 2002; 99: 1038–1043.

    Article  CAS  PubMed  Google Scholar 

  24. Mateo V, Lagneaux L, Bron D, Biron G, Armant M, Delespesse G et al. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat Med 1999; 5: 1277–1284.

    Article  CAS  PubMed  Google Scholar 

  25. Nagy ZA, Hubner B, Lohning C, Rauchenberger R, Reiffert S, Thomassen-Wolf E, Zahn S et al. Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells. Nat Med 2002; 8: 801–807.

    Article  CAS  PubMed  Google Scholar 

  26. Mone AP, Huang P, Pelicano H, Cheney CM, Green JM, Tso JY . Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells. Blood 2004; 103: 1846–1854.

    Article  CAS  PubMed  Google Scholar 

  27. Suzuki T, Kiyokawa N, Taguchi T, Sekino T, Katagiri YU, Fujimoto J . CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system. J Immunol 2001; 166: 5567–5577.

    Article  CAS  PubMed  Google Scholar 

  28. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996; 87: 4990–4997.

    CAS  PubMed  Google Scholar 

  29. Weitkamp JH, Crowe Jr JE . Blood donor leukocyte reduction filters as a source of human B lymphocytes. Biotechniques 2001; 31: 464–466.

    Article  CAS  PubMed  Google Scholar 

  30. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH . Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415–1420.

    CAS  PubMed  Google Scholar 

  31. Byrd JC, Lucas DM, Mone A, Kitner JB, Drabick JJ, Grever MR . KRN5500: a novel therapeutic agent with in vitro activity against human B-cell chronic lymphocytic leukemia cells mediates cytotoxicity via the intrinsic pathway of apoptosis. Blood 2003; 101: 4547–4550.

    Article  CAS  PubMed  Google Scholar 

  32. Byrd JC, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, Nguyen PL et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 1998; 92: 3804–3816.

    CAS  PubMed  Google Scholar 

  33. Goebel J, Forrest K, Flynn D, Rao R, Roszman TL . Lipid rafts, major histocompatibility complex molecules, and immune regulation. Hum Immunol 2002; 63: 813–820.

    Article  CAS  PubMed  Google Scholar 

  34. Cinek T, Horejsi V . The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol 1992; 149: 2262–2270.

    CAS  PubMed  Google Scholar 

  35. Harder T, Simons K . Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid cells: accumulation of actin regulated by local tyrosine phosphorylation. Eur J Immunol 1999; 29: 556–562.

    Article  CAS  PubMed  Google Scholar 

  36. Fra AM, Williamson E, Simons K, Parton RG . Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem 1994; 269: 30745–30748.

    CAS  PubMed  Google Scholar 

  37. Harder T, Scheiffele P, Verkade P, Simons K . Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 1998; 141: 929–942.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Fishman PH . Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 1982; 69: 85–97.

    Article  CAS  PubMed  Google Scholar 

  39. Cheng PC, Dykstra ML, Mitchell RN, Pierce SK . A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J Exp Med 1999; 190: 1549–1560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Janes PW, Ley SC, Magee AI . Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol 1999; 147: 447–461.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Edmonds SD, Ostergaard HL . Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes. J Immunol 2002; 169: 5036–5042.

    Article  PubMed  Google Scholar 

  42. Aman MJ, Tosello-Trampont AC, Ravichandran K . Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts. J Biol Chem 2001; 276: 46371–46378.

    Article  CAS  PubMed  Google Scholar 

  43. Edidin M . The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 2003; 32: 257–283.

    Article  CAS  PubMed  Google Scholar 

  44. Munro S . Lipid rafts: elusive or illusive? Cell 2003; 115: 377–388.

    Article  CAS  PubMed  Google Scholar 

  45. Simons K, Ikonen E . Functional rafts in cell membranes. Nature 1997; 387 (6633): 569–572.

    Article  CAS  PubMed  Google Scholar 

  46. Brown DA, London E . Functions of lipid Rafts in Biological Membranes. Annual Review of Cell Developemental Biology 1998; 14: 111–136.

    Article  CAS  Google Scholar 

  47. Spiegel S, Kassis S, Wilchek M, Fishman PH . Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes. J Cell Biol 1984; 99: 1575–1581.

    Article  CAS  PubMed  Google Scholar 

  48. Kellie S, Patel B, Pierce EJ, Critchley DR . Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin. J Cell Biol 1983; 97: 447–454.

    Article  CAS  PubMed  Google Scholar 

  49. Godal T, Henriksen A, Iversen JG, Landaas TO, Lindmo T . Altered membrane-associated functions in chronic lymphocytic leukemia cells. Int J Cancer 1978; 21: 561–569.

    Article  CAS  PubMed  Google Scholar 

  50. Keller P, Simons K . Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol 1998; 140: 1357–1367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kilsdonk EP, Yancey PG, Stoudt GW, Bangerter FW, Johnson WJ, Phillips MC et al. Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem 1995; 270: 17250–17256.

    Article  CAS  PubMed  Google Scholar 

  52. Truman JP, Ericson ML, Choqueux-Seebold CJ, Charron DJ, Mooney NA . Lymphocyte programmed cell death is mediated via HLA class II DR. Int Immunol 1994; 6: 887–896.

    Article  CAS  PubMed  Google Scholar 

  53. Mateo V, Brown EJ, Biron G, Rubio M, Fischer A, Deist FL et al. Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization. Blood 2002; 100: 2882–2890.

    Article  CAS  PubMed  Google Scholar 

  54. Deans JP, Li H, Polyak MJ . CD20-mediated apoptosis: signalling through lipid rafts. Immunology 2002; 107: 176–182.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Garcia A, Cayla X, Fleischer A, Guergnon J, Alvarez-Franco Canas F, Rebollo MP et al. Rafts: a simple way to control apoptosis by subcellular redistribution. Biochimie 2003; 85: 727–731.

    Article  CAS  PubMed  Google Scholar 

  56. Semac I, Palomba C, Kulangara K, Klages N, van Echten-Deckert G, Borisch B et al. Anti-CD20 therapeutic antibody rituximab modifies the functional organization of rafts/microdomains of B lymphoma cells. Cancer Res 2003; 63: 534–540.

    CAS  PubMed  Google Scholar 

  57. Li H, Ayer LM, Lytton J, Deans JP . Store-operated cation entry mediated by CD20 in membrane rafts. J Biol Chem 2003; 278: 42427–42434.

    Article  CAS  PubMed  Google Scholar 

  58. Craig MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW et al. Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 2003; 101: 1045–1052.

    Article  Google Scholar 

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Acknowledgements

This work was supported by the National Cancer Institute (P01 CA95426), The Leukemia and Lymphoma Society and The D Warren Brown Foundation.

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Authors and Affiliations

  1. Division of Hematology-Oncology, Department of Medicine, The Ohio State University, Columbus, OH, USA

    A P Mone, C Cheney, A L Banks, N Mehter, S Guster, T Lin, C F Eisenbeis, D C Young & J C Byrd

  2. Division of Pulmonary Medicine, Department of Medicine, The Ohio State University, Columbus, OH, USA

    S Tridandapani

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  1. A P Mone
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Mone, A., Cheney, C., Banks, A. et al. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism. Leukemia 20, 272–279 (2006). https://doi.org/10.1038/sj.leu.2404014

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