, Volume 6, Issue 6, pp 419–429

Mechanism of apoptosis induced by zinc deficiency in peripheral blood T lymphocytes

  • V. M. Kolenko
  • R. G. Uzzo
  • N. Dulin
  • E. Hauzman
  • R. Bukowski
  • J. H. Finke


Alterations in intracellular Zn2+ concentrations are believed to play a crucial role in modulating apoptosis. The observation that Zn2+ deficiency can induce cell death both in vivo and in vitro has been attributed to the fact that exchange of Zn2+ for Ca2+ and Mg2+ within the nuclei may directly activate endogenous endonucleases therefore inducing DNA fragmentation independent of cytoplasmic factors. Here we show that the membrane-permeable zinc chelator, N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces translocation of cytochrome c from the mitochondrial intramembranous space into the cytosol in human peripheral blood T lymphocytes (PBL) with subsequent activation of caspases-3, -8, and -9. Pretreatment of T lymphocytes with caspase inhibitors Z-VAD.fmk or DEVD.fmk prevented DNA fragmentation in response to TPEN indicating that apoptosis triggered by zinc deficiency is entirely dependent on activation of caspase family members. The release of cytochrome c and activation of downstream caspases precedes changes in the mitochondrial transmembrane potential (Δ Ψm). Therefore, cytoplasmic and mitochondrial events are critical to this process.

apoptosis caspases cytochrome c T lymphocytes zinc 


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  1. 1.
    Martin S, Mazdai G, Strain J, et al. Programmed cell death (apoptosis) in lymphoid and myeloid cell lines during zinc deficiency. Clin Exp Immunol 1991; 83: 338-343.Google Scholar
  2. 2.
    Zalewski P, Forbes I, Giannakis C, Betts W. Regulation of protein kinase C by Zn(2+)-dependent interaction with actin. Biochem Int 1991; 24: 1103-1110.Google Scholar
  3. 3.
    Cohen J, Duke R. Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J Immunol 1984; 132: 38-42.Google Scholar
  4. 4.
    Giannakis C, Forbes I, Zalewski P. Ca2+/Mg(2+)-dependent nuclease: Tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+. Biochem Biophys Res Commun 1991; 181: 915-920.Google Scholar
  5. 5.
    Zhivotovsky B, Wade D, Gahm A, et al. Formation of 50 kbp chromat in fragments in isolated liver nuclei is mediated by protease and endonuclease activation. FEBS Lett 1994; 351:150-154.Google Scholar
  6. 6.
    Stennicke H, Salvesen G. Biochemical characteristics of caspases-3,-6,-7, and-8. J Biol Chem 1997; 272: 25719-25723.Google Scholar
  7. 7.
    McCabe MJ, Jiang S, Orrenius S. Chelation of intracellular zinc triggers apoptosis in mature thymocytes. Lab Invest 1993; 69: 101-110.Google Scholar
  8. 8.
    Sunderman FJ. The influence of zinc on apoptosis. Ann Clin Lab Sci 1995; 25:134-142.Google Scholar
  9. 9.
    Green D, Kroemer G. The central executioners of apoptosis: Caspases or mitochondria? Trends Cell Biol 1998; 8: 267-271.Google Scholar
  10. 10.
    Green D. Apoptotic pathways: The roads to ruin. Cell 1998; 94: 695-698.Google Scholar
  11. 11.
    Ashkenazi A, Dixit V. Death receptors: Signaling and modulation. Science 1998; 281: 1305-1308.Google Scholar
  12. 12.
    Fernandes-Alnemri T, Armstrong R, Krebs J, et al. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci USA 1996; 93: 7464-7469.Google Scholar
  13. 13.
    Muzio M, Chinnaiyan A, Kischkel F, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 1996; 85: 817-827.Google Scholar
  14. 14.
    Medema J, Scaffidi C, Kischkel F, et al. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J 1997; 16: 2794-2804.Google Scholar
  15. 15.
    Hirata H, Takahashi A, Kobayashi S, et al. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J Exp Med 1998; 187: 587-600.Google Scholar
  16. 16.
    Muzio M, Salvesen G, Dixit V. FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J Biol Chem 1997; 272: 2952-2956.Google Scholar
  17. 17.
    Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998; 17: 1675-1687.Google Scholar
  18. 18.
    Dumont A, Hehner S, Hofmann T, et al. Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB. Oncogene 1999; 18: 747-757.Google Scholar
  19. 19.
    Ferrari D, Stepczynska A, Los M, et al. Differential regulation and ATP requirement for caspase-8 and caspase-3 activation during CD95-and anticancer drug-induced apoptosis. J Exp Med 1998; 188: 979-984.Google Scholar
  20. 20.
    Fulda S, Friesen C, Los M, et al. Betulinic acid triggers CD95 (APO-1/Fas)-and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res 1997; 57: 4956-4964.Google Scholar
  21. 21.
    Green D, Reed J. Mitochondria and apoptosis. Science 1998; 281: 1309-1312.Google Scholar
  22. 22.
    Lemasters JV. Necrapoptosis and the mitochondrial permeability transition: Shared pathways to necrosis and apoptosis. Am J Physiol 1999; 276: 1-6.Google Scholar
  23. 23.
    Susin S, Zamzami N, Castedo M, et al. The central executioner of apoptosis: Multiple connections between protease activation and mitochondria in Fas/APO-1/CD95-and ceramide-induced apoptosis. J Exp Med 1997; 186: 25-37.Google Scholar
  24. 24.
    Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479-489.Google Scholar
  25. 25.
    Slee E, Harte M, Kluck R, et al. Ordering the cytochrome c-initiated caspase cascade: Hierarchical activation of caspases-2,-3,-6,-7,-8, and-10 in a caspase-9-dependent manner. J Cell Biol 1999; 144: 281-292.Google Scholar
  26. 26.
    Srinivasula S, Ahmad M, Fernandes-Alnemri T, Alnemri E. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1998; 1: 949-957.Google Scholar
  27. 27.
    Csermely P, Szamel M, Resch K, Somogyi J. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes. J Biol Chem 1988; 263: 6487-6490.Google Scholar
  28. 28.
    Treves S, Trentini P, Ascanelli M, et al. Apoptosis is dependent on intracellular zinc and independent of intracellular calcium in lymphocytes. Exp Cell Res 1994; 211: 339-343.Google Scholar
  29. 29.
    Finke J, Zea A, Stanley J, et al. Loss of T-cell receptor zeta chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res 1993; 53: 5613-5616.Google Scholar
  30. 30.
    Wang Q, Stanley J, Kudoh S, et al. T cells infiltrating non-Hodgkin's B cell lymphomas show altered tyrosine phosphorylation pattern even though T cell receptor/CD3-associated kinases are present. J Immunol 1995; 155: 1382-1392.Google Scholar
  31. 31.
    Zamzami N, Marchetti P, Castedo M, et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 1995; 181: 1661-1672.Google Scholar
  32. 32.
    Ahn Y, Kim Y, Hong S, Koh J. Depletion of intracellular zinc induces protein synthesis-dependent neuronal apoptosis in mouse cortical culture. Exp Neurol 1998; 154: 47-56.Google Scholar
  33. 33.
    Kolenko V, Uzzo R, Bukowski R, et al. Dead or dying: Necrosis versus apoptosis in caspase-deficient human renal cell carcinoma. Cancer Res 1999; 59: 2838-2842.Google Scholar
  34. 34.
    Bradham C, Qian T, Streetz K, et al. The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol Cell Biol 1998; 18: 6353-6364.Google Scholar
  35. 35.
    Chinnaiyan A, Orth K, O'Rourke K, et al. 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.Google Scholar
  36. 36.
    Enari M, Talanian R, Wong W, Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 1996; 380: 723-726.Google Scholar
  37. 37.
    Longthorne V, Williams G. Caspase activity is required for commitment to Fas-mediated apoptosis. EMBO J 1997; 16: 3805-3812.Google Scholar
  38. 38.
    Aragane Y, Kulms D, Metze D, et al. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. J Cell Biol 1998; 140: 171-182.Google Scholar
  39. 39.
    Froelich C, Orth K, Turbov J, et al. New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. J Biol Chem 1996; 271: 29073-29079.Google Scholar
  40. 40.
    Talanian R, Yang X, Turbov J, et al. Granule-mediated killing: Pathways for granzyme B-initiated apoptosis. J Exp Med 1997; 186: 1323-1331.Google Scholar
  41. 41.
    Garcia-Calvo M, Peterson E, Leiting B, et al. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 1998; 273: 32608-32613.Google Scholar
  42. 42.
    Thornberry N, Rano T, Peterson E, et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 1997; 272: 17907-17911.Google Scholar
  43. 43.
    Fernandes-Alnemri T, Takahashi A, Armstrong R, et al. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res 1995; 55: 6045-6052.Google Scholar
  44. 44.
    Nicholson D, Ali A, Thornberry N, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 1995; 376: 37-43.Google Scholar
  45. 45.
    Tewari M, Quan L, O'Rourke K, et al. 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.Google Scholar
  46. 46.
    Liu X, Kim C, Yang J, et al. Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 1996; 86: 147-157.Google Scholar
  47. 47.
    Zou H, Henzel W, Liu X, Lut et al. Apaf-1, a human protein homologous to C. Elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997; 90: 405-413.Google Scholar
  48. 48.
    Pan G, Humke E, Dixit V. Activation of caspases triggered by cytochrome c in vitro. FEBS Lett 1998; 426: 151-154.Google Scholar
  49. 49.
    MacDonald G, Shi L, Vande VC, et al. Mitochondria-dependent and-independent regulation of Granzyme B-induced apoptosis. J Exp Med 1999; 189: 131-144.Google Scholar
  50. 50.
    Bernardi P, Broekemeier K, Pfeiffer D. Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane. J Bioenerg Biomembr 1994; 26: 509-517.Google Scholar
  51. 51.
    Petit P, Susin S, Zamzami N, et al. Mitochondria and programmed cell death: Back to the future. FEBS Lett 1996; 396: 7-13.Google Scholar
  52. 52.
    Zoratti M, Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1995; 1241: 139-176.Google Scholar
  53. 53.
    Hirsch T, Marchetti P, Susin S, et al. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 1997; 15: 1573-1581.Google Scholar
  54. 54.
    Krippner A, Matsuno-Yagi A, Gottlieb R, Babior B. Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis. J Biol Chem 1996; 271: 21629-21636.Google Scholar
  55. 55.
    Vayssiere J, Petit P, Risler Y, Mignotte B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40. Proc Natl Acad Sci USA 1994; 91: 11752-11756.Google Scholar
  56. 56.
    Pastorino J, Chen S, Tafani MS, et al. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J Biol Chem 1998; 273: 7770-7775.Google Scholar
  57. 57.
    Kluck R, Bossy-Wetzel E, Green D, Newmeyer D. The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 1997; 275: 1132-1136.Google Scholar
  58. 58.
    Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science 1997; 275: 1129-1132.Google Scholar
  59. 59.
    Kayagaki N, Kawasaki A, Ebata T, et al. Metalloproteinase-mediated release of human Fas ligand. J Exp Med 1995; 182: 1777-1783.Google Scholar
  60. 60.
    Nakagawa T, Kubota T, Kabuto M, Kodera T. Captopril inhibits glioma cell invasion in vitro: Involvement of matrix metalloproteinases. Anticancer Res 1995; 15: 1985-1989.Google Scholar
  61. 61.
    Wojtowicz-Praga S, Dickson R, Hawkins M. Matrix metalloproteinase inhibitors. Invest New Drugs 1997; 15: 61-75.Google Scholar
  62. 62.
    Suda T, Hashimoto H, Tanaka M, et al. Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing. J Exp Med 1997; 186: 2045-2050.Google Scholar
  63. 63.
    Delneste Y, Jeannin P, Sebille E, et al. Thiols prevent Fas (CD95)-mediated T cell apoptosis by down-regulating membrane Fas expression. Eur J Immunol 1996; 26: 2981-2988.Google Scholar
  64. 64.
    Kluck R, Martin S, Hoffman B, et al. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J 1997; 16: 4639-4649.Google Scholar
  65. 65.
    Jurgensmeier J, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 1998; 95: 4997-5002.Google Scholar
  66. 66.
    Li H, Zhu H, Xu C, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998; 94: 491-501.Google Scholar
  67. 67.
    Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 1998; 94: 481-490.Google Scholar
  68. 68.
    Susin S, Lorenzo H, Zamzami N, et al. Mitochondrial release of caspase-2 and-9 during the apoptotic process. J Exp Med 1999; 189: 381-394.Google Scholar
  69. 69.
    Susin S, Lorenzo H, Zamzami N, et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397: 441-446.Google Scholar
  70. 70.
    Petit P, Goubern M, Diolez P, et al. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: The impact of irreversible permeability transition. FEBS Lett 1998; 426: 111-116.Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • V. M. Kolenko
    • 1
    • 2
  • R. G. Uzzo
    • 1
    • 3
  • N. Dulin
    • 4
  • E. Hauzman
    • 5
  • R. Bukowski
    • 1
    • 2
  • J. H. Finke
    • 1
    • 3
    • 2
  1. 1.Department of ImmunologyThe Cleveland Clinic FoundationClevelandUSA
  2. 2.Experimental Therapeutics ProgramThe Cleveland Clinic FoundationClevelandUSA
  3. 3.Department of UrologyThe Cleveland Clinic FoundationClevelandUSA
  4. 4.Department of PharmacologyUniversity of Illinois at ChicagoChicagoUSA
  5. 5.Boston Biomedical Research InstituteWatertownUSA

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