Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (ΔΨm). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of ΔΨm, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of ΔΨm and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of ΔΨm. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis. Cell Death and Differentiation (2000) 7, 1090–1100

[1]  S. Wahl,et al.  Apoptosis induction by a novel anti-prostate cancer compound, BMD188 (a fatty acid-containing hydroxamic acid), requires the mitochondrial respiratory chain. , 1999, Cancer research.

[2]  Dean P. Jones,et al.  Prevention of Apoptosis by Bcl-2: Release of Cytochrome c from Mitochondria Blocked , 1997, Science.

[3]  N. Thornberry,et al.  Caspases: killer proteases. , 1997, Trends in biochemical sciences.

[4]  R. Armstrong,et al.  Cell-specific Induction of Apoptosis by Microinjection of Cytochrome c , 1997, The Journal of Biological Chemistry.

[5]  M. Latterich,et al.  The AAA team: related ATPases with diverse functions. , 1998, Trends in cell biology.

[6]  M. V. Heiden,et al.  Bcl-xL Regulates the Membrane Potential and Volume Homeostasis of Mitochondria , 1997, Cell.

[7]  V. Skulachev.  Cytochrome c in the apoptotic and antioxidant cascades , 1998, FEBS letters.

[8]  Sten Orrenius,et al.  Caspases: their intracellular localization and translocation during apoptosis , 1999, Cell Death and Differentiation.

[9]  Walter Neupert,et al.  Prohibitins Regulate Membrane Protein Degradation by the m-AAA Protease in Mitochondria , 1999, Molecular and Cellular Biology.

[10]  Y. Lazebnik,et al.  Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE , 1994, Nature.

[11]  N. Thornberry,et al.  Control of apoptosis by proteases. , 1997, Advances in pharmacology.

[12]  A. Perl,et al.  Elevation of mitochondrial transmembrane potential and reactive oxygen intermediate levels are early events and occur independently from activation of caspases in Fas signaling. , 1999, Journal of immunology.

[13]  D. Green,et al.  The Release of Cytochrome c from Mitochondria: A Primary Site for Bcl-2 Regulation of Apoptosis , 1997, Science.

[14]  D. Vaux,et al.  CED-4—The Third Horseman of Apoptosis , 1997, Cell.

[15]  D. Green,et al.  Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD‐specific caspase activation and independently of mitochondrial transmembrane depolarization , 1998, The EMBO journal.

[16]  Y. Lazebnik,et al.  Caspases: enemies within. , 1998, Science.

[17]  D. Green,et al.  p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release* , 2000, The Journal of Biological Chemistry.

[18]  S. Srinivasula,et al.  Cytochrome c and dATP-Dependent Formation of Apaf-1/Caspase-9 Complex Initiates an Apoptotic Protease Cascade , 1997, Cell.

[19]  P. Tawa,et al.  Hsp60 accelerates the maturation of pro‐caspase‐3 by upstream activator proteases during apoptosis , 1999, The EMBO journal.

[20]  A. Lehninger,et al.  Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. , 1985, Archives of biochemistry and biophysics.

[21]  W. Neupert,et al.  The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m‐AAA protease , 1998, The EMBO journal.

[22]  Gerard I. Evan,et al.  The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant , 2000, Nature Cell Biology.

[23]  Miguel Ángel Martínez,et al.  Tumor Necrosis Factor-α Increases the Steady-state Reduction of Cytochrome b of the Mitochondrial Respiratory Chain in Metabolically Inhibited L929 Cells* , 2000, The Journal of Biological Chemistry.

[24]  T. Langer,et al.  AAA proteases: cellular machines for degrading membrane proteins. , 2000, Trends in biochemical sciences.

[25]  R. Gottlieb,et al.  Loss of Function of Cytochrome c in Jurkat Cells Undergoing Fas-mediated Apoptosis* , 1996, The Journal of Biological Chemistry.

[26]  J C Reed,et al.  Mitochondria and apoptosis. , 1998, Science.

[27]  Y. Hatefi.  The mitochondrial electron transport and oxidative phosphorylation system. , 1985, Annual review of biochemistry.

[28]  G. Salvesen,et al.  Caspases - controlling intracellular signals by protease zymogen activation. , 2000, Biochimica et biophysica acta.

[29]  J. Turrens,et al.  Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. , 1980, The Biochemical journal.

[30]  Xiaodong Wang,et al.  Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c , 1996, Cell.

[31]  Z. Darżynkiewicz,et al.  During apoptosis of HL-60 and U-937 cells caspases are activated independently of dissipation of mitochondrial electrochemical potential. , 2000, Experimental cell research.

[32]  G. Steele,et al.  Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Junying Yuan,et al.  Human ICE/CED-3 Protease Nomenclature , 1996, Cell.

[34]  Seamus J. Martin,et al.  Cytochrome c activation of CPP32‐like proteolysis plays a critical role in a Xenopus cell‐free apoptosis system , 1997, The EMBO journal.

[35]  B. Zhivotovsky,et al.  Presence of a pre‐apoptotic complex of pro‐caspase‐3, Hsp60 and Hsp10 in the mitochondrial fraction of Jurkat cells , 1999, The EMBO journal.

[36]  J. Bancroft,et al.  Theory and Practice of Histological Techniques , 1990 .

[37]  N. Thornberry,et al.  The Caspase-3 Precursor Has a Cytosolic and Mitochondrial Distribution: Implications for Apoptotic Signaling , 1998, The Journal of cell biology.

[38]  C. Franceschi,et al.  JC‐1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis , 1997, FEBS letters.

[39]  Xiaodong Wang,et al.  Apaf-1, a Human Protein Homologous to C. elegans CED-4, Participates in Cytochrome c–Dependent Activation of Caspase-3 , 1997, Cell.