Characterization of Caspase Processing and Activation in HL-60 Cell Cytosol Under Cell-free Conditions

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 °C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitroand in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5′-adenylyl methylenediphosphate, 5′-adenylyl imidodiphosphate, or 5′-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn2+, aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin,N α-p-tosyl-l-phenylalanine chloromethyl ketone,N α-p-tosyl-l-lysine chloromethyl ketone, andN-(N α-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.

[1]  A. Fornace,et al.  Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells , 1999, Oncogene.

[2]  W. Earnshaw,et al.  Comparison of Paclitaxel-, 5-Fluoro-2′-deoxyuridine-, and Epidermal Growth Factor (EGF)-induced Apoptosis , 1999, The Journal of Biological Chemistry.

[3]  J C Reed,et al.  Caspase-9 Can Be Activated without Proteolytic Processing* , 1999, The Journal of Biological Chemistry.

[4]  M MacFarlane,et al.  Distinct Caspase Cascades Are Initiated in Receptor-mediated and Chemical-induced Apoptosis* , 1999, The Journal of Biological Chemistry.

[5]  Emad S. Alnemri,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 , 1999, The Journal of cell biology.

[6]  M. Prevost,et al.  Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process , 1999, The Journal of experimental medicine.

[7]  S H Kaufmann,et al.  Mammalian caspases: structure, activation, substrates, and functions during apoptosis. , 1999, Annual review of biochemistry.

[8]  N. Thornberry,et al.  Inhibition of Human Caspases by Peptide-based and Macromolecular Inhibitors* , 1998, The Journal of Biological Chemistry.

[9]  John Calvin Reed,et al.  Regulation of cell death protease caspase-9 by phosphorylation. , 1998, Science.

[10]  W. Earnshaw,et al.  Phosphorylated forms of activated caspases are present in cytosol from HL-60 cells during etoposide-induced apoptosis. , 1998, Blood.

[11]  M. Hengartner.  Death by Crowd Control , 1998, Science.

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

[13]  Keisuke Kuida,et al.  Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9 , 1998, Cell.

[14]  R. Bertrand,et al.  Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities. , 1998, Cancer research.

[15]  S. Srinivasula,et al.  Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. , 1998, Molecular cell.

[16]  V. Cryns,et al.  Proteases to die for. , 1998, Genes & development.

[17]  U. Rawat,et al.  Interactions of chaperone alpha-crystallin with the molten globule state of xylose reductase. Implications for reconstitution of the active enzyme. , 1998, The Journal of biological chemistry.

[18]  J C Reed,et al.  IAPs block apoptotic events induced by caspase‐8 and cytochrome c by direct inhibition of distinct caspases , 1998, The EMBO journal.

[19]  V. Dixit,et al.  Activation of caspases triggered by cytochrome c in vitro 1 , 1998, FEBS letters.

[20]  V. Kidd,et al.  Proteolytic activities that mediate apoptosis. , 1998, Annual review of physiology.

[21]  John Calvin Reed,et al.  The c‐IAP‐1 and c‐IAP‐2 proteins are direct inhibitors of specific caspases , 1997, The EMBO journal.

[22]  W. Earnshaw,et al.  Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. , 1997, Blood.

[23]  C. Bortner,et al.  Intracellular K+ Suppresses the Activation of Apoptosis in Lymphocytes* , 1997, The Journal of Biological Chemistry.

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

[25]  G. Salvesen,et al.  Biochemical Characteristics of Caspases-3, -6, -7, and -8* , 1997, The Journal of Biological Chemistry.

[26]  L. Greene,et al.  Inhibitors of Trypsin‐Like Serine Proteases Inhibit Processing of the Caspase Nedd‐2 and Protect PC12 Cells and Sympathetic Neurons from Death Evoked by Withdrawal of Trophic Support , 1997, Journal of neurochemistry.

[27]  E. Koonin,et al.  Role of CED-4 in the activation of CED-3 , 1997, Nature.

[28]  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.

[29]  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.

[30]  Y. Pommier,et al.  Camptothecin-induced apoptosis in p53-null human leukemia HL60 cells and their isolated nuclei: effects of the protease inhibitors Z-VAD-fmk and dichloroisocoumarin suggest an involvement of both caspases and serine proteases , 1997, Leukemia.

[31]  W. Earnshaw,et al.  Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. , 1997, Blood.

[32]  Y. Hannun,et al.  Zinc Is a Potent Inhibitor of the Apoptotic Protease, Caspase-3 , 1997, The Journal of Biological Chemistry.

[33]  S. Seshagiri,et al.  Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced , 1997, Current Biology.

[34]  P. W. Mesner,et al.  Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8 , 1997, Oncogene.

[35]  H. Morii,et al.  Hydrolysis of AMPPNP by the motor domain of ncd, a kinesin‐related protein , 1997, FEBS letters.

[36]  Eric A. Hendrickson,et al.  A Sequential Two-Step Mechanism for the Production of the Mature p17:p12 Form of Caspase-3 in Vitro * , 1997, The Journal of Biological Chemistry.

[37]  Y. Tsujimoto,et al.  Intracellular ATP levels determine cell death fate by apoptosis or necrosis. , 1997, Cancer research.

[38]  Yuri Lazebnik,et al.  Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells , 1997, The EMBO journal.

[39]  P. Nicotera,et al.  Intracellular Adenosine Triphosphate (ATP) Concentration: A Switch in the Decision Between Apoptosis and Necrosis , 1997, The Journal of experimental medicine.

[40]  J. Yuan,et al.  Transducing signals of life and death. , 1997, Current opinion in cell biology.

[41]  Y. Hannun,et al.  Apoptosis and the dilemma of cancer chemotherapy. , 1997, Blood.

[42]  W. Earnshaw,et al.  Activation of Multiple Interleukin-1β Converting Enzyme Homologues in Cytosol and Nuclei of HL-60 Cells during Etoposide-induced Apoptosis* , 1997, The Journal of Biological Chemistry.

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

[44]  S. Nagata,et al.  Apoptosis by Death Factor , 1997, Cell.

[45]  A. Eastman,et al.  Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease , 1997, Cell Death and Differentiation.

[46]  J. Lotem,et al.  Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[47]  E. Solary,et al.  Pivotal role of a DEVD‐sensitive step in etoposide‐induced and Fas‐mediated apoptotic pathways. , 1996, The EMBO journal.

[48]  E. Alnemri,et al.  Activation of the CPP32 protease in apoptosis induced by 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents. , 1996, Blood.

[49]  Y. Lazebnik,et al.  Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

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

[51]  G. Evan,et al.  A License to Kill , 1996, Cell.

[52]  G. Cohen,et al.  Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. , 1996, The Biochemical journal.

[53]  J. Mankovich,et al.  Protease Activity of in Vitro Transcribed and Translated Caenorhabditis elegans Cell Death Gene (ced-3) Product (*) , 1996, The Journal of Biological Chemistry.

[54]  E. Alnemri,et al.  Mch3, a novel human apoptotic cysteine protease highly related to CPP32. , 1995, Cancer research.

[55]  D. Przywara,et al.  Deoxynucleoside Induces Neuronal Apoptosis Independent of Neurotrophic Factors (*) , 1995, The Journal of Biological Chemistry.

[56]  K. Wang,et al.  Aurintricarboxylic acid is an inhibitor of mu- and m-calpain. , 1995, Biochemistry and molecular biology international.

[57]  G. Cohen,et al.  A pre‐existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli , 1995, FEBS letters.

[58]  N. Thornberry,et al.  Interleukin‐1βconverting enzyme: A novel cysteine protease required for IL‐1β production and implicated in programmed cell death , 1995, Protein science : a publication of the Protein Society.

[59]  R. Capaldi,et al.  ATP binding causes a conformational change in the gamma subunit of the Escherichia coli F1ATPase which is reversed on bond cleavage. , 1994, Biochemistry.

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

[61]  P. Walker,et al.  Role of proteolysis in apoptosis: involvement of serine proteases in internucleosomal DNA fragmentation in immature thymocytes. , 1993, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[62]  N. Davidson,et al.  Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. , 1993, Cancer research.

[63]  A. Sartorelli,et al.  Evidence for a relationship between intracellular GTP levels and the induction of HL-60 leukemia cell differentiation by 5,10-dideazatetrahydrofolic acid (DDATHF). , 1993, Oncology research.

[64]  S. Kaufmann,et al.  Metabolic effects and kill of human T-cell leukemia by 5-deazaacyclotetrahydrofolate, a specific inhibitor of glycineamide ribonucleotide transformylase. , 1992, Cancer research.

[65]  R. A. Swank,et al.  Staurosporine is a potent inhibitor of p34cdc2 and p34cdc2-like kinases. , 1992, Biochemical and biophysical research communications.

[66]  R. Cohen,et al.  Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues. , 1991, Biochemistry.

[67]  C. Prives,et al.  The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides , 1991, Journal of virology.

[68]  A. Wyllie,et al.  Apoptosis: mechanisms and roles in pathology. , 1991, International review of experimental pathology.

[69]  T. Haystead,et al.  Use of okadaic acid to inhibit protein phosphatases in intact cells. , 1991, Methods in enzymology.

[70]  Y. Banai,et al.  Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf. , 1990, The Journal of biological chemistry.

[71]  E. Shaw,et al.  Cysteinyl proteinases and their selective inactivation. , 1990, Advances in enzymology and related areas of molecular biology.

[72]  S. Kaufmann.  Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note. , 1989, Cancer research.

[73]  K. Johnson,et al.  Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein. , 1989, Biochemistry.

[74]  S. Orrenius,et al.  Calcium‐activated DNA fragmentation kills immature thymocytes , 1989, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[75]  M. Shibuya,et al.  Genistein, a specific inhibitor of tyrosine-specific protein kinases. , 1987, The Journal of biological chemistry.

[76]  N. Thompson,et al.  Decrease of cellular ATP by dihexanoylglycerol may limit responses to protein kinase C activation , 1987, FEBS letters.

[77]  M. Webb,et al.  Absence of a phosphorylated intermediate during ATP hydrolysis by Escherichia coli transcription termination protein rho. , 1986, The Journal of biological chemistry.

[78]  R. Hough,et al.  Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. , 1986, The Journal of biological chemistry.

[79]  J. Harper,et al.  Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor. , 1985, Biochemistry.

[80]  R. Sekura,et al.  Adenine nucleotides directly stimulate pertussis toxin. , 1985, The Journal of biological chemistry.

[81]  H. Swarts,et al.  Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg2+ by (Na+ + K+)-activated ATPase. , 1983, Biochimica et biophysica acta.

[82]  J. Cohen,et al.  Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[83]  G. Swarup,et al.  Phosphotyrosyl-protein phosphatase of TCRC-2 cells. , 1982, The Journal of biological chemistry.

[84]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[85]  C. Goodno.  Inhibition of myosin ATPase by vanadate ion. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[86]  R. Warner,et al.  Vanadate is a potent (Na,K)-ATPase inhibitor found in ATP derived from muscle. , 1977, The Journal of biological chemistry.

[87]  P. Gray,et al.  Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation. , 1977, Nucleic acids research.

[88]  A. Barrett.  Human cathepsin B1. Purification and some properties of the enzyme. , 1973, The Biochemical journal.

[89]  A. Gold,et al.  Sulfonyl Fluorides as Inhibitors of Esterases. I. Rates of Reaction with Acetylcholinesterase, α-Chymotrypsin, and Trypsin , 1963 .

[90]  E. Shaw,et al.  Direct evidence for the presence of histidine in the active center of chymotrypsin. , 1963, Biochemistry.