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

NUCLEOTIDE REQUIREMENT AND INHIBITOR PROFILE*
Open AccessPublished:August 06, 1999DOI:https://doi.org/10.1074/jbc.274.32.22635
      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.
      Apoptosis is a morphologically distinct form of physiological cell death that is widely observed in nature (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ,
      • Arends M.J.
      • Wyllie A.H.
      ). Studies performed over the past 5 years have revealed that many of the changes observed in apoptotic cells result from the action of a family of cysteine-dependent aspartate-directed intracellular proteases (now termed caspases) on their substrates (
      • Hannun Y.A.
      ,
      • Cryns V.
      • Yuan J.
      ,
      • Kidd V.J.
      ,
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ). As a result, considerable attention is now focused on understanding the factors that control caspase activation and activity.
      Several pathways of caspase activation have been identified. One involves ligation of certain receptors (e.g. CD95 or the type 1 tumor necrosis factor-α receptor), recruitment of adaptor proteins such as FADD/Mort1, binding of procaspases-8 and -10 to the adaptor molecules, activation of these initiator caspases, and subsequent proteolytic activation of the downstream caspases-3, -6, and -7 (
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ,
      • Fraser A.
      • Evan G.
      ,
      • Yuan J.
      , ). This canonical pathway appears to account, at least in broadstroke, for the events initiated by ligation of a number of death receptors.
      An alternative pathway of caspase activation (
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ,
      • Hengartner M.
      ) appears to be involved in other apoptotic deaths (
      • Eischen C.M.
      • Kottke T.J.
      • Martins L.M.
      • Basi G.S.
      • Tung J.S.
      • Earnshaw W.C.
      • Leibson P.J.
      • Kaufmann S.H.
      ,
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ). Many proapoptotic stimuli cause mitochondria to release cytochrome c to the cytosol, where it binds to a docking protein called Apaf-1, inducing a conformational change in Apaf-1 that facilitates binding and activation of procaspase-9 (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ,
      • Yang J.
      • Liu X.
      • Bhalla K.
      • Kim C.N.
      • Ibrado A.M.
      • Cai J.
      • Peng T.-I.
      • Jones D.P.
      • Wang X.
      ,
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ,
      • Zou H.
      • Henzel W.J.
      • Liu X.
      • Lutsch A.
      • Wang X.
      ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Stennicke H.R.
      • Deveraux Q.L.
      • Humke E.W.
      • Reed J.C.
      • Dixit V.M.
      • Salvesen G.S.
      ). Caspase-9 then proteolytically activates caspases-3 and -7; the former activates caspase-6 (
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ).
      Previous studies have indicated that this cytochromec/Apaf-1/caspase-9 pathway can be reconstituted in vitro by incubating cytosol from nonapoptotic cells with purified cytochrome c and dATP (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ,
      • Deveraux Q.L.
      • Roy N.
      • Stennicke H.R.
      • Van Arsdale T.
      • Zhou Q.
      • Srinivasula S.M.
      • Alnemri E.S.
      • Salvesen G.S.
      • Reed J.C.
      ,
      • Slee E.A.
      • Harte M.T.
      • Kluck R.M.
      • Wolf B.B.
      • Casiano C.A.
      • Newmeyer D.D.
      • Wang H.-G.
      • Reed J.C.
      • Nicholson D.W.
      • Alnemri E.S.
      • Green D.R.
      • Martin S.J.
      ). Although this pathway has been intensively studied, several aspects remain poorly understood. First, the role of ATP or dATP is unclear. Because mutation of the ATP-binding site abolishes the caspase activating activity of the Apaf-1 homolog ced-4 (
      • Seshagiri S.
      • Miller L.K.
      ,
      • Chinnaiyan A.M.
      • Chaudhary D.
      • O'Rourke K.
      • Koonin E.V.
      • Dixit V.M.
      ), it has generally been assumed that the nucleoside triphosphate is hydrolyzed during caspase activation. Consistent with this possibility, ATP depletion has been shown to abrogate caspase activation and subsequent apoptotic events in damaged cells (
      • Leist M.
      • Single B.
      • Castoldi A.F.
      • Kuhnle S.
      • Nicotera P.
      ,
      • Eguchi Y.
      • Shimizu S.
      • Tsujimoto Y.
      ). Srinivasula et al. (
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ), however, observed that a fragment of Apaf-1 can facilitate caspase-9 activation in a nucleotide-independent fashion. More recently, Kuida et al.(
      • Kuida K.
      • Haydar T.F.
      • Kuan C.-Y.
      • Gu Y.
      • Taya C.
      • Karasuyama H.
      • Su M.S.-S.
      • Rakic P.
      • Flaveli R.A.
      ) reported that caspase activation can occur upon incubation of brain cytosol in the absence of exogenous dATP and cytochromec. These observations raise the possibility that nucleotide hydrolysis might not be required for caspase activation. Second, because the vast majority of studies have focused upon the activation of caspase-3, it is unclear how faithfully the events produced in vitro reflect the selective activation of a subset of the available procaspases that is observed in situ. Finally, the effects of various inhibitors on this caspase activation pathway remain to be elucidated.
      A number of inhibitors of the apoptotic process have been previously identified. Early experiments established that Zn2+ (
      • Duke R.C.
      • Chervenak R.
      • Cohen J.J.
      ) and ATA
      The abbreviations used are: AMPPCP, 5′-adenylyl methylenediphosphate; AMPPNP, 5′-adenylyl imidodiphosphate; ATA, aurintricarboxylic acid; ATPγS, adenylyl-5′-O-(3-thiotriphosphate); DCI, 3,4-dichloroisocoumarin; DEVD-AFC, N-(N α-acetylaspartylglutamylvalinyl)aspartate 7-amino-4-trifluoromethylcoumarin; DEVD-pNA, N-(N α-acetylaspartylglutamylvalinyl)aspartatep-nitroanilide; DTT, dithiothreitol; LEHD-AFC, N-(N α-acetylleucinylglutamylhistidyl)aspartate 7-amino-4-trifluoromethylcoumarin; PMSF, α-phenylmethylsulfonyl fluoride; pNA, p-nitroaniline; TLCK, N α-p-tosyl-l-lysine chloromethyl ketone; TPCK, N α-p-tosyl-l-phenylalanine chloromethyl ketone; VEID-AFC, N-(N α-acetylvalinylglutamylisoleucyl)aspartate 7-amino-4-trifluoromethylcoumarin; ZEK(bio)d-aomk, N-(N α-benzyloxycarbonylglutamyl-N ε-biotinyllysyl) aspartic acid [(2, 6-dimethylbenzoyl)oxy]methyl ketone; ZVAD-fmk, N-(N α-benzyloxycarbonylvalinylalanyl)aspartate fluoromethyl ketone; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline
      1The abbreviations used are: AMPPCP, 5′-adenylyl methylenediphosphate; AMPPNP, 5′-adenylyl imidodiphosphate; ATA, aurintricarboxylic acid; ATPγS, adenylyl-5′-O-(3-thiotriphosphate); DCI, 3,4-dichloroisocoumarin; DEVD-AFC, N-(N α-acetylaspartylglutamylvalinyl)aspartate 7-amino-4-trifluoromethylcoumarin; DEVD-pNA, N-(N α-acetylaspartylglutamylvalinyl)aspartatep-nitroanilide; DTT, dithiothreitol; LEHD-AFC, N-(N α-acetylleucinylglutamylhistidyl)aspartate 7-amino-4-trifluoromethylcoumarin; PMSF, α-phenylmethylsulfonyl fluoride; pNA, p-nitroaniline; TLCK, N α-p-tosyl-l-lysine chloromethyl ketone; TPCK, N α-p-tosyl-l-phenylalanine chloromethyl ketone; VEID-AFC, N-(N α-acetylvalinylglutamylisoleucyl)aspartate 7-amino-4-trifluoromethylcoumarin; ZEK(bio)d-aomk, N-(N α-benzyloxycarbonylglutamyl-N ε-biotinyllysyl) aspartic acid [(2, 6-dimethylbenzoyl)oxy]methyl ketone; ZVAD-fmk, N-(N α-benzyloxycarbonylvalinylalanyl)aspartate fluoromethyl ketone; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline
      (
      • McConkey D.J.
      • Hartzell P.
      • Nicotera P.
      • Orrenius S.
      ) decrease thymocyte apoptosis. Although these effects were initially attributed to inhibition of apoptotic nucleases, subsequent observations have indicated that Zn2+ inhibits active caspases (
      • Takahashi A.
      • Alnemri E.S.
      • Lazebnik Y.A.
      • Fernandes-Alnemri T.
      • Litwack G.
      • Moir R.D.
      • Goldman R.D.
      • Poirier G.G.
      • Kaufmann S.H.
      • Earnshaw W.C.
      ,
      • Stennicke H.R.
      • Salvesen G.S.
      ,
      • Perry D.K.
      • Smyth M.J.
      • Stennicke H.R.
      • Salvesen G.S.
      • Duriez P.
      • Poirier G.G.
      • Hannun Y.A.
      ) or apoptotic events further upstream (
      • Wolf C.M.
      • Morana S.J.
      • Eastman A.
      ). A variety of additional compounds have been observed to inhibit apoptosis in intact cells. These include the caspase inhibitor ZVAD-fmk (
      • Slee E.A.
      • Zhu H.
      • Chow S.C.
      • MacFarlane M.
      • Nicholson D.W.
      • Cohen G.M.
      ); agents such as TLCK, TPCK, and DCI (
      • Bruno S.
      • Del Bino G.
      • Lassota P.
      • Giaretti W.
      • Darzynkiewicz Z.
      ,
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ,
      • Weaver V.M.
      • Lach B.
      • Walker P.R.
      • Silorska M.
      ,
      • Fearnhead H.O.
      • Rivett A.J.
      • Dinsdale D.
      • Cohen G.M.
      ,
      • Dubrez L.
      • Savoy I.
      • Hamman A.
      • Solary E.
      ,
      • Yoshida A.
      • Takauji R.
      • Inuzuka M.
      • Ueda T.
      • Nakamura T.
      ,
      • Lotem J.
      • Sachs L.
      ,
      • Stefanis L.
      • Troy C.M.
      • Qi H.
      • Greene L.A.
      ,
      • Shimizu T.
      • Pommier Y.
      ,
      • Sane A.T.
      • Bertrand R.
      ,
      • Huang Y.
      • Sheikh M.S.
      • Fornace Jr., A.J.
      • Holbrook N.J.
      ), which are often regarded as serine protease inhibitors; and ZFA-fmk (
      • Lotem J.
      • Sachs L.
      ), which is considered a specific inhibitor of sulfhydryl-dependent cathepsins. Although it is now clear that ZVAD-fmk can inhibit caspase-9 activation (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Deveraux Q.L.
      • Roy N.
      • Stennicke H.R.
      • Van Arsdale T.
      • Zhou Q.
      • Srinivasula S.M.
      • Alnemri E.S.
      • Salvesen G.S.
      • Reed J.C.
      ) and activity (
      • Garcia-Calvo M.
      • Peterson E.P.
      • Leiting B.
      • Ruel R.
      • Nicholson D.W.
      • Thornberry N.A.
      ), the possibility that the other agents inhibit caspase activation has not been previously explored.
      In the present study, we have compared caspase activation in HL-60 cells in situ and in HL-60 cytosol in vitro.Earlier studies established that etoposide-induced apoptosis in HL-60 cells is accompanied by cleavage of multiple caspase substrates (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ,
      • Kaufmann S.H.
      ,
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ) and that this cleavage can be inhibited by treatment of cells with TPCK, TLCK, or Zn2+ (
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ). Subsequent reports have demonstrated that a subset of the available caspase precursors is activated during the course of apoptosis in this model system (
      • Martins L.M.
      • Kottke T.J.
      • Mesner P.W.
      • Basi G.S.
      • Sinha S.
      • Frigon Jr., N.
      • Tatar E.
      • Tung J.S.
      • Bryant K.
      • Takahashi A.
      • Svingen P.A.
      • Madden B.J.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ) and that cytochrome c release from mitochondria precedes caspase activation (
      • Yang J.
      • Liu X.
      • Bhalla K.
      • Kim C.N.
      • Ibrado A.M.
      • Cai J.
      • Peng T.-I.
      • Jones D.P.
      • Wang X.
      ,
      • Martins L.M.
      • Mesner P.W.
      • Kottke T.J.
      • Basi G.S.
      • Sinha S.
      • Tung J.S.
      • Svingen P.A.
      • Madden B.J.
      • Takahashi A.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ). In the present study, the cohort of caspases activated by treatment of HL-60 cytosol with cytochrome cand ATP in vitro has been compared with that activatedin situ during etoposide-induced apoptosis. In addition, the nucleotide requirements in vitro have been compared with the nucleotide pools available in intact cells. Finally, the cell-free system was utilized to explore the possibility that small molecule inhibitors of apoptosis, including ATA and protease inhibitors that are usually thought to inhibit other types of proteases, might act by inhibiting caspase activation.

      DISCUSSION

      Recent studies from several laboratories have established that addition of cytochrome c and adenine nucleoside triphosphates to cytosol from control cells results in activation of caspase-3 through a pathway that involves Apaf-1-mediated activation of procaspase-9 (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Deveraux Q.L.
      • Roy N.
      • Stennicke H.R.
      • Van Arsdale T.
      • Zhou Q.
      • Srinivasula S.M.
      • Alnemri E.S.
      • Salvesen G.S.
      • Reed J.C.
      ). In the present study, we have extended these observations by contrasting the cohorts of active caspases that are generated in vitro and in situ, comparing the nucleotide requirements in vitro to the adenosine nucleotide triphosphate levels available in intact cells, assessing the effects of poorly hydrolyzable ATP analogs, and determining the effects of selected inhibitors in the caspase activation process. These experiments address a number of unresolved questions about the activation of caspases under cell-free conditions.
      Before embarking on these studies, we confirmed that the caspase activation process in HL-60 cytosol in vitro was similar to that observed in other model systems. Our studies demonstrated that the process generated active forms of caspases-3, -6, and -7 in vitro (Fig. 1 A) but did not generate active forms of caspases-2 or -8 (Fig. 1 A and Fig. 2 B). This selectivity mirrors the activation process in intact HL-60 cells (Fig.1 C). Failure to activate caspase-2 (
      • Roy N.
      • Deveraus Q.L.
      • Takahashi R.
      • Salvesen G.S.
      • Reed J.C.
      ,
      • Pan G.
      • Humke E.W.
      • Dixit V.M.
      ) and caspase-8 (
      • Pan G.
      • Humke E.W.
      • Dixit V.M.
      ) has been reported in other cell-free systems, although it appears that this selectivity might be cell type-dependent (
      • Slee E.A.
      • Harte M.T.
      • Kluck R.M.
      • Wolf B.B.
      • Casiano C.A.
      • Newmeyer D.D.
      • Wang H.-G.
      • Reed J.C.
      • Nicholson D.W.
      • Alnemri E.S.
      • Green D.R.
      • Martin S.J.
      ). Our studies demonstrated that the activation process in dialyzed HL-60 cytosol was dependent upon addition of exogenous cytochromec and dATP or ATP (Fig. 3), consistent with results observed in other laboratories (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ,
      • Deveraux Q.L.
      • Roy N.
      • Stennicke H.R.
      • Van Arsdale T.
      • Zhou Q.
      • Srinivasula S.M.
      • Alnemri E.S.
      • Salvesen G.S.
      • Reed J.C.
      ,
      • Slee E.A.
      • Harte M.T.
      • Kluck R.M.
      • Wolf B.B.
      • Casiano C.A.
      • Newmeyer D.D.
      • Wang H.-G.
      • Reed J.C.
      • Nicholson D.W.
      • Alnemri E.S.
      • Green D.R.
      • Martin S.J.
      ). A number of other nucleotides, including AMP, ADP-ribose, CMP, CTP, or UTP, could not substitute for ATP or dATP (Fig. 3 A), in agreement with the results of Liuet al. (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ). Finally, our studies demonstrated that the activation process was inhibited by 60–80 mm NaCl or KCl, consistent with the results obtained in rat thymocyte cytosol (
      • Hughes Jr., F.M.
      • Bortner C.D.
      • Purdy G.D.
      • Cidlowski J.A.
      ). Based on these similarities, it appears that the caspase activation process in HL-60 cytosol is similar to that observed in other cell-free systems.
      Some of the results obtained using the HL-60 cell-free system raise questions about the relationship between conditions that result in caspase activation in vitro and in situ. First, as noted above, caspase activation in vitro is strongly inhibited by buffers of physiological ionic strength. Second, the activation process is more efficient in vitro at 30 °C than at 37 °C. Third, results in Fig. 2 B indicate that the cohort of active caspase species detected by affinity labeling after activation in vitro differs significantly from that detected after activation in situ, possibly reflecting differences in reactions that remove the caspase prodomains and alter caspase phosphorylation. These observations raise the possibility that one or more factors that modulate caspase activation in situat physiological ionic strength and temperature might have been lost during the cell fractionation procedure. The similarities between the results observed in HL-60 cytosol and other model systems (see above) suggest that similar concerns might apply to many of the recently described cell-free systems for caspase activation. Further studies are required to evaluate the cause of the differences between caspase activation in vitro and in vivo.
      With these limitations in mind, the HL-60 system was utilized to study nucleotide requirements and inhibitor sensitivity of caspase activation. Studies employing cytosol from HeLa cells had previously suggested that dATP might be specifically required for activation of caspases (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ). In particular, ATP was initially reported to be inactivein vitro, whereas dATP was reportedly active, leading to the speculation that elevations in dATP levels might occur after drug treatment and contribute to the caspase activation process (
      • Liu X.
      • Kim C.N.
      • Yang J.
      • Jemmerson R.
      • Wang X.
      ). Consistent with this hypothesis, Wakade et al. (
      • Wakade A.R.
      • Przywara D.A.
      • Palmer K.C.
      • Kulkarni J.S.
      • Wakade T.D.
      ) observed a 40-fold increase in dATP levels when chick embryo sympathetic neurons were induced to undergo apoptosis by treatment with 2-deoxyadenosine. In the present study, nucleotide levels required for caspase activation in cytosol under cell-free conditions were directly compared with nucleotide pools in the same cell line as it underwent apoptosis. Results of these studies indicated that caspase activation in HL-60 cytosol in vitro required as little as 10 μmdATP or 100 μm ATP. In intact HL-60 cells, neither dATP nor ATP increased during the induction of apoptosis by etoposide (Fig.4 B). Instead, base-line levels of both nucleotides were far above those required for caspase activation in vitro. In fact, levels of dATP observed in HL-60 cells are substantially higher than previously reported in another leukemia cell line (
      • Smith G.K.
      • Duch D.S.
      • Dev I.K.
      • Kaufmann S.H.
      ), raising the interesting possibility that the exquisite sensitivity of HL-60 cells to a variety of proapoptotic agents might be related to the high base-line dATP levels.
      In subsequent experiments, ATP was replaced with AMPPCP, AMPPNP, or ATPγS. Results of these experiments help clarify the role of the nucleotide in the caspase activation process. In ATP-requiring processes that depend on ATP binding rather than hydrolysis (e.g. allosteric activation of pertussis toxin (
      • Lim L.K.
      • Sekura R.D.
      • Kaslow H.R.
      ), the release of eukaryotic initiation factor-2 from its ternary complex (
      • Gonsky R.
      • Lebendiker M.A.
      • Harary R.
      • Banai Y.
      • Kaempfer R.
      ), the binding of polyoma virus T antigen to its DNA-binding site (
      • Lorimer H.E.
      • Wang E.H.
      • Prives C.
      ), the activation of ubiquitin protein ligase (
      • Johnston N.L.
      • Cohen R.E.
      ), or release of xylose reductase from its protein chaperone (
      • Rawat U.
      • Rao M.
      )), one or more of these poorly hydrolyzable analogs has been observed to substitute fully for ATP. In contrast, our experiments revealed that these analogs were poor substitutes for ATP over a wide range of concentrations. In particular, AMPPCP and AMPPNP permitted only slow caspase activation (Fig.3 B), and ATPγS did not facilitate caspase activation at all (Fig. 3 C). Although these compounds are frequently considered “nonhydrolyzable,” previous studies have demonstrated that various ATPases can hydrolyze these analogs at rates that range from 0.0007 to 0.25 times the corresponding rates of ATP hydrolysis (
      • Schuurmans Stekhoven F.M.
      • Swarts H.G.
      • De Pont J.J.
      • Bonting S.L.
      ,
      • Stitt B.L.
      • Webb M.R.
      ,
      • Shimizu T.
      • Katsura T.
      • Domanico P.L.
      • Marchese-Ragona S.P.
      • Johnson K.A.
      ,
      • Turina P.
      • Capaldi R.A.
      ,
      • Suzuki Y.
      • Shimizu T.
      • Morii H.
      • Tanokura M.
      ). The slowing of caspase activation in the presence of these poorly hydrolyzable analogs (Fig. 3, B andC) is consistent with a process that requires hydrolysis of the terminal phosphodiester bond. Likewise, the inhibition of caspase activation by millimolar concentrations of Na3VO4 (Fig. 5 B), which have previously been shown to inhibit ATPases (
      • Cantley Jr., L.C.
      • Josephson L.
      • Warner R.
      • Yanagisawa M.
      • Lechene C.
      • Guidotti G.
      ,
      • Goodno C.C.
      ), is consistent with the suggestion that ATP or dATP hydrolysis is involved in this process.
      In further experiments, the effects of a variety of inhibitors were analyzed. A major advantage of the cell-free system is the lack of permeability barriers that have been postulated to inhibit the action of certain caspase inhibitors (
      • Thornberry N.A.
      • Molineaux S.M.
      ). Results obtained with the cell-free system were compared with effects observed when recombinant caspases were treated with the same inhibitors. For these experiments, we utilized antisera that recognized caspase large subunits as well as an antiserum raised against a neoepitope present in mature caspase-9 but not the zymogen. This latter antiserum recognized caspase-9 but not other caspases (Fig. 8 B). In addition, this serum recognized multiple species in a preparation of purified recombinant caspase-9, including several 30–35-kDa species that correspond in molecular weight to the reported sizes of active caspase-9 species (
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ) as well as a unique M r ∼18,000 species that appears to represent further processing of the large subunit (Fig. 8 A). Interestingly, the major species detected by this antiserum after caspase activation in vitro (Fig. 8 C) or in intact HL-60 cells (Fig. 8 D) was theM r ∼18,000 species. A similarM r ∼18,000 product of procaspase-9 activation was recently observed by Susin et al. (
      • Susin S.A.
      • Lorenzo H.K.
      • Zamzami N.
      • Marzo I.
      • Brenner C.
      • Larochette N.
      • Prevost M.C.
      • Alzari P.M.
      • Kroemer G.
      ) but not by others (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Deveraux Q.L.
      • Roy N.
      • Stennicke H.R.
      • Van Arsdale T.
      • Zhou Q.
      • Srinivasula S.M.
      • Alnemri E.S.
      • Salvesen G.S.
      • Reed J.C.
      ,
      • Slee E.A.
      • Harte M.T.
      • Kluck R.M.
      • Wolf B.B.
      • Casiano C.A.
      • Newmeyer D.D.
      • Wang H.-G.
      • Reed J.C.
      • Nicholson D.W.
      • Alnemri E.S.
      • Green D.R.
      • Martin S.J.
      ). Further studies are required to understand the nature and significance of the processing event that gives rise to this species. Nonetheless, this reagent allowed us to examine caspase-9 activation under cell-free conditions.
      Based on recent observations that some active caspases are phosphoproteins (
      • Martins L.M.
      • Kottke T.J.
      • Kaufmann S.H.
      • Earnshaw W.C.
      ), we examined the effect of broad spectrum kinase and phosphatase inhibitors on caspase activation. At concentrations that selectively inhibit kinases or phosphatases, these reagents had little if any effect on caspase activation (Fig. 5 B), suggesting that phosphorylation or dephosphorylation of the effector caspases does not play a major role in this process in vitro.
      In contrast, a variety of other agents that have previously been reported to diminish apoptosis in intact cells inhibited caspase activation in vitro. For example, Zn2+, which was originally introduced into the apoptosis literature as a nuclease inhibitor, inhibited caspase-9 activity (Fig. 6 B) and caspase activation (Fig. 5 A). Although previous studies have identified Zn2+ as a potential caspase inhibitor (
      • Takahashi A.
      • Alnemri E.S.
      • Lazebnik Y.A.
      • Fernandes-Alnemri T.
      • Litwack G.
      • Moir R.D.
      • Goldman R.D.
      • Poirier G.G.
      • Kaufmann S.H.
      • Earnshaw W.C.
      ,
      • Stennicke H.R.
      • Salvesen G.S.
      ,
      • Perry D.K.
      • Smyth M.J.
      • Stennicke H.R.
      • Salvesen G.S.
      • Duriez P.
      • Poirier G.G.
      • Hannun Y.A.
      ), the present study provides the first evidence that caspase-9 is a pertinent target of this cation. Consistent with this hypothesis, additional experiments have demonstrated that activation of caspase-9 and its downstream target caspase-3 is inhibited by Zn2+ in intact cells.
      T. J. Kottke and S. H. Kaufmann, unpublished observations.
      Similarly, our data raise the possibility that ATA might inhibit apoptosis by acting as a caspase inhibitor. Although ATA is known to inhibit endonucleases (
      • Hallick R.B.
      • Chelm B.K.
      • Gray P.W.
      • Orozco E.M.
      ), studies performed over the past 20 years have indicated that ATA can also inhibit proteases, including serine proteases (
      • Bina-Stein M.
      • Tritton T.R.
      ), the proteosome (
      • Hough R.
      • Pratt G.
      • Rechsteiner M.
      ), calpains (
      • Hugunin M.
      • Quintal L.J.
      • Mankovich J.A.
      • Ghayur T.
      ), and certain caspases (
      • Posner A.
      • Raser K.J.
      • Hajimohammadreza I.
      • Yuen P.W.
      • Wang K.K.
      ). Our results extend these previous studies by demonstrating that ATA not only inhibits recombinant caspases-3, -6, -7, and -9 in vitro (Figs. 6 and 7) but also abrogates caspase activation under cell-free conditions (Fig. 5 A).
      The same analysis revealed that various caspase inhibitors prevented caspase activation (Figs. 5 C and 8 E). Consistent with results that were published as the present studies were nearing completion (
      • Sun X.-M.
      • MacFarlane M.
      • Zhuang J.
      • Wolf B.B.
      • Green D.R.
      • Cohen G.M.
      ,
      • Garcia-Calvo M.
      • Peterson E.P.
      • Leiting B.
      • Ruel R.
      • Nicholson D.W.
      • Thornberry N.A.
      ), we observed that ZVAD-fmk inhibited caspase-9 activity (Fig. 6) and activation (Figs. 5 C and8 E). ZVAD(OMe)-fmk exhibited lower potency as an inhibitorin vitro, presumably because esterases that activate this agent in intact cells were not available to deesterify it after preparation of cytosol in the presence of the serine esterase inhibitor PMSF (
      • Fahrney D.E.
      • Gold A.M.
      ). Interestingly, DEVD-fmk was only 10-fold less potent than ZVAD-fmk as an inhibitor of caspase activation (Figs. 5 C and8 E) and caspase-9 activity (Fig. 6). Although it has often been argued that the anti-apoptotic effects of DEVD-fmk result from the inhibition of caspase-3, these results raise the possibility DEVD-fmk might also be acting by inhibiting caspase-9 at the 10–300 μm concentrations used in many experiments.
      Caspase activation was also inhibited by protease inhibitors that are not traditionally viewed as caspase inhibitors. The chloromethyl ketones TPCK and TLCK, which have been reported to inhibit caspases-3 and -7 in crude bacterial lysates (
      • Fernandes-Alnemri T.
      • Takahashi A.
      • Armstrong R.
      • Krebs J.
      • Fritz L.
      • Tomaselli K.J.
      • Wang L.
      • Yu Z.
      • Croce C.M.
      • Salveson G.
      • Earnshaw W.C.
      • Litwack G.
      • Alnemri E.S.
      ), inhibited caspase activation (Figs. 5 C and 8 E). DCI and ZFA-fmk also prevented caspase activation under cell-free conditions (Figs. 5, Cand D, and 8 E). Further analysis revealed that all of these reagents could inhibit purified caspases-3, -6, -7, and -9 as well (Figs. 6 and 7). Collectively, the results in Figs. Figure 5, Figure 6, Figure 7, Figure 8 raise the possibility that these agents might be affecting apoptosis through effects on caspase activation and activity rather than effects on noncaspase proteases. In view of these results, previous studies that utilized these inhibitors to support the view that noncaspase proteases play a role in apoptosis might need to be reinterpreted.

      Acknowledgments

      We gratefully acknowledge Gregory Gores for advice and discussions, Guy Salvesen for the caspase-6 and caspase-8 used to raise antisera, and Deb Strauss for secretarial assistance.

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