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Activation of Multiple Interleukin-1β Converting Enzyme Homologues in Cytosol and Nuclei of HL-60 Cells during Etoposide-induced Apoptosis*

Open AccessPublished:March 14, 1997DOI:https://doi.org/10.1074/jbc.272.11.7421
      Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.

      INTRODUCTION

      Recent studies (reviewed in
      • Sen S.
      • D'Incalci M.
      ,
      • Dive C.
      • Evans C.A.
      • Whetton A.D.
      ,
      • Sachs L.
      • Lotem J.
      ,
      • Kerr J.F.
      • Winterford C.M.
      • Harmon B.V.
      ,
      • Kaufmann S.H.
      ) indicate that the cytotoxicity of virtually all chemotherapeutic agents is accompanied by apoptosis in susceptible cell lines. Likewise, experiments in animals (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      • Martin D.S.
      • Stolfi R.L.
      • Colofiore J.R.
      • Nord L.D.
      • Sternberg S.
      ) and studies of circulating blasts from leukemia patients (
      • Li X.
      • Gong J.
      • Feldman E.
      • Seiter K.
      • Traganos F.
      • Darzynkiewicz Z.
      ) have provided evidence that chemotherapy is accompanied by apoptosis in vivo Moreover, it has been suggested that resistance to the cytotoxic effects of chemotherapeutic agents can result from resistance to chemotherapy-induced apoptosis (
      • Lowe S.W.
      • Bodis S.
      • McClatchey A.
      • Remington L.
      • Ruley H.E.
      • Fisher D.E.
      • Housman D.E.
      • Jacks T.
      ,
      • Miyashita T.
      • Reed J.C.
      ,
      • Bedi A.
      • Barber J.P.
      • Bedi G.C.
      • el-Deiry W.S.
      • Sidransky D.
      • Vala M.S.
      • Akhtar A.J.
      • Hilton J.
      • Jones R.J.
      ). These observations highlight the potential importance of understanding the factors that control apoptosis.
      A variety of experimental results suggest that ICE
      The abbreviations used are: ICE
      interleukin-1β converting enzyme
      AFC
      7-amino-4-trifluoromethylcoumarin
      AMC
      7-amino-4-methyl-coumarin
      cmk
      chloromethyl ketone
      caspase
      cysteine-dependent aspartate-directed protease
      DTT
      dithiothreitol
      E64
      trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane
      fmk
      fluoromethylketone
      Fmoc
      N-(9-fluorenyl)methoxycarbonyl
      IRP
      ICE-related protease
      MOI
      multiplicity of infection
      PAGE
      polyacrylamide gel electrophoresis
      PARP
      poly(ADP-ribose) polymerase
      PCR
      polymerase chain reaction
      PMSF
      α-phenylmethylsulfonyl fluoride
      RT-PCR
      reverse transcription-polymerase chain reaction
      Z-EK(bio)D-aomk
      N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone
      Z-
      benzyloxycarbonyl
      PIPES
      1,4-piperazinediethanesulfonic acid
      HPLC
      high pressure liquid chromatography
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      TLCK
      1-chloro-3-tosylamido-7-amino-2-haptanone
      aomk
      Z-Glu-Lys-Asp-(α-((arylacyl)oxy)methyl ketone).
      family proteases (now termed caspases)
      In accordance with recent recommendations (
      • Alnemri E.S.
      • Livingston D.J.
      • Nicholson D.W.
      • Salveson G.
      • Thornberry N.A.
      • Wong W.W.
      • Yuan J.
      ) members of the ICE family are now called caspases. Previous names of the proteases described in this manuscript are as follows: ICE (caspase-1), Ich-1 (caspase-2), CPP32/apopain (caspase-3), ICE-relII/Tx (caspase-4), ICE-relIII/TY (caspase-5), Mch2 (caspase-6), Mch3 (caspase-7), FLICE/Mach/Mch5 (caspase-8), and Mch4 (caspase-10).
      might play a critical role in initiating and sustaining the biochemical events that result in apoptotic cell death (reviewed in
      • Kaufmann S.H.
      and
      • Earnshaw W.C.
      ,
      • Martin S.J.
      • Green D.R.
      ,
      • Nicholson D.W.
      ). Caspases are unusual in several respects. First, although they are cysteine-dependent proteases, the members that have been examined are insensitive to antipain and E64, two broad spectrum inhibitors of sulfhydryl proteases (
      • Black R.A.
      • Kronheim S.R.
      • Sleath P.R.
      ,
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ). Second, caspases cleave at the carboxyl side of aspartate residues (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Sleath P.R.
      • Hendrickson R.C.
      • Kronheim S.R.
      • March C.J.
      • Black R.A.
      ,
      • Howard A.D.
      • Kostura M.J.
      • Thornberry N.
      • Ding G.J.
      • Limjuco G.
      • Weidner J.
      • Salley J.P.
      • Hogquist K.A.
      • Chaplin D.D.
      • Mumford R.A.
      ,
      • 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.
      ), a specificity that is unusual for mammalian proteases. Third, activation of caspases appears to require cleavage at Asp-X sequences in the proenzymes to yield the large and small subunits that are present in the active α2β2 tetramer (reviewed in
      • Nicholson D.W.
      ,
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      , and
      • Thornberry N.A.
      • Bull H.G.
      • Calaycay J.R.
      • Chapman K.T.
      • Howard A.D.
      • Kostura M.J.
      • Miller D.K.
      • Molineaux S.M.
      • Weidner J.R.
      • Aunins J.
      ). The last two observations raise the possibility that caspases might undergo autoactivation and/or activate each other in a cell death cascade (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ,
      • Fraser A.
      • Evan G.
      ).
      In Caenorhabditis elegans, deletion of the ced-3 gene, which encodes the single caspase known for this organism, abolishes all developmentally regulated cell death (
      • Ellis H.M.
      • Horvitz H.R.
      ,
      • Yuan J.
      • Shaham S.
      • Ledoux S.
      • Ellis H.M.
      • Horvitz H.R.
      ). In higher eukaryotes, the situation appears to be more complex. First, proenzyme forms of multiple caspases are expressed in many cell types (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ,
      • 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.
      ,
      • Chinnaiyan A.M.
      • Orth K.
      • O'Rourke K.
      • Duan H.
      • Poirier G.G.
      • Dixit V.M.
      ). Second, deletion of the genes for several of these proteases does not appear to alter the features of apoptosis induced by many stimuli, perhaps because of functional redundancy in the substrate recognition properties of these proteases (
      • Martin S.J.
      • Green D.R.
      ,
      • Patel T.
      • Gores G.J.
      • Kaufmann S.H.
      ).
      Previous studies have also identified a number of cellular polypeptides that are cleaved during apoptosis (reviewed in
      • Kaufmann S.H.
      ,
      • Martin S.J.
      • Green D.R.
      ,
      • Nicholson D.W.
      , and
      • Patel T.
      • Gores G.J.
      • Kaufmann S.H.
      ). These include the DNA damage recognition protein PARP, which is cleaved at the sequence DEVD↓G (
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ), and the lamins, which are cleaved by a protease that recognizes the sequence VEID↓N (
      • 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.
      ). After treatment of HL-60 human acute myelomonocytic leukemia cells with a variety of chemotherapeutic agents, PARP and lamin B1 are cleaved just prior to or concomitant with the appearance of other apoptotic changes (
      • Kaufmann S.H.
      ,
      • Kaufmann S.H.
      • Desnoyers S.
      • Ottaviano Y.
      • Davidson N.E.
      • Poirier G.G.
      ). Other polypeptides that appear to be cleaved by caspases during apoptosis in various cell types include the DNA-activated protein kinase DNA-PK, the retinoblastoma gene product, the 70-kDa polypeptide subunit of U1 small nuclear ribonucleoprotein complexes, steroid response element binding proteins, protein kinase Cδ, actin, and the intermediate filament-associated polypeptide Gas2 (reviewed in
      • Kaufmann S.H.
      and
      • Nicholson D.W.
      ). The individual proteases responsible for these cleavages in situ have not been conclusively identified. Recent studies have indicated that PARP can be cleaved in vitro by a variety of caspases including caspase-1, −2, −3, −4, −6, −7, and −8 (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ,
      • 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.
      ,
      • Tewari M.
      • Quan L.T.
      • O'Rourke K.
      • Desnoyers S.
      • Zeng Z.
      • Beidler D.R.
      • Poirier G.G.
      • Salveson G.S.
      • Dixit V.M.
      ,
      • Gu Y.
      • Sarnecki C.
      • Aldape R.A.
      • Livingston D.J.
      • Su M.S.-S.
      ,
      • Muzio M.
      • Chinnaiyan A.M.
      • Kischkel F.C.
      • O'Rourke K.
      • Shevchenko A.
      • Ni J.
      • Scaffidi C.
      • Bretz J.D.
      • Zhang M.
      • Gentz R.
      • Mann M.
      • Krammer P.H.
      • Peter M.E.
      • Dixit V.M.
      ), although cleavage of expressed fragments of PARP by caspase-1, −2, and −4 appears to require enzyme concentrations that are stoichiometric rather than catalytic (
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Lazebnik Y.A.
      • Kaufmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ,
      • Gu Y.
      • Sarnecki C.
      • Aldape R.A.
      • Livingston D.J.
      • Su M.S.-S.
      ). To date, caspase-6 (previously termed Mch2) is the only caspase known to be capable of cleaving the lamins (
      • 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.
      ,
      • Orth K.
      • Chinnaiyan A.M.
      • Garg M.
      • Froelich C.J.
      • Dixit V.M.
      ).
      In the present study, we have examined HL-60 cells undergoing apoptosis after treatment with the topoisomerase II-directed agent etoposide (reviewed in
      • Froelich-Ammon S.J.
      • Osheroff N.
      ). These studies have focused on determining 1) the number of caspases that are activated during etoposide-induced apoptosis, 2) the subcellular distribution of these active caspases, and 3) the identity of the active caspases. In order to address these questions, a novel affinity label for active caspases was synthesized and characterized, and a new fluorogenic assay for caspase-6-like protease activity was developed.

      DISCUSSION

      Previous studies have suggested that caspase activation plays a crucial role in the initiation or propagation of apoptotic events. Recent reports also indicate that genes encoding as many as nine caspases are simultaneously expressed in certain human leukemia cell lines. However, it has been unclear whether all of these proteases participate in apoptotic events. Two factors contribute to this uncertainty. First, activation of caspases, which involves proteolytic cleavage of zymogen precursors at Asp-X bonds to yield the large and small subunits of the active heterotetrameric proteases, is poorly understood (reviewed in
      • Nicholson D.W.
      and
      • Patel T.
      • Gores G.J.
      • Kaufmann S.H.
      ). Second, high titer antibodies that are specific for the subunits of mature proteases are not available for all of the caspases. As a result, it has remained unclear whether all of the proenzymes are activated simultaneously, whether the appearance of active enzymes involves de novo synthesis of caspases or activation of preexisting proenzymes, and whether active caspases are localized exclusively to the cytoplasm or are found in other cellular compartments. The present study sheds light on all of these issues.
      Analysis of affinity labeling experiments by unidimensional SDS-PAGE revealed that HL-60 cells undergoing etoposide-induced apoptosis contain four distinct bands that covalently label with Z-EK(bio)D-aomk (Fig. 4). Although these polypeptides appear sequentially rather than simultaneously, the apparent stability of the larger bands (IRP1-3) at later time points suggests that the lower bands are not derived from the upper band(s) by proteolytic cleavage. Instead, these polypeptides appear to correspond to the larger subunits of the active forms of multiple discrete caspases. A similar repertoire of five endogenous caspases with distinct cleavage preferences was recently demonstrated by us in apoptosis-inducing S/M extracts prepared from chicken hepatoma cells (
      • 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.
      ). Thus, apoptosis in higher eukaryotic cells differs in detail from developmental cell death in C. elegans, where a single caspase appears to be required (
      • Ellis H.M.
      • Horvitz H.R.
      ,
      • Yuan J.
      • Shaham S.
      • Ledoux S.
      • Ellis H.M.
      • Horvitz H.R.
      ).
      The results obtained with Z-EK(bio)D-aomk provide the first direct evidence that a number of caspases are activated during apoptosis in HL-60 cells. This caspase activation is accompanied by the appearance of at least two distinct enzyme activities. These activities cleave DEVD-AFC (Fig. 2A) and VEID-AMC (Fig. 2C) but display different inhibitor sensitivities (Fig. 2, B and D). In contrast, activation of a caspase-1-like activity was not observed during etoposide-induced apoptosis in HL-60 cells (Fig. 2E), a result that differs from the recent observation that caspase-1-like protease activity is elevated early in the course of Fas-mediated apoptosis in T cells (
      • Enari M.
      • Talanian R.V.
      • Wong W.W.
      • Nagata S.
      ). Whether these disparate findings reflect differences between various cell types or variations in the pathways activated by different apoptotic stimuli requires further investigation.
      The observation that several recombinant caspase polypeptides expressed in Sf9 cells comigrate with the IRPs present in HL-60 extracts following labeling with Z-EK(bio)D-aomk enables us to propose a tentative identification of the proteases acting in the course of etoposide-induced HL-60 apoptosis. IRP2 and IRP4 appear to correspond to caspase-3 (Fig. 7B). The appearance of these active species occurs with a time course (Fig. 4) that parallels the appearance of DEVD-AFC cleavage activity (Fig. 2A) and disappearance of procaspase-3 from immunoblots (Fig. 6C). Our demonstration that caspase-3 is enzymatically active in HL-60 apoptosis (i.e. covalently labels with Z-EK(bio)D-aomk) confirms and extends recent claims of caspase-3 activation based solely on appearance of immunoreactive P17 subunit (
      • Chinnaiyan A.M.
      • Orth K.
      • O'Rourke K.
      • Duan H.
      • Poirier G.G.
      • Dixit V.M.
      ,
      • Ibrado A.M.
      • Huang Y.
      • Fang G.
      • Bhalla K.
      ).
      The two-dimensional analysis presented in Fig. 7 provides the first evidence for the presence of multiple active forms of caspase-3 during apoptosis. Spots A1, C1, and C2 all comigrate with species observed in Sf9 cells expressing human caspase-3. The various forms of the enzyme could arise from alternative splicing of the transcripts (
      • Alnemri E.S.
      • Fernandes-Alnemri T.
      • Pomerenke K.
      • Robertson N.M.
      • Dudley K.
      • DuBois G.C.
      • Litwack G.
      ), alternative processing of the proenzymes, and/or posttranslational modification of the processed subunits. Whether these species correspond to functionally distinct subpopulations of caspase-3 that differ in their substrate recognition properties, time of activation during apoptosis, or intracellular location requires further study. Nonetheless, the observation that cells contain multiple active species of this caspase suggests that the spectrum of active caspases in apoptosis could be even more complicated than previously thought.
      Measurements of enzyme activity also indicate that a discrete VEID-AMC cleavage activity appears in etoposide-treated HL-60 cells beginning ∼2 h after the addition of etoposide (Fig. 2, C and D). Analysis by one- and two-dimensional isoelectric focusing/SDS-PAGE supports this observation by indicating that IRP3/spot B has the same mobility as one species of active caspase-6 (Fig. 7). Caspase-6 was recently shown to be unique among caspases characterized to date in being able to cleave the VEID-X sequence (
      • 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.
      ). Although analysis by unidimensional SDS-PAGE strongly suggested that IRP1 might correspond to a second active species of caspase-6 (Fig. 7A), this assignment could not be confirmed by two-dimensional isoelectric focusing/SDS-PAGE because IRP1 did not focus in the two-dimensional gels (Fig. 7, B and C).
      Two-dimensional analysis of HL-60 cytosol also revealed the presence of multiple additional Z-EK(bio)D-aomk-reactive species (Fig. 7C) that did not comigrate with any of the recombinant caspases tested to date. Among these, spots C3 and C4 have mobilities on SDS-PAGE similar to species tentatively identified as caspase-3 but have different isoelectric points, suggesting that they might be posttranslationally modified versions of the caspase-3 large subunit. Alternatively, these minor species could correspond to other human caspases, either known or yet to be characterized.
      In addition to the caspases detected in cytosol, Z-EK(bio)D-aomk also selectively labeled two polypeptides in nuclei of etoposide-treated HL-60 cells (Fig. 5B). Despite the fact that several of the known caspase substrates are nuclear proteins (reviewed in
      • Nicholson D.W.
      and
      • Patel T.
      • Gores G.J.
      • Kaufmann S.H.
      ), this is the first demonstration of multiple active caspases in nuclei. It is unlikely that the nuclear enzymes represent contaminating cytosolic proteins, since the isolated nuclei were shown to lack significant cytoplasmic contamination as detected either by electron microscopy or assays for the cytosolic marker enzyme lactate dehydrogenase. The comigration of the active caspase subunits in cytosol and nuclei (Fig. 5B) suggests the possibility that some (but not all) of the species activated in the cytosol might be transported into nuclei. Alternatively, we cannot rule out the possibility that certain caspase precursors present in nuclei are activated during the course of apoptosis. Further experiments are required to distinguish between these possibilities.
      The present observations also shed light on the origin of the active caspases. We were unable to detect either DEVD-X cleavage activity (Fig. 2A) or Z-EK(bio)D-aomk-labeled polypeptides corresponding to the large subunit of active caspases in cytosol from control HL-60 cells (Fig. 4A). These observations argue against the possibility that caspase-3 (or any related protease) is preactivated in the cytosol of HL-60 cells and effectively rule out a model in which the activity of preactivated proteases is controlled in these cells by regulation of the levels of endogenous inhibitors.
      An alternative possibility is that caspases are synthesized de novo in response to apoptotic stimuli (
      • Boudreau N.
      • Sympson C.J.
      • Werb Z.
      • Bissell M.J.
      ). We did not observe a reproducible etoposide-induced increase in content of any of the four caspases for which antibodies are currently available (Fig. 6 and data not shown). Moreover, the observation that high concentrations of the protein synthesis inhibitors cycloheximide and puromycin failed to prevent the appearance of DEVD-AFC cleavage activities in etoposide-treated HL-60 cells (Fig. 3) argues against the possibility that this activity results from de novo synthesis of the proenzyme. Instead, it appears that multiple caspase proenzymes are present in HL-60 cells (and several other human cell lines) prior to the application of apoptotic stimuli (Fig. 6; see also
      • Nicholson D.W.
      • Ali A.
      • Thornberry N.A.
      • Vaillancourt J.P.
      • Ding C.K.
      • Gallant M.
      • Gareau Y.
      • Griffin P.R.
      • Labelle M.
      • Lazebnik Y.A.
      ,
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ,
      • 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.
      ,
      • Chinnaiyan A.M.
      • Orth K.
      • O'Rourke K.
      • Duan H.
      • Poirier G.G.
      • Dixit V.M.
      , and
      • Fernandes-Alnemri T.
      • Litwack G.
      • Alnemri E.S.
      ).
      In support of this conclusion, RT-PCR (Fig. 1) indicated that untreated HL-60 cells contain transcripts for at least nine caspases. Although procaspase polypeptide levels might not always reflect the levels of the corresponding transcripts, it is reasonable to assume that as many as nine procaspase polypeptides are expressed in HL-60 cells. This does not mean, however, that all nine procaspases are activated during the course of apoptosis. We failed to detect an increase in caspase-1 enzymatic activity (YVAD-AFC cleavage) after etoposide treatment (Fig. 2E). Likewise, immunoblotting indicated that the bulk of procaspase-2 present in HL-60 cells remained intact during apoptosis (Fig. 6D), and two-dimensional analysis failed to detect a Z-EK(bio)D-aomk-labeled spot corresponding to active caspase-2 (Fig. 7C). This is in contrast to the extensive cleavage (Fig. 6C) and activation (Fig. 7C) of procaspase-3. The mechanism for selective apoptotic activation of some caspase precursors (e.g. procaspase-3) and not others (e.g. procaspase-2) is currently unclear and requires further investigation.

      Acknowledgments

      We thank Dr. Ivan Lieberburg for making this collaboration possible; Michael Power for DNA sequence confirmation of clones and constructs used in this work; Alastair Mackay and Joe Lewis for help with the two-dimensional gel analysis; and Deb Strauss for secretarial assistance.

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