Doxorubicin Treatment Activates a Z-VAD-Sensitive Caspase, Which Causes titi m Loss, Caspase-9 Activity, and Apoptosis in Jurkat Cells
Susana Gamen,* Alberto Anel,* Patricia Pe´rez-Gala´n,* Pilar Lasierra,† Daniel Johnson,‡ Andre´s Pin˜eiro,* and Javier Naval*,1
*Departamento de Bioquı´mica y Biologı´a Molecular y Celular, Facultad de Ciencias, †Servicio de Imunologı´a, Hospital Clı´nico Universitario, Universidad de Zaragoza, 50009 Zaragoza, Spain; and ‡Department of Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania 15123

ease, sarcomas, and breast cancer. Despite their gen-

Doxorubicin induces caspase-3 activation and apop- tosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analy- sis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (titi m), phospa- tidilserine exposure, and cell death. Western blot anal- ysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential or- der of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activa- tion of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD- fmk after doxorubicin treatment, but prior to the ap- pearance of cells displaying a low titi m, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jur- kat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause titi m loss, release of apoptogenic factors from mitochondria, and cell death. © 2000 Academic Press
Key Words: caspases; doxorubicin; apoptosis; mito- chondria; leukemia.

INTRODUCTION
Doxorubicin and daunorubicin are currently used drugs in treatment of acute leukemias, Hodgkin’s dis-
1To whom reprint requests should be addressed. Fax: (34)-976 762123; E-mail: [email protected].
eralized use for more than 30 years, their mechanisms of cytotoxicity have been a long-time matter of debate. During the past 20 years, several hypotheses have been formulated, including DNA intercalation/binding, inhibition of topoisomerase II, free-radical generation, and damage to cell membranes [1]. In recent years, it has been found that doxorubicin and other chemother- apeutic drugs induce the intrinsic program of cell death known as apoptosis [2–5]. Apoptosis induced by doxorubicin was initially proposed to be mediated by ceramide, either synthesized de novo [6] or generated from sphingomyelin [7]. However, the role of ceramide in this and other apoptotic process remains unclear [8–10]. Other authors proposed that doxorubicin in- duced apoptosis through the up-regulation of the death receptor Fas and the gene induction of its ligand, FasL, leading to Fas/FasL interaction and cell death [11, 12]. However, although treatment with doxorubicin may be associated, in some cases, with the up-regulation of membrane Fas expression, doxorubicin induces apop- tosis in a Fas-independent way [13–18].
It has been recently described that doxorubicin treat- ment induces disruption of inner mitochondrial mem- brane potential (titi m)2 that precedes the nuclear signs of apoptosis [19]. Both loss of titi m and apoptosis were prevented by Bcl-2 overexpression [19, 20], suggesting an important role for mitochondria in doxorubicin-in- duced apoptosis. On the other hand, treatment with doxorubicin of sensitive Jurkat cells induced caspase-3 activation [13], but cell death was not blocked by Ac-

2Abbreviations used: titi m, mitochondrial transmembrane potential; DiOC6(3), 3,3ti-dihexyloxacarbocyanine iodide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; PS, phosphatidylserine; MTT, 3-[4,5-di- methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp aldehyde; Z-VAD-fmk, benzyloxycarbonyl- Val-Ala-Asp-fluoromethylketone; Z-IETD-fmk, benzyloxycarbonyl-Ile- Glu-Thr-Asp-fluoromethylketone; Z-LEHD-fmk, benzyloxycarbonyl- Leu-Glu-His-Asp-fluoromethylketone; Z-VDVAD-fmk, benzyloxycar- bonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone; Ac-YVAD-cmk, N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone; Z-FA-fmk, benzyloxy- carbonyl-Phe-Ala-fluoromethylketone.

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DEVD-CHO, an efficient inhibitor of this caspase. More- over, MCF-7 breast carcinoma cells, which lack caspase-3 expression, and are sensitive to doxorubicin [17] and HUT78 lymphoma cells, which express signif- icant amounts of caspase-3, are relatively resistant to this drug (unpublished results and [13]). All these data, taken together, suggest that another different caspase(s) might be the key mediator of doxorubicin- induced apoptosis. The aim of the present work was to identify such a caspase. To do this, we have analyzed the sequential order of caspase activation, the relative contribution of each type of caspases to cell death, and the relationship between caspase activation and loss of mitochondrial integrity. Results indicate that caspases from the three different subfamilies are activated fol- lowing doxorubicin treatment of Jurkat cells. All the morphological and biochemical signs of apoptosis, in- cluding titi m loss, could be prevented by cotreatment of cells with Z-VAD-fmk, by overexpression of Bcl-2, or through inhibition of protein synthesis with cyclohex- imide. Treatment with the more selective caspase in- hibitors Ac-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk, and Z-VDVAD-fmk or overexpression of CrmA pre- vented some biochemical and morphological features of apoptosis but did not block cell death. Taken together, these results suggest that the de novo expression and activity of a Z-VAD-sensitive caspase (caspase-X), dis- tinct from caspases 2, 3, 4, 6, 7, 8, 9, and 10, induces titi m loss and release of apoptogenic proteins from mi- tochondria and is a key step in the mechanism of doxorubicin-induced apoptosis in Jurkat cells.

MATERIALS AND METHODS

Materials. Doxorubicin, daunorubicin, actinomycin D, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and 3-[4,5-dimethylthia- zol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) were from Sigma (Madrid, Spain). Mouse IgG2a (clone 19) anti human CPP32 (caspase-3), which recognizes the 32-kDa proenzyme was from Transduction Laboratories (Madrid, Spain). Rabbit polyclonal anti- bodies anti human ICH-1L (caspase-2), goat polyclonal antibodies anti-human TX (caspase-4), Mch-2 (caspase-6), FLICE (caspase-8), and Mch-4 (caspase-10) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-human caspase-9 antibody (Cat. No. 68086E), which recognizes both the 46/48-kDa unprocessed pro- caspase-9 and the 35/37-kDa large subunit of active enzyme [21], was from Pharmingen (Madrid, Spain). Mouse monoclonal anti-caspase-9 antibody, recognizing the small subunit of active enzyme (clone B40, Pharmingen), was also used. Benzyloxycarbonyl-Val-Ala-Asp-flu- oromethylketone (Z-VAD-fmk), N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk) were from Bachem (Bubendorf, Switzerland). Benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone (Z-VD- VAD-fmk) and benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl-ke- tone (Z-IETD-fmk) were from Enzyme Systems Products (Dublin, CA), and benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethylketone (Z-LEHD-fmk) and benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-FA-fmk) from Calbiochem (Madrid, Spain). Annexin V-FITC was

from Bender (Barcelona, Spain), and 3,3ti-dihexyloxacarbocyanine iodide (DiOC6(3)) from Molecular Probes (Leiden, The Netherlands).
Cell proliferation assays. The human T-cell leukemia Jurkat (Clone E6.1, ATCC TIB-152) was routinely cultured at 37°C in RPMI 1640 medium supplemented with 10% fetal calf serum, L-glutamine, and penicillin/streptomycin (hereafter, complete medium), using standard cell culture procedures. Jurkat cell lines stably overex- pressing CrmA protein, CrmA/Jurkat (clone L), and Bcl-2 protein, Bcl-2/Jurkat (clone 4), as well as vector-transfected cells, Vector/
Jurkat (clone A), were generated as described [22]. Transfected cell lines were maintained in complete medium containing 0.5 mg/ml of G418 (Life Technologies, Barcelona). In proliferation assays, cells (3–5 ti 105 cells/ml) were treated with doxorubicin in complete me- dium, in flat-bottom, 96-well plates (100 til/well) for different times, as indicated. In assays of apoptosis inhibition, cells were incubated with one of the following inhibitors: 600 tiM Ac-DEVD-CHO for 3 h, or for 1 h with 300 tiM YVAD-cmk, 100 tiM Z-VAD-fmk, 80 tiM Z-IETD- fmk, 100 tiM Z-VDVAD-fmk, 75–150 tiM Z-LEHD-fmk, or 100 tiM Z-FA-fmk prior to the addition of doxorubicin. Control cultures were treated with the appropriate amount of DMSO (0.1%, final concen- tration), used as solvent for peptide inhibitors. In other experiments, the effect of actinomycin D (20–40 ng/ml), an inhibitor of transcrip- tion, or cycloheximide (0.1–5 tig/ml), an inhibitor of translation, was also evaluated. Cell proliferation was determined by a modification of the MTT reduction method of Mosmann [23] and viability by microscopical evaluation of Trypan blue-stained cells. In addition to stained cells, cells exhibiting a blebbing morphology, defined by the appearance of distinct protrusions of the plasma membrane and/or vacuolization, were scored as nonviable. Chromatin condensation and nuclear fragmentation during apoptosis were analyzed by label- ing cell nuclei with p-phenylenediamine (PPDA) in oxidized glycerol and visualized by fluorescence microscopy as described [24].
Flow cytometry analysis. Phosphatidylserine (PS) exposure dur- ing apoptosis was evaluated by annexin V-FITC binding [25]. Briefly, cells (1.5 ti 105) were washed with PBS and incubated in a solution of 0.5 tig/ml FITC-labeled annexin V in binding buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM Hepes/NaOH, pH 7.4), at 4°C for 30 min. Then, cells were washed and resuspended in 1 ml binding buffer and at least 5000 cells per sample analyzed in an Epics XL-MCL (Coulter, Spain) flow cytometer. To evaluate titi m, the cationic li- pophilic fluorochrome DiOC6(3) was used [26]. Cells (1.5 ti 105 in 100 til) were incubated with 20 nM DiOC6(3) for 15 min at 37°C. DiOC6(3) was prepared from a 40 tiM stock solution in DMSO. This solution was diluted with sterile PBS, pH 7.4, to a 400 nM working solution, followed by a further dilution with the medium containing cells. As a negative control, cells were treated in parallel cultures with the protonophore uncoupling agent CCCP at a final concentra- tion of 50 tiM (stock solution, 10 mM in ethanol). Cells were diluted with PBS to a final volume of 1 ml and analyzed by flow cytometry.
Analysis of caspase activation. Caspase activation was evaluated by Western blot analysis of cell homogenates using specific anti- caspase antibodies. Cells (5 ti 105 cells/ml) were treated in complete medium with doxorubicin (1 tiM), daunorubicin (1 tiM), actinomycin D (0.5–1 tig/ml), methotrexate (100 ng/ml), or vincristine (10 ng/ml) for the times indicated, and cell viability was determined by Trypan blue staining and the MTT assay. For Western blot analysis, 2 ti 106 Trypan blue-negative cells were processed for each lane. Cells were recovered by centrifugation, washed with RPMI 1640 medium, and lysed in 1 ml of lysis buffer (0.15 M NaCl, 1 mM EDTA, 30 mM NaF, 10 tig/ml leupeptin, 1 mM PMSF, 50 mM Tris/HCl, pH 7.6) contain- ing 1% Triton X-100. Solubilized proteins were resolved by SDS– PAGE and transferred to nitrocellulose membranes, which were incubated with one of the following antibodies diluted in PBS con- taining 5% skimmed milk: 0.25 tig/ml anti caspase-3; 5 tig/ml mouse monoclonal anti-caspase-9; 1 tig/ml of anti-caspase-2, -4, -6, -8, or -10 antibodies and a 1/3000 dilution of rabbit polyclonal anticaspase-9. This was followed by washing with blocking buffer and incubation

with 0.2 tig/ml of the corresponding secondary antibody, immunoad- sorbed with human serum proteins (Sigma). Western blots were revealed with the BCIP/NBT substrate, as described [13]. In some cases, the lower part of blotting membranes was cut after the posi- tion of the 20-kDa marker and the portion of blotting membrane containing proteins of lower Mr was further developed for 6 h to allow detection of active caspase subunits. In blots of caspases 6, 8, and 10, some extra bands appeared that did not hamper the detection of caspases. These bands were due to proteins of different Mr recog- nized by the corresponding polyclonal antibodies, since omission of primary, but not secondary, antibody gave no bands.

RESULTS

Activation of Caspases during Doxorubicin-Induced
Apoptosis
We previously showed that doxorubicin, daunorubi- cin, and actinomycin D induce activation of caspase-3 and apoptosis in Jurkat cells [13]. Chromatin conden- sation and nuclear fragmentation were prevented by treatment with the caspase-3 inhibitor Ac-DEVD-CHO or the general caspase inhibitor Z-VAD-fmk, but only the latter prevented cell death. We have now analyzed the activation of other caspases during drug-induced apoptosis by Western blot analysis with specific anti- bodies. Treatment with doxorubicin, daunorubicin, or actinomycin D activated caspases 2, 3, 4, 6, 7, 8, and 10 in Jurkat cells (Fig. 1). These cells also expressed sig- nificant amounts of procaspase-9 (46–48 kDa protein), as revealed by a specific polyclonal anti-caspase-9 an- tibody (Fig. 2). Treatment of cells with doxorubicin increased the intensity of a protein doublet at 35/37 kDa (large subunit) and ti14 kDa (proteolytic frag- ment). Activation of caspase-9 was also confirmed by Western blot analysis with a different antibody recog- nizing the small subunit (10 kDa) of the active caspase-9 (data not shown).

Effect of Inhibitors on Doxorubicin-Induced Apoptosis

To evaluate the relative contribution of the different caspase subfamilies to doxorubicin-induced apoptosis, we analyzed the effect of different peptide inhibitors on apoptosis. As reported [13], treatment of Jurkat cells with doxorubicin in the presence of Ac-DEVD-CHO, which primarily inhibits caspases 3, 7, and 8 [27, 28], prevented nuclear apoptotic morphology but not cell death. Similarly, the peptide Z-IETD-fmk, which pref- erentially inhibits caspases 6, 8, 9, and 10 [28, 29], blocked nuclear apoptosis (Fig. 3) but did not inhibit PS exposure, loss of titi m, and cell death (see later). Treatment with a combination of Ac-DEVD-CHO and Z-IETD-fmk did not improve cell survival. The mini- mal-length inhibitor of caspase-2, Z-VDVAD-fmk, which also inhibits caspases 3 and 7 [30], prevented

FIG. 1. Drug-induced activation of caspases 2, 3, 4, 6, 7, 8, and 10 in Jurkat cells. Jurkat cells (5 ti 105 cells/ml) were left un- treated (1) or incubated with 1 tiM doxorubicin for 16 h (2), 1 tiM daunorubicin for 9 h (3) or 16 h (4) or 1 tig/ml actinomycin D for 8 h (5). Cell proteins (2 ti 106 per sample) were extracted and sepa- rated by SDS–12% PAGE and caspase activation was analyzed by Western blotting using the corresponding antibodies. The relative molecular mass of the fragments obtained is indicated. Activation of caspase-2 generated three fragments of 17, 12, and 10 kDa. Activation of caspase-3 was assessed by the disappearance of the 32-kDa band. Activation of caspase-4 caused the appearance of a fragment of 36 kDa (arrow) and three small fragments of 17, 14, and 12 kDa (not shown). Activation of caspase-6 was characterized by the appearance of an 18-kDa fragment. Caspase-7 activation led to a reduction in the intensity of the 35-kDa proenzyme and the appearance of a 12-kDa band. Activation of caspases 8 and 10 was characterized by a moderate reduction in the intensity of the proforms (53/55 kDa and 55 kDa, respectively) and the appear- ance of a band at around 17 kDa.

(61% of cell death in cells treated with 1 tiM doxorubi- cin alone versus 35% of cell death in cells treated with doxorubicin and actinomycin D).

Effect of Peptide Inhibitors on Caspase Activation
To determine the sequential order of caspase activa- tion, we analyzed by Western blotting their proteolytic processing in cells treated with doxorubicin in the pres- ence of different peptide inhibitors. Inhibition of

FIG. 2. Drug treatment induces caspase-9 activation. Jurkat cells were left untreated (lane 1) or treated with different drugs (lanes 2–6) and analyzed as indicated in the legend of Fig. 1, by immunoblotting with an anti-caspase-9 antibody. Lane 2, doxorubi- cin (1 tiM, 16 h); lane 3, daunomycin (1 tiM, 16 h); lane 4, actinomy- cin D (0.5 tig/ml, 13 h); lane 5, methotrexate (100 ng/ml, 18 h); lane 6, vincristine (10 ng/ml, 18 h).

doxorubicin-induced nuclear apoptosis (not shown), but not cell death (Fig. 4A). Treatment with the opti- mal sequence inhibitor of caspase-9, Z-LEHD-fmk [31], prevented again the appearance of apoptotic morphol- ogy but did not inhibit cell death (Fig. 4A). Ac-YVAD- cmk, an inhibitor of ICE-like proteases, offered no pro- tection (not shown). All the morphological and biochemical manifestations of apoptosis, as well as cell death, were abrogated by the broad-specificity caspase inhibitor Z-VAD-fmk [13, 18]. The cytoprotective effect of Z-VAD-fmk was still maintained after 48 h of treat- ment, although cell growth was significantly inhibited (not shown). After 72 h of incubation with doxorubicin, Jurkat cells begun to reduce their viability (Fig. 4B). Treatment with higher doses of doxorubicin (ti10 tiM), led to a necrotic phenotype (enlarged nuclei and cell size, loss of membrane integrity), which was not pre- vented by Z-VAD-fmk (not shown).
It has been recently reported [32] that Z-VAD-fmk can inhibit, in cell-free systems, the activity of cathep- sin B, a lysosomal cysteine protease. We therefore eval- uated whether cytoprotective effects of Z-VAD-fmk were mediated by the inhibition of this enzyme. Jurkat cells were treated with doxorubicin in the presence or absence of Z-FA-fmk, an inhibitor of cathepsin B that did not inhibit caspases. As shown in Fig. 4A, treat- ment with Z-FA-fmk alone (100 tiM) inhibited cell growth and offered no significant protection from doxo- rubicin-induced apoptosis. On the other hand, the toxic effects of doxorubicin were dependent on de novo pro- tein synthesis, since cotreatment of Jurkat cells with cycloheximide prevented, in great extent, cell death (Fig. 4C). Treatment with low doses of actinomycin D (30–40 ng/ml), which did not cause apoptosis per se, also protected significantly from doxorubicin toxicity

FIG. 3. Z-IETD-fmk prevents doxorubicin-induced nuclear apop- tosis, but not cell death. Jurkat cells were incubated for 18 h with (A) 80 tiM Z-IETD-fmk, (B) 1 tiM doxorubicin, or (C) 80 tiM Z-IETD-fmk plus 1 tiM doxorubicin. Nuclear morphology was analyzed by fluo- rescence microscopy of PPDA-stained cells. Original magnification, ti400.

gated the characteristic degradation of lamin B, a marker of caspase-6 activity [33, 34]. In cells incubated with doxorubicin and Ac-DEVD-CHO, no activation of caspase-2, caspase-8, and caspase-10 was noticed (Fig. 6). It is known that procaspase-3 is processed by an upstream caspase to yield two subunits of 20 and 12 kDa. The 20-kDa subunit is then autoproteolyzed to 19- and 17-kDa fragments. Accordingly, the major sub- unit detected in cells treated with doxorubicin alone was p17 but in the presence of Ac-DEVD-CHO, only the p20 subunit of caspase-3 appeared (Fig. 6). However, since Ac-DEVD-CHO efficiently inhibits caspases 3 and 8 [28, 35], these experiments did not allow to distin- guish which of these caspases was first activated. To solve this question, we used cells overexpressing CrmA (CrmA/Jurkat cells). As previously reported [20], treat- ment with doxorubicin caused apoptosis in CrmA/Jur- kat cells, which increased with time, and activation of caspases 3 and 8 (Fig. 7). Apoptosis in CrmA/Jurkat cells was prevented by Z-VAD-fmk. However, in CrmA/
Jurkat cells treated with cytotoxic anti-Fas antibodies no activation of caspases 8 and 3 could be observed by Western blotting (data not shown), according to their known resistance to Fas-induced apoptosis [22]. Since CrmA is a strong inhibitor of caspase-8 but it does not inhibit caspase-3 [27, 28], these results indicate that caspase-3 was first activated in doxorubicin-induced apoptosis and was responsible for the proteolytic pro-

FIG. 4. Effect of peptide inhibitors and cycloheximide on doxo- rubicin-induced apoptosis. (A) Jurkat cells (5 ti 105 cells/ml) were treated for 22 h with 100 tiM Z-VDVAD-fmk, 100 tiM Z-FA-fmk, or Z-LEHD-fmk in the presence (tiDox) or absence (tiDox) of 1 tiM doxorubicin and cell viability determined by the MTT-assay. (B) Cells were incubated with 1 tiM doxorubicin for the times indicated in the presence or absence of 100 tiM Z-VAD-fmk. Cell death was evaluated by Trypan-blue staining and microscopical examination of cells. (C) Cells were preincubated with the indicated doses of cyclo- heximide for 1 h prior to the addition of doxorubicin and further incubated for 18 h. Cell viability was determined by the MTT assay. Results are the mean of four individual values, each from three independent experiments. Vertical bars indicate SD.

caspases 8, 9, and 10 with Z-IETD-fmk (Fig. 5) or Z-LEHD-fmk (not shown) blocked caspase-2, caspase-3, and caspase-4 activation. This also abro-

FIG. 5. Z-IETD-fmk inhibits the activation of caspases 2, 3, and 4, and lamin B degradation. Jurkat cells were preincubated for 1 h with 80 tiM Z-IETD-fmk and then with 1 tiM doxorubicin for 16 h more. The activation of caspases was analyzed by Western blot, using anti-caspase-2, anti-caspase-3, anti-caspase-4, and anti-lamin B antibodies, as indicated. 1, control cells; 2, ti doxorubicin; 3, ti doxorubicin ti Z-IETD-fmk.

displaying a reduced titi m and PS translocation (Fig. 9B and see below).

Effect of Doxorubicin Treatment on
Phosphatidylserine Translocation and titi m Loss
An early event of many types of apoptosis is the opening of mitochondrial pores or megachannels, a pro-

FIG. 6. Ac-DEVD-CHO blocks caspase-3 activity and prevents activation of caspases 2, 8, and 10. Jurkat cells were preincubated for 3 h with 600 tiM Ac-DEVD-CHO and then 1 tiM doxorubicin was added and incubated for 16 h. Caspase-3 activation was analyzed with a mAb raised against the proenzyme. Caspase-2, -8, and -10 activation was analyzed with polyclonal anti-caspase antibodies raised against the p20 subunit. The fragments obtained are the same as shown in Fig. 1: Jurkat; 2: Jurkat ti doxorubicin; 3: Jurkat ti Ac-DEVD-CHO ti doxorubicin.

cessing of caspase-8. Doxorubicin also induced activa- tion of caspase-9 in CrmA/Jurkat cells (Fig. 8).
Activation of caspase-9 was blocked in Jurkat cells treated with doxorubicin in the presence of Z-VAD- fmk, according to the absence of apoptosis. In cells cotreated with Ac-DEVD-CHO, only the 35-kDa band was present (Fig. 8). Thus, Ac-DEVD-CHO inhibited the cleavage of caspase-9, mediated by caspase-3, but not the self-cleavage of caspase-9, as recently reported [36, 37]. Z-IETD-fmk blocked caspase-9 processing, al- though this inhibitor was unable to prevent cell death. On the other hand, Jurkat cells overexpressing Bcl-2 gene (Bcl-2/Jurkat) were fully resistant to doxorubicin toxicity (Fig. 7A), according to previous reports [19, 20]. In doxorubicin-treated Bcl-2/Jurkat cells, no acti- vation of caspases 3, 8, and 9 was observed (Figs. 7 and 8). Time-course analysis of caspase-9 activation re- vealed that activation was first detected in Jurkat cell homogenates after 12–13 h of incubation with doxoru- bicin (Fig. 9A), at the time of the appearance of cells

FIG. 7. Effect of doxorubicin on caspase activation and cell death in Jurkat transfectants. (A) Cells overexpressing CrmA (JCrmA, 5 ti 105 cells/ml), Bcl-2 (JBcl-2, 3 ti 105 cells/ml), and vector-transfected cells (Jvector, 5 ti 105 cells/ml) were incubated with 1 tiM doxorubi- cin for different periods of time in the presence or absence of 100 tiM Z-VAD-fmk or 600 tiM Ac-DEVD-CHO, as indicated. Cell death was evaluated by Trypan-blue staining. Results are the mean values from four different cultures and vertical bars indicate SD. (B) Doxo- rubicin induced activation of caspases 3 and 8 in CrmA/Jurkat but not in Bcl-2/Jurkat cells. Jurkat transfectants were incubated for 30 h in complete medium (C) or in medium containing 1 tiM doxo- rubicin (ti Dox) and activation of caspase-3 and caspase-8 was de- termined by Western blot analysis of cell extracts (2 ti 106 cells/lane) using the corresponding antibodies. The position of proenzymes and activation fragments is indicated by arrows.

expression did not prevent doxorubicin-induced titi loss, but cells transfected with the Bcl-2 gene main- tained a normal titi m after doxorubicin treatment (not shown), according to previous reports [19].

FIG. 8. Effect of caspase inhibitors and Bcl-2 on doxorubicin- induced caspase-9 activation. (A) Jurkat cells were treated with doxorubicin for 22 h in the presence of different peptide caspase inhibitors and activation of caspase-9 was analyzed by Western blotting with an anti-caspase-9 antibody. 1, untreated cells; 2, ti doxorubicin; 3, doxorubicin ti 600 tiM Ac-DEVD-CHO; 4, doxorubi- cin ti 100 tiM Z-VAD-fmk; 5, doxorubicin ti 80 tiM Z-IETD-fmk. (B) Jurkat transfectants were left untreated or treated for 30 h with doxorubicin, as indicated, and caspase-9 activation was analyzed by Western blotting.

cess known as the mitochondrial permeability transi- tion (PT) [38]. One of the consequences of PT is the disruption of the mitochondrial inner transmembrane potential (titi m). We used the potential-sensitive probe DiOC6(3) for the cytofluorometric determination of titi m during doxorubicin-induced apoptosis. As a nega- tive control, cells were preincubated with the uncou- pling protonophore CCCP, prior to DiOC6(3) labeling (Fig. 10A). Treatment of Jurkat cells with doxorubicin caused the collapse of titi m, as detected by a reduction in the amount of incorporated probe. The titi m loss was first appreciable after 12–13 h of incubation with the drug and increased afterwards (Fig. 9B). Doxorubicin- induced loss of titi m was prevented by coincubation with Z-VAD-fmk, but not with Ac-DEVD-CHO, Z-IETD- fmk (Fig. 10), Z-LEHD-fmk, or Z-VDVAD-fmk (data not shown). In 24-h incubations, the percentage of cells displaying a reduced titi m correlated quite well in all cases with the percentage of cell death estimated by the annexin V staining (Fig. 10). In the case of cells treated with doxorubicin in the presence of Ac-DEVD-CHO or Z-IETD-fmk, there was a population of cells display- ing an intermediate transmembrane mitochondrial po- tential (titi m)int, absent in cells treated with the drug alone (Fig. 10). This may represent cells that lose more slowly their titi m due to the inhibition of caspases located downstream of mitochondria [19]. CrmA over-

FIG. 9. Doxorubicin causes titi m disruption and PS exposure in Jurkat cells, which parallels caspase-3 and -9 activation. (A) Cells were incubated for different periods, as indicated, with 1 tiM doxorubicin and the processing of caspase-3 and caspase-9 was analyzed by Western blotting. Mitochondrial membrane potential (titi m) and PS exposure was determined by flow cytometric analysis of cells labeled with DiOC6(3) and annexin V-FITC, respectively, and is indicated in each case. (B) Time course of PS exposure and titi m loss induced by doxoru- bicin treatment. The corresponding values for cells incubated in the absence of doxorubicin were subtracted in each point. Results corre- spond to a single representative experiment out of three. Results of the three experiments were comparable except for the time at which PS exposure and titi m loss came to be evident (11–13 h).

FIG. 10. Z-VAD-fmk, but not Ac-DEVD-CHO or Z-IETD-fmk, inhibits titi m loss induced by doxorubicin. Jurkat cells (5 ti 105 cells/ml) were preincubated with Z-VAD-fmk (100 tiM, 1 h), Ac-DEVD-CHO (600 tiM, 3 h), or Z-IETD-fmk (80 tiM, 1 h) and then treated with doxorubicin for 18 h. Three cell populations exhibiting different titi m could be distinguished after doxorubicin treatment in the presence of caspase inhibitors: high (titi m)high, intermediate (titi m)int, and low (titi m)low. Cells incubated with inhibitors alone were used as controls. (A) Flow cytometry profiles of DiOC6(3) staining. Figures in the boxes indicate the percent of cell death, evaluated by Trypan-blue staining. (B) Relative distribution of titi m in the cell subpopulations showed in (A). (C) Plasma membrane PS exposure of cells after the different treatments, as determined by annexin V-FITC binding.

Translocation of PS to the outer layer of the plasma membrane, an early apoptotic event that seems to de- pend on caspase activity [39], was coincident in time or slightly preceded the appearance of cells displaying a reduced titi m (Fig. 9). Both parameters were compara- ble at longer incubation times (Figs. 9 and 10). Since only Z-VAD-fmk, but not other more selective caspase inhibitors, prevented doxorubicin-induced cell death, we next analyzed whether the key caspase inhibited by Z-VAD-fmk acted at pre- or postmitochondrial level. In particular, we evaluated whether Z-VAD-fmk could
block apoptosis in cells that have undergone titi m loss. After 13 h of incubation, doxorubicin induced a specific reduction in titi m in approximately 10% of cells, as determined by DiOC6(3) labeling (Fig. 9). This number increased up to 20% after around 14–15 h. At this time, virtually all cells excluded Trypan blue. Addition of Z-VAD-fmk 1 h before or together with doxorubicin prevented, for the most part, the loss of titi m and cell death, determined after 24 h of incubation with doxo- rubicin. Addition of Z-VAD-fmk 2, 4, or 6 h after doxo- rubicin addition was also equally protective (Fig. 11).

CASPASE ACTIVATION IN DOXORUBICIN APOPTOSIS 231

FIG. 11. Effect of delayed addition of Z-VAD-fmk in doxorubicin- induced titi m loss and apoptosis. Jurkat cells (5 ti 105 cells/ml) were incubated with 1 tiM doxorubicin for 24 h and the percentage of cells with low titi m and cell death was estimated in each case by DiOC6(3) and Trypan-blue staining, respectively. Z-VAD-fmk (100 tiM) was added at different intervals from 1 h before to 15 h after the addition of doxorubicin, as indicated. Cells treated with 0.1% DMSO or Z-VAD-fmk alone were used as controls.

Addition of Z-VAD-fmk after 15 h of incubation with doxorubicin, rescued from apoptosis cells still display- ing a normal titi m, since, after 24 h of treatment, the percentage of cell death was similar to the percentage of cells displaying a low titi m at 15 h (Fig. 9). This indicates that cells with low titi m are irreversibly com- mitted to die, even if Z-VAD-fmk inhibits the activity of all caspases downstream of mitochondria. These re- sults suggest the existence of a Z-VAD-sensitive caspase, acting at a premitochondrial level, whose ac- tivity would cause the titi m loss.

DISCUSSION
Doxorubicin is one of the chemotherapeutic drugs more widely used, but its mechanism of toxicity still remains unclear. Recent works indicate that different mechanisms of DNA damage occur depending on doxo- rubicin concentration [1, 40]. Concentrations between 1 and 100 tiM achieved similar cytotoxic effects in a human leukemia. However, at concentrations greater than 3 tiM (pharmacologically inachievable), doxorubi- cin induced free-radical generation, lipid peroxidation, and cell death without internucleosomal DNA frag- mentation [11, 40]. Maximal apoptosis was observed at 1 tiM doxorubicin, congruent with concentrations that can be reached in vivo [41, 42]. Doxorubicin-induced apoptosis has been proposed to be mediated through the activation of the Fas/FasL system [11, 12]. How- ever, although in some systems doxorubicin may in- duce Fas and FasL upregulation, it has been unambig- uously proved that apoptosis induced by doxorubicin is Fas-independent [13–18, 43]. Recently, it has been shown that apoptosis induced by doxorubicin causes disruption of titi m [19] and caspase activation [13, 20,
44]. In sensitive leukemia cells, doxorubicin induces caspase-3 activation and apoptosis. However, selective inhibition of caspase-3 did not prevent cell death [13], indicating that the activity of other caspase is critical for apoptosis.
According to their substrate specificity, sequence ho- mology, and biochemical function, the known human caspases may be classified into three major subfami- lies: ICE-like (caspases 1, 4, 5, and 13), activator (caspases 8, 9, 10), and effector (caspases 2, 3, 6, 7) caspases [45]. While ICE-like enzymes seem to be mainly involved in inflammation, the two latter sub- families are implicated in apoptosis. Optimal se- quences for peptide-based caspase inhibitors have been determined to be (I/V/L)EXD for activator caspases, DEXD for effector caspases, and (W/L)EHD for ICE- like caspases [30, 31]. Peptide-based inhibitors have been proven useful to evaluate the implication of caspases in apoptosis, although their specificity seem to be less strict than previously recognized [28], since some peptide fluoro- and chloromethylketones can also react with lysosomal cysteine proteases [32]. Another inhibitor, the serpin CrmA, efficiently inhibits caspases 1, 8, and 10, but it does not inhibit caspases 2, 3, 6, 7, and 9 (present work and [28, 46, 47]).
We show here that doxorubicin treatment induces activation of caspases 2, 3, 4, 6, 7, 8, 9, and 10 in Jurkat cells. Caspase activation and apoptosis was completely blocked by Z-VAD-fmk, an extremely efficient inhibitor of all known caspases [28, 35, 46], with the possible exception of caspase-2 [30]. More selective inhibitors, such as Z-IETD-fmk (which inhibits caspases 8, 9, and 10), Ac-DEVD-CHO (which inhibits caspases 3, 7, and 8), Z-VDVAD-fmk (a minimal inhibitor of caspase-2), and Z-LEHD-fmk (optimal inhibitor for caspase-9) only pre- vented morphological features of apoptosis (i.e., cell shrinking, chromatin condensation, nuclear fragmen- tation, formation of apoptotic bodies), but did not block cell death. Overexpression of CrmA was also insuffi- cient to prevent cell death. Treatment with Ac- DEVD-CHO, Z-LEHD-fmk, or Z-IETD-fmk abrogated nevertheless the cleavage and activation of caspases 2, 3, 4, 6, 8, 9, and 10. Thus, only caspase inhibitors halting the apoptotic process before titi m loss (Z-VAD- fmk), but not those inhibiting caspases activated after titi m loss (Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk, Ac-DEVD-CHO), were actually cytoprotective. Since Z- IETD-fmk or Z-LEHD-fmk also blocked the activation of all caspases examined, these results suggest that while caspases activated after titi m loss were respon- sible for the development of the apoptotic phenotype, a Z-VAD-sensitive caspase (caspase-X), acting at a pre- mitochondrial level, was the key mediator of cell death. The inhibition of titi m loss and cell death by cyclohex- imide and the cytoprotection offered by the delayed administration of Z-VAD-fmk suggest that the expres-

sion and/or the activity of this caspase-X may be in- duced de novo by doxorubicin. It has been reported that inhibition of protein synthesis blocks doxorubicin-in- duced apoptosis, both in vitro [3, 4, 11, 40] and in vivo [48, 49]. Anyway, the induction of caspase-X activity is not dependent on p53, since Jurkat cells express inac- tive mutant or no p53 protein [50].
Comparable results have been found in other cell types or with other drugs. Treatment of promyelocytic HL-60 cells with clinically achievable doses of etopo- side (0.1–1 tiM) caused caspase-9 activation and apop- tosis. Apoptosis was blocked by overexpression of Bcl-2 or cotreatment with Z-VAD-fmk but not with Z-IETD- fmk [51]. However, Z-IETD-CHO was able to block pro- cessing of caspases 3, 7, 8, and 9 in a Jurkat cell lysate [52]. In HL-60 cells, caspase-3 and -6 were activated during etoposide-induced apoptosis and their activity was blocked by caspase inhibitors [34]. Moreover, over- expression of Bcl-2, but not CrmA, protected murine WEHI-231 leukemia or human CEM-7H2 lymphoma cells from doxorubicin-induced apoptosis [19]. Com- plete inhibition of apoptotic fenotype and cell death by Z-VAD-fmk was also observed in promonocytic THP.1 cells treated with etoposide. Etoposide caused titi m loss, PS translocation, and activation of caspases 2, 3, 6, and 7 [53]. Etoposide-induced activation of caspases 3, 7, 8, and 9 in IMR90E1A cells expressing a domi- nant-negative mutant of caspase-9 was blocked, but cells still died [54].
In the past 2 years, the existence of two essentially different apoptotic pathways has emerged [55]. The first one, the “cytosolic pathway,” is triggered by extra- cellular death messengers and involves the activation of a cytosolic caspase cascade. The activity of these caspases will ultimately lead to the development of the apoptotic phenotype and cell death. The second, the so-called “mitochondrial pathway,” is rather triggered by intracellular signals. The pathway is initiated when mitochondria, in response to cell damage or other sig- nals, release several apoptogenic factors, including cy- tochrome c [56, 57], the protein AIF [58], and pro- caspases 2, 3, and 9 [59, 60]. Cytochrome c binds to a cytosolic adaptor protein, Apaf-1, and the complex binds to and activates procaspase-9 through limited autoproteolysis [61]. Active caspase-9 proteolyzes and activates caspase-3, which in turn hydrolyzes caspase-9 [36]. Caspase-3 may then activate other downstream caspases causing the apoptotic phenotype. Bcl-2 proteins inhibit this pathway probably by block- ing cytochrome c release from mitochondria, thus pre- venting binding to Apaf-1 [62, 63]. The existence of these two pathways explains why apoptosis induced by extracellular death messengers is, in general, not blocked by Bcl-2 or Bcl-xL overexpression but apoptosis induced by glucocorticoids, drugs, or irradiation is blocked by Bcl-2 or Bcl-xL. Thus, in the cytosolic path-

FIG. 12. Hypothetical schema for doxorubicin-induced apoptosis in Jurkat cells. Doxorubicin causes DNA damage and gene expres- sion leading to the de novo synthesis of an unidentified caspase (caspase-X). Caspase-X may then act on mitochondria either directly or indirectly, through the proteolysis of a cytosolic substrate. This would lead to titi m loss and release of apoptogenic factors from mitochondria, including AIF, which would cause nuclear apoptosis, and cytochrome c (cyt c), which would bind to cytosolic Apaf-1 and activate caspase-9. Caspase-9 would then cleave and activate caspase-7 and caspase-3, which, in turn, would hydrolyze caspase-9, engaging in a feedback amplification loop. Caspase-3 would also activate caspases 2, 4, 6, 8, and 10 and their concerted actions will ultimately cause the apoptotic phenotype. The possible sites of inhi- bition of Bcl-2 protein and of the different caspase inhibitors are indicated.

way, caspase-3 activity precedes titi m loss; in the mi- tochondrial pathway, titi m loss precedes caspase-3 ac- tivation [55]. From these models and our present results, we would suggest a hypothetical scenario to explain the role of caspases in doxorubicin-induced apoptosis in Jurkat cells (Fig. 12). When doxorubicin enters cells it reaches the nucleus and causes genotoxic damage, which may induce the activity of an unchar- acterized, Z-VAD-sensitive caspase (caspase-X). This may be due to the de novo expression of the caspase or another protein that would cause activation of that

caspase. Caspase-X would then act on mitochondria and induce titi m loss and release of apoptogenic factors, including cytochrome c and AIF to cytoplasm [58, 64]. Alternatively, caspase-X may cause titi m loss indirectly through the cleavage and activation of a Bid-like pro- tein [65]. Caspase-9 may then be activated through binding to cytosolic cytochrome c/Apaf-1 complex. Ac- tive caspase-9 will in turn directly activate caspase-3 and caspase-7, but not caspases 2, 4, 8, and 10 [66] and cleave and activate caspase-6. Caspases 8, 10, 2, and 4 may be either directly activated by caspase-3 or indi- rectly via caspase-6 [37]. Inhibition of all caspases, downstream of mitochondria, may prevent the devel- opment of the classical apoptotic phenotype but not cell death, since the activity of AIF is not blocked by caspase inhibitors [58]. AIF induces an atypical nu- clear apoptosis through a caspase-independent mech- anism [58]. Bcl-2 overexpression may block apoptosis by direct binding to mitochondrial PT complex [67], preventing either direct action of caspase-X or the in- sertion of a Bid-like protein. Thus, caspase-9 activity triggers the execution phase of apoptosis and controls the development of apoptotic morphology but is not essential for cell death. Consistent with this, death induced by doxorubicin is not blocked, in thymocytes and splenocytes from caspase-9 knock-out mice [68, 69]. Our present results are not incompatible, never- theless, with the existence of caspase-independent mechanisms for titi m loss and release of apoptogenic factors from mitochondria in response to other cyto- toxic drugs or in other cell types [21, 70].
In brief, the activity of a still unidentified Z-VAD- sensitive caspase, which induces titi m loss and release of apoptogenic proteins from mitochondria, seems to be the key step in the mechanism of doxorubicin-induced apoptosis in Jurkat cells. The precise identification of this caspase and its targets would be useful to improve prognosis and chemotherapy of leukemia.

This work was supported by Grants P74/98 from Diputacio´n Gen- eral de Arago´n and 99/1250 from Fondo de Investigacio´n Sanitaria. S.G. was recipient of a fellowship from Fundacio´n Cientı´fica de la Asociacio´n Espan˜ola contra el Cancer, and P.P.-G. from Ministerio de Educacio´n y Cultura (Spain).

REFERENCES
1.Gewitz, D. A. (1999). A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57, 727–741.
2.Barry, M. A., Behnke, C. A., and Eastman, A. (1990). Activation of programmed cell death (apoptosis) by cisplatin, other anti- cancer drugs, toxins and hyperthermia. Biochem. Pharmacol. 40, 2353–2362.
3.Skladanowski, A., and Konopa, J. (1993). Adriamycin and daunomycin induce programmed cell death (apoptosis) in tumor cells. Biochem. Pharmacol. 46, 375–382.

4.Zaleskis, G., Berleth, E., Verstovsek, S., Ehrke, M. J., and Mihich, E. (1994). Doxorubicin-induced DNA degradation in murine thymocytes. Mol. Pharmacol. 46, 901–908.
5.Hannun, Y. A. (1997). Apoptosis and the dilemma of cancer chemotherapy. Blood 89, 1845–1853.
6.Bose, R., Verheij, M., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995). Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals. Cell 82, 405–414.
7.Jaffre´zou, J. P., Levade, T., Bettaı¨eb, A., Andrieu, N., Bezombes, C., Maestre, N., Vermeersch, S., Rousse, A., and Laurent, G. (1996). Daunorubicin-induced apoptosis: Trigger- ing of ceramide generation through sphingomyelin hydrolysis. EMBO J. 15, 2417–2424.
8.Dbaibo, G. S., Perry, D. K., Gamard, C. J., Platt, R., Poirier, G. G., Obeid, L. M., and Hannun, Y. A. (1997). Cytokine re- sponse modifier A (CrmA) inhibits ceramide formation in re- sponse to tumour necrosis factor (TNF)-ti: CrmA and Bcl-2 target distinct components in the apoptotic pathway. J. Exp. Med. 185, 481–490.
9.Gamen, S., Anel, A., Pin˜eiro, A., and Naval, J. (1998). Caspases are the main executioniers of Fas-mediated apoptosis, irrespec- tive of the ceramide-signaling pathway. Cell Death Differ. 5, 241–249.
10.Hofmann, K., and Dixit, V. M. (1998). Ceramide in apoptosis— Does it really matter? Trends Biochem. Sci. 23, 374–377.
11.Friesen, C., Herr, I., Krammer, P. H., and Debatin, K. M. (1996). Involvement of the CD95 (APO-1/Fas) receptor/ligand system in drug-induced apoptosis of leukemia cells. Nature Med. 2, 574–577.
12.Fulda, S., Sieverts, H., Friesen, C., Herr, I., and Debatin, K. M. (1997). The CD95 (APO-1/Fas) system mediates drug-induced apoptosis in neuroblastoma cells. Cancer Res. 57, 3823–3829.
13.Gamen, S., Anel, A., Lasierra, P., Alava, M. A., Martinez- Lorenzo, M. J., Pin˜eiro, A., and Naval, J. (1997). Doxorubicin- induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way. FEBS Lett. 417, 360–364.
14.Eischen, C. M., Kottke, T. J., Martins, L. M., Basi, G. S., Tung, J. S., Earnshaw, W. C., Leibson, P. J., and Kaufmann, S. H. (1997). Comparison of apoptosis in wild-type and Fas-resistant cells: Chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. Blood 90, 935–943.
15.Villunger, A., Egle, A., Kos, M., Hartmann, B. L., Geley, S., Kofler, R., and Greil, R. (1997). Drug-induced apoptosis is as- sociated with enhanced Fas (Apo-1/CD95) ligand expression but occurs independently of Fas signaling in human T-acute lym- phatic leukemia cells. Cancer Res. 57, 3331–3334.
16.Yeh, W. C., de la Pompa, J. L., McCurrach, M. E., Shu, H. B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998). FADD: Essential for embryo development and signaling from some, but not all, inducers of apoptosis. Science 279, 1954–1958.
17.Ruiz-Ruiz, M. C., and Lo´pez-Rivas, A. (1999). p53-mediated upregulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells. Cell Death Differ. 6, 271–280.
18.Wesselborg, S., Engels, I. H., Rossman, E., Los, M., and Schulze-Osthoff, K. (1999). Anticancer drugs induce caspase-8/
FLICE activation and apoptosis in the absence of CD95 recep- tor/ligand interaction. Blood 93, 3053–3063.
19.Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R., and Kroemer, G. (1997). Bcl-2 and Bcl-XL

234 GAMEN ET AL.

antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res. 57, 62–67.
20.Antoku, K., Liu, Z., and Johnson, D. E. (1997). Inhibition of caspase proteases by CrmA enhances the resistance of human leukemic cells to multiple chemotherapeutic agents. Leukemia 11, 1665–1672.
21.Sun, X. M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R., and Cohen, G. M. (1999). Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J. Biol. Chem. 274, 5053–5060.
22.Dou, Q. P., An, B., Antoku, K., and Johnson, D. E. (1997). Fas stimulation induces RB dephosphorylation and proteolysis that is blocked by inhibitors of the ICE protease family. J. Cell. Biochem. 64, 586–594.
23.Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czer- winski, M. J., Fine, D., Abbot, B. J., Mayo, J. G., Shoemaker, R. H., and Boyd, M. R. (1988). Sensibility of drug screening with panels of human tumor cell lines using microculture tetrazo- lium assay. Cancer Res. 48, 589–601.
24.Anel, A., Gamen, S., Alava, M. A., Schmitt-Verhulst, A. M., Pin˜eiro, A., and Naval, J. (1996). Role of oxidative damage and ICE-like proteases in Fas-based cytotoxicity exerted by effector T cells. Int. Immunol. 8, 1173–1183.
25.Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J. A., van Schie, R. C. A. A., LaFace, D. M., and Green, D. R. (1995). Early redistribution of plasma membrane phos- phatydilserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545–1556.
26.Petit, P. X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., and Gougeon, M. L. (1995). Alterations in mitochondrial struc- ture and function are early events of dexamethsone-induced thymocyte apoptosis. J. Cell Biol. 130, 157–167.
27.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., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T. T., Yu, V. L., and Miller, D. K. (1995). Identification and inhibition of the ICE/CED-3 protease necessary for mam- malian apoptosis. Nature 376, 37–43.
28.Garcı´a-Calvo, M., Peterson, E. P., Leiting, B., Ruel, R., Nichol- son, D. W., and Thornberry, N. A. (1998). Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273, 32608–32613.
29.Medema, J. P., Scaffidi, C., Kischkel, F. C., Shevchenko, A., Mann, M., Krammer, P. H., and Peter, M. E. (1997). FLICE is activated by association with the CD95 death-inducing signal- ing complex (DISC). EMBO J. 16, 2794–2804.
30.Talanian, R. V., Quinlan, C., Trautz, S., Hackett, M. C., Man- kovich, J. A., Banach, D., Ghayur, T., Brady, K. D., and Wong, W. W. (1997). Substrate specificity of caspase family proteases. J. Biol. Chem. 272, 9677–9682.
31.Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nichol- son, D. W. (1997). 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. 272, 17907–17911.
32.Schotte, P., Declercq, W., Van Huffel, S., Vandenabeele, P., and Beyaert, R. (1999). Non-specific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett. 442, 117–121.
33.Lazebnik, Y. A., Takahashi, A., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshw, W. C. (1995).
Studies of the lamin protease reveal multiple parallel biochem- ical pathways during apoptotic execution. Proc. Natl. Acad. Sci. USA 92, 9042–9046.
34.Martins, L. M., Kottke, T., Mesner, P. W., Basi, G. S., Sinha, S., Frigon, N., Tatar, E., Tung, J. S., Bryant, K., Takahashi, A., Svingen, P. A., Madden, B. J., McCormick, D. J., Earnshaw, W. C., and Kaufmann, S. H. (1997). Activation of multiple interleukin-1ti converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis. J. Biol. Chem. 272, 7421–7430.
35.Muzio, M., Chinnayian, 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., and Dixit, V. M. (1996). FLICE, a novel FADD-homologous ICE/CED-3- like protease, is recruited to the CD95 (Fas/APO-1) death-in- ducing signaling complex. Cell 85, 817–827.
36.Srinivasula, S., Ahmad, M., Fernandes-Alnemri, T., and Al- nemri, E. S. (1998). Autoactivation of procaspase-9 by Apaf-1- mediated oligomerization. Mol. Cell. 1, 949–957.
37.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., and Martin, S. J. (1999). Ordering the cytochrome c-initiated caspase cascade: Hierar- chical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell Biol. 144, 281–292.
38.Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssie`re, J. L., Petit, P. X., and Kroemer, G. (1995). Reduction in mito- chondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661– 1672.
39.Naito, M., Nagashima, K., Mashima, T., and Tsuruo, T. (1997). Phosphatidylserine externalization is a downstream event of interleukin-1ti-converting enzyme family protease activation during apoptosis. Blood 89, 2060–2066.
40.Mu¨ller, I., Jenner, A., Bruchelt, G., Niethammer, D., and Hal- liwell, B. (1997). Effect of concentration on the cytotoxic mech- anism of doxorubicin-apoptosis and oxidative DNA damage. Biochem. Biophys. Res. Commun. 230, 254–257.
41.Muller, C., Chatelut, E., Gualano, V., De Forni, M., Huguet, F., Attal, M., Canal, P., and Laurent, G. (1993). Cellular pharma- cokinetics of doxorubicin in patients with chronic lymphocytic leukemia: Comparison of bolus administration and continuous infusion. Cancer Chemother. Pharmacol. 32, 379–384.
42.Speth, P. A., Linssen, P. C., Boezeman, J. B., Wessels, H. M., and Haanen, C. (1987). Cellular and plasma adriamycin con- centrations in long-term infusion therapy of leukemia patients. Cancer Chemother. Pharmacol. 20, 305–310.
43.Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., and Peter, M. E. (1998). Apoptosis signaling by death receptors. Eur. J. Biochem. 254, 439–459.
44.Martı´nez-Lorenzo, M. J., Gamen, S., Etxeberria, J., Lasierra, P., Larrad, L., Pin˜eiro, A., Anel, A., Naval, J., and Alava, M. A. (1998). Resistance to apoptosis correlates with a highly prolif- erative phenotype and loss of Fas and CPP32 (caspase-3) ex- pression in human leukemias. Int. J. Cancer. 75, 473–481.
45.Nicholson, D. W., and Thornberry, N. A. (1997). Caspases: Killer proteases. Trends Biochem. Sci. 22, 299–306.
46.Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1996). Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE- like cysteine proteases. Proc. Natl. Acad. Sci. USA 93, 14486– 14491.

47.Hedge, R., Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., and Alnemri, E. S. (1998). Blk, a BH3-containing mouse protein that interacts with Bcl-2 and Bcl-xL, is a potent death agonist. J. Biol. Chem. 273, 7783–7786.
48.Thakkar, N. S., and Potten, C. S. (1992). Abrogation of adria- mycin toxicity in vivo by cycloheximide. Biochem. Pharmacol. 43, 1683–1691.
49.Furusawa, S., Nakano, S., Kosaka, K., Takayanagi, M., Takay- anagi, Y., and Sasaki, K. (1995). Inhibition of doxorubicin- induced cell death in vitro and in vivo by cycloheximide. Biol. Pharm. Bull. 18, 1367–1372.
50.Yamato, K., Yamamoto, M., Hirano, Y., and Tsuchida, N. (1995). A human temperature-sensitive p53 mutant p53Val- 138: Modulation of the cell cycle, viability and expression of p53-responsive genes. Oncogene 11, 1–6.
51.Perkins, C., Kim, C. N., and Bhalla, K. N. (1998). Overexpres- sion of Apaf-1 promotes apoptosis of untreated and paclitaxel- or etoposide-treated HL-60 cells. Cancer Res. 58, 4561–4566.
52.Sun, D. Y., Jiang, S., Zheng, L. M., Ojcius, D. M., and Young, J. D. E. (1994). Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. J. Exp. Med. 179, 559–568.
53.McFarlane, M., Cain, K., Sun, X. M., Alnemri, E. S., and Cohen, G. M. (1997). Processing/activation of at least four interleu- kin-1ti converting enzyme-like proteases occurs during the ex- ecution phase of apoptosis in human momocytic tumor cells. J. Cell Biol. 137, 469–479.
54.Fearnhead, H. O., Rodriguez, J., Govek, E. E., Guo, W., Koba- yashi, R., Hannon, G., and Lazebnik, Y. A. (1998). Oncogene- dependent apoptosis is mediated by caspase-9. Proc. Natl. Acad. Sci. USA 95, 13664–13669.
55.Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Toma- selli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998). Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17, 1675–1687.
56.Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., and Wang, X. (1997). Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science 275, 1129–1132.
57.Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997). The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 275, 1132–1136.
58.Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., and Kroemer, G. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446.

Received February 7, 2000
Revised version received April 5, 2000

59.Mancini, M., Nicholson, D. W., Roy, S., Thornberry, N. A., Peterson, E. P., Casciola-Rosen, L. A., and Rosen, A. (1998). The caspase-3 precursor has a cytosolic and mitochondrial distribu- tion: Implications for apoptotic signaling. J. Cell Biol. 140, 1485–1495.
60.Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Pre´vost, M. C., Alzari, P. M., and Kroemer, G. (1999). Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med. 189, 381–393.
61.Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997). Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex ini- tiates apoptotic protease cascade. Cell 91, 479–489.
62.Zhivotovsky, B., Orrenius, S., Brustugun, O. T., and Doskeland, S. O. (1998). Injected cytochrome c induces apoptosis. Nature 391, 449–450.
63.Rosse´, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., and Borner, C. (1998). Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 391, 496– 499.
64.Marzo, I., Susin, S. A., Petit, P. X., Ravagnan, L., Brenner, C., Larochette, N., Zamzami, N., and Kroemer, G. (1998). Caspases disrupt mitochondrial barrier function. FEBS Lett. 427, 198– 202.
65.Bossy-Wetzel, E., and Green, D. R. (1999). Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. J. Biol. Chem. 274, 17484–17490.
66.Pan, G., Humke, E. W., and Dixit, V. M. (1998). Activation of caspases triggered by cytochrome c in vitro. FEBS Lett. 426, 151–154.
67.Marzo, I., Brenner, C., Zamzami, N., Susin, S. A., Beutner, G., Brdiczka, D., Re´my, R., Xie, Z. H., Reed, J. C., and Kroemer, G. (1998). The permeability transition pore complex: A target for apoptosis regulation by caspases and Bcl-2-related proteins. J. Exp. Med. 187, 1261–1271.
68.Kuida, K., Haydar, T. F., Kuan, C. Y., Gu, Y., Taya, C., Kara- suyama, H., Su, M. S. S., Rakic, P., and Flavell, R. A. (1998). Reduced apoptosis and cytochrome c-mediated caspase activa- tion in mice lacking caspase 9. Cell 94, 325–337.
69.Hakem, R., Hakem, A., Duncan, G. S., Henderson, J. T., Woo, M., Soengas, M. S., Elia, A., de la Pompa, J. L., Kagi, D., Khoo, W., Potter, J., Yoshida, W., Kaufman, S. A., Lowe, S. W., Pen- ninger, J. M., and Mak, T. W. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352.
70.McCarthy, N. J., Whyte, M. K. B., Gilbert, C. S., and Evan, G. I. (1997). Inhibition of Ced-3/ICE-related proteases does not pre- vent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell. Biol. 136, 215–227.