Caspase-3-like activity is necessary but not sufficient for ... - Nature

Leukemia (1999) 13, 1056–1061  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu

Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells KJ Turnbull1, BL Brown2 and PRM Dobson1 Divisions of 1Oncology and Cellular Pathology, and 2Biochemical and Musculoskeletal Medicine, University of Sheffield Medical School, Sheffield, UK

In the present study we have shown that the cancer therapeutic drug, daunorubicin, induces apoptosis in the human lymphoblastic leukemia cell line Jurkat E6.1. This effect was both doseand time-dependent with nuclear fragmentation detectable by 8 h. Caspases have been implicated in pro-apoptotic events. By utilizing synthetic fluorochrome-linked substrates of the caspases, we observed that a caspase-3-like enzyme had dramatically increased activity (3340 ± 130% with respect to basal levels) in response to daunorubicin treatment. Furthermore, by using an inhibitor to caspase-3, Ac-DEVD-CHO, we have shown that activation of a caspase-3-like enzyme appears to be necessary for nuclear fragmentation and apoptotic body formation, but is not required for chromatin condensation. In contrast, a general caspase inhibitor, Z-VAD-fmk, inhibited all apoptotic parameters measured. Ceramide has been implicated in daunorubicin-induced apoptosis in human myeloid leukemia cells. However, in Jurkat cells, caspase activation does not appear to be a consequence of ceramide generation since, although ceramide levels were elevated through the action of ceramide synthase in response to daunorubicin treatment, this occurred with slower kinetics than either nuclear fragmentation or caspase activation. In contrast, caspase inhibitors abrogated ceramide elevation induced by DNR treatment, suggesting that ceramide synthase may be a downstream target for caspase action. Therefore, daunorubicin-induced apoptosis does not appear to be mediated by ceramide in the lymphoblastic leukemia cell line, Jurkat E6.1. Instead, caspase 3 activity appears to be necessary, but not sufficient for this process. Keywords: daunorubicin; caspases; ceramide; apoptosis; Jurkat cells

Introduction Apoptosis has been shown to be central to both physiological and pathophysiological processes. Apoptosis, unlike necrosis, occurs in the absence of an inflammatory response. Apoptosis can be initiated by various stimuli including growth factor withdrawal, some cytokines, ionizing radiation and cytotoxic drugs.1 Despite the diverse modes of induction of this process, evidence has amassed suggesting that the signal transduction pathways may converge in one, or a limited number, of common death pathways. One family of enzymes which may play a crucial role in a common death pathway is the caspases. The caspases represent a rapidly expanding family of aspartatedirected cysteine proteases identified as having homology to CED-3, a protein necessary for cell death in Caenorhabditis elegans.2 Within this family of 11 reported members to date, caspases-1, -4 and -5 are believed to be involved in procytokine activation with the remaining members involved in mediating apoptosis. All caspases are synthesized as catalytically inactive proenzymes comprising a large and a small subunit with a variable length amino-terminal prodomain. On

activation, the prodomain is lost by catalytic cleavage carboxy-terminal to an aspartate residue, with heterodimerization of the large and small subunits to form the active enzyme,3 which according to crystallographic studies, exists as a tetramer of two such heterodimers.4 Since caspases themselves cleave carboxy-terminal to an aspartate residue, they have the inherent capacity to activate each other allowing the potential for a caspase cascade to exist. Indeed, caspases have been shown to activate each other in vitro5 with some evidence suggesting that a temporal relationship exists in the sequential activation of different caspases in vivo.6 Although several reports suggest that caspase activation, in particular that of the terminator caspase, caspase 3, is sufficient to mediate the apoptotic response characterized by membrane blebbing, chromatin condensation, nuclear fragmentation and apoptotic body formation, other reports contradict this, suggesting that other factors are also required. The chemotherapeutic anthracycline, daunorubicin (DNR), can intercalate into DNA and inhibit topoisomerase II activity. Until recently, this property, or enzymatic redox cycling of the hydroxyanthraquinone group generating reactive oxygen species, were thought to be responsible for the cytotoxicity of this drug. However, recently, the putative second messenger, ceramide, has also been implicated in daunorubicin-induced apoptosis in human myeloid leukemia cells. It has been suggested that either ceramide synthase7 or sphingomyelinase8 are involved in this drug-induced ceramide generation. Although several targets of ceramide have been elucidated, direct links between ceramide generation and the activation of a common death pathway remain elusive. In light of the evidence for a crucial role for the caspases in apoptosis, we have investigated whether caspase activation was sufficient for daunorubicin-induced apoptosis in the human lymphoblastic leukemic T cell line, Jurkat E6.1. Furthermore, we attempted to determine whether ceramide generation was a critical upstream signal in this pathway. Here, we report that caspase activation was sufficient to mediate some of the hallmarks of the apoptotic response, but although caspase-3-like activity was necessary for the induction of several features of apoptosis, it was not responsible for the entire apoptotic phenotype. Ceramide generation was not a prerequisite for caspase activation in Jurkat cells and may actually be a downstream event. Ceramide generation, through modulation of the activity of ceramide synthase, may be a direct or indirect target for caspase action. Materials and methods

Cell culture Correspondence: PRM Dobson, Institute of Endocrinology, Division of Oncology & Cellular Pathology, University of Sheffield Medical School, Sheffield S10 2RX, UK; Fax: 44 114 271 3515 Received 22 October 1998; accepted 2 February 1999

Jurkat E6.1 cells, a human lymphoblastic leukemic T cell line, were obtained from the ECACC (European Collection of Animal Cell Cultures, Porton Down, UK). The cells were main-

Caspases and daunorubicin-induced apoptosis KJ Turnbull et al

tained in RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 ␮g/ml), L-glutamine (2 mM), and 10% heat-inactivated fetal calf serum (FCS) (all supplied by Gibco BRL, Paisley, UK) at 37°C in a 5% CO2 atmosphere. Subculturing was performed twice weekly maintaining the cells in log phase of proliferation between a density of 2 × 105 cells/ml and 1 × 106 cells/ml.

Nuclear morphological analysis Cells were fixed with 4% paraformaldehyde and permeabilised overnight with 70% ethanol. Cells were washed with phosphate-buffered saline (PBS), cytospun (200 g, 3 min) on to glass slides and stained with the DNA-specific fluorochrome, 4, 6-diamidino-2-phenylindole (DAPI) (5 ␮g/ml). Cellular nuclei were examined under a 63 × apochromat lens (Zeiss, Welwyn Garden City, UK) with a fluorescent microscope (Axioskop; Zeiss). Images were captured by a KAF 1400 CCD camera (Photometrics, Tucson, AZ, USA) using Smart Capture software (Digital Scientific, Cambridge, UK) and analysed with IPLab Spectrum software (Signal Analytics, Vienna, VA, USA).

In situ nick translation (ISNT) Detection of DNA strand breaks associated with apoptosis was performed as described by Gorczyca and colleagues9 with quantification by fluorescent-activated cell sorter analysis (FACS Vantage cell sorter; Becton Dickinson, Oxford, UK) using Cellquest software (Becton Dickinson).

Fluorimetric analysis of caspase-3-like activity Cells (2.5 × 106) were washed in PBS and incubated for 20 min on ice with lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 10 mM EGTA, 0.5% CHAPS, 1 mM phenylmethylsulfonylfluoride (PMSF) and 1 mM phenanthroline). Each sample was centrifuged at 13 000 g for 10 min at 4°C and protein concentrations determined using the Bio-Rad (Hemel Hempstead, UK) protein assay system. Caspase activity was determined by amino-4-methylcoumarin (AMC) released from the synthetic caspase substrate DEVD-AMC (Alexis, Nottingham, UK). Each sample (100 ␮l protein) was incubated for 10 min at 37°C in a buffer containing 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 10 mM EGTA and 5 mM L-cysteine. DEVD-AMC (40 ␮M) was added and the fluorescence due to AMC was measured at 0 and 60 min with an excitation wavelength of 380 nm and an emission wavelength of 440 nm using a LS-5B luminescence spectrometer (Perkin-Elmer, Beaconsfield, UK). Results were expressed as fluorescence intensity (AMC released) standardised for protein concentration.

Detection of poly (ADP ribose) polymerase (PARP) cleavage For the detection of PARP cleavage, cell extracts were prepared by first washing the cells with ice-cold PBS and then resuspending the cells in lysis buffer (1% (v/v) Nonidet P40, 20 mM Tris-base; pH 8, 137 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 5 mM EDTA, 1 mM sodium orthovanadate, 10 ␮g/ml aprotinin, 4 ␮M iodoacetamide and 1 mM PMSF) for 30 min

on ice. Cell extracts were then centrifuged at 13 000 g for 10 min at 4°C. Supernatant protein content was quantitated using the Bio-Rad protein assay system. Total cellular proteins (50 ␮g) were electrophoresed under denaturing conditions through a 10% SDS-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membrane (Electran; BDH, Poole, UK). Antibodies used for immunoblotting were mouse anti-PARP monoclonal antibody (1:2000 C2-10 clone; Pharmingen, Oxford, UK) followed by horseradish peroxidase-conjugated sheep anti-mouse Ig antiserum (1:5000; Amersham, Little Chalfont, UK). Bound antibody was detected by enhanced chemiluminescence following the manufacturer’s instructions (ECL; Amersham).

Measurement of cellular ceramide For the quantitation of total cellular ceramide, 2.5 × 105 cells/ml were incubated with media supplemented with 75 kBq/ml [9, 10 (n)-3H]-palmitic acid (Amersham) for at least 24 h. Cells were resuspended in fresh media supplemented with 75 kbq/ml [9, 10 (n)-3H]-palmitic acid and treated with or without daunorubicin. Where applicable, cells were pretreated with inhibitors for 1 h prior to addition of daunorubicin. Lipids were extracted from harvested cells and separated by high performance thin layer chromatography (HP-KF silica gel plates; Whatman, Maidstone, UK) using chloroform/methanol/acetic acid/water (80:3.34:3.8:0.2 (v/v)) as a developing solvent system as described by Quintans and colleagues.10 Plates were sprayed with En3Hance (Amersham) and autoradiography performed. Radioactive ceramide spots were identified by comigration with C16-ceramide (N-palmitoyl sphingosine; Sigma, Poole, UK) upon exposure to iodine vapour, scraped into Ultima Gold XR scintillation fluid (Packard, Pangbourne, UK) and quantified by liquid scintillation counting. Results were expressed as % basal levels relative to radioactive lipid which did not migrate. Results and discussion

Daunorubicin-induced apoptosis in Jurkat E6.1 cells A dose-dependent induction of apoptosis in the human acute lymphoblastic leukemia cell line, Jurkat E6.1, was observed on treatment with the anthracycline daunorubicin (DNR). Apoptosis, as demonstrated by nuclear fragmentation measured utilizing an in situ nick-translation assay and flow cytometry, was evident with daunorubicin concentrations as low as 0.02 ␮M (Figure la). This was verified by staining paraformaldehyde-fixed cells with the DNA-specific fluorochrome DAPI and quantifying on the basis of the typical morphological changes evident during apoptosis by fluorescence microscopy (data not shown). Increases in apoptosis were detected 6 h after stimulation with 1 ␮M DNR, whereas 0.1 ␮M did not induce detectable apoptosis until 8 h posttreatment (Figure 1b), suggesting that intracellular accumulation of daunorubicin, either in the nucleus where it can intercalate into DNA, or in the cytosol, where its metabolism could generate reactive oxygen species, may be a contributory factor in the potency of this drug. Indeed, daunorubicin treatment for only 1 h resulted in a lower level of apoptosis when apoptosis was measured 24 hours later than continuous treatment with DNR for this period (data not shown). Cell cycle analysis showed that non-apoptotic cells accumulated late in

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Figure 1 Effect of the chemotherapeutic drug daunorubicin on apoptosis. Cells were stimulated (a) with the stated concentrations of daunorubicin for 24 h or (b) with 0.1 ␮M or 1 ␮M for the stated period. Apoptosis was measured by ISNT. Error bars represent mean ± s.e.m. from three independent experiments.

G2-phase of the cell cycle (data not shown), consistent with results published elsewhere.11

Daunorubicin-induced activation of a caspase 3-like protease To ascertain the potential role of the caspases in DNR-induced apoptosis, the synthetic fluorochrome linked substrate DEVDAMC was utilized. This was originally thought to be a specific substrate for caspase-3 but has recently also been shown12 to be cleaved by caspases -6, -7 and -8. DNR-induced the cleavage of this substrate in a dose- and time-dependent manner (Figures 2a and b), with caspase activation appearing to precede nuclear fragmentation. However, DNR did not induce cleavage of the caspase-1 substrate, YVAD-AMC (Alexis) (data not shown). To determine the specificity of the DEVD-AMC cleavage, the cells were pretreated for 1 h with the caspase3 inhibitor, Ac-DEVD-CHO (Alexis) before stimulation with 0.05 ␮M daunorubicin, a dose which induced sub-maximal levels of apoptosis after 24 h treatment. DEVD-AMC cleavage

Figure 2 Effect of daunorubicin on caspase-3 activity. Cells were stimulated with (a) the stated concentration of daunorubicin for 24 h or (b) 0.1 ␮M for the stated period. Caspase-3 activity was measured by fluorimetric detection of cleavage of the artificial substrate DEVDAMC. Error bars represent mean ± s.e.m. from three independent experiments.

was significantly inhibited in the presence of the inhibitor in a dose-dependent fashion, but was not reduced to basal levels at the concentrations of inhibitor used (Figure 3a). This was likely due to the reversible nature of this inhibitor or another caspase, possibly -6, -7 or -8, retaining catalytic activity in the presence of the inhibitor. The broader-spectrum irreversible caspase inhibitors, Z-Asp-2, 6-dichlorobenzoyloxymethylketone (ICE inhibitor III; Alexis) and Z-VAD-fmk (Alexis), proved to be more potent inhibitors of DEVD-AMC cleavage, resulting in inhibition of caspase activity to less than basal levels (Figure 3a). ICE inhibitor III did in fact inhibit DEVDAMC cleavage directly when DNR-stimulated cell extracts were pretreated with inhibitor prior to the addition of substrate (data not shown) indicating that the caspase(s) involved are directly inhibited by this compound. In addition, preincubation with all three of these inhibitors abrogated DNRinduced in vivo cleavage of the caspase-3 substrate PARP (Figure 3b). To determine whether caspase-3-like activity was necessary for daunorubicin-induced nuclear fragmentation, Jurkat cells


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Figure 3 Effect of caspase inhibitors on daunorubicin-induced (a) caspase-3 activity, (b) in vivo PARP cleavage, (c) nuclear fragmentation and (d) nuclear morphology. Cells were preincubated with or without 200 ␮M Ac-DEVD-CHO (DEVD), 150 ␮M ICE inhibitor III (ICE) or 150 ␮M Z-VAD-fmk (VAD) for 1 h before 23 h stimulation with or without 0.05 ␮M DNR. (a) Caspase-3 activity was measured by fluorimetric detection of cleavage of the artificial substrate DEVD-AMC. Error bars represent mean ± s.e.m. from three independent experiments. (b) PARP cleavage was detected by separating proteins by SDS PAGE with subsequent Western blot analysis. (c) Nuclear fragmentation was measured by ISNT. Error bars represent mean ± s.e.m. from three independent experiments. (d) Nuclear morphology was determined by DAPI staining with examination using fluorescent microscopy. A represents untreated cells; B, D, F and H were treated with 0.05 M DNR; C and D were pretreated with Ac-DEVD-CHO; E and F were pretreated with ICE inhibitor III; G and H were pretreated with Z-VAD-fmk. Results are representative of at least three independent experiments.

were pretreated with caspase inhibitors before stimulation with 0.05 ␮M daunorubicin for 24 h. The inhibitors DEVDCHO, ICE inhibitor III and Z-VAD-fmk all inhibited nuclear fragmentation in a dose-dependent manner (Figure 3c). These results were consistent with other studies since caspase-3 cleaves an inhibitor of caspase-activated deoxyribonuclease

(ICAD),13,14 inducing its dissociation from cytosolic caspaseactivated deoxyribonuclease (CAD), thereby allowing CAD to translocate to the nucleus and degrade DNA. In addition, caspase-3 cleaves and activates p21-activated kinase 2 (PAK2) which has recently been shown to be involved in apoptotic body formation.15 Morphological analysis revealed that,


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although both Ac-DEVD-CHO and ICE inhibitor III inhibited nuclear fragmentation and apoptotic body formation, only ZVAD-fmk had the additional ability to prevent chromatin condensation characterized by an increase in nuclear secondary structure (Figure 3d). This may suggest that the caspases inhibited by Ac-DEVD-CHO and ICE inhibitor III, presumably caspase-3 and possibly -6, -7 and -8, were necessary for certain processes in the apoptotic pathway, but not sufficient for the entire apoptotic response to DNR treatment. Furthermore, measurement of phosphatidylserine (PS) flipping from the intracellular to the extracellular leaf of the plasma membrane using a FITC-annexin V assay with PI dual staining, showed that DNR increased the percentage of labelled cells (45.9 ± 1.98% vs control 6.11 ± 2.90%, n = 4). Daunorubicin-induced PS-labelling was inhibited by Ac-DEVD-CHO (14.66 ± 6.16%), ICE inhibitor III (15.44 ± 6.13%), and by Z-VAD fmk (6.75 ± 3.23%). Recently, Wang and colleagues16 proposed a mechanism for non-receptor-mediated caspase activation whereby an apoptosome is assembled. In this model, the binding of dATP and cytochrome C released from mitochondria induce a conformational change in the caspase recruitment domain (CARD) of Apaf-1, rendering it more accessible to the CARD of procaspase-9. This permits protein–protein binding and Apaf-1induced procaspase-9 autoactivation, with subsequent caspase-9-mediated cleavage and activation of caspase-3, whose presence has been reported to be a prerequisite for microinjected cytochrome C-induced caspase activation and cell death in some mammalian cells.17 In our system, this would imply that either caspase-9 itself, or another activated caspase not inhibited by either Ac-DEVD-CHO or ICE inhibitor III, is necessary for chromatin condensation. Alternatively, another apoptosome-like complex may exist, such as the endoplasmic reticulum-based p28 Bap31 intracellular caspase activating complex which recruits procaspase-8.18

DNR-induced ceramide synthase activation Ceramide has been postulated as a mediator of apoptosis in certain cell types in response to various inducers of apoptosis. Indeed, ceramide has been reported to be involved in DNRinduced apoptosis in the acute human myeloid leukemia cell line, U937. Bose and colleagues7 reported that ceramide was synthesized de novo by ceramide synthase with ceramide lev´ els detectable 6 h after treatment. In contrast, Jaffrezou and 8 co-workers reported transient ceramide elevation within 4– 10 min after treatment through sphingomyelin hydrolysis by neutral sphingomyelinase. To determine whether ceramide was upstream of caspase activation and its possible role in commitment to DNR-induced apoptosis, we measured intracellular ceramide levels after DNR stimulation. DNR did not induce any detectable increase in intracellular ceramide either over a short time course or up to 10 h, but a time-dependent increase in ceramide was detected 12 h post-treatment (data not shown). This elevation was inhibited by the specific inhibitor of ceramide synthase, Fumonisin B1 (Figure 4a), but the slower kinetics of ceramide elevation in comparison to caspase activation or nuclear fragmentation implied that it was not necessary for DNR-induced caspase activation or nuclear fragmentation. The fact that treatment with Fumonisin B1 did not completely abrogate ceramide generation may indicate that a proportion of ceramide may be produced via activation of sphingomyelinase. In addition, Fumonisin B1 prevented neither caspase activation (Figure 4b) nor nuclear

Figure 4 Effect of the ceramide synthase inhibitor, Fumonisin B1, on daunorubicin-induced (a) ceramide generation, (b) apoptosis and (c) caspase-3 like activity. (a) Quantitation of ceramide levels was performed as described in ‘Materials and methods’. Cells were preincubated with 25 ␮M Fumonisin B1 or 0.2% methanol vehicle for 1 h before 23 h stimulation with 0.05 ␮M daunorubicin. Results are expressed as a percentage of basal ceramide levels. Error bars represent mean ± s.e.m. of three independent experiments performed in triplicate. (b) Apoptosis was measured by ISNT. Error bars represent mean ± s.e.m. from three independent experiments. (c) Caspase-3-like activity was measured by fluorimetric detection of cleavage of the artificial substrate DEVD-AMC. Error bars represent mean ± s.e.m. from three independent experiments.


References

Figure 5 Effect of caspase inhibitors on daunorubicin-induced ceramide generation. Quantitation of ceramide levels was performed as described in ‘Materials and methods’. Cells were preincubated with or without 200 ␮M Ac-DEVD-CHO (DEVD) or 150 ␮M Z-VAD-fmk (VAD) for 1 h before 23 h stimulation with or without 0.05 ␮M DNR. Results are expressed as a percentage of basal ceramide levels. Error bars represent mean ± s.e.m. of four independent experiments performed in triplicate.

fragmentation (Figure 4c). In contrast, caspase inhibitors inhibited ceramide elevation induced by DNR treatment (Figure 5) raising the intriguing possibility that ceramide synthase may be a downstream target for caspase action. This supports the hypothesis that ceramide does not appear to have a role in the initiation of daunorubicin-induced apoptosis in Jurkat cells. In conclusion, we have shown that daunorubicin induces apoptosis in the human lymphoblastic leukemic cell line, Jurkat E6.1. This appears to be mediated by multiple caspases, of which, a caspase-3-like protease is sufficient for nuclear fragmentation and apoptotic body formation, but not for chromatin condensation. Elevation of intracellular ceramide levels does not appear to be a prerequsite for these processes and indeed, ceramide synthase, the rate-limiting enzyme in de novo synthesis of ceramide, may itself be a downstream target of the caspases. Future studies should elucidate the components of the caspase family necessary for apoptosis, upstream signals leading to their regulation and critical targets for their action.

Acknowledgements We thank Yorkshire Cancer Research and the University of Sheffield Cancer Research Fund for providing funding for this work and Dr J Lawry and Ms O Smith for technical assistance with FACS analysis.

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