Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.     

Apoptosis-resistant mitochondria in T cells selected for resistance to Fas signaling

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.     

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.     

Stress-induced apoptosis: Toward a symmetry with receptor-mediated cell death

Apoptosis is a form of programmed cell death executed by caspases activated along signalling pathways initiated by ligation of cell-surface death receptors ( extrinsic pathway ) or by perturbation of the mithocondrial membrane promoted by physical or chemical stress agents ( intrinsic pathway ). In metazoans, this evolutionary conserved, genetically controlled process has a role in a variety of physiological settings, as development, homeostasis of tissues and maintenance of the organism integrity. When deranged by impaired regulation or inappropriate activation apoptosis contributes to the pathogenesis of diseases as autoimmunity, cancer, restenosis, ischaemia, heart failure and neurodegenerative disorders. In this review we will present a survey of the stress-induced intrinsic, mithochondrial, pathway and, based on recent experimental data, we will propose a view compatible with an emergent conceptual symmetry between the two apoptogenic extrinsic and intrinsic pathways. Elements of symmetry present in both the apoptogenic signalling pathways include: early activation of initiator caspases (feed-forwarded by a direct or post-mitocondrial effector caspase-mediated amplification loop in some cell types) and mitochondrial membrane permeabilization with required release of antagonists of active caspase inhibitors (IAPs) in high-level IAPs-expressing cells and apoptosome-mediated amplification of the caspase cascade more or less needed in different cell types.     

Mitochondrial membrane dynamics, cristae remodelling and apoptosis

Mitochondria form a highly dynamic reticular network in living cells, and undergo continuous fusion/fission events and changes in ultrastructural architecture. Although significant progress has been made in elucidating the molecular events underlying these processes, their relevance to normal cell function remains largely unexplored. Emerging evidence, however, suggests an important role for mitochondrial dynamics in cellular apoptosis. The mitochondria is at the core of the intrinsic apoptosis pathway, and provides a reservoir for protein factors that induce caspase activation and chromosome fragmentation. Additionally, mitochondria modulate Ca2+ homeostasis and are a source of various metabolites, including reactive oxygen species, that have the potential to function as second messengers in response to apoptotic stimuli. One of the mitochondrial factors required for activation of caspases in most intrinsic apoptotic pathways, cytochrome c, is largely sequestered within the intracristae compartment, and must migrate into the boundary intermembrane space in order to allow passage across the outer membrane to the cytosol. Recent evidence argues that inner mitochondrial membrane dynamics regulate this process. Here, we review the contribution of mitochondrial dynamics to the intrinsic apoptosis pathway, with emphasis on the inner membrane.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

The heat shock protein 27 (Hsp27) operates predominantly by blocking the mitochondrial-independent/extrinsic pathway of cellular apoptosis

Heat shock protein 27 (Hsp27) is a molecular chaperone protein which regulates cell apoptosis by interacting directly with the caspase activation components in the apoptotic pathways. With the assistance of the Tat protein transduction domain we directly delivered the Hsp27 into the myocardial cell line, H9c2 and demonstrate that this protein can reverse hypoxia-induced apoptosis of cells. In order to characterize the contribution of Hsp27 in blocking the two major apoptotic pathways operational within cells, we exposed H9c2 cells to staurosporine and cobalt chloride, agents that induce mitochondria-dependent (intrinsic) and -independent (extrinsic) pathways of apoptosis in cells respectively. The Tat-Hsp27 fusion protein showed a greater propensity to inhibit the effect induced by the cobalt chloride treatment. These data suggest that the Hsp27 predominantly exerts its protective effect by interfering with the components of the extrinsic pathway of apoptosis. These authors contributed equally to this work.     

Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways

Activated immune cells secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interferon–gamma (IFN-gamma) and Fas ligand (FasL) and these cytokines have been reported to induce apoptosis in numerous cell types. Apoptotic cell death has been associated with the progression of numerous autoimmune diseases. Proinflammatory cytokines are reportedly involved in apoptosis in the salivary glands of patients with Sjögren’s syndrome (SS); an autoimmune disorder characterized by the destruction of salivary and lachrymal glands. In this study, we used the HSG cell line to determine if exposure to proinflammatory cytokines induces apoptosis in human salivary gland cells. In addition, we identified the mediators controlling the apoptotic process in response to TNF alpha and IFN gamma. TNF-alpha and IFN-gamma induced apoptosis in HSG cells and resulted in the activation of caspase 8 and the “death receptor” pathway. We further determined that caspase 9 and the “mitochondrial” pathway was also activated. Induction of the intrinsic and extrinsic pathways in HSG cells resulted in substrate cleavage by effector caspases, in particular the cleavage of alpha II spectrin, an autoantigen in Sjögren’s syndrome. Our results suggest that HSG cells provide a model system to study processes regulating proinflammatory cytokine-induced apoptotic cell death.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

XIAP activity dictates Apaf-1 dependency for caspase 9 activation

The current model for the intrinsic apoptotic pathway holds that mitochondrial activation of caspases in response to cytotoxic drugs requires both Apaf-1-induced dimerization of procaspase 9 and Smac/Diablo-mediated sequestration of inhibitors of apoptosis proteins (IAPs). Here, we showed that either pathway can independently promote caspase 9 activation in response to apoptotic stimuli. In drug-treated Apaf-1(-/-) primary myoblasts, but not fibroblasts, Smac/Diablo accumulates in the cytosol and sequesters X-linked IAP (XIAP), which is expressed at lower levels in myoblasts than in fibroblasts. Consequently, caspase 9 activation proceeds in Apaf-1(-/-) myoblasts; concomitant ablation of Apaf-1 and Smac is required to prevent caspase 9 activation and the onset of apoptosis. Conversely, in stimulated Apaf-1(-/-) fibroblasts, the ratio of XIAP to Smac/Diablo is high compared to that for myoblasts and procaspase 9 is not activated. Suppressing XIAP with exogenous Smac/Diablo or a pharmacological inhibitor can still induce caspase 9 in drug-treated Apaf-1-null fibroblasts. Thus, caspase 9 activation in response to intrinsic apoptotic stimuli can be uncoupled from Apaf-1 in vivo by XIAP antagonists.     

Caspase-9 Activation by the Apoptosome Is Not Required for Fas-mediated Apoptosis in Type II Jurkat Cells

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-x_L were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-x_L-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.     

Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway

Accumulation of misfolded proteins and alterations in Ca2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1(-/-) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems

Heat shock protein 70 (hsp70) is a stress-inducible protein that prevents apoptosis induced by a wide range of cytotoxic agents by an as yet undefined mechanism. The caspase family of cysteine proteases have been attributed a central role in the execution of apoptosis. However, several cases of caspase-independent apoptosis have been recently reported, suggesting that caspases may not be necessary for apoptosis in all cells. This study examines the protective role of hsp70 in both caspase-dependent and -independent apoptosis. Hydrogen peroxide (H2O2) used at low and high concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. A hsp70-transfected Jurkat clone was used to observe the protection mediated by hsp70 during these two forms of apoptosis. Results reveal that hsp70 inhibits both caspase-dependent and -independent apoptosis. Furthermore, measurement of caspase-3 activity during caspase-dependent apoptosis revealed that caspase activation was inhibited in hsp70 transfectants. Early apoptotic events, such as mitochondrial depolarization, cytochrome c release, and increased intracellular calcium, were demonstrated to be common to both caspase-dependent and -independent H2O2-induced apoptosis. The inhibition of these events by hsp70 suggests that hsp70 may be an important anti-apoptotic regulator, functioning at a very early stage in the apoptotic pathway.