Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis

Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic volume decrease (AVD), is necessary for the progression of apoptotic cell death, we sought to evaluate the relationship between GSH transport and ionic homeostasis during Fas ligand (FasL)-induced apoptosis in Jurkat cells. GSH depletion in response to FasL was paralleled by distinct degrees of AVD identified by differences in cellular forward scatter and electronic impedance analysis. Inhibition of GSH efflux prevented AVD, K+ loss, and the activation of two distinct ionic conductances, mediated by Kv1.3 and outward rectifying Cl- channels. Reciprocally, stimulation of GSH loss accelerated the loss of K+, AVD, and consequently the progression of the execution phase of apoptosis. Although high extracellular K+ inhibited FasL-induced apoptosis, GSH depletion was largely independent of K+ loss. These results suggest that deregulation of GSH and ionic homeostasis converge in the regulation of apoptosis in lymphoid cells.     

Glutathione export during apoptosis requires functional multidrug resistance-associated proteins

GSH is released in cells undergoing apoptosis, and the present study indicates that the multidrug resistance-associated proteins (MRPs/ABCC) are responsible for this GSH release. Jurkat cells released approximately 75-80% of their total intracellular GSH during both Fas antibody- and staurosporine-induced apoptosis. In contrast, Raji cells, a lymphocyte cell line that is deficient in phosphatidylserine externalization, did not release GSH during apoptosis, and other apoptotic features appeared more slowly in these cells. Jurkat and Raji cell lines expressed comparable MRP and OATP/SLCO (organic anion-transporting polypeptide) mRNA levels, and MRP1 protein levels; however, differences existed in MRP1 localization and function. In Jurkat cells, MRP1 was largely localized to the plasma membrane, and these cells exported the MRP substrate calcein. Calcein release was enhanced during apoptosis. In contrast, Raji cells had little MRP1 at the plasma membrane and did not export calcein under basal or apoptotic conditions, indicating that these cells lack functional MRPs at the plasma membrane. GSH release in Jurkat cells undergoing apoptosis was inhibited by the organic anion transport inhibitors MK571, sulfinpyrazone, and probenecid, supporting a role for the MRP transporters in this process. Furthermore, when MRP1 expression was decreased with RNA interference, GSH release was lower under both basal and apoptotic conditions, providing direct evidence that MRP1 is involved in GSH export.     

Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1

Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Specific efflux of glutathione from the basolateral membrane domain in polarized MDCK cells during ricin-induced apoptosis.

Although the depletion of reduced glutathione (GSH) has been observed in a variety of apoptotic systems, little is known about the mechanism of GSH depletion. In this study we used polarized MDCK cells to study the GSH flux during ricin-induced apoptosis. Here we report that the specific accumulation of GSH occurred in the basolateral medium during ricin treatment with similar kinetics to in apoptotic changes such as an increase in caspase-3 like activity and DNA fragmentation, while there was no significant increase in the GSH level in apical medium. These results suggest that GSH efflux occurred through a GSH-specific channel or transporter located in the basolateral membrane domain of polarized MDCK cells undergoing apoptosis. Treatment with other protein toxins such as modeccin, Pseudomonas toxin, and diphtheria toxin, which can induce apoptotic cell death, also resulted in selective GSH efflux from the basolateral side. Thus, GSH efflux through a specific transporter may be a common step of apoptosis induced by these toxins, while these toxins have different intoxication mechanisms leading to protein synthesis inhibition. Pretreatment of cells with Z-Asp-CH(2)-DCB, a caspase family inhibitor, inhibited ricin-induced basolateral GSH efflux as well as DNA fragmentation, suggesting that the activation of caspases, i.e. those that are inhibited by Z-Asp-CH(2)-DCB, is implicated in the opening of the GSH transporter.     

Functional characterization of Jurkat T cells rescued from CD95/Fas-induced apoptosis through the inhibition of caspases

The caspases are known to play a pivotal role in the triggering and execution of apoptosis in virtually all cell types. Because inappropriate apoptosis is a prominent feature of many human diseases, the caspases are attractive targets for therapeutic intervention. In the present study we investigated whether Jurkat T lymphocytes rescued from Fas-induced cell death through the inhibition of caspases are functional. Here we show that the pan-caspase, tripeptide inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Ome) fluoromethylketone (z-VAD-FMK), inhibited the activation of caspase-2, -3, -7, and -8, and subsequently apoptosis in Jurkat T lymphocytes induced by agonistic anti-Fas. The apoptotic signals induced by the cross-linking of the Fas antigen have a relatively long half-life, as z-VAD-FMK had to be continuously present in the culture medium for 72 h after Fas stimulation in order to maintain cell survival. After 72 h, the z-VAD-FMK-rescued cells proliferate normally and responded to activation induced cell death after phytohaemaglutinin treatment, and readily undergo apoptosis when restimulated with agonistic Fas antibodies. Taken together, our results demonstrate that Jurkat T cells rescued from Fas-mediated cell death through the inhibition of caspases are functional.     

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.     

Effects of dopamine on the glutathione metabolism of cultured astroglial cells: implications for Parkinson's disease

To investigate the effects of dopamine (DA) on the release of glutathione (GSH) from astrocytes, we used astroglia-rich primary cultures from the brains of newborn rats. In the absence of DA, GSH accumulated in the medium of these cultures with a constant rate. In contrast, during incubation of the cells with 50 micro m DA extracellular GSH was not detectable anymore. This disappearance of extracellular GSH was prevented by superoxide dismutase, indicating that DA does not affect GSH release but rather reacts with the released GSH in a superoxide-dependent reaction. Incubation of astroglial cultures with 0.5 and 1 mm DA established almost constant extracellular concentrations of H2O2 of 5 microm and 15 microm, respectively. Under these conditions astroglial cultures release glutathione disulphide (GSSG). This GSSG export was blocked by catalase and by MK571, an inhibitor of the multidrug resistance protein 1. The effects of DA on the extracellular accumulations of GSH and GSSG were not modulated by inhibitors of DA receptors, DA transport, and monoamine oxidases. The other catecholamines adrenaline and noradrenaline showed similar effects on the accumulation of GSH and GSSG in the medium compared with those obtained for DA. In conclusion, the data presented demonstrate that DA affects astroglial GSH metabolism by two mechanisms: (i) directly by chemical reaction with extracellular GSH, and (ii) indirectly by generation of hydrogen peroxide that leads to the efflux of GSSG from astroglial cells. These observations are discussed in the context of the brain's GSH metabolism in Parkinson's disease.     

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.     

Role of reduced glutathione efflux in apoptosis of immortalized human keratinocytes induced by UVA

We have investigated the role played by GSH efflux in apoptosis of human HaCaT keratinocytes induced by UVA irradiation. UVA irradiation of HaCaT cells caused a rapid rise in GSH efflux across the intact cell membrane, followed by an increase in apoptosis. GSH efflux was stimulated by glucose and was reduced by the addition of exogenous GSH and intracellular GSH depletion by buthionine sulfoximine, suggesting that GSH transport is active and is influenced by the GSH concentration gradient across the cell membrane. Verapamil and cyclosporin A, blockers of the multidrug resistance-associated protein, decreased UVA-induced GSH efflux. GSH efflux occurred within 2 h of UVA irradiation, suggesting that the stimulation of GSH efflux is due to an increase in the activity of pre-existing multidrug resistance-associated protein transporter carrier. Although inhibition of GSH efflux did not affect caspase activation and DNA fragmentation, it delayed the gradual increase in plasma membrane permeability and reduced phosphatidylserine translocation in HaCaT cells. It is therefore likely that upon UVA irradiation, GSH efflux increased the intracellular oxidative stress without intervention of reactive oxygen species, thus resulting in more phosphatidylserine externalization and membrane rearrangement. These provide targets for macrophage recognition and phagocytosis and thus minimize the potential to invoke inflammation or neoplastic transformation.     

Hypericin photo-induced apoptosis involves the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and activation of caspase-8

Hypericin (HYP) is a photosensitizing pigment from Hypericum perforatum that displays cytotoxic effects in neoplastic cell lines. Therefore, HYP is presently under consideration as a new anticancer drug in photodynamic therapy. Here, we investigated the mechanism of action of HYP photo-induced apoptosis of Jurkat cells compared to the cytostatic drug paclitaxel (PXL). Both photoactivated HYP and PXL similarly increased the activity of caspase-8 and caspase-3, and drug-induced apoptosis of Jurkat cells was completely blocked by inhibitors of caspase-8 (Z-IETD-FMK) and caspase-3 (Z-DEVD-FMK). The involvement of death receptors was analyzed using neutralizing monoclonal antibodies against Fas (SM1/23), FasL (NOK-2) and TNF-R1 (MAB225), and a polyclonal rabbit anti-human TNF-related apoptosis-inducing ligand (TRAIL) antiserum. TRAIL antibody blocked TRAIL-induced and HYP photo-induced, but not PXL-induced apoptosis of Jurkat cells. In contrast, PXL-induced, but not HYP-induced apoptosis was blocked by the SM1/23 and NOK-2 antibodies. Anti-TNF-R1 antibody had no effect. These findings suggest that HYP photo-induced apoptosis of Jurkat cells is mediated in part by the TRAIL/TRAIL-receptor system and subsequent activation of upstream caspases.     

Mechanisms of N-acetylcysteine-driven enhancement of MK886-induced apoptosis

N-Acetylcysteine (NAC), besides being a precursor of glutathione, has an array of other effects including an ability to scavenge free radicals, modulate gene expression and signal transduction pathways, and regulate cell survival and apoptosis. At concentrations lower than 20 mmol/L, NAC is nontoxic to cultured cells and can protect against apoptosis induced by a number of agents. A few recent reports, however, have indicated that NAC can also increase apoptosis. MK886, a 5-lipoxygenase activating protein (FLAP) inhibitor, induces apoptosis in many cell lines by an unknown mechanism that is independent of FLAP and lipoxygenase activity but is possibly related to effects on kinases such as Akt. In Jurkat T lymphocytes, NAC pretreatment (10 mmol/L) enhanced MK886-induced apoptosis by 2.4-fold. Following NAC-MK886 treatment, there was a significant increase in caspase-3 activity, and a decrease in mitochondrial transmembrane potential compared to MK886 alone. However, the extent of cytochrome c release was comparable between MK886 alone and MK886-NAC treatments. The enhancement of MK886-induced apoptosis by 10 mmol/L NAC appears to be partly related to a decrease in pH caused by this concentration of NAC, because an acidic environment favors activation of effector caspases and triggering of mitochondrial apoptosis. However, because neutralized NAC also enhanced apoptosis (1.6-fold), a direct role for NAC in augmenting the apoptotic pathways initiated by MK886 is suggested.     

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.     

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.     

Diphenyleneiodonium triggers the efflux of glutathione from cultured cells

Diphenyleneiodonium (DPI) is a broad-spectrum flavoprotein inhibitor commonly used to inhibit oxidant production by the NADPH oxidase of phagocytic and nonphagocytic cells. A previous study has shown that DPI can sensitize T24 bladder carcinoma cells to Fas-mediated apoptosis. We observed DPI to deplete intracellular reduced glutathione (GSH) in T24 cells and a range of other primary and transformed cell types. The effect was immediate, with 50% loss of intracellular GSH within 2 h of treatment with DPI. The glutathione was quantitatively recovered in the extracellular medium, indicating that efflux was occurring. The loss of GSH was blocked with bromosulfophthalein, an inhibitor of the canalicular GSH transporters. We conclude that DPI induces a dramatic efflux of cellular GSH from T24 cells via a specific transport channel. This provides a potential mechanism for its proapoptotic effect, and it also has important implications for the regulation of glutathione homeostasis in cells.     

Multidrug-resistance-associated protein plays a protective role in menadione-induced oxidative stress in endothelial cells

AimsMenadione, a redox-cycling quinone known to cause oxidative stress, binds to reduced glutathione (GSH) to form glutathione S-conjugate. Glutathione S-conjugates efflux is often mediated by multidrug-resistance-associated protein (MRP). We investigated the effect of a transporter inhibitor, MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), on menadione-induced oxidative stress in bovine aortic endothelial cells (BAECs).Main methodsBAECs were treated with menadione and MK571, and cell viability was measured. Modulation of intracellular GSH levels was performed with buthionine sulfoximine and GSH ethyl ester treatments. Intracellular superoxide was estimated by dihydroethidium oxidation using fluorescence microscopy or flow cytometry. Expression of MRP was determined by flow cytometry using phycoerythrin-conjugated anti-MRP monoclonal antibody.Key findingsIntracellular GSH depletion by buthionine sulfoximine promoted the loss of viability of BAECs exposed to menadione. Exogenous GSH, which does not permeate the cell membrane, or GSH ethyl ester protected BAECs against the loss of viability induced by menadione. The results suggest that GSH binds to menadione outside the cells as well as inside. Pretreatment of BAECs with MK571 dramatically increased intracellular levels of superoxide generated from menadione, indicating that menadione may accumulate in the intracellular milieu. Finally, we found that MK571 aggravated menadione-induced toxicity in BAECs and that MRP levels were increased in menadione-treated cells.SignificanceWe conclude that MRP plays a vital role in protecting BAECs against menadione-induced oxidative stress, presumably due to its ability to transport glutathione S-conjugate.     

Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with G?6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and G?6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with G?6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.     

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.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

A serine protease is involved in the initiation of DNA damage-induced apoptosis

Caspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.