Nuclear factor-kappaB and caspases co-operatively regulate the activation and apoptosis of human macrophages

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor-kappaB (NF-kappaB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12-myristate 13-acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro-caspase-8 and pro-caspase-3 in U937, but not apoptosis or intracellular caspase-3 activity. PMA also increased the expression of X-chromosome-linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA-stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF-kappaB activity in PMA-stimulated U937. When a potent NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase-3, which was induced by caspase-8 activation. XIAP expression was markedly suppressed in PMA-treated U937 in the presence of PDTC. The inhibitors of caspase-8 and caspase-3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co-operatively regulated by the use of NF-kappaB and caspase inhibitors, thus enabling the control of macrophage function and number.     

Inhibition of T cell apoptosis by IFN-beta rapidly reverses nuclear translocation of protein kinase C-delta.

Type I interferons rescue activated human T cells from cytokine deprivation-induced apoptosis. Our data now show that IFN-beta also rapidly inhibits apoptotic signals induced through the Fas receptor (CD95) in human T cells. To identify upstream signaling elements that could be targets of IFN-beta, we have studied protein kinase C (PKC). PKC-delta is actively involved in the regulation of apoptosis and immunofluorescence staining revealed that early in apoptosis PKC-delta accumulated in the nucleus. Addition of IFN-beta to T cells already deprived of survival factors or treated with anti-Fas antibody caused a rapid retranslocation of PKC-delta away from the nucleus. Furthermore, the generation of a constitutively active catalytic fragment by cleavage of PKC-delta by caspase 3 occurred only after translocation of full-length PKC-delta to the nucleus. IFN-beta also inhibited caspase 3 and the proteolytic activation of PKC-delta. We conclude from these studies that nuclear translocation of PKC-delta is an early event in T cell apoptosis and that IFN-beta rapidly reverses this process.     

Release of Cytochrome c, Bax Migration, Bid Cleavage, and Activation of Caspases 2, 3, 6, 7, 8, and 9 during Endothelial Cell Apoptosis

Although the executioner phase of apoptosis has been well defined in many cell types, the subcellular events leading to apoptosis in endothelial cells remain undefined. In the current study, apoptosis was induced in primary human umbilical venous endothelial cells by the photosensitizer verteporfin and light. Release of mitochondrial cytochrome c into the cytosol was detectable immediately and accumulated over 2 hours after treatment while cytosolic levels of the proapoptotic Bcl-2 family member, Bax, decreased reciprocally over the same time period. Cleavage of another proapoptotic Bcl-2 family member, Bid, was observed by 2 hours after treatment. Although Bid cleavage has been shown to occur as an upstream event responsible for inducing cytochrome c release, we demonstrate that Bid cleavage can also occur after cytochrome c release. Activation of caspases 2, 3, 6, 7, 8, and 9 occurred following the release of cytochrome c, and cleavage of downstream substrates was observed. In summary, endothelial cell death involves the cellular redistribution of Bax and cytochrome c, followed by the activation of multiple caspases which manifest the apoptotic phenotype.     

GAMMA-INTERFERON (IFN-γ) AUGMENTS APOPTOTIC RESPONSE TO MISTLETOE LECTIN-II VIA UPREGULATION OF FAS/FAS L EXPRESSION AND CASPASE ACTIVATION IN HUMAN MYELOID U937 CELLS

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

Role of PKC-delta during hypoxia in heart-derived H9c2 cells

In the present study, we investigated the role of protein kinase C (PKC) isoforms during hypoxia in heart-derived H9c2 cells. Hypoxia caused a rapid translocation of PKC-delta from soluble to particulate fraction and a downregulation of PKC-epsilon and PKC-zeta, whereas PKC-alpha and PKC-beta I remained unaltered. When H9c2 cells were pretreated with PKC-delta inhibitor rottlerin (3 microM), hypoxia-induced apoptotic and necrotic cell death were significantly increased. Hypoxic insult also caused an activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK with no change in c-Jun NH(2)-terminal protein kinase (JNK) phosphorylation. Hypoxia-induced cell death was increased by treatment with ERK1/2 inhibitor U0126 (10 microM), but attenuated by p38 MAPK inhibitor SB202190 (10 microM). Treatment with rottlerin completely blocked the hypoxia-induced ERK phosphorylation, whereas it significantly increased p38 MAPK phosphorylation. The hypoxia-induced translocation of PKC-delta was not altered by U0126 and/or SB202190. From these results, it is suggested that hypoxia causes a rapid translocation of PKC-delta and subsequently ERK activation and p38 inactivation, rendering H9c2 cells resistant to hypoxia-induced cell death.     

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

Lytic infection with vaccinia virus activates caspases in a Bcl-2-inhibitable manner

Vaccinia virus (VV) is considered to cause lytic infection of most cells, with lysis being regarded equivalent to necrosis. Activation of caspases has not been associated with necrosis. However, we observed the activation and activity of caspases in epithelial cells HeLa G and BSC-40 lytically infected with VV. Using three different flow-cytometric approaches, we characterized the distinct stages of caspase cascade in VV-infected cells: a cleaved, activated form of caspases detected using a fluorescent pan-caspase inhibitor; caspase activity assayed by cleavage of a non-fluorescent substrate into a fluorescent product; caspase-specific cleavage of death substrates characterized by a fluorescent antibody detecting a neo-epitope in cytokeratin-18. All of these approaches yielded an increased fluorescent signal in VV-infected cells compared to mock-infected controls. Additionally, the signal was decreased by the expression of Bcl-2. The cleavage of cytokeratin-18 was confirmed by western blotting, but another key protein involved in apoptosis, PARP, was not cleaved in VV-infected lytic cells. The necrotic phenotype of the cells was confirmed by increased cell membrane permeability and/or decreased mitochondrial membrane potential. In conclusion, our data suggest that VV infection of the epithelial cells HeLa G and BSC-40 initiates the apoptotic program, however, apoptosis is not completed and switches into necrosis.     

The Notch ligand, Delta-1, reduces TNF-alpha-induced growth suppression and apoptosis by decreasing activation of caspases in U937 cells

The Notch signaling pathway plays an important role in the regulation of self-renewal and differentiation of hematopoietic progenitors. Tumor necrosis factor (TNF)-alpha induces apoptosis through activation of caspase pathway. A monoblastic leukemia cell line, U937, undergoes apoptosis following stimulation with TNF-alpha. We found that Notch activation induced by a recombinant Notch ligand, Delta-1, reduced the TNF-alpha-induced growth suppression and apoptosis in U937 cells. As the molecular mechanism involved, we showed Delta-1 stimulation partially suppressed the sequential activation of caspase-8, caspase-3, and, PARP induced by TNF-alpha. The TNF-alpha-induced activation of c-Jun N-terminal kinase (JNK), p38, and NF-kappaB was not affected by Delta-1 stimulation. The cells needed to be exposed to Delta-1 prior to TNF-alpha stimulation to reduce the suppressive effect of TNF-alpha. Therefore, we thought that Delta-1 stimulation might reduce the expression of TNF-receptor (R) 1 and proteins to modulate the activation of caspases such as FLIP and XIAP. However, Delta-1 stimulation did not affect their expression. The precise mechanism by which Notch signaling suppresses caspase activation has yet to be determined. This is the first report to show the relationship between Notch activation and TNF-R1 signaling. The findings suggest possible mechanisms by which Notch activation supports cell survival.     

Se-methylselenocysteine enhances PMA-mediated CD11c expression via phospholipase D1 activation in U937 cells

CD11c/CD18 is expressed primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages, and is also a receptor for fibrinogen and lipopolysaccharide (LPS). We focused on the molecular mechanisms leading to the activation of CD11c expression in differentiating U937 cells. During phorbol myristate acetate (PMA)-induced differentiation of U937 cells, we found that the mRNA expression of CD11c was increased. Se-methylselenocysteine (Se-MSC) potentiated up-regulation of CD11c expression and its promoter activity and increased PLD1 activity without affecting the level of PLD1 protein in PMA-treated cells. To examine the regulation mechanism of PMA and Se-MSC on CD11c gene expression through the activation of PLD1, we analyzed changes in the CD11c mRNA level and the promoter activity following treatment of a selective PLD inhibitor n-butanol. The combinatory effect of PMA and Se-MSC on CD11c gene expression was abolished by n-butanol in a dose-dependent manner. Further, introduction of PLD1 gene into U937 cells increased CD11c mRNA expression and activated CD11c promoter activity in a dose-dependent manner. These results showed that Se-MSC increased PMA-induced CD11c expression through the activation of PLD1 signaling pathway. To our knowledge, this is the first report that expression of the CD11c gene is regulated by PLD1 and is enhanced by Se-MSC during PMA-induced U937 differentiation.     

Inhibition of caspases but not of calpains temporarily protect against C2-ceramide-induced death of CAD cells

Evidence has implicated apoptosis as a mechanism underlying cell death in diverse neurodegenerative diseases including Parkinson's disease (PD). Endogenous agents such as TNF-alpha, INF-gamma, IL-1beta and others stress signals activate the sphingomyelin pathway increasing ceramide levels. Ceramide triggers apoptotic pathways while inhibiting survival signalling, and is involved in the regulation of intracellular Ca(2+) homeostasis and compartmentalisation. The contribution of caspases in neuronal apoptosis has been highlighted by the increased survival exerted by caspase inhibition, but the involvement of calpains during neuronal apoptosis and the potential benefit of their inhibition is still controversial. In the present paper, we have analysed the contribution of caspases and calpains to cell death of CAD cells, a catecholaminergic cell line of mesencephalic origin, following C2-ceramide exposure. Ceramide caused CAD cell death by a dose and time dependant mechanism. 25microM of C2-ceramide caused apoptosis. Analysis of activation of caspases and calpains by differential cleavage of alpha-fodrin showed that although calpains are activated before caspases following C2-ceramide exposure, only caspase inhibition increased cell survival. These results demonstrate the activation of caspases and calpains in C2-ceramide-induced cell death, and support the role of caspase inhibition as a neuroprotective strategy and a plausible therapeutic approach to decrease catecholaminergic cell death.     

Association of mitochondrial calpain activation with increased expression and autolysis of calpain small subunit in an early stage of apoptosis

Various stimuli including anticancer drugs are capable of initiating the apoptotic death program in human tumor cells via activation of caspases. Mitochondria play an essential role for cell apoptotic commitment. Previous studies have shown a potential role of calpain activation in apoptosis, however, the involved molecular mechanisms remain to be defined. In the current study, we have examined the expression and activation of mitochondrial calpain in Jurkat T leukemia cells, MCF-7 breast carcinoma and LNCaP prostate cancer cells during apoptosis induced by an anticancer drug (VP-16, tamoxifen) or the specific p38 kinase inhibitor PD-169316. Our results suggest that increased expression and autolysis of the mitochondrial calpain small subunit are tightly associated with calpain activation in an early stage of apoptosis. In contrast, there were no correlations observed between the early calpain activation and changes in levels of mitochondrial calpain large subunit and the endogenous calpain inhibitor calpastatin. Furthermore, pretreatment with the specific pharmacological calpain inhibitor calpeptin blocked the drug-induced calpain small subunit autolysis and calpain activation in mitochondria and inhibited apoptosis-associated caspase-3 activation, demonstrating that mitochondrial calpain activation through small subunit cleavage is an essential step for inducing tumor cell apoptosis by various anticancer drugs.     

MST1-JNK promotes apoptosis via caspase-dependent and independent pathways

BACKGROUND: MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C-terminal regulatory domain, increasing the kinase activity of the MST1 N-terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified. RESULTS: In this study, we found that the expression of MST1 resulted in caspase-3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co-expression of a dominant-negative mutant of JNK inhibited MST1-induced morphological changes as well as caspase activation. In contrast, neither a dominant-negative p38 nor the p38 inhibitor SB203580 inhibited them. MST1 induced nucleosomal DNA fragmentation, which was suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase-Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation were not inhibited by caspase inhibitors. CONCLUSION: These results suggest that MST1 most likely promotes two events through JNK activation; first, MST1 induces the activation of caspases, resulting in CAD-mediated DNA fragmentation, and second, MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases.     

Poliovirus induces apoptosis in the human U937 promonocytic cell line

The human promonocytic U937 cell line, which is moderately susceptible to poliovirus infection, has been used to investigate the induction of apoptosis by this virus. Infection of U937 cells with poliovirus induces morphological changes typical of apoptosis. Poliovirus-resistant U937 cells (PRU) have been isolated that are resistant to apoptosis induced by poliovirus, but that undergo apoptosis after treatment with TNF plus cycloheximide. Despite the fact that poliovirus triggers nitric oxide production in U937 cells, the inhibitor of inducible nitric oxide (NO) synthase, N(omega)-monomethyl-l-arginine, did not hinder apoptosis after infection, suggesting that NO does not play a direct role in this process. Finally, poliovirus infection of U937 cells led to the cleavage of pro-caspase-3 and poly(ADP-ribose)polymerase, indicating the activation of the CPP32 ICE-like cysteine protease in the induction of apoptosis. Our findings suggest that cellular death takes place in U937 cells productively infected by poliovirus as a result of apoptosis and involves caspase activation.     

The Notch ligand, Delta-1, partially inhibits GM-CSF-induced differentiation and apoptosis along with reducing the cleavage of PARP in U937 cells

Notch signaling plays an important role in the regulation of self-renewal and differentiation of hematopoietic cells. Human monoblastic U937 cells undergo differentiation into macrophage-like cells, growth suppression, and apoptosis following stimulation with GM-CSF. We examined the effects of Notch activation induced by Notch ligands on GM-CSF-induced differentiation and apoptosis in U937 cells. Furthermore, the molecular mechanism of the effects was investigated. A recombinant Notch ligand, Delta-1 protein did not affect the growth of U937 cells by itself. GM-CSF-induced growth suppression and apoptosis of U937 cells were partially rescued by incubation with Delta-1. Delta-1 also reduced the GM-CSF-induced differentiation. Incubation with Delta-1 did not affect the expression of GM-CSF receptor. GM-CSF stimulation induced the phosphorylation of ERK1/2 and STAT5 and the cleavage of caspase-8, which were not affected by Delta-1 incubation, either. GM-CSF stimulation induced the cleavage of PARP, which is the key molecule for differentiation and apoptosis. We found that incubation with Delta-1 significantly suppressed the GM-CSF-induced cleavage of PARP. Taken together, we found that Notch activation induced by Delta-1 partially inhibited GM-CSF-induced differentiation, growth suppression, and apoptosis, along with reducing the GM-CSF-induced cleavage of PARP. These findings suggest one of the mechanisms by which Notch activation inhibits differentiation and apoptosis.     

Changes in nuclear morphology during apoptosis correlate with vimentin cleavage by different caspases located either upstream or downstream of Bcl-2 action

BACKGROUND: Upon Fas stimulation, procaspase-8 is recruited to the death-inducing signalling complex where autoactivation of caspase-8 occurs. Active caspase-8 can directly activate downstream caspases (e.g. caspase-3, 6, and 7) for the execution of apoptosis (mitochondria-independent pathway), while caspase-8 can also lead to executioner caspase activation through mitochondrial damage (mitochondria-dependent pathway). Caspase activation results in the dismantling of intracellular structure through specific proteolysis. RESULTS: We have found that an intermediate filament protein, vimentin, is cleaved at multiple sites by caspases at an early stage of apoptosis in Jurkat cells. The sequences of the two major cleavage sites in vimentin (IDVD/V and DSVD/F) suggested that these sites are cleaved by caspase-8 and caspase-3, respectively, or by close homologues of these proteases. The IDVD/V site can be cleaved by caspase-8 in vitro, and its cleavage is less sensitive to DEVD-CHO and Bcl-2 over-expression than that of the DSVD/F site in Jurkat cells. Over-expression of a mutant vimentin which was insensitive to caspase cleavage at these sites delayed the appearance of apoptotic nuclei in Jurkat cells. CONCLUSION: The specific cleavage of vimentin can be used as an apoptotic marker of both apical- and mitochondria-dependent caspase activation. Apoptotic cleavage of vimentin most likely results in disruption of its filamentous structure, which may facilitate nuclear condensation and subsequent fragmentation through disruption of the cytoskeletal network.     

Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231.

B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades.     

Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration

We previously reported that dieldrin, one of the potential environmental risk factors for development of Parkinson's disease, induces apoptosis in dopaminergic cells by generating oxidative stress. Here, we demonstrate that the caspase-3-dependent proteolytic activation of protein kinase Cdelta (PKCdelta) mediates as well as regulates the dieldrin-induced apoptotic cascade in dopaminergic cells. Exposure of PC12 cells to dieldrin (100-300 microM) results in the rapid release of cytochrome C, followed by the activation of caspase-9 and caspase-3 in a time- and dose-dependent manner. The superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride significantly attenuates dieldrin-induced cytochrome C release, indicating that reactive oxygen species may contribute to the activation of pro-apoptotic factors. Interestingly, dieldrin proteolytically cleaves native PKCdelta into a 41 kDa catalytic subunit and a 38 kDa regulatory subunit to activate the kinase. The dieldrin-induced proteolytic cleavage of PKCdelta and induction of kinase activity are completely inhibited by pretreatment with 50-100 microM concentrations of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), indicating that the proteolytic activation of PKCdelta is caspase-3-dependent. Additionally, Z-VAD-FMK, Z-DEVD-FMK or the PKCdelta specific inhibitor rottlerin almost completely block dieldrin-induced DNA fragmentation. Because dieldrin dramatically increases (40-80-fold) caspase-3 activity, we examined whether proteolytically activated PKCdelta amplifies caspase-3 via positive feedback activation. The PKCdelta inhibitor rottlerin (3-20 microM) dose-dependently attenuates dieldrin-induced caspase-3 activity, suggesting positive feedback activation of caspase-3 by PKCdelta. Indeed, delivery of catalytically active recombinant PKCdelta via a protein delivery system significantly activates caspase-3 in PC12 cells. Finally, overexpression of the kinase-inactive PKCdelta(K376R) mutant in rat mesencephalic dopaminergic neuronal cells attenuates dieldrin-induced caspase-3 activity and DNA fragmentation, further confirming the pro-apoptotic function of PKCdelta in dopaminergic cells. Together, we conclude that caspase-3-dependent proteolytic activation of PKCdelta is a critical event in dieldrin-induced apoptotic cell death in dopaminergic cells.     

Bax cleavage implicates caspase-dependent H2O2-induced apoptosis of hepatocytes

Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.