Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.56 (caspase-3)
 35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some granule neurons naturally undergo apoptosis in the external granular layer (EGL) of the postnatally developing cerebellum. In the present study, we examined the involvement of caspase-3 in this apoptosis using an organotypic slice culture system of postnatal rat cerebellum and an antibody specific for the active form of caspase-3 (p20/17). Double staining by immunohistochemistry against p20/17 and in situ nick-end labeling showed that p20/17 was present in some of the apoptotic EGL neurons. A similar staining pattern was also observed in the postnatal cerebellum in vivo. Double positive cells were observed more frequently when T7 DNA polymerase was used for the DNA fragmentation labeling in place of terminal deoxynucleotidyl transferase, by which apoptotic cells at earlier stages were thought to be labeled. Taken together, whereas caspase-3 was shown to be activated in some of the apoptotic EGL neurons in the developing cerebellum, activation of caspase-3 in some apoptotic EGL neurons may occur before they become positive on DNA fragmentation labeling. In addition, there may be another mechanism of EGL neuron apoptosis that is independent of caspase-3.
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PMID:In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo. 1087 36

Nuclear morphological changes during apoptosis are very distinct and effector caspases have been implicated to play a central role in these processes. To investigate this in greater detail we examined the effect of blocking caspase activity and its activation on the nuclear morphological change in Jurkat T cells undergoing apoptosis after staurosporine treatment. In the presence of caspase inhibitors, like benzyloxycarbonyl-Val-Ala-Asp fluoro-methylketone (z-VAD-FMK), N-acetyl Tyr-Val-Ala-Asp chloromethylketone (Ac-YVAD-CMK) and benzyloxy-carbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-FMK), staurosporine-treated Jurkat cells displayed a nuclear morphological change distinct from that of normal and apoptotic cells. This nuclear morphological change is an early event, characterised by convoluted nuclei with cavitations, and clumps of chromatin abutting to inner regions of the nuclear envelope between the nuclear pores. Both the nuclear envelope and endoplasmic reticulum were grossly dilated. This pre-apoptotic nuclear change precedes the externalisation of phosphatidylserine, chromatin condensation and DNA laddering, and can be dissociated from the formation of high molecular weight DNA fragments and cell shrinkage. Although cytochrome c efflux from the mitochondria and the processing of caspase-3 were observed in Jurkat cells with pre-apoptotic nuclear morphology, caspase-2, -6, -7 and -8 were not activated. In the presence of z-DEVD-FMK or Ac-YVAD-CMK, caspase-3 was processed to both the p17 and p20 fragments in staurosporine-treated cells, but only to p20 fragment in the presence of z-VAD-FMK. However, the caspase-3 substrate, poly(ADP ribose) polymerase was not cleaved in the presence of z-VAD-FMK, despite >70% of the cells have pre-apoptotic nuclei. In addition, caspase-3 null MCF-7 cells also undergo pre-apoptotic nuclear change when treated with staurosporine in the presence of caspase inhibitors, indicating that caspase-3 is not required for the early nuclear morphological change in cells undergoing apoptosis. Although cell death in staurosporine-treated Jurkat cells was markedly delayed, they eventually die without discernible downstream apoptotic features. Other apoptotic stimuli like etoposide and the heavy metal chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine also induced this nuclear morphological change in Jurkat cells in the presence of z-VAD-FMK. In summary, the effector caspases are not involved in early nuclear morphological change, which precedes the conventional hallmark morphological changes associated with chemical-induced apoptosis.
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PMID:Effector caspases are dispensable for the early nuclear morphological changes during chemical-induced apoptosis. 1093 34

Cell death from spinal cord injury is mediated in part by apoptotic mechanisms involving downstream caspases (e.g., caspase-3). Upstream mechanisms may involve other caspases such as procaspase-8, a 55 kDa apical caspase, which we found constitutively expressed within spinal cord neurons along with Fas. As early as 1.5 hr after transient ischemia, activated caspase-8 (p18) and caspase-8 mRNA appeared within neurons in intermediate gray matter and in medial ventral horn. We also detected evidence for an increase in death receptor complex by co-immunoprecipitation using Fas and anti-procaspase-8 after ischemia. At early time points, Fas and p18 were co-expressed within individual neurons, as were activated caspase-8 and caspase-3. Moreover, we detected p18 in cells before procaspase-3 cleavage product (p20), suggesting sequential activation. The appearance of cytosolic cytochrome c and gelsolin cleavage after ischemia was consistent with mitochondrial release and caspase-3 activation, respectively. Numerous terminal deoxynucleotidyl transferase-mediated DNA nick end-labeling-positive neurons contained p18 or p20 (65 and 80%, respectively), thereby supporting the idea that cells undergoing cell death contain both processed caspases. Our data are consistent with the idea that transient spinal cord ischemia induces the formation of a death-inducing signaling complex, which may participate in caspase-8 activation and sequential caspase-3 cleavage. Death receptors as well as downstream caspases may be useful therapeutic targets for limiting the death of cells in spinal cord.
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PMID:Fas receptor and neuronal cell death after spinal cord ischemia. 1099 32

Although apoptosis is a well-recognized phenomenon in chronic atherosclerotic disease, its role in sudden coronary death, in particular, acute plaque rupture is unknown. Culprit lesions from 40 cases of sudden coronary death were evaluated. Cases were divided into two mechanisms of death: ruptured plaques with acute thrombosis (n = 25) and stable plaques with and without healed myocardial infarction (n = 15). Apoptotic cells were identified by staining of fragmented DNA and confirmed in select cases by gold conjugate labeling combined with ultrastructural analysis. Additional studies were performed to examine the expression and activation of two inducers of apoptosis, caspases-1 and -3. Ruptured plaques showed extensive macrophage infiltration of the fibrous cap, in particular at rupture sites contrary to stable lesions, which contained fewer inflammatory cells. Among the culprit lesions, the overall incidence of apoptosis in fibrous caps was significantly greater in ruptured plaques (P < 0.001) and was predominantly localized to the CD68-positive macrophages. Furthermore, apoptosis at plaque rupture sites was more frequent than in areas of intact fibrous cap (P = 0. 028). Plaque rupture sites demonstrated a strong immunoreactivity to caspase-1 within the apoptotic macrophages; staining for caspase-3 was weak. Immunoblot analysis of ruptured plaques demonstrated caspase-1 up-regulation and the presence of its active p20 subunit whereas stable lesions showed only the precursor; nonatherosclerotic control segments were negative for both precursor and active enzyme. These findings demonstrate extensive apoptosis of macrophages limited to the site of plaque rupture. The proteolytic cleavage of caspase-1 in ruptured plaques suggests activation of this apoptotic precursor. Whether macrophage apoptosis is essential to acute plaque rupture or is a response to the rupture itself remains to be determined.
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PMID:Localization of apoptotic macrophages at the site of plaque rupture in sudden coronary death. 1102 30

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).
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PMID:Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells. 1107 88

Apoptosis is an important process in normal animal development as well as in diseases, and inhibitor of apoptosis protein (IAP) is one of the important factors that regulate apoptotic cell death. We found that lipopolysaccharide (LPS) enhances the expression of mRNA and protein of cellular IAP-2 (cIAP2) in human monoblastic U937 cells differentiated by phorbol ester pretreatment. cIAP2 mRNA was not detected in undifferentiated U937 cells. mRNAs of cIAP1 and X-chromosome-linked IAP (XIAP) were expressed constitutively and not affected by LPS in both undifferentiated and differentiated cells. LPS stimulated the expression of cIAP2 mRNA and protein in time- and concentration-dependent manners. LPS enhanced the expression of cIAP2 mRNA and protein in human monocyte-derived macrophages, which was associated with the inhibition of the caspase-3 activation, i.e., decrease in active p17 fragment of caspase-3 with simultaneous accumulation of precursor p20 fragment. We conclude that LPS may inhibit apoptosis of macrophages, at least in part, through the induction of cIAP2.
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PMID:Lipopolysaccharide induces the expression of cellular inhibitor of apoptosis protein-2 in human macrophages. 1111 65

The cytotoxicity of cocaine (0 - 1000 microM), was studied on parameters related to the mitochondrial role and the cascade of events that lead to apoptosis in hepatocyte cultures from phenobarbitone (PB) pretreated rats. Cytotoxicity was dose-dependent and LDH leakage was significantly enhanced above 100 microM cocaine. Apoptosis was visualized by DNA fragmentation on agarose gel, and appeared at 50 and 100 microM cocaine. Cocaine induced biphasic changes in mitochondrial transmembrane potential and significantly increased the mitochondrial release of cytochrome c, the caspase-3 like DEVDase activity and the level of 20 kDa subunit, a product of pro-caspase-3 cleavage. The protective effect of N-acetylcysteine (NAC) and deferoxamine (DFO) on all these parameters confirmed the involvement of oxygen radicals in cocaine-induced necrosis/apoptosis. We conclude: first, that the biphasic changes recorded in mitochondrial inner membrane potential by the effect of cocaine, were parallel to apoptosis; second, that caspase-3 activity and cleavage to it p20 subunit increased sharply in parallel to the translocation of cytochrome c from mitochondria to cytosol; and third, that the antioxidants, NAC or DFO exerted a noticeable protective role in counteracting the cytotoxicity of cocaine, these effects being more pronounced in the case of DFO than NAC. These findings demonstrate that cocaine cytotoxicity involves mitochondrial damage.
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PMID:Mitochondrial involvement in cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine. 1122 37

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.
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PMID:Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice. 1145 82

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.
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PMID:Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein. 1180 95

The mitochondrial pathway is critical for the efficient execution of death receptor-initiated apoptosis in certain cell types. Questions remain as to why the mitochondria are required in that scenario. We investigated the molecular events that determined the need for the mitochondria by using an in vivo model of anti-Fas-induced hepatocyte apoptosis. In wild-type mice, Fas stimulation resulted in normal activation of caspase-3, with the generation of the active p19-p12 complex. In bid-deficient mice, caspase-3 activation was arrested after the initial cleavage at Asp(175). This allowed the generation of the p12 small subunit, but the p20 large subunit could not be further processed to the p19 subunit. The p20-p12 complex generated by Fas stimulation in bid-deficient hepatocytes was inactive, arresting the death program. Failure of p20/p12 caspase-3 to mature and to exhibit activity was because of the inhibition by the inhibitor-of-apoptosis proteins (IAPs), such as XIAP, and also to a low caspase-8 activity. This block could be overcome in wild-type mice by two mechanisms. Smac was released from mitochondria early following Fas activation and was competitively bound to the IAPs to reverse their effects. XIAP could also be cleaved, and this occurred later and was likely mediated by enhanced caspase activities. Both mechanisms were dependent on Bid and thus were not operative in bid-deficient hepatocytes. In conclusion, mitochondrial activation by Bid is required for reversing the IAP inhibition through Smac release. It is also required for the alternative activation of caspases through cytochrome c release, as demonstrated previously. Together, these events ensure a successful progression of the death program initiated by the death receptor activation in the hepatocyte.
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PMID:Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. 1268 80


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