Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Identification of the processing/activation of multiple interleukin-1beta converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3alpha but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3alpha. was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3alpha or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3alpha, Mch2alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.
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PMID:Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. 912 56

Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1beta converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.
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PMID:Protease activation is required for glucocorticoid-induced apoptosis in chronic lymphocytic leukemic lymphocytes. 934 52

c-Myc is a transcriptional activator implicated in the control of cell proliferation, differentiation and transformation, but is also involved in the regulation of programmed cell death, apoptosis. Despite intensive research, the molecular mechanisms by which c-Myc triggers and executes cell death remain still elusive. Here, we made use of Rat 1A MycER cells expressing a conditionally active c-Myc protein and tested first the hypothesis that ornithine decarboxylase (ODC), which is a transcriptional target of c-Myc, were a mediator of c-Myc-induced apoptosis. However, our results show that the activity of ODC is not required for the c-Myc-mediated apoptosis to occur in these cells. We also found that the expression of p53, p21waf1/cip1, Bcl-2, Bax, Bcl-xL, Bad and cyclins D1, E, A and B did not show any significant changes following c-Myc induction. But, our studies revealed that the c-Myc induced apoptosis is associated with a specific cleavage of poly(ADPribose) polymerase (PARP), suggesting that a cysteine protease of the ICE/CED-3 family is involved. Moreover, we found that the cysteine protease CPP32/Caspase-3, which is known to cleave PARP, is processed from its inactive form to an active protease composed of 17 and 12 kDa subunits; whilst Ich-1/Caspase-2 belonging to another subset of this protease family was not processed/ activated following c-Myc activation. The activation of CPP32 and apoptotic cell death were inhibited by addition of Z-VAD-fmk, a universal inhibitor of ICE-like proteases. Further, a selective inhibitor of CPP32-like proteases (Z-DEVD-fmk) partly inhibited apoptosis. These results provide evidence that the ICE/CED3-family proteases, CPP32 and likely others, play a critical role in the execution of a nuclear proto-oncogene, c-Myc-induced apoptosis.
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PMID:Involvement of CPP32/Caspase-3 in c-Myc-induced apoptosis. 946 64

Though p53-induced apoptosis plays an important role in tumor suppression, the mechanism(s) by which p53 induces apoptosis is still unclear. To elucidate the p53-induced apoptotic pathway, we examined the role of p53 transactivation activity and caspase in J138V5C cells carrying a human temperature-sensitive (ts) p53 mutant (138Ala-->Val). The results showed that p53-induced apoptosis was not blocked by cycloheximide, which effectively prevented the expression of p53 target genes, indicating that transactivation was not essential for p53-induced apoptosis in this system. Western blot analysis showed that PARP, CPP32 and ICH-1 precursors were cleaved during apoptosis. The CPP32-preferential tetrapeptide inhibitor Ac-DEVD-CHO blocked the cleavage of ICH-1 and PARP precursors, suggesting that CPP32 or some other DEVD-sensitive caspase(s) is the upstream activator of ICH-1. We also examined the role of the Fas pathway by using Fas and Fas ligand-neutralizing antibodies. Both antibodies failed to block p53-induced apoptosis, suggesting that the Fas pathway was not essential for p53-induced apoptosis in this system. Taken together, our results indicate that p53-induced, transactivation-independent apoptosis in Jurkat cells involves sequential activation of CPP32 or some other DEVD-sensitive caspase(s) and ICH-1, via a Fas-independent pathway.
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PMID:Activation of caspases in p53-induced transactivation-independent apoptosis. 1018 88

The B cell lymphoma WEHI-231 has been used as a model to study immature B cell tolerance, based on its capacity to undergo growth arrest and programmed cell death on B cell receptor (BCR) cross-linking. Using this model to identify the molecular mechanisms underlying these processes, we found that BCR cross-linking results in the selective activation of caspase 7/Mch3, but not of the other two members of the CPP32 family, caspase 2/Nedd2 and caspase 3/CPP32. This was evidenced by the induction of proteolytic activity against the substrate for the CPP32 subfamily of caspases (z-DVED-AMC) in vitro, as well as PARP proteolysis in vivo and by the processing of the 35 kDa Mch3 into a 32 kDa species, which was later further proteolyzed. The general caspase inhibitor z-VAD-fmk, but not the CPP32 family inhibitor Ac-DEVD-CHO, blocked anti- micro-induced apoptosis, indicating that a caspase not belonging to the CPP32-like family is also implicated in anti- micro-triggered apoptosis. In contrast, z-VAD-fmk was not able to counteract growth arrest induced by anti- micro treatment, suggesting that caspase activation is not necessary for induction of growth arrest. Neither of the inhibitors prevented Mch3 processing; however, z-VAD-fmk prevented proteolysis of the p32 subunit, suggesting that further processing of this subunit is associated with apoptosis. Bcl-2 overexpression prevented anti- micro induction of CPP32-like activity and apoptosis, and blocked further processing of the Mch3 p32 subunit. In contrast, CD40 stimulation completely blocked the appearance of the p32 subunit in addition to blocking CPP32-like activity and apoptosis induced by BCR cross-linking. Moreover, only CD40 stimulation was able to prevent anti- micro-induced growth arrest, which was correlated with inhibition of retinoblastoma and of cyclin A down-regulation. In splenic B cells, Mch3 is also specifically proteolyzed ex vivo after induction of apoptosis by BCR cross-linking, demonstrating the specific involvement of caspase-7/Mch3 in apoptosis induced in B cell tolerance.
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PMID:Caspase activation by BCR cross-linking in immature B cells: differential effects on growth arrest and apoptosis. 1022 36

Caspases play crucial roles in the inflammatory response and in the cell pathway leading to apoptosis. Caspase 1 (ICE), 2 (Nedd2), 3 (CPP32), 6 (Mch2) and 8 (Mch5, FLICE) expression was examined using immunohistochemistry in the brains of rats and gerbils following systemic administration of kainic acid (KA). The distribution of caspase expression was compared with the distribution of c-Fos expression, a transcription factor that is produced in response to the excitotoxic insult. Strong caspase 2 immunoreactivity was found in microglia up to 6 h following KA administration. Focal strong expression of caspases 1, 2, 3, 6 and 8 was observed in astrocytes and neurons, from 12 to 48 h after KA injection, in areas in which a number of neurons were committed to die. This distribution was in contrast with the generalised distribution of c-Fos expression following KA administration. Only a minority of neurons in the entorhinal cortex, amygdala and hilus, but a majority of neurons in selected thalamic nuclei, exhibited strong caspase expression in KA-treated rats. Similar findings, although minimised, were observed in KA-treated gerbils. Double-labelling caspase immunohistochemistry and in situ end-labelling of nuclear DNA fragmentation disclosed co-localisation of strong caspase expression and nuclear DNA breaks in a small percentage of neurons but no co-localisation in astrocytes. Western blots of entorhinal cortex and neocortex homogenates showed cleavage of certain caspase substrates in KA-treated rats. The intensity of the bands corresponding to lamin B and protein kinase C-delta was decreased in the entorhinal cortex following KA administration. Several bands appeared in the entorhinal cortex and neocortex paragraph signin Western blots processed for the demonstration of poly(ADP-ribose) polymerase (PARP), thus indicating that other proteases, in addition to caspases, cleaved PARP following KA administration. Taken together, these findings indicate that KA excitotoxicity triggers caspase expression which, although predominant in regions subjected to irreversible cell damage, has only a weak association with the presence of nuclear DNA breaks and neuron cell death. Although these results suggest caspase activation, further studies have to be performed to elucidate whether caspase activation plays a crucial role in KA excitotoxicity.
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PMID:Differential c-Fos and caspase expression following kainic acid excitotoxicity. 1066 66

Apoptotic cell death is induced in SH-SY5Y neuroblastoma cells following exposure to the protein kinase inhibitors staurosporine (100 nM) and 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine: H-7 (100 microM). This is associated with reduced levels of PARP 117 kDa and with the concomitant formation of PARP-cleaved products of 89 kDa that result from caspase-3 activation. The process is inhibited with DEVD-fmk, a potent caspase-3 (and caspase-8) inhibitor, thus indicating that staurosporine- and H-7-induced cell death in SH-SY5Y is mediated by caspase activation. Increased caspase-2- and caspase-3-like activities, but not caspase-9-like activity, were demonstrated by monitoring proteolysis of the corresponding colorimetric substrates. Caspase-2 activity peaked at 6 h, whereas caspase-3 peaked at 12 h in parallel with the maximal loss of cell viability. No modifications in the expression levels of Fas and Fas-L were observed by Western blotting. Furthermore, no activation of caspase-8 was elicited by colorimetric assays through the process of apoptosis of neuroblastoma cells. These findings indicate that the Fas/Fas-L-caspase-8 pathway of cell death signaling is not involved in staurosporine- and H-7-induced apoptosis in SH-SY5Y neuroblastoma cells.
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PMID:Staurosporine- and H-7-induced cell death in SH-SY5Y neuroblastoma cells is associated with caspase-2 and caspase-3 activation, but not with activation of the FAS/FAS-L-caspase-8 signaling pathway. 1114 7

Apoptotic death is a physiological process with regulatory mechanisms that are under the control of different molecules such as caspases. These are classified as initiators, such as caspases-8 and -9, and effectors, such as caspases-3 and -7. The participation of caspase-2 in the effector phase of apoptosis has been commonly observed in many cell types; however, it is able to act as an initiator caspase, depending on the apoptotic stimulus. Cerebellar granule cells (CGCs) undergo apoptosis when they are transferred from high potassium (K25) to low potassium (K5); this process seems to be mediated by caspase-3 activation. Staurosporine (STS), a full strength inhibitor of kinase proteins, also induces apoptosis in these cells. To characterize the caspase cascade induced by two stimuli in the same cell type we studied the activation of different caspases in CGCs treated with STS or K5. We found that both K5 and STS induce the activation of caspase-3. This result was confirmed by the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. Caspase-2 was activated preferentially by STS, which showed a temporal course suggesting that this caspase was induced before caspase-3. The initiator caspase-9 was also activated by both K5 and STS, as well as cytochrome-c release. The results obtained in this study suggest that STS and K5 induced different activation caspase pathways for apoptotic cell death of CGCs.
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PMID:Caspase activation pathways induced by staurosporine and low potassium: role of caspase-2. 1252 27

Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of mitochondria to promote Cyt c release, but caspase-2 is already maximally activated 6 h after 4 microM DAU or TT13 treatments, whereas DAU- or TT-induced caspase-8 and -9 activities peak at 9 h. Pre-treatments with 15 microM of the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally block DAU- and TT13-induced caspase-2, -8 and -9 activities, whereas pre-treatments with 15 microM of the caspase-8 inhibitor z-Ile-Glu-Thr-Asp (IETD)-fmk prevent DAU and TT13 from inducing caspase-8 activities without affecting their caspase-2- and -9-inducing activities, suggesting that the induction of apical caspase-2 activity by these drugs may be a critical upstream event required for the activation of other downstream caspases, including caspase-9 and the mitochondrial amplification loop through caspase-8. However, the mechanisms by which DAU and TT13 induce the release of mitochondrial Cyt c appear to be caspase-independent since they are both insensitive to similar pre-treatments with 100 microM of these specific caspase-2 and -8 inhibitors. Moreover, pre-treatments with 10 microg/ml of the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb are all unable to prevent DAU and TT13 from inducing Cyt c release and caspase-2, -8 and -9 activities, suggesting that the Fas-FasL signaling pathway is not involved in the mechanism by which these quinone antitumor drugs trigger apoptosis in HL-60 cells.
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PMID:Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling. 1551 62

A novel small molecule inhibitor, 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB), competes with the Bak BH3 peptide to bind Bcl-2 protein with a binding affinity of IC(50) = 0.70 microM, as assessed by a fluorescence polarization based binding assay. HL-60 cells express the highest levels of Bcl-2 among the cell lines examined. Treated with 5 microM of MNB only for 6 h, 85% of HL-60 cells were detected to undergo apoptosis. Pan-caspase inhibitor, Z-VAD-FMK, blocks MNB-induced apoptosis in HL-60 cells. Caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, and PARP activation were observed at as early as 4 to 6 h of MNB treatment. In addition, it has been confirmed that the caspase-3 specific inhibitor, Z-DEVD-FMK, blocks the activation of caspase-8 in MNB-treated HL-60 cells. MNB treatment does not change Bcl-2 or Bax expression level in HL-60 cells, but causes Bid cleavage. Further experiments have illustrated that MNB inhibits the heterodimerization of Bcl-2 with Bax or Bid, reduces the mitochondrial membrane potential (DeltaPsimt), and induces cytochrome c release from mitochondria in HL-60 cells. These results suggest that MNB induces apoptosis in HL-60 by inhibiting the heterodimerization of Bcl-2 with pro-apoptosis Bcl-2 members, resulting in a decrease in the mitochondrial membrane potential and cytochrome c release, activation of caspases and PARP; it is a caspase-dependent process in which the activation of caspase-8 is dependent on the mitochondrial apoptosis signal transduction pathway. MNB prolongs the life spans of HL-60 bearing mice, potently kills fresh AML and ALL cells, indicating that it has the potential to be developed to treat leukemia.
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PMID:A novel Bcl-2 small molecule inhibitor 4-(3-methoxy-phenylsulfannyl)-7-nitro-benzofurazan-3-oxide (MNB)-induced apoptosis in leukemia cells. 1739 62


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