Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The prevention of apoptosis by Zn2+ has generally been attributed to its inhibition of an endonuclease acting in the late phase of apoptosis. In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the "death substrate" poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leukemia cells with micromolar concentrations of Zn2+ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn2+ inhibited caspase-3 with an IC50 of 0.1 microM. These results identify caspase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machinery.
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PMID:Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. 922 15

We report the identification of the large subunit of the DNA replication factor, DSEB/RF-C140, as a new substrate for caspase-3 (CPP32/YAMA), or a very closely related protease activated during Fas-induced apoptosis in Jurkat T cells. DSEB/RF-C140 is a multifunctional DNA-binding protein with sequence homology to poly(ADP-ribose) polymerase (PARP). This similarity includes a consensus DEVD/G cleavage site for caspase-3. Cleavage of DSEB/RF-C140 is predicted to occurs between Asp706 and Gly707, generating 87-kDa and 53-kDa fragments. An antiserum raised against the amino-terminal domain of DSEB/RF-C140 detects a new 87-kDa protein in Jurkat T cells in which apoptosis is activated by a monoclonal antibody to Fas. This cleavage occurs shortly after PARP cleavage. In vitro translated DSEB/RF-C140 is specifically cleaved into the predicted fragments when incubated with a cytoplasmic extract from Fas antibody-treated cells. Proteolytic cleavage was prevented by substituting Asp706 by an alanine in the DEVD706/G caspase-3 cleavage site. The cleavage of DSEB/RF-C140 is prevented by iodoacetamide and the specific caspase-3 inhibitor, tetrapeptide aldehyde Ac-DEVD-CHO, but not by the specific ICE (interleukin-1-converting enzyme) inhibitors: CrmA and Ac-YVAD-CHO, indicating that the protease responsible for the cleavage of DSEB/RF-C140 during Fas-induced apoptosis in Jurkat cells is caspase-3, or a closely related protease. This conclusion is reinforced by the fact that recombinant caspase-3 but not caspase-1 reproduced the "in vivo" cleavage. Inasmuch as the cleavage of DSEB/RF-C140 separates its DNA binding from its association domain, required for replication complex formation, we propose that such a cleavage will impair DNA replication. Recent in vitro mutagenesis support this proposal (Uhlmann, F., Cai, J., Gibbs, E., O'Donnel, M., and Hurwitz, J. (1997) J. Biol. Chem. 272, 10058-10064).
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PMID:The large subunit of the DNA replication complex C (DSEB/RF-C140) cleaved and inactivated by caspase-3 (CPP32/YAMA) during Fas-induced apoptosis. 923 61

The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1. Likewise, affinity labeling with N-(N(alpha)-benzyloxycarbonylglutamyl-N(epsilon)-biotinyllysyl+ ++)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK(bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or gamma-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.
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PMID:Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. 924 21

Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.
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PMID:Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. 927 9

Proteases that are members of the caspase (or interleukin-1beta converting enzyme (ICE)) protease family have been shown to be important mediators of apoptosis induced by Fas activation, neurotrophic factor withdrawal, and detachment from extracellular matrix. In this report we have investigated the potential importance of caspase proteases in apoptosis induced by multiple chemotherapeutic agents. Human T leukemic cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases, were studied for their resistance to 1-beta-D-arabinofurasosyl-cytosine (Ara-C), etoposide (VP-16), doxorubicin (DOX), and cis-dichlorodiammine platinum (CP). Overexpression of CrmA dramatically inhibited drug-induced activation of caspases, as measured by processing of the inactive precursor form of caspase-3 and cleavage of caspase substrate proteins poly(ADP-ribose) polymerase (PARP) and lamin B. CrmA also significantly inhibited the kinetics of cell death induced by each of the four drugs. Moreover, when examined several days or weeks after initial exposure to drug, cultures of CrmA-expressing cells were found to have recovered and repopulated, whereas vector-transfected control cells did not. These studies demonstrate that caspase proteases play an important role in conferring sensitivity to multiple chemotherapy drugs, and that constitutive downmodulation of caspase activities can enhance chemoresistance.
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PMID:Inhibition of caspase proteases by CrmA enhances the resistance of human leukemic cells to multiple chemotherapeutic agents. 932 87

Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.
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PMID:Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus. 934 8

Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homology with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.
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PMID:The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during Fas-mediated apoptosis. 935 17

Betulinic acid (BA), a melanoma-specific cytotoxic agent, induced apoptosis in neuroectodermal tumors, such as neuroblastoma, medulloblastoma, and Ewing's sarcoma, representing the most common solid tumors of childhood. BA triggered an apoptosis pathway different from the one previously identified for standard chemotherapeutic drugs. BA-induced apoptosis was independent of CD95-ligand/receptor interaction and accumulation of wild-type p53 protein, but it critically depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3-like proteases). FLICE/MACH (caspase-8), considered to be an upstream protease in the caspase cascade, and the downstream caspase CPP32/YAMA/Apopain (caspase-3) were activated, resulting in cleavage of the prototype substrate of caspases PARP. The broad-spectrum peptide inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cleavage of FLICE and PARP, also completely abrogated BA-triggered apoptosis. Cleavage of caspases was preceded by disturbance of mitochondrial membrane potential and by generation of reactive oxygen species. Overexpression of Bcl-2 and Bcl-XL conferred resistance to BA at the level of mitochondrial dysfunction, protease activation, and nuclear fragmentation. This suggested that mitochondrial alterations were involved in BA-induced activation of caspases. Furthermore, Bax and Bcl-xs, two death-promoting proteins of the Bcl-2 family, were up-regulated following BA treatment. Most importantly, neuroblastoma cells resistant to CD95- and doxorubicin-mediated apoptosis were sensitive to treatment with BA, suggesting that BA may bypass some forms of drug resistance. Because BA exhibited significant antitumor activity on patients' derived neuroblastoma cells ex vivo, BA may be a promising new agent for the treatment of neuroectodermal tumors in vivo.
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PMID:Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. 986 49

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.
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PMID:Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis. 937 39

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP-/- and PARP+/- mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP-/- and 3-AB-treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD--the substrate of PARP--were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP-/- mice and in 3-AB-treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end-labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP-/- mice or in 3-AB-treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.
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PMID:Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. 939 Jun 45


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