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)

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.
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PMID:Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells. 979 25

The effects of dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the apoptotic response of U937 monocytic leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) were examined. After a 6-h exposure to 1 microM ara-C, cells stably transfected with a p21WAF1/CIP1 antisense construct were significantly more sensitive to the induction of classic apoptotic morphology, DNA fragmentation, caspase-3 activation, poly(ADP-ribose) polymerase degradation, and underphosphorylation of the retinoblastoma protein (pRb) than their empty-vector counterparts. Enhanced susceptibility of antisense-expressing cells to ara-C was accompanied by a corresponding reduction in clonogenic and suspension culture growth. The increased sensitivity of these cells to ara-C-mediated lethality could not be attributed to cytokinetic perturbations, nor did ara-CTP formation or (ara-C)DNA incorporation differ significantly between the cell lines. Moreover, synchronization of p21 antisense-expressing cells in S-phase by aphidicolin block resulted in a further increase in ara-C-mediated apoptosis, suggesting enhanced drug sensitivity of the S-phase cell fraction. After exposure to ara-C, p21 antisense-expressing cells displayed a greater decline in mitochondrial membrane potential (deltapsi(m)) and generation of reactive oxygen species than their empty-vector counterparts, as well as early potentiation (e.g., within 2-4 h) of cytochrome c release into the cytosolic S-100 fraction. Lastly, ara-C-mediated increases in mitogen-activated protein kinase activity over basal levels were attenuated in p21 antisense-expressing cells. Collectively, these findings indicate that dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 increases the susceptibility of U937 human leukemia cells to ara-C-related lethality, and this phenomenon occurs as a relatively early event that is independent of cell cycle or pharmacodynamic factors and is associated with mitochondrial perturbations implicated in activation of the apoptotic protease cascade.
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PMID:Dysregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/MDA6 increases the susceptibility of human leukemia cells (U937) to 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial dysfunction and apoptosis. 1009 57

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.
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PMID:Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest. 1036 53

Recent studies of transient focal ischemia have focused interest on apoptotic mechanisms of neuronal cell death involving constitutive pro-apoptotic proteins. The finding of specific patterns of novel gene expression might indicate the activation of pro-apoptotic genes in previously ischemic areas. Thus, we investigated gene expression for the pro-apoptotic regulators, Bax and caspase-3, after transient focal brain ischemia, together with the p53-regulated cell cycle inhibitor, p21/WAF1/CIP1. Reversible occlusion of the middle cerebral artery for 2 h was carried out in halothane-anesthetized rats using the poly-L-lysine coated filament method. In situ hybridization was performed at 0, 1, 3, 6 h and 1, 3 and 7 d of recirculation and in sham controls. Radioactive antisense probes served for detection of bax, p21 and caspase-3 mRNAs on brain sections, and quantitative film autoradiography was combined with image-averaging techniques. Bax mRNA tended to decline after focal brain ischemia within 1 d. p21 mRNA was upregulated with a perifocal pattern at 3 h and 1 d after ischemia whereas the ischemic regions themselves failed to show significant upregulation. Caspase-3 mRNA was elevated in the resistant dorsomedial cortex at 1 d. A pro-apoptotic pattern of novel gene expression, involving Bax and caspase-3, was not observed after transient focal brain ischemia. Rather, the perifocal expression of p21 and caspase-3 mRNAs observed at 1 d after ischemia points to reactive changes in resistant brain areas.
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PMID:Differential changes of bax, caspase-3 and p21 mRNA expression after transient focal brain ischemia in the rat. 1092 46

Butyric acid, an extracellular metabolite from periodontopathic bacteria, induces apoptosis in murine thymocytes, splenic T-cells, and human Jurkat T-cells. The present study examines the contributions of apoptosis-related proteins (Bcl-2, Bcl-XL, Bax, and p21WAF1/CIP1) in the regulation of T-cell death induced by butyric acid, using p53 knock-out (p53-/-) and wild-type (p53+/+) mice. The results of a DNA fragmentation assay indicated that thymocytes, splenic T-cells, and B-cells from p53-/- mice were susceptible to butyric-acid-induced apoptosis to a degree similar to those from p53+/+ mice. Moreover, butyric acid significantly induced apoptosis in lymphocytes from both p53+/+ and p53-/- mice in a concentration- and time-dependent fashion. Experiments with fractionated subpopulations of splenic T-cells revealed that DNA fragmentation was equally observed in CD4+ and CD8+ splenic T-cells from both p53+/+ and p53-/- lymphocytes. Activation of caspase-3, caspase-6, and caspase-8, but not of caspase-1, in butyric-acid-induced T-cell apoptosis occurred regardless of the presence of p53. Western blotting analysis of splenic T-cells showed that butyric acid treatment decreased Bcl-2 and Bcl-XL expressions in p53+/+ and p53-/- cells. Splenic T-cells had barely detectable Bax and p21WAF1/CIP1, regardless of whether butyric acid and/or p53 was present. These results suggest that butyric-acid-mediated apoptosis of murine T-cells takes place via a pathway that is independent of p53, and is followed by the p53-regulated proteins Bax and p21WAF1/CIP1, which lower the levels of the apoptosis antagonists Bcl-2 and Bcl-XL in cells.
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PMID:Butyric-acid-induced apoptosis in murine thymocytes and splenic T- and B-cells occurs in the absence of p53. 1120 Oct 44

Transient expression of the tumor suppressor gene p53 via adenoviral-mediated gene transfer induces apoptosis in glioma cells expressing mutant p53, while causing cell cycle arrest in cells with wild-type p53. To determine whether a change in p53 status of a wild-type p53-expressing cell line such as U-87 MG would alter its apoptotic resistant phenotype in response to Ad-p53 infection, we generated cell lines U-87-175.4 and U-87-175.13 via retroviral-mediated gene transfer of the p53 (175H) mutant into the U-87 MG parental line. Control cell lines U-87-Lux.6 and U-87-Lux.8 were also generated and express the reporter gene luciferase. Both U-87-175.4 and U-87-175.13, but not control cell lines, exhibited morphology characteristic of apoptosis after Ad-p53 infection. Furthermore, expression of other p53 mutants (248W, 273H) in U-87 MG also sensitized cells to Ad-p53-induced apoptosis. Apoptosis was confirmed by TUNEL and cell cycle analysis. Several p53 response genes were examined in cells infected with Ad-p53, and among these, BCL2, p21WAF1/CIP1, CPP32/caspase 3, and PARP showed differences in expression between U87-175 and U87-Lux cell lines. Taken together, our data demonstrate that the introduction of p53 mutants in U-87 MG promotes an apoptotic response in association with adenoviral-mediated wild-type p53 gene transfer. These results underscore the importance of glioma p53 genotype for predicting tumor response to p53-based gene therapy.
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PMID:Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis. 1129 82

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1-/-) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1-0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1-/- cells and by PN in only WT cells. These two groups of cells shared similar cytochrome c release, caspase-3 activation, and p21(WAF1/CIP1) cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1-/- cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1-/- than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.
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PMID:Opposite roles of selenium-dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxynitrite-induced apoptosis and signaling. 1156 67

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.
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PMID:The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax. 1157 43

The lack of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivity to acute renal failure produced by the chemotherapeutic agent cisplatin. We used primary cultures of mouse renal proximal tubular cells (MPTC) grown in optimized culture conditions to investigate the cellular basis for increased apoptosis in p21 knockout mice. Cisplatin (15 microM) activated caspase-3 but not caspase-8 or caspase-9 and produced phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation in wild-type [p21(+/+)] MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treated MPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21(+/+) MPTC, cisplatin activated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3 activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis in p21(+/+) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activity and decreased chromatin condensation by 51% in p21(-/-) but not in p21(+/+) MPTC. However, cisplatin-induced phosphatidylserine externalization was not inhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1) in the presence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4) MPTC apoptosis is caspase independent in the presence of p21 but partially dependent on caspases in the absence of p21.
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PMID:Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells. 1274 56

To investigate apoptosis induced by selenite in hepatocytes in vivo, rats received a single injection of sodium selenite immediately after partial hepatectomy. Characteristic DNA fragmentation in gel electrophoresis and in situ end-labeling and the increase in caspase-3 activity were observed at 4 h after partial hepatectomy with selenite injection. The activation of Jun N-terminal kinase (JNK) was observed as early as 15 min and increased to about 10-fold the maximal level of the control at 1 and 2 h after partial hepatectomy in selenite-injected rats, while a transient increase was observed at 1 h in the control. Western blot analysis revealed that the c-Jun and the phosphorylated c-Jun protein markedly increased after 30 min and reached a maximal level at 1 and 2 h after partial hepatectomy with selenite injection, although c-Jun and a faint band of the phosphorylated c-Jun were observed after 1 h in the control. The levels of c-jun mRNA and c-Fos protein and mRNA in selenite-injected rats also increased more than in the control. The rise in the p53 protein level after partial hepatectomy with selenite injection was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein expression. These results suggested that selenite induced apoptosis accompanied by the activation of caspase-3 and JNK and the upregulation of c-jun, c-fos, p53 and p21(WAF1/CIP1) at the early stage of liver regeneration.
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PMID:Jun N-terminal kinase activation and upregulation of p53 and p21(WAF1/CIP1) in selenite-induced apoptosis of regenerating liver. 1280 46


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