UNIPROT:P42574 - FACTA Search

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Query: UNIPROT:P42574 (caspase-3)
45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A tumor-suppressor gene, p16(INK4), which is deleted or mutated in tumors, regulates cell-cycle progression through a G(1)-S restriction point by inhibiting CDK4(CDK6)/cyclin-D-mediated phosphorylation of pRb. We have found that ectopic p16(INK4) expression increased cellular sensitivity of human non-small-cell-lung-cancer (NSCLC) A549 cells to a selective growth-inhibitory effect induced by the topoisomerase-I inhibitor 11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT-11) in vitro. In this study, we observed enhanced apoptosis characterized by DNA fragmentation in A549 cells transfected with p16(INK4) cDNA (A549/p16-1) and treated with CPT-11. This apoptosis was suppressed by the inhibitor of interleukin-1beta-converting enzyme (ICE/caspase-1) or ICE-like proteases, Z-Asp-CH2-DCB, as determined by DNA fragmentation and proteolytic cleavage of poly(ADP-ribose) polymerase, a natural substrate for CPP32/caspase-3. In A549/p16-1 cells, cytosolic peptidase activities that cleaved Z-DEVD-7-amino-4-trifluoromethylcoumarin increased during CPT-11-induced apoptosis and were suppressed by a highly specific caspase-3 and caspase-3-like inhibitor, Z-DEVD-fluoromethylketone. These findings indicate that p16(INK) is positively involved in the activation pathway of the caspase-3 induced by CPT-11. The increased delay in S-phase progression and subsequent induction of apoptosis were observed in CPT-11-treated A549/p16-1 cells on the basis of DNA histograms. Specific down-regulation of the cyclin-A protein level in A549/p16-1 cells was observed after CPT-11-treatment, whereas cyclin B, cdk2, and cdc2 protein levels were unaffected. These results suggest that ectopic p16(INK4) expression inappropriately decreases cyclin A and thereby terminates CPT-11-induced G(2)/M accumulation, which is followed by increased apoptosis in p16(INK4)-expressing A549 cells.
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PMID:Ectopic p16(ink4) expression enhances CPT-11-induced apoptosis through increased delay in S-phase progression in human non-small-cell-lung-cancer cells. 1073 46

Poly (ADP-ribose) polymerase (PARP) is an abundant chromatin associated protein important in DNA repair, maintenance of chromosomal stability and programmed cell death. Here we report that an increase in caspase 3-activity and cleavage of PARP serves as an early execution phase signal in human neuroblastoma. Human neuroblastoma SK-N-SH cells were exposed to a protein kinase inhibitor, staurosporine, or a topoisomerase II inhibitor, etoposide, at various concentrations and time points. Cells exposed to staurosporine (0.1 microM) for 30 min showed an increase in caspase 3-activity and by 1 h an increase in PARP 116-kDa band and an 85-kDa cleavage product, which further increased in density with time after treatment. Quantitative analysis for condensed chromatin material using bisbenzimide, and DNA fragmentation enzyme immunoassays showed a significant increase in apoptosis 5 h after staurosporine treatment. This was further confirmed with a Klenow fragment of DNA polymerase I assay which primarily detects single-stranded DNA breaks. A significant decrease in mitochondrial metabolism occurred within 8-12 h after treatment. Studies using Trypan Blue exclusion, and lactic dehydrogenase (LDH) release revealed a significant increase in membrane permeability 8 h after staurosporine (0.1 microM) or etoposide (10 microM) treatments. Cleavage of lamin B1, a protein important in maintaining the nuclear envelope integrity was observed 12 h after staurosporine treatment. Our results show that activation of caspase 3 followed by PARP cleavage occur at much earlier time point than any other morphological or biochemical parameters of apoptosis or cytotoxicity.
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PMID:Poly (ADP-ribose) polymerase induction is an early signal of apoptosis in human neuroblastoma. 1076 13

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.
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PMID:Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells. 1076 78

Ultraviolet (UV) light is a strong apoptotic trigger that can induce a caspase-dependent biochemical change in cells. We previously showed that UV irradiation can elicit caspase-3 activation and the subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. We report that genistein, an isoflavone compound with known inhibitory activities to protein tyrosine kinases (PTKs) and topoisomerase-II (topo-II), can prevent UV irradiation-induced apoptotic biochemical changes (DNA fragmentation, caspase-3 activation, and cleavage/activation of PAK2) in A431 cells. Surprisingly, two typical PTK inhibitors (tyrphostin A47 and herbimycin A) and three known topo-II inhibitors (etoposide, daunorubicin, and novomycin) had no effect on UV irradiation-induced apoptotic biochemical changes, suggesting that the inhibitory effect of genistein is not dependent on its property as a PTK/topo-II inhibitor. In contrast, azide, a reactive oxygen species (ROS) scavenger, could effectively block the UV irradiation-induced apoptotic cell responses. Flow cytometric analysis using the cell-permeable dye 2',7'-dichlorofluorescin diacetate as an indicator of the generation of ROS showed that UV irradiation caused increase of the intracellular oxidative stress and that this increase could be abolished by azide, suggesting that oxidative stress plays an important role in mediating the apoptotic effect of UV irradiation. Importantly, the UV irradiation-induced oxidative stress in cells could be significantly attenuated by genistein, suggesting that impairment of ROS formation during UV irradiation is responsible for the antiapoptotic effect of genistein. Collectively, our results demonstrate the involvement of oxidative stress in the UV irradiation-induced caspase activation and the subsequent apoptotic biochemical changes and show that genistein is a potent inhibitor for this process.
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PMID:Inhibition of UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein. 1079 67

Ascididemin (ASC) is a pentacyclic DNA-intercalating agent isolated from the Mediterranean ascidian Cystodytes dellechiajei. This marine alkaloid exhibits marked cytotoxic activities against a range of tumor cells, but its mechanism of action remains poorly understood. We investigated the effects of ASC on DNA cleavage by human topoisomerases I and II. Relaxation assays using supercoiled DNA showed that ASC stimulated double-stranded cleavage of DNA by topoisomerase II, but exerted only a very weak effect on topoisomerase I. ASC is a conventional topoisomerase II poison that significantly promoted DNA cleavage, essentially at sites having a C on the 3' side of the cleaved bond (-1 position), as observed with etoposide. The stimulation of DNA cleavage by topoisomerase I in the presence of ASC was considerably weaker than that observed with camptothecin. Cytotoxicity measurements showed that ASC was even less toxic to P388 leukemia cells than to P388CPT5 cells resistant to camptothecin. In addition, the marine alkaloid was found to be equally toxic to HL-60 leukemia cells sensitive or resistant to mitoxantrone. It is therefore unlikely that topoisomerases are the main cellular targets for ASC. This alkaloid was found to strongly induce apoptosis in HL-60 and P388 leukemia cells. Cell cycle analysis showed that ASC treatment was associated with a loss of cells in the G1 phase accompanied with a large increase in the sub-G1 region. Cleavage experiments with poly(ADP-ribose) polymerase (PARP) revealed that caspase-3 was a mediator of the apoptotic pathway induced by ASC. The DNA of ASC-treated cells was severely fragmented. Collectively, these findings indicate that ASC is a potent inducer of apoptosis in leukemia cells.
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PMID:Inhibition of topoisomerase II by the marine alkaloid ascididemin and induction of apoptosis in leukemia cells. 1087 27

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.
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PMID:Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells. 1094 Mar 16

Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD.FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation.
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PMID:Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells. 1104 71

The main objective of this study to analyze which of 31 cellular factors (resistance proteins, proliferative factors, apoptotic factors, angiogenic factors, proto-oncogenes) most accurately predict the resistance of non-small cell lung carcinomas. To this purpose, we used a short-term in vitro test that measures changes in the rate at which radioactive nucleic acid precursors are incorporated into tumor cells after the addition of doxorubicin to determine the response to doxorubicin in 94 non-small cell lung carcinomas. The results obtained by the short-term test were related to the various cellular factors which were in turn determined by immunohistochemistry and flow cytometry. A significant correlation was found between the data obtained by the short-term test and the expression of P-glycoprotein 170 (P = 0.00004), glutathione-S-transferase-pi (P = 0.0002), metallothionein (P = 0.0008), thymidylate synthase (P = 0.002), O6-methylguanine-DNA-methyltransferase (P = 0.008) and lung resistance-related protein (LRP, P = 0.03). There was only a weak correlation between heat shock proteins (HSP70) and no correlation between the expression of topoisomerase II or catalase and the short-term test results. To measure the proliferative activity, the following were determined: PCNA, cyclin A, cyclin D and cdk2. Only a weak relationship was found between the expression of cdk2 (P = 0.04) and PCNA (P = 0.05) and the doxorubicin response in vitro. Of the investigated pro-apoptotic factors (Fas/CD95, Fas ligand, caspase-3), only Fas/CD95 is significantly associated with the drug response (P = 0.007). The apoptotic index also reveals a significant correlation (P = 0.03). Angiogenesis, as measured by the microvessel density and the angiogenic factors, is inversely correlated to the resistance of non-small cell lung cancer. Platelet-derived endothelial cell growth factor (PD-ECGF) and vascular endothelial growth factor (VEGF) exhibit a significant relationship to the drug resistance (P = 0.0006 and P = 0.004, respectively). Of the investigated proto-oncogenes (Fos, Jun, ErbB-1, ErbB-2, Myc, Ras), only ErbB-2 is weakly associated with the in vitro short term test. In order to determine whether combining factors can result in improved predictive information, combinations of the factors (pairs, triplets) were analyzed. The systematic investigation of these combinations yields an improvement in the predictive information. With one factor up to 76.6% of the tumors, with two factors up to 85.4% and with three factors up to 89.5% of the tumors could be correctly diagnosed.
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PMID:Cellular predictive factors for the drug response of lung cancer. 1113 47

DNA topoisomerase (topo) IIalpha gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better understand the mechanisms by which topoIIalpha expression levels are modulated, we examined topoIIalpha transcriptional regulation in ICRF-187-sensitive and ICRF-187-resistant human leukemic cell lines that express an increased amount of topoIIalpha protein and mRNA. Transient transfections of luciferase reporter plasmids containing either the full-length human topoIIalpha promoter or fragments of it revealed that topoIIalpha transcriptional activity was significantly increased in the drug-resistant CEM/ICRF-8 cells, compared with CEM cells. Specifically, the transcriptional activity of the full-length topoIIalpha promoter (nucleotides -557 to +90) was doubled in CEM/ICRF-8 compared with CEM cells. Serial deletion of the topoIIalpha promoter permitted localization of the region responsible for its up-regulation in the drug-resistant cells between nucleotides -557 and -162, which includes the last three inverted CCAAT elements (ICE) 3 to 5. Note that construction of a point mutation in ICE3 resulted in a significant increase in transcriptional activity of the topoIIalpha promoter in the drug-sensitive CEM cells. In addition, by electrophoretic mobility shift assay, ICE3 was recognized by a protein complex containing NF-YB that was present at reduced levels in the topoIIalpha-overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoIIalpha gene expression. This is the first study to show that topoIIalpha transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoIIalpha promoter.
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PMID:Role of an inverted CCAAT element in human topoisomerase IIalpha gene expression in ICRF-187-sensitive and -resistant CEM leukemic cells. 1116 Aug 54

Peroxisomicine A(1) (T-514) is a dimeric anthracenone first isolated from the plant Karwinskia humboldtiana. The compound presents a high and selective toxicity toward liver and skin cell cultures and is currently the subject of preclinical studies as an antitumor drug. To date, the molecular basis for its diverse biological effects remains poorly understood. To elucidate its mechanism of action, we studied its interaction with DNA and its effects on human DNA topoisomerases. Practically no interaction with DNA was detected. Peroxisomicine was found to inhibit topoisomerase II but not topoisomerase I. DNA relaxation and decatenation assays indicated that the drug interferes with the catalytic activity of topoisomerase II but does not stimulate DNA cleavage, in contrast to conventional topoisomerase poisons such as etoposide. Two human leukemia cell lines sensitive or resistant to mitoxantrone were used to assess the cytotoxicity of the toxin and its effect on the cell cycle. In both cases, peroxisomicine treatment was associated with a loss of cells from every phase of the cell cycle and was accompanied by a large increase in the sub-G1 region which is characteristic of apoptotic cells. The cell cycle changes were more pronounced with the sensitive HL-60 cells than with the resistant HL-60/MX2 cells (with reduced topoisomerase II activity), in agreement with the cytotoxicity measurements. Treatment of HL-60 cells with T-514 stimulated the cleavage of the poly(ADP-ribose) polymerase by intracellular proteases such as caspase-3. The cytometry and Western blot analyses reveal that peroxisomicine induces apoptosis in leukemia cells. In addition, we characterized a catabolite of peroxisomicine, named T-510R, in the form of a highly stable radical metabolite. The electron spin resonance and mass spectrometry data are consistent with the formation of an anionic semiquinonic radical. The oxidized product T-510R inhibits topoisomerase II with a reduced efficiency compared to the parent toxin and was found to be about 3-4 times less toxic to both the sensitive and resistant leukemia cell lines than T-514. Collectively, the results suggest that topoisomerase II inhibition plays a role in the cytotoxicity of the plant toxin peroxisomicine. Inhibition of topoisomerase II may serve as an inducing signal triggering the apoptotic cell death of leukemia cells exposed to the toxin. The dihydroxyanthracenone unit may represent a useful chemotype for the preparation of topoisomerase II-targeted anticancer agents.
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PMID:DNA topoisomerase II inhibition by peroxisomicine A(1) and its radical metabolite induces apoptotic cell death of HL-60 and HL-60/MX2 human leukemia cells. 1117 May 4

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