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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Events accompanying sequential exposure of U937 leukemic cells to the deoxycytidine (dCyd) analogs 1-[beta-D-arabinofuranosyl]cytosine (ara-C) or 2',2'-difluorodeoxycytidine (gemcitabine; dFdC) followed by two protein kinase C (PKC) activators [bryostatin 1 (BRY) or phorbol 12'-myristate 13'-acetate (PMA)] exhibiting disparate differentiation-inducing abilities were characterized. A 24-hr exposure to 10 nM BRY or PMA after a 6-hr incubation with 1 microM ara-C or 100 nM dFdC resulted in equivalent increases in apoptosis, caspase-3 activation, and polyADP-ribose polymerase degradation, as well as identical DNA cleavage patterns. BRY and PMA did not modify retention of the lethal ara-C metabolite ara-CTP or alter ara-CTP/dCTP ratios. Unexpectedly, pretreatment of cells with ara-C or dFdC opposed BRY- and PMA-related induction of the cyclin-dependent kinase inhibitors (CDKIs) p21CIP1 and/or p27KIP1. These effects were not mimicked by the DNA polymerase inhibitor aphidicolin or by VP-16, a potent inducer of apoptosis. Inhibition of PKC activator-induced CDKI expression by ara-C and dFdC did not lead to redistribution of proliferating cell nuclear antigen but was accompanied by sub-additive or antagonistic effects on leukemic cell differentiation. Sequential exposure of cells to ara-C followed by BRY or PMA led to substantial reductions in clonogenicity that could not be attributed solely to apoptosis. Finally, pretreatment of cells with ara-C attenuated PMA- and BRY-mediated activation of mitogen-activated protein kinase, an enzyme implicated in CDKI induction. Collectively, these findings suggest that pretreatment of leukemic cells with certain dCyd analogs interferes with CDKI induction by the PKC activators PMA and BRY, and that this action may contribute to modulation of apoptosis and differentiation in cells exposed sequentially to these agents.
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PMID:Inhibition of protein kinase C activator-mediated induction of p21CIP1 and p27KIP1 by deoxycytidine analogs in human leukemia cells: relationship to apoptosis and differentiation. 1040 25

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

Human DNA polymerase epsilon (pol epsilon) normally contains a 261-kDa catalytic subunit (p261), but from some sources it is isolated as a 140-kDa catalytic core of p261. This shortened form possesses normal or somewhat enhanced polymerase activity and its significance is unknown. We report here that caspase-3 and calpain can form p140 from p261 in vitro and in vivo and that during early stages of apoptosis induced in Jurkat cells by staurosporine or anti-Fas-activating antibody, p261 is cleaved into p140 by caspase-3. At later stages, activated calpain might also contribute to this conversion. The sites of cleavage by caspase-3 have been identified, and mutations at these 'DEAD boxes' resulted in cleavage-resistant enzyme. Cleavage at these sites separates the 'N-terminal catalytic core' from the 'C-terminal' regions described for p261. Cleavage does not occur during necrosis or following exposure to H(2)O(2) or methanesulfonic acid methyl ester. p140 is unlikely to be able to functionally replace p261 in vivo, since it does not bind to PCNA or the other pol epsilon subunits.
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PMID:Proteolysis of the human DNA polymerase epsilon catalytic subunit by caspase-3 and calpain specifically during apoptosis. 1105 15

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. Here we demonstrate that treatment of promonocytic U937 cells with arsenic trioxide leads to G2/M arrest which was associated with a dramatic increase in the levels of cyclin B and cyclin B-dependent kinase and apoptosis. We further show that apoptosis occurs after bcl-2 phosphorylation and caspase-3 activation followed by cleavage of PARP and PLC-gamma1 degradation and DNA fragmentation. The arsenic trioxide-induced apoptosis could be blocked by the protein synthesis inhibitor cycloheximide. In addition, pretreatment of U937 cells with the DNA polymerase inhibitor aphidicolin also blocked apoptosis, but did not cause the arrest of cells in the G2/M phase. The findings suggest that arsenic trioxide exerts its growth-inhibitory effects by modulating expression and/or activity of several key G2/M regulatory proteins. Furthermore, arsenic trioxide-mediated G2/M arrest correlates with the onset of apoptosis.
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PMID:Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells. 1152 58

Cells deficient in DNA polymerase beta (beta-pol) are impaired in base excision repair (BER) and hypersensitive to various DNA damaging agents, including methylating mutagens. Hypersensitivity of beta-pol-deficient cells to methylating agents is because of induction of apoptosis (Ochs et al., Cancer Res., 59: 1544-1551, 1999), indicating incompletely repaired DNA damage to trigger the response. Here we show that defective BER in beta-pol-null cells results in an early and transient increase in the frequency of DNA single-strand breaks on treatment with methyl methanesulfonate. These breaks arising as repair intermediates are not likely to trigger apoptosis directly because they were repaired efficiently and generated both in resting and proliferating cells, whereas only proliferating cells underwent with high frequency apoptosis after methylation. Therefore, we propose that single-strand breaks are converted into another kind of critical apoptosis-triggering lesion during replication. These critical secondary DNA lesions are likely to be non-repaired DNA double-strand breaks (DSBs), which are formed at higher frequency in beta-pol-null than in wild-type cells. Apoptosis was a late response not detectable before 24 h after methylation and was preceded by DSBs formation, extensive chromosomal breakage, and decline in Bcl-2 level and caspase-9 and caspase-3 activation. Caspase-8 was not significantly activated. Transfection of beta-pol-null cells with bcl-2 protected against methylation-induced apoptosis, indicating Bcl-2 to be causally involved. Overall, the data demonstrate that in cells lacking beta-pol, defective BER results in incompletely repaired DNA damage, which triggers apoptosis in a replication-dependent way by activating the mitochondrial death pathway. It is suggested that DSBs act as a critical ultimate apoptosis-inducing lesion.
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PMID:Deficiency in DNA polymerase beta provokes replication-dependent apoptosis via DNA breakage, Bcl-2 decline and caspase-3/9 activation. 1188 30

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase gamma-mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [(3)H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [(3)H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.
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PMID:Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver. 1293 98

The goal of this study was to examine the effect of ursolic acid, a pentacyclic triterpenoid compound, on growth of the endometrial cancer cell line SNG-II. We found that ursolic acid strongly inhibited the growth of SNG-II cells in a dose- and time-dependent manner. Morpholgical changes characteristic of apoptosis were observed in treated cells, such as the presence of apoptotic bodies and fragmentation of DNA into oligonucleosomal-sized fragments. We also investigated the active forms of caspase-3, -8 and -9 in ursolic acid-treated SNG-II cells. At 25 and 50 microM strength, ursolic acid induced marked increases in caspase-3 activity to approximately 5-fold that of control cells. Levels of cleaved caspase-3 increased in a time- and dose-dependent manner. Activation of caspases also led to the cleavage of target proteins, such as PARP. Ursolic acid treatment also resulted in a cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Testing whether caspase-3 activation and DNA polymerase activity were inhibited by addition of Ac-DEDV-HCO during ursolic acid treatment showed that 50 microM Ac-DEDV-HCO inhibited caspase-3 activity in treated cells. Although DNA fragmentation was observed after ursolic acid treatment, DNA fragmentation did not occur in SNG II cells treated with both Ac-DEDV-HCO and ursolic acid. Because some researchers have suggested that mitochondrial pathways are involved in ursolic acid-induced apoptosis secondary to induction of mitochondrial cytochrome c release, we studied mitochondrial events in ursolic acid-induced apoptosis in these cell lines. After ursolic acid treatment, the anti-apoptotic Bcl-2 protein decreased and Bax expression was enhanced. Our results indicated that ursolic acid induced apoptotic processes in the endometrial cancer SNG-II cell line through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.
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PMID:Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer SNG-II cells. 1558 1

We studied the effect of ursolic acid, a pentacyclic triterpene acid, on the growth of poorly differentiated type endometrial cancer HEC108 cells in vitro. Ursolic acid strongly inhibited the growth of HEC108 cells in a dose- and time-dependent manner. Morphological changes characteristic of apoptosis were observed in ursolic acid-treated cells, such as the presence of apoptotic bodies and fragmentation of DNA to oligonucleosomal-sized fragments. Investigation of caspase activity in ursolic acid-treated HEC108 cells showed that exposure at 50, 75 or 100 microM induced marked increases in caspase-3 activity (after 24 h) to 5.00, 11.76 or 12.75 times that of control levels, while cleaved caspase-3 levels increased in dose-dependent manner after 24 h. Activation of caspase was shown to lead to the cleavage of target proteins such as PARP. Ursolic acid treatment also resulted in a cleavage of poly(ADP-ribose) polymerase in a dose-dependent manner. Testing whether caspase-3 activation and DNA polymerase activity were inhibited by the addition of Ac-DEDV-HOC during ursolic acid treatment showed that 50 microM Ac-DEDV-HOC inhibited caspase-3 activity in treated cells. A mitochondrial pathway has been suggested to be involved in ursolic acid-induced apoptosis because the treatment induces mitochondria cytochrome c release. Experimentally, we found that anti-apoptotic Bcl-2 protein levels decreased after ursolic acid treatment, while Bax expression increased. Our results indicated that ursolic acid induced apoptotic processes in these poorly differentiated endometrial cancer cells occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.
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PMID:Molecular mechanism of ursolic acid induced apoptosis in poorly differentiated endometrial cancer HEC108 cells. 1601 38

Modified bases, such as O6-methylguanines, are produced in cells exposed to alkylating agents and cause apoptosis. In human cells treated with N-methyl-N-nitrosourea, we detected a protein complex composed of MutSalpha, MutLalpha and PCNA on damaged DNA by immunoprecipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical crosslinking. Time course experiments revealed that MutSalpha, consisting of MSH2 and MSH6 proteins, and PCNA bind to DNA to form an initial complex, and MutLalpha, composed of MLH1 and PMS2, binds to the complex when the DNA is damaged. This sequential mode of binding was further confirmed by the findings that the association of PCNA-MutSalpha complex on chromatin was observed even in the cells that lack MLH1, whereas in the absence of MSH2 no association of MutLalpha with the chromatin was achieved. Moreover, reduction in the PCNA content by small-interfering RNA or inhibition of DNA replication by aphidicolin, an inhibitor of DNA polymerase, significantly reduced the levels of the PCNA-MutSalpha-MutLalpha complex and also suppressed an increase in the caspase-3 activity, a hallmark for the induction of apoptosis. These observations imply that the induction of apoptosis is coupled with the progression of DNA replication through the action of PCNA.
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PMID:PCNA-MutSalpha-mediated binding of MutLalpha to replicative DNA with mismatched bases to induce apoptosis in human cells. 1620 60


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