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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.
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PMID:Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay. 164 64

Apoptosis is now recognized as one of the major processes regulating the size of cell populations. However, despite intensive investigations the biochemical and enzymological mechanisms involved in apoptosis remain unclear. In the present study we demonstrate activation of a Ca2+/Mg(2+)-dependent endonuclease during VP-16-induced apoptosis in MCF7 cells. Nuclease activation occurred prior to the appearance of internucleosomal DNA fragmentation, suggesting that this activation may be an early and possibly critical step in drug-induced apoptosis. Analysis of the internucleosomal DNA fragments showed that they contained phosphorylated 5'-ends, indicating that they were produced by a Ca2+/Mg(2+)-dependent endonuclease.
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PMID:Ca2+/Mg(2+)-dependent endonuclease activation is an early event in VP-16-induced apoptosis of human breast cancer MCF7 cells in vitro. 774 78

The mechanism by which etoposide, a topoisomerase II inhibitor, killed replicating mouse L929 fibroblasts was investigated. Etoposide at 10 microM killed 70% of the cells within 4 days, a result that was accompanied by DNA fragmentation. A characteristic "ladder" pattern of DNA fragmentation was confirmed by agarose gel electrophoresis. Simultaneous exposure of the cells to 10 microM etoposide plus 1 microM cycloheximide reduced both the extent of cell killing and the fragmentation of DNA. Delayed addition of cycloheximide protected cells only if cycloheximide was added 1-6 hr after exposure to etoposide. When added 6-24 hr after treatment with etoposide, cycloheximide lost the ability to protect cells. Cell growth was completely inhibited by either etoposide or cycloheximide. Furthermore, DNA synthesis was inhibited by either etoposide or cycloheximide within 6 hr. Protein synthesis, however, was not inhibited by etoposide. Thus, the ability of cycloheximide to protect cells correlated with inhibition of protein synthesis, rather than inhibition of DNA synthesis. A 1-hr exposure to 2.5 mM N-methyl-N-nitrosourea similarly inhibited DNA synthesis within 6 hr. without affecting protein synthesis. However, no loss of viability accompanied N-methyl-N-nitrosourea treatment. Thus, an imbalance between protein synthesis and DNA synthesis cannot explain the cell killing by etoposide. H-7, a protein kinase C inhibitor, prevented the cell killing and DNA fragmentation, whereas aurintricarboxylic acid, an endonuclease inhibitor, reduced the extent of DNA fragmentation but did not have an effect on cell killing. The data document that the killing of replicating mouse fibroblasts by etoposide represents an example of programmed cell death (apoptosis) that depends on protein synthesis. Although protein synthesis is required during the first 24 hr of exposure to etoposide, cell death is delayed until several days later.
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PMID:Programmed cell death (apoptosis) of mouse fibroblasts is induced by the topoisomerase II inhibitor etoposide. 796 76

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.
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PMID:Changes in nuclear chromatin precede internucleosomal DNA cleavage in the induction of apoptosis by etoposide. 830 63

The DNA fragmentation, a parameter of apoptosis, in non-small (NSCLC) and small (SCLC) cell lung cancer cell lines (N231 and PC-9) was evaluated. The DNA fragmentation in SCLC lines, but not in NSCLC lines, was observed in overgrown cells without exposure to anticancer drugs. In etoposide (VP-16)-treated N231 but not PC-9 cells, DNA fragmentation continued to increase up to 42 h, and the increase was dependent on the concentration of VP-16. The endonuclease activity of VP-16-treated N231, but not PC-9, cells required both Ca2+ and Mg2+ for full activity. It was elevated in a time- and concentration-dependent manner. As this activity was not affected by addition of cycloheximide, the activation of the endonuclease activity without protein synthesis may be involved in VP-16-induced cytotoxicity in N231.
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PMID:Apoptosis induced by etoposide in small-cell lung cancer cell lines. 830 12

The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.
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PMID:Topoisomerase inhibitors induce apoptosis in thymocytes. 838 Mar 39

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.
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PMID:Activation of poly(ADP-ribose)polymerase in apoptotic human cells. 852 93

The multifunctional DNA repair enzyme (APEX nuclease) having apurinic/apyrimidinic (AP) endonuclease, 3'-5' exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities is thought to be involved in repair of AP sites and single-strand breaks with 3'-blocked termini. To investigate the biological role of the enzyme, we studied the correlation between APEX AP endonuclease activity in several human glioma cell lines having various degree of its expression and cellular susceptibility to cytotoxic agents such as methyl methanesulfonate (MMS), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), cis-diamminedichloroplatinum(II) (CDDP), etoposide (VP-16), hydrogen peroxide (H2O2), hyperthermia and X-ray. The cell lines having lower APEX expression showed higher sensitivity to MMS and H2O2 which are known to induce AP sites and single strand breaks on DNA, respectively. The cellular susceptibility to the other agents tested was not significantly correlated to the APEX expression. The present results are thought to support the notion that APEX nuclease plays an important role on repair of AP sites and single-strand DNA breaks with 3'-blocked termini in mammalian cells.
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PMID:Relationship between expression of a major apurinic/apyrimidinic endonuclease (APEX nuclease) and susceptibility to genotoxic agents in human glioma cell lines. 859 68

Higher order DNA fragmentation may be an essential signal in apoptosis. We found that etoposide (VP-16) induced apoptosis in human DU-145 prostatic carcinoma cells in a time- and concentration-dependent manner. Chromatin condensation was morphologically evident only when cells detached from the monolayer; untreated or VP-16-treated attached cells retained a normal morphology. We describe a radiolabeled alu-I sequence-based quantitative field inversion gel electrophoresis (QFIGE) method that permitted observation and quantification of discrete high molecular weight DNA fragments in detached (apoptotic) and attached (preapoptotic) DU-145 cells. The DNA fragments generated during the apoptotic death of these cells were > or = 1 (mega-base pairs) mbp, 450-600 (kilo-base pairs) kbp, and 30-50 kbp; we observed that these DNA fragments increased 9 +/- 2-, 8 +/- 2-, and 25 +/- 11-fold versus control, respectively, with a 24-hr exposure to 30 microM VP-16 in attached cell populations. In detached VP-16-treated cells, there was accrual of 30-50-kbp DNA fragments with a concomitant loss of the > or = 1-mbp and 450-600-kbp fragments; internucleosomal DNA cleavage was never observed. This pattern of high molecular weight DNA fragmentation was inhibited by cycloheximide treatment and was common to other apoptotic agents, including melphalan and bleomycin. These findings suggest that the > or = 1-mbp and 450-600-kbp DNA fragments are products of endonuclease activation and are not topoisomerase II/DNA interactions. Finally, the generation of the 30-50-kbp DNA fragments may mediate chromatin condensation, which characterizes apoptosis.
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PMID:Genesis of discrete higher order DNA fragments in apoptotic human prostatic carcinoma cells. 863 56

A single amino acid change transforms restriction enzyme NaeI to a topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI-L43K, implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K. This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.
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PMID:Changing a leucine to a lysine residue makes NaeI endonuclease hypersensitive to DNA intercalative drugs. 875 63


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