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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A common strongly ordered multi-step-pattern of endogenous DNA degradation was induced in rat liver nuclei and intact thymocytes, prepared in the presence of chelating agents and incubated in the presence of CaCl2 and/or MgCl2. It consisted of sequential generation of 0.3 Mbp, then 0.05 Mbp DNA fragments and finally of oligo- and mononucleosomal DNA. Oligonucleosomal DNA was generated when the genome had already been disintegrated into 0.05 Mbp DNA fragments. ZnCl2 completely inhibited advanced genome cleavage to oligo- and mononucleosomal DNA without affecting the initial generation of large DNA fragments. Therefore, the endonucleolytic activity which produce large DNA fragments is different from Ca2+/Mg2+ endonuclease. The similar pattern of DNA degradation was observed in thymocytes treated with dexamethasone and with the topoisomerase II inhibitor VM-26, the agents known to induce apoptosis. The effect of VM-26 strongly suggests the involvement of topoisomerase II in generation of large DNA fragments. Multi-level organization and regulation of the chromatin structure determine the stepwise process of genome degradation. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation thus leading to cell death in terminal differentiation or stress-induced apoptosis.
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PMID:Comparative study of induction of endogenous DNA degradation in rat liver nuclei and intact thymocytes. 774 35

Aurintricarboxylic acid (ATA) is a polyanionic, polyaromatic compound which has been shown to inhibit apoptotic cell death in various cell types induced by a variety of factors. Since ATA is known to be a general inhibitor of nuclease activities in vitro (ID50S ranging from 2 to 50 microM), the in vivo effects are usually attributed to inhibition of endogenous endonuclease activities. We show herein that ATA is a potent inhibitor of the nuclear enzyme DNA topoisomerase II. ATA inhibits the catalytic activity of purified yeast topoisomerase II with an ID50 of approx. 75nM as measured by relaxation assays. ATA does not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex") as do other inhibitors of this enzyme such as 4'-(9-acridinylamino)-methane sulfon-m-anisidide (amsacrime), 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-gluco pyr anoside) (etoposide) and ellipticines. In contrast, cleavable complex formation induced by amsacrine and etoposide is trongly inhibited in the presence of ATA. ATA also prevents the binding of topoisomerase II to DNA and inhibits topoisomerase II-catalysed ATP hydrolysis. The ability of ATA to interfere with more than one step in t he catalytic cycle of DNA topoisomerase II may explain its unusual potency as an inhibitor of this enzyme. ATA reduces the number of amsacrine-induced DNA-protein complexes in intact DC-3F Chinese hamster fibrosarcoma cells and protects these cells from the cytotoxic action of amsacrine. The effects of ATA on DNA-protein complex formation in living cells appear to be due to the direct interaction of the drug with topoisomerase II, since similar results are found when nuclei from untreated DC-3F cells are exposed to amsacrine after a short preincubation with ATA. Cells resistant to 9-hydroxyellipticine, which have been shown to possess altered topoisomerase II activity, are approx. 5-fold more resistant to ATA than the sensitive parental cells as shown by colony formation essays. We conclude that ATA is a potent inhibitor of topoisomerase II and that the drug interacts with topoisomerase II in living cells. Our findings raise the possibility that the protective effects of ATA towards apoptotic cell death might, at least in part, involve DNA topoisomerase II.
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PMID:Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells. 785 17

Nae I endonuclease must bind to two DNA sequences for cleavage. Examination of the amino acid sequence of Nae I uncovered similarity to the active site of human DNA ligase I, except for leucine 43 in Nae I instead of the lysine essential for ligase activity. Changing leucine 43 to lysine 43 (L43K) changed Nae I activity: Nae I-L43K relaxed supercoiled DNA to yield DNA topoisomers and recombined DNA to give dimeric molecules. Interruption of the reactions of Nae I and Nae I-L43K with DNA demonstrated transient protein-DNA covalent complexes. These findings imply coupled endonuclease and ligase domains and link Nae I endonuclease to the topoisomerase and recombinase protein families.
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PMID:DNA topoisomerase and recombinase activities in Nae I restriction endonuclease. 789 5

DNA topoisomerase I isolated from the lower eukaryote Neurospora crassa mitochondria was characterized. Molar mass of the enzyme in the native state is 120 kDa and 60-65 kDa when denatured. The pH optimum of the enzyme is 7.8 and the KCl optimum concentration is 40 mmol/L. This topoisomerase is independent of ATP and Mg2+. N-Ethylmaleimide, 4-chloromercuribenzoate, SDS, guanidinium chloride, polyethylene glycol, heparin and ethidium bromide inhibit its activity, while novobiocin, nalidixic acid, Triton X-100 and chloroquine do not. Polyamines and histone H1 stimulate the topoisomerase activity. We classify this DNA topoisomerase as type I and eukaryotic. Conversion of the topoisomerase to a nonspecific endonuclease at increased temperature is proposed.
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PMID:Characterization of mitochondrial DNA topoisomerase I from Neurospora crassa. 795 26

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

DNA topoisomerase I inhibitor camptothecin (CAM), topoisomerase II inhibitors teniposide (TN) and amsacrine (m-AMSA) induce apoptosis of HL-60 cells. One of the early events of apoptosis is DNA degradation, which occurs as a result of activation of the specific endonuclease. DNA strand breaks generated during this process were revealed, in the present study, by the in situ nick translation assay which was adapted to flow cytometry. In this assay, the incorporation of biotinylated dUTP by apoptotic cells was detected by the use of fluorescinated avidin, whereas simultaneous staining of DNA with propidium iodide made it possible to correlate the appearance of DNA strand breaks with cell position in the cell cycle. The breaks were detected as early as 90 min after the initial cell contact with CAM, and they were limited to cells in the S phase of the cell cycle. At that early stage of apoptosis DNA was not yet extractable from the cells; the loss of DNA from S-phase cells could not be seen, by flow cytometry, during the initial 2 h of incubation with CAM. DNA strand breaks induced by TN and m-AMSA also occurred preferentially in S-phase cells. The data indicate that DNA strand breaks resulting from activation of endonuclease in HL-60 cells treated with DNA topoisomerase I or II inhibitors can be conveniently measured using the in situ nick translation assay. This assay has certain advantages over other methods of identification of apoptotic cells by flow cytometry, such as providing direct evidence of DNA damage and offering the opportunity to correlate DNA damage with cell position in the cell cycle. The method may be of interest in clinical oncology where testing tumor response (by DNA degradation) to DNA topoisomerase inhibitors or other treatments may be of prognostic value.
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PMID:Apoptosis of S-phase HL-60 cells induced by DNA topoisomerase inhibitors: detection of DNA strand breaks by flow cytometry using the in situ nick translation assay. 838 87

Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both physiological stimuli and cytotoxic treatment with many anticancer agents. This digestion has generally been considered to be mediated by a Ca2+/Mg(2+)-dependent endonuclease that is activated by increases in intracellular Ca2+. However, we suggest that an alternate endonuclease, DNase II, may be a more likely candidate. In these studies, apoptosis was induced in human HL-60 cells by a 30-min incubation with the topoisomerase II inhibitor etoposide. DNA digestion characteristic of apoptosis began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells underwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progressed to the G2 phase and arrested there for at least 48 h. Intracellular Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of up to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on the basis of intracellular pH and only the acidic cells showed the morphology and DNA digestion characteristic of apoptosis. These results demonstrate the involvement of DNase II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis as regulators of apoptosis.
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PMID:Etoposide-induced apoptosis in human HL-60 cells is associated with intracellular acidification. 838 92

The effect of the topoisomerase II inhibitor doxorubicin and its non-cross-resistant analogue annamycin on DNA degradation and programmed cell death was examined in murine leukemia P388 cells. P388 parental cells exposed to various concentrations of doxorubicin and annamycin for 24 h displayed dose-dependent DNA cleavage: at 1 microM, both doxorubicin and annamycin were effective in inducing DNA breakdown, but at 10 microM, the effect was markedly decreased or totally absent. In multidrug-resistant P388/Dox cells, doxorubicin did not cause DNA cleavage, while 10 microM annamycin had a significant effect. By agarose gel analysis, drug-induced DNA fragmentation showed the characteristic pattern of internucleosomal ladder. Morphologically, P388 cells treated with 1 microM doxorubicin or annamycin for 24 h showed a reduction in cell volume and condensation of nuclear structures. Similar changes were observed in P388/Dox cells exposed to 10 microM annamycin for 24 h but not in cells exposed to 10 microM doxorubicin. Time course studies demonstrated that DNA fragmentation was detected 12 h after incubation with 1 microM doxorubicin or annamycin, while loss of membrane integrity appeared at 24 h, thus indicating that DNA degradation was a preceding event. DNA fragmentation caused by doxorubicin and annamycin was inhibited by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, and the endonuclease inhibitor aurintricarboxylic acid. Drug-induced cell death was partially prevented by cycloheximide and aurintricarboxylic acid, thus suggesting that the apoptotic process caused by these drugs requires gene expression, synthesis of new proteins, and activation of endogenous nucleases. In contrast, DNA cleavage was not affected by incubating cells with 1 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, thus indicating that intracellular calcium depletion does not affect anthracycline-induced apoptosis. The results obtained demonstrate that the cell killing effect of anthracyclines is mediated, at least in part, by the induction of apoptosis.
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PMID:Apoptosis induced by anthracycline antibiotics in P388 parent and multidrug-resistant cells. 846 4

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays. During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope. In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA. Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle. The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay. Camptothecin, teniposide, and 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle. In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle. The method of detection of DNA strand breaks (3'-hydroxyl termini) in individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker. The protein-associated DNA breaks in the "cleavable" DNA-topoisomerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods.
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PMID:Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. 846 13

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis.
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PMID:Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. 859 91


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