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)

To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.
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PMID:DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes. 900 Jan 72

A mitochondrial DNA topoisomerase (type I, ATP-independent) can be biochemically distinguished from the nuclear enzyme DNA topoisomerase I. This conclusion is based on the subcellular localization of the mitochondrial enzyme, its optimal reaction conditions and sensitivity to enzyme inhibitors. Unlike its nuclear counterpart, the mitochondrial DNA topoisomerase exhibits an absolute requirement for a divalent cation (Mg2+ and Ca2+ work equally well in vitro). In addition, it is slightly more sensitive to monovalent salts, with optimal activity obtained in 50-100 mM KCl. The mitochondrial enzyme is equally active at pH 7.5 or pH 9.5, but unlike its nuclear equivalent, is inactivated at higher pH values. The mitochondrial DNA topoisomerase is sensitive to coumermycin, berenil, camptothecin and 2,2,5,5-tetramethyl-4-imidazolidinone, a chemical that has no inhibitory effect on DNA topoisomerase I. Immunoblotting studies show that mitochondrial DNA topoisomerase activity is associated with a polypeptide (M(r) approximately 79,000) that cross-reacts with the antiserum against DNA topoisomerase I. Thus, the mitochondrial DNA topoisomerase may be derived by the differential expression of the DNA topoisomerase I gene or from the expression of a gene that is homologous to the DNA topoisomerase I gene.
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PMID:Mitochondrial DNA topoisomerase I of Saccharomyces cerevisiae. 931 Jan 83

The subcellular compartmentalization of ions is perturbed during the process of apoptosis. In this work, we investigated the impact of K+ on the apoptotic process in thymocytes and T cell hybridoma cells. Irrespective of the death-inducing stimulus (glucocorticoids, topoisomerase inhibition, or Fas-crosslinking), a significant K+ outflow was observed during apoptosis, as determined on the single-cell level by means of the K+-sensitive fluorochrome, benzofuran isophtalate. This loss of cytosolic K+ only occurs in cells that have completely disrupted their inner mitochondrial transmembrane potential. Inhibition of this mitochondrial transmembrane potential loss by Bcl-2 or by specific inhibitors acting on the mitochondrial permeability transition pore (bongkrekic acid, cyclosporin A) prevents K+ leakage. K+ drops at the same stage at which cells expose phosphatidylserine residues on the outer leaflet of the membrane and reduce the levels of nonoxidized glutathione, but before they hyperproduce reactive oxygen species, undergo massive Ca2+ influx, shrink, and lyse. In a cell-free system of apoptosis, isolated nuclei exposed to the supernatant of mitochondria that have undergone permeability transition only manifest chromatinolysis when the K+ concentration is lowered from physiologic to apoptotic levels. Accordingly, massive DNA fragmentation causing subdiploidy is confined to cells that have undergone K+ leakage. Together, these data point to the step-wise acquisition of membrane dysfunction in apoptosis and indicate an important role for the disruption of normal K+ homeostasis in apoptotic degradation. Derepression of endonucleases due to low K+ concentrations may be a decisive prerequisite for end-stage DNA fragmentation.
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PMID:Potassium leakage during the apoptotic degradation phase. 960 66

Tumor cell resistance to inhibitors of topoisomerase II (topo II) is associated frequently with the overexpression of P-glycoprotein (PGP), and strategies to overcome resistance are focused on restoring defects in drug accumulation. Inhibitors of calcium-calmodulin-dependent enzymes sensitize resistant tumor cells to the topo II poison etoposide (VP-16) by enhancing DNA damage and an apoptotic response. In the present study, we have investigated the consequences of buffering intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra(acetoxy-methyl) ester (BAPTA-AM) on the sensitizing effects of the calmodulin-dependent protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-piperazine (KN-62) in etoposide-resistant human leukemia HL-60 (HL-60/ADR0.05) cells. In cells pretreated with 20 microM BAPTA-AM for 2 hr, extracellular ATP failed to trigger intracellular calcium transients, and no effects on the accumulation of VP-16 were apparent. Also, the effect of KN-62 in significantly (P=0.002 to 0.042) enhancing the accumulation of VP-16 in HL-60/ADR0.05 cells was unaffected due to pretreatment with BAPTA-AM. In contrast, pretreatment with BAPTA-AM reduced the DNA damage induced by VP-16, and significantly (P=0.038) reversed the enhancement by KN-62 of VP-16-stabilized topo II-mediated DNA cleavable complex formation. The pretreatment of HL-60/ADR0.05 cells with BAPTA-AM was also associated with the hypophosphorylation of topo IIalpha. Consistent with the ability of BAPTA-AM to circumvent the potentiation by KN-62 of VP-16-induced DNA damage, survival of cells treated with 40 microM VP-16 in the absence of KN-62 and 10 microM VP-16 in the presence of KN-62 was significantly (P=0.026 to 0.031) higher due to BAPTA-AM pretreatment. Results demonstrate that intracellular calcium transients could play a key role in the sensitization of etoposide-resistant tumor cells by inhibitors of calcium-calmodulin-dependent enzymes.
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PMID:Tumor cell resistance to topoisomerase II poisons: role for intracellular free calcium in the sensitization by inhibitors or calcium-calmodulin-dependent enzymes. 974 72

The amine-carboxyboranes were shown to be synergistic with tumor necrosis factor alpha (TNF alpha) in cytotoxicity and inhibition of DNA synthesis in select types of cancer cells depending on the presence of a TNF alpha high affinity receptor on the membrane of the cell. Initially both TNF alpha and the amine-carboxyboranes reduce the influx of calcium but later cause a significant increase intracellularly. This influx is not linked with the amine-carboxyborane activating the calcitonin receptor in the tumor cells. Neither the agents nor TNF alpha directly inhibits DNA topoisomerase II activity but both did cause decreased phosphorylation of the enzyme by protein kinase C (PKC). The two agents caused synergistic inhibition. This event correlated with increased DNA protein linked breaks, DNA fragmentation and cell death. These protein linked breaks are additive with etoposide's effects but the latter agent's mechanism is different than phosphorylation of topoisomerase II. There was no evidence that the DNA fragmentation was caused by a calcium induced endonuclease enzyme in these cancer cells. The low-molecular weight amine-carboxyboranes appear to play an identical function as TNF alpha in its role to cause DNA breaks and fragmentation to cause apoptosis.
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PMID:Relationship between amine-carboxyboranes and TNF alpha for the regulation of cell growth in different tumor cell lines. 975 12

A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.
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PMID:Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions. 1006 92

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.
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PMID:Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix. 1032 34

The DNA sequence selectivity of topoisomerase II (top2)-DNA cleavage complexes was examined for the human (top2alpha), yeast, and Escherichia coli (i.e. gyrase) enzymes in the absence or presence of anticancer or antibacterial drugs. Species-specific differences were observed for calcium-promoted DNA cleavage. Similarities and differences in DNA cleavage patterns and nucleic acid sequence preferences were also observed between the human, yeast, and E. coli top2 enzymes in the presence of the non-intercalators fluoroquinolone CP-115,953, etoposide, and azatoxin and the intercalators amsacrine and mitoxantrone. Additional base preferences were generally observed for the yeast when compared with the human top2alpha enzyme. Preferences in the immediate flanks of the top2-mediated DNA cleavage sites are, however, consistent with the drug stacking model for both enzymes. We also analyzed and compared homologous mutations in yeast and human top2, i.e. Ser(740) --> Trp and Ser(763) --> Trp, respectively. Both mutations decreased the reversibility of the etoposide-stabilized cleavage sites and produced consistent base sequence preference changes. These data demonstrate similarities and differences between human and yeast top2 enzymes. They also indicate that the structure of the enzyme/DNA interface plays a key role in determining the specificity of top2 poisons and cleavage sites for both the intercalating and non-intercalating drugs.
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PMID:Molecular analysis of yeast and human type II topoisomerases. Enzyme-DNA and drug interactions. 1049 80

Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.
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PMID:A new function for CD38/ADP-ribosyl cyclase in nuclear Ca2+ homeostasis. 1055 84

Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3-0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed.
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PMID:Relation between intracellular acidification and camptothecin-induced apoptosis in leukemia cells. 1072 78


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