Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytotoxic effect of the 9-azaellipticine derivative pazelliptine in combination with gamma-ray irradiation was investigated using Chinese hamster V-79 cells in culture. gamma-ray irradiation and drug treatment (1-h drug exposure) were applied at 1-h intervals for partial DNA damage recovery in growth medium. Isobologram analysis of the clonogenic potential gave evidence of supraadditive interaction in the radiation----drug sequence with 10% survival as an endpoint. No synergistic potentiation was observed at higher survival or as pazelliptine was applied first. Pazelliptine abolished the low-dose shoulder characteristic of asynchronous cell response to gamma-rays. Although rejoining of radiation-induced DNA strand breaks was completed at the time of drug exposure, pazelliptine brought about a larger amount of DNA strand breaks in preirradiated than in nonirradiated cells. The time and dose dependencies of DNA strand break formation and repair with radiation and/or pazelliptine were analyzed by neutral and alkaline filter elution. Pazelliptine in the micromolar range showed the same pattern of double-stranded cleavable complex formation as expected of a DNA topoisomerase II-targeting agent. At a low concentration of pazelliptine, however, protein-concealed breaks were mostly in the form of single-stranded adducts. Such single-stranded complexes have been reported to occur with some topoisomerase II-targeting drugs; their properties are also reminiscent of those induced by the topoisomerase I poison, camptothecin. It is proposed that topoisomerase poisoning interacts with the repair of radiation-induced lesions.
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PMID:Additive and supraadditive interaction between ionizing radiation and pazelliptine, a DNA topoisomerase inhibitor, in Chinese hamster V-79 fibroblasts. 164 14

The conventional laboratory approach to study the mechanisms of drug resistance has been the selection of drug-resistant cell lines by continuous exposure to cytotoxic agents. Such lines, which are selected for resistance to a single agent, frequently display cross-resistance to a number of cytotoxic agents that are unrelated in both structure and proposed mechanism of action. Multidrug-resistant cells display reduced drug accumulation, which is the result of overexpression of a surface glycoprotein (P170). Although resistance to multiple antitumor agents is a common clinical problem in the treatment of cancer, the precise role of the P-glycoprotein-mediated mechanism in human tumors remains to be established. Many alterations in multidrug-resistant cells selected in vitro have been identified. The concomitant expression of multiple phenotypic differences, which appear to be favored by continued and prolonged drug exposure, makes analysis of critical individual resistance pathways more difficult. However, multiple factors may also be involved in the development of clinical resistance. Recent studies have identified alterations in DNA topoisomerase II activity and function as an alternative mechanism that contributes to the multidrug-resistance phenomenon or is responsible for a different type of drug resistance. The precise nature of these changes remains unclear. Available evidence supports the view that expression of the enzyme is an important determinant of cell sensitivity to DNA topoisomerase poisons, but that other changes involved in regulation of enzyme function and/or in the cellular processing of drug-induced DNA damage may be critical in determining the differential pattern of cell response to antitumor agents.
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PMID:The role of topoisomerase II in drug resistance. 164 58

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.
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PMID:Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes. 164 93

To study the mechanism of illegitimate recombination in mammalian cells, we have developed a shuttle vector, pNK1, that contains three bacterial markers, amp (ApR), galK, and neo (KmR). The frequency of deletions occurring in autonomously replicating pNK1 DNA during the growth of monkey COS1 cells was measured by transfecting the plasmid into Escherichia coli cells and counting the number of galK- ApS double mutants among total KmR cells. This method allowed us to test the effects of topoisomerase inhibitors on deletion formation in mammalian cells. The DNA topoisomerase II (TopII) inhibitor, 4'-dimethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM26), stimulated deletions in pNK1 DNA in monkey cells. Since VM26 does not inhibit the strand-break activity of TopII, but rather stabilizes an enzyme-DNA complex in which DNA is cleaved upon treatment with sodium dodecyl sulfate, it is implicated that TopII participates in the deletion process in mammalian cells.
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PMID:A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency. 164 63

Exposure of promyelocytic leukemic HL-60 cells to 3-60 nM of the DNA topoisomerase I inhibitor camptothecin (CAM) or to 30-450 nM and 0.12-1.5 microM of DNA topoisomerase II inhibitors teniposide (TN) and 4-(9-acridynylamino)-3-methanesulfon-m-anisidide (m-AMSA), respectively, resulted in two distinct kinetic effects: (1) the cells entered S phase but the rate of DNA replication was reduced in proportion to the inhibitor concentration; (2) the transition from G2 to M was impaired, approximately 1 h after addition of the inhibitor. As a consequence, the cells accumulated in the S (preferentially in early S) and in G2 phases of the cell cycle. Whereas CAM was more efficient in suppressing cell progression through S phase, TN and m-AMSA were more potent G2 blockers. At these low inhibitor concentrations no signs of immediate cytotoxicity or DNA degradation were apparent. However, above 145 nM of CAM, 900 nM of TN, or 2 microM of m-AMSA extensive DNA degradation in nuclei of S phase cells was evident within 6 h of addition of the inhibitor, resulting in the loss of S and G2 + M cells from these cultures. The data indicate that depending on concentration, mechanisms mediating the cytostatic/cytotoxic activity of both DNA topoisomerase I and II inhibitors may be quite different. Suppression of the DNA replication and the G2 to M transition, seen at low inhibitor concentrations, is compatible with the assumption that the inhibitor-induced stabilization of the topoisomerase-DNA cleavable complexes interferes with DNA replication and chromosome condensation/segregation, respectively. Above the threshold concentration for each inhibitor, an endonucleolytic activity is triggered, resulting in rapid DNA degradation in nuclei of S and G2 phase cells. The endonucleolytic effect is not only cell cycle phase-specific but is also modulated by tissue-specific factors because it cannot be observed, e.g., in the lymphocytic leukemic cell lines.
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PMID:The concentration-dependent diversity of effects of DNA topoisomerase I and II inhibitors on the cell cycle of HL-60 cells. 164 59

We have isolated two etoposide (VP16)-resistant cell lines, KB/VP-1 and KB/VP-2, from human cancer KB cells after stepwise exposure to increasing doses of VP16. KB/VP-1 and KB/VP-2 showed 30- and 50-fold higher resistance to VP16 and also 20- and 30-fold higher resistance to teniposide than the parent cell line. Furthermore, both resistant cell lines showed more than 2-fold cross-resistance to Adriamycin and daunomycin than KB cells. The levels of accumulation and outward transport of radioactive VP16 were similar in KB/VP-1, KB/VP-2, and KB. The activity of nuclear extracts of DNA topoisomerase II for both KB/VP-1 and KB/VP-2 assayed by decatenation of kinetoplast DNA was consistently similar to that of KB. However, in both immunoblot assay with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA, cellular levels of topoisomerase II in both resistant cell lines were less than one-tenth the level in KB. The cellular levels of DNA topoisomerase I, however, were similar between the mutants and their parent. A quantitative precipitation assay of covalent DNA-topoisomerase II complexes showed greatly reduced VP16-induced cleavages of 3'-32P-DNA by nuclear extracts of KB/VP-1 or KB/VP-2 cells in comparison with KB cells. The relative specific phosphorylation of DNA topoisomerase II was about 14- to 18-fold higher in the mutants than in the parental cells. Phosphoamino acid analysis of DNA topoisomerase II showed that serine was the phosphorylated amino acid in all three cell lines, KB, KB/VP-1, and KB/VP-2. These data suggest that reduced expression of DNA-topoisomerase II might account for the acquired VP16 resistance and reduced VP16-induced cleavages of DNA-topoisomerase II complexes in both VP16-resistant variants.
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PMID:Increased phosphorylation of DNA topoisomerase II in etoposide-resistant mutants of human cancer KB cells. 164 96

New details of the molecular interactions of quinolones with their target DNA gyrase and DNA have come from the nucleotide sequences of the gyrA genes from resistant mutants of Escherichia coli and wild-type strains of other bacteria and studies of gyrase A tryptic fragments, all suggesting the importance of an amino-terminal domain in quinolone action. Alterations in DNA supertwisting were also associated with altered quinolone susceptibility, possibly by indirect effects on DNA gyrase expression. Specific binding of relevant concentrations of norfloxacin to a complex of DNA gyrase and DNA in the presence of ATP, the cooperativity of DNA binding, and the crystalline structure of nalidixic acid have led to a model in which quinolones bind cooperatively to a pocket of single-strand DNA created by DNA gyrase. Quinolones vary in their relative activity against DNA gyrase and its eukaryotic homolog topoisomerase II, and in some assays increased action against the eukaryotic enzyme was associated with genotoxicity. Inhibition of bacterial DNA synthesis by quinolones may correlate with MICs in some species, but comparisons of drug accumulation and inhibition of DNA synthesis in permeabilized cells among species have been difficult to interpret. The specific factors necessary for bacterial killing by quinolones in addition to interaction with DNA gyrase have remained elusive, but include oxygen and new protein synthesis. The coordinate expression of the SOS proteins appears not to be necessary for quinolone lethality. Two independent mutants with selective reduced killing by quinolones and beta-lactams indicate overlap in the pathways of bactericidal activity of these classes of agents with distinct targets.
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PMID:Mode of action of the new quinolones: new data. 165 Jun 98

CPT-11, a derivative of camptothecin, has drawn attention to cancer chemotherapy because of the specific mode of action, and the clinical study is now under progress. Liu et al. proved that camptothecin was a DNA topoisomerase I inhibitor, and some kinds of antitumor agents have been recognized as DNA topoisomerase II inhibitors. Based on these findings, DNA topoisomerases have emerged as target enzymes of antitumor agents in cancer chemotherapy. This paper dealt with investigation on the cytotoxic effects induced by combined use of DNA topoisomerase targeting antitumor agents, especially using CPT-11 as a core antitumor agent. Synchronous administration of CPT-11 with other antitumor agents induced cytotoxic effects less than metachronous administration of CPT-11 with other antitumor agents, especially preceding use of CPT-11. Dose of antitumor agents was not necessarily correlated to the cytotoxic effects. In some instances, small doses of the agents showed better therapeutic effects than large doses. The cytotoxic effects of vincristine, vindesine, and hydroxyurea were reduced by combination with CPT-11. On the other hand, non-cytotoxic agents such as aphidicolin, novobiocin, propentofylline, pentoxifylline, norfloxacin, and tosufloxacin enhanced the cytotoxic effects of CPT-11. Hypothetical consideration of cell killing and acquisition of drug resistance was proposed.
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PMID:[Combination cancer chemotherapy using a DNA topoisomerase inhibitor CPT-11, as a core agent--the in vitro evaluation]. 165 82

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta,gamma-imido]triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by Walker et al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G----A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.
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PMID:Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. 165 58

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.
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PMID:A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus. 165 24


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