Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.
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PMID:Negative supercoiling of DNA by eukaryotic DNA topoisomerase II and dextran sulfate. 165 Jul 78

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

DNA topoisomerases interconvert various topological isomers of DNA and play key roles in replication and gene expression. The possible involvement of the 2',5'-oligoadenylates (2-5A) system in cell growth, regulation, and cell differentiation led us to investigate the effects of 2-5A on mammalian topoisomerases. We found that the calf thymus type I topoisomerase was inhibited by a variety of 2-5A compounds. The level of inhibition was dependent upon the number of residues and the degree of phosphorylation at the 5' terminus. The 5'-triphosphorylated 2',5' hexamer, ppp(Ap)5A, was the most effective, strongly reducing relaxation at less than micromolar concentrations. These results raise the possibility that physiological concentrations of 2-5A of sufficient chain length may be capable of regulating gene expression by virtue of a direct inhibition of DNA topoisomerase I.
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PMID:2',5'-oligoadenylates inhibit relaxation of supercoiled DNA by calf thymus DNA topoisomerase I. 165 15

An etoposide-resistant K562 cell line (K/eto) was obtained by stepwise exposure, in culture, to increasing concentrations of etoposide, without the use of mutagens. This cell line was resistant to etoposide, and slightly resistant to adriamycin, but sensitive to anti-cancer drugs such as camptothecin, vincristine, actinomycin D and so on. P-Glycoprotein, the mdr1 gene product, was not detected in this cell line, as assessed by immunocytochemistry, immunoprecipitation and flow cytometry. Overexpression of mdr1 mRNA was also not found. Interestingly, expression of 85 kD protein recognized by MRK 20 monoclonal antibody was noted. The level of DNA topoisomerase II protein, detected by antibody staining, decreased concomitantly with a general decrease in DNA topoisomerase II unknotting activity, while DNA topoisomerase I activity was not affected. Cellular accumulation of [3H]etoposide was reduced by 75% in the resistant line compared with parental K562. Karyotype analysis showed that the number of chromosomes in K/eto was 55 and neither a homogeneous staining region nor double-minute chromosomes were detected. These results indicate that this resistance is not due to an altered interaction between the drug and cellular transport machinery, i.e. MDR1, associated with the "classic" multiple drug resistance phenotype, but rather is due to the existence of other mechanism(s) of resistance, decreased transport of the drug and decreased target enzyme, DNA topoisomerase II.
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PMID:Characterization of an etoposide-resistant human K562 cell line, K/eto. 165 46

In order to investigate the mechanism of topoisomerase I inhibition by camptothecin, we studied the induction of DNA cleavage by purified mammalian DNA topoisomerase I in a series of oligonucleotides and analyzed the DNA sequence locations of preferred cleavage sites in the SV40 genome. The oligonucleotides were derived from the sequence of the major camptothecin-induced cleavage site in SV40 DNA (Jaxel, C., Kohn, K. W., and Pommier, Y. (1988) Nucleic Acids Res. 16, 11157 to 11170) with the cleaved bond in their center. DNA length was critical since cleavage was detectable only in 30 and 20 base pair-(bp) oligonucleotides, but not in a 12-bp oligonucleotide. Cleavage was at the same position in the oligonucleotides as in SV40 DNA. Its intensity was greater in the 30- than in the 20-bp oligonucleotide, indicating that sequences more than 10 bp away from the cleavage site may influence intensity. Camptothecin-induced DNA cleavage required duplex DNA since none of the single-stranded oligonucleotides were cleaved. Analysis of base preferences around topoisomerase I cleavage sites in SV40 DNA indicated that camptothecin stabilized topoisomerase I preferentially at sites having a G immediately 3' to the cleaved bond. Experiments with 30-bp oligonucleotides showed that camptothecin produced most intense cleavage in a complementary duplex having a G immediately 3' to the cleavage site. Weaker cleavage was observed in a complementary duplex in which the 3'G was replaced with a T. The identity of the 3' base, however, did not affect topoisomerase I-induced DNA cleavage in the absence of drug. These results indicate that camptothecin traps preferentially a subset of the enzyme cleavage sites, those having a G immediately 3' to the cleaved bond. This strong preference suggests that camptothecin binds reversibly to the DNA at topoisomerase I cleavage sites, in analogy to a model previously proposed for inhibitors of topoisomerase II (Capranico, G., Kohn, K.W., and Pommier, Y. (1990) Nucleic Acids Res. 18, 6611-6619).
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PMID:Effect of local DNA sequence on topoisomerase I cleavage in the presence or absence of camptothecin. 165 24

Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi. As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se. The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA. Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage. To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced. In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners. Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage. Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision. The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.
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PMID:Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. 165 96

DNA topoisomerase II was isolated from mouse leukemia L1210 cells and the activity was determined by using P4 phage knotted DNA and pBR 322 DNA as the substrates. Based on these results, a method for screening antitumor agents by using DNA topoisomerase II as a target was established. The experiments showed that DNA topoisomerase II catalyzed pBR 322 DNA breaking and relaxing which were reversible and dependent on ATP. The activity was increased 2-4 times in the presence of ATP 1 mmol.L-1. In contrast with type II enzyme, the activity of DNA topoisomerase I was completely inhibited in the presence of ATP 1 mmol.L-1 and had full activity in the absence of ATP. Type II enzyme also showed the unknotting activity by using p4 phage knotted DNA as a substrate. DNA cleavage and relaxing reaction induced by type II enzyme increased 5-fold in the presence of Doxorubicin (Dox) 1 microgram.ml-1 or daunorubicin (Dau). Etoposide (Eto) and aclarubicin B (Acl B) also stimulated the reaction at 100 micrograms.ml-1. The cleavage reaction resulted from topoisomerase II was inhibited by other agents, such as frankincense extracts, terpenic compounds (BC series).
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PMID:Determination of DNA topoisomerase II activity from L1210 cells--a target for screening antitumor agents. 166 90

The activity of DNA topoisomerase I present in the nuclear extract of yeast, Saccharomyces cerevisiae, was inhibited by additions of NAD, the substrate of poly (ADP-ribose) polymerase. This NAD-inhibited topoisomerase activity was restored to the normal level in a dose-dependent manner by adding 3-aminobenzamide (3-AB), an inhibitor of the polymerase. The 3-AB sensitive polymerase enzyme activity, as determined by the rate of incorporation of the radiolabelled NAD in permeabilized cells, increased by treatment of cells with methyl methanesulfonate (MMS) in a dose-dependent manner. While the additions of MMS increased the polymerase activity, it has caused a decrease in cell survival. However, this cell killing activity of MMS was markedly potentiated by adding benzamide, another inhibitor of polymerase. Thus, these results suggest that the mode of modification of nuclear proteins by altering the poly(ADP-ribosylation) in S. cerevisiae resembles with those observed in mammalian cells.
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PMID:Inhibition of topoisomerase I by NAD and enhancement of cytotoxicity of MMS by inhibitors of poly(ADP-ribose) polymerase in Saccharomyces cerevisiae. 166 35

Cleavage of linear duplex DNA by purified vaccinia virus DNA topoisomerase I occurs at a conserved sequence element (5'-C/T)CCTT decreases) in the incised DNA strand. Oligonucleotides spanning the high affinity cleavage site CCCTT at nucleotide 2457 in pUC19 DNA are cleaved efficiently in vitro, but only when hybridized to a complementary DNA molecule. As few as 6 nucleotides proximal to the cleavage site and 6 nucleotides downstream of the site are sufficient to support exclusive cleavage at the high affinity site (position +1). Single nucleotide substitutions within the consensus pentamer have deleterious effects on the equilibria of the topoisomerase binding and DNA cleavage reactions. The effects of base mismatch within the pentamer are more dramatic than are the effects of mutations that preserve base complementarity. Competition experiments indicate that topoisomerase binds preferentially to DNA sites containing the wild-type pentamer element. Single-stranded DNA containing the sequence CCCTT in the cleaved stand is a more effective competitor than is single-stranded DNA containing the complementary sequence in the noncleaved strand.
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PMID:Site-specific DNA cleavage by vaccinia virus DNA topoisomerase I. Role of nucleotide sequence and DNA secondary structure. 184 64


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