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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Used for centuries in traditional Chinese medicine, camptothecin was rediscovered in the 1950s during a search for compounds that could be used as a source for steroid synthesis. Due to its limited water solubility, a sodium salt was used in the early clinical trials. The severe toxicity and erratic absorption relegated this compound to the research laboratory until the 1980s when the topoisomerase enzyme was identified as the cellular target of camptothecin, the topoisomerase enzyme was found to be overexpressed in cancer cells and a structure-activity relationship was determined for camptothecin. These new developments brought the camptothecins back to the clinical setting for further testing. The various analogues that have been most studied to date include: irinotecan (CPT-11), and its derivative SN-38, topotecan, and 9-aminocamptothecin. Numerous trials have been conducted in an attempt to establish the efficacy in various tumour types, to determine the dose-limiting toxicity and to define the optimal schedule of administration. It seems that large doses of these drugs given on intermittent schedules are not effective. Our hypothesis is that the camptothecins require a prolonged schedule of administration given continuously at low doses or frequent intermittent dosing schedules to be most effective. With these schedules, normal haematopoietic cells and mucosal progenitor cells with low topoisomerase I levels may be spared, while efficacy is preserved.
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PMID:Camptothecins: a review of their development and schedules of administration. 989 20

DACA [N-[2-(dimethylamino)ethyl]acridine-4-carboxamide], an acridine derivative that is highly active against solid tumours in mice, is currently in clinical trial. The ability of DACA to overcome "atypical" (topoisomerase II-mediated) multidrug resistance has been hypothesised to stem from its dual topoisomerase I/II specificity. We investigated the topoisomerase specificity of DACA and its 7-chloro derivative (C1-DACA) using camptothecin and amsacrine as control compounds. In cell-free assays employing supercoiled plasmid DNA, C1-DACA at 5 microM induced topoisomerase I-mediated DNA breakage, indicating cleavable complex formation (poisoning), and at 10 microM it inhibited relaxation of DNA, consistent with suppression (self-inhibition) of poisoning. In this assay, DACA provided no evidence of poisoning of this enzyme but inhibited its function at concentrations above 10 microM. In DNA cleavage assays utilising purified topoisomerase II, DACA induced breakage of supercoiled plasmid DNA at 5 microM whereas C1-DACA showed very weak poisoning at 1 microM and inhibition at 5 microM. Under conditions required for the assay of DNA relaxation, C1-DACA, but not DACA, inhibited topoisomerase II action at 5 microM. The actions of DACA and C1-DACA could also be distinguished by their ability to form DNA-protein cross-links in H460 human lung carcinoma cells as measured by precipitation of DNA-protein complexes with sodium dodecyl sulfate and potassium chloride. Both drugs stimulated the formation of complexes at low concentrations but inhibited formation at high concentrations. In survival assays with H460 cells, both drugs demonstrated biphasic responses with self-inhibition of cytotoxicity at intermediate drug concentrations. It was concluded that although both drugs have dual topoisomerase I/II specificity, DACA preferentially poisons topoisomerase II and C1-DACA preferentially poisons topoisomerase I. In addition, drug-induced inhibition of topoisomerase action at higher drug concentrations may mask poisoning in the cell-free assays as well as masking cytotoxicity in cultured cells. A model in which drug binding occludes topoisomerase-binding sites on the DNA can explain this self-inhibition of cytotoxic action.
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PMID:Mechanism of cytotoxicity of N-[2-(dimethylamino)ethyl] acridine-4-carboxamide and of its 7-chloro derivative: the roles of topoisomerases I and II. 1007 81

The Chinese hamster V79 lung cell in vitro micronucleus assay was adapted to detect and quantify phototoxicity and photogenotoxicity of fluoroquinolones. Using this assay, the quinolones were ranked in terms of decreasing phototoxicity: clinafloxacin >> lomefloxacin, sparfloxacin >> trovafloxacin, nalidixic acid, ofloxacin, ciprofloxacin > enoxacin, norfloxacin. This rank order agrees well with published studies utilizing various other phototoxicity models and establishes this approach as a fast and sensitive way to characterize the phototoxic potential of quinolones. Nearly complete inhibition of phototoxicity was observed if the cells were pretreated for as little as 1 min with 10-20 mM sodium azide prior to the addition of quinolone. An identical azide effect was seen in unirradiated quinolone- and etoposide-treated cells. These findings are consistent with a model in which sodium azide renders DNA topoisomerase II catalytically inactive. In this state, topoisomerase II cannot initiate DNA strand cleavage and the DNA/topoisomerase complex becomes insensitive to quinolones and other topoisomerase II inhibitors. The fact that azide reduces both UV-dependent and UV-independent toxicity and clastogenicity strongly suggests a common mechanism of toxicity dependent on the formation of topoisomerase-induced DNA double-strand breaks.
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PMID:Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II. 1008 19

Increased levels of DNA-protein cross-links (DNAPC) have been observed in vitro and in vivo following treatment with a number of chemotherapeutic alkylating agents and topoisomerase II inhibitors, that is, agents that have also been associated with the development of bone marrow depression and acute myelogenous leukemia. The current studies were undertaken to examine the effect of benzene, a bone marrow toxin and human leukemogen, on DNAPC levels in mouse bone marrow cells. Using a K+/sodium dodecyl sulfate (SDS) precipitation assay for DNAPC determination, the results indicate increased DNA-protein cross-link levels in mouse bone marrow cells at 2 and 4 but not 8 h after a single ip injection of 440 mg/kg benzene. Following the administration of multiple hematotoxic benzene doses (440 or 880 mg/kg, 2x/d for 2 d), increases in DNA-protein cross-link levels were either slight or not present. These results suggest that DNAPC induced by benzene are neither cumulative nor persistent lesions. The toxicity of benzene is mediated by a number of number of ring-hydroxylated and ring-opened compounds; therefore the present studies also examined DNAPC levels in mice administered trans,trans-muconaldehyde (MUC), a ring-opened hematotoxic and genotoxic metabolite of benzene. No marked increases in DNAPC levels were observed in CD- mouse bone marrow cells 1-12 h following a single ip injection of 3 mg/kg muconaldehyde. It is possible that multiple doses of MUC are required to induce elevated DNAPC levels in bone marrow cells of mice, since multiple doses are required for MUC-induced hematotoxicity. Other reactive metabolites and/or an interaction of reactive intermediates may also be involved in DNAPC induced by benzene.
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PMID:DNA-protein cross-link levels in bone marrow cells of mice treated with benzene or trans,trans-muconaldehyde. 1009 61

The purpose of these studies was to determine whether interferon-alpha (IFN-alpha) could enhance the sensitivity of human osteosarcoma cells to the cytotoxic actions of etoposide (VP-16). Cytostasis was determined using a [3H]thymidine incorporation assay, whereas cytotoxicity was quantified by a colony-formation assay. Low concentrations (0.1-5 U/ml) of IFN-alpha enhanced the cytostatic activity of VP-16 against MG-63, SAOS-2, and TE-85 osteosarcoma cells. The cytostatic activity of 1 microM VP-16 rose from 11% to 64%, 9% to 31%, and 10% to 71%, respectively, in the three cell lines when IFN-alpha was present. Survival fraction was also decreased when the osteosarcoma cells were treated with VP-16 + IFN-alpha as compared to either agent alone. The interaction between these two agents was determined to be synergistic rather than additive by interaction index analysis. Similar effects on cytostasis and cytotoxicity were observed when IFN-alpha was combined with Adriamycin but not cisplatin, actinomycin D, vinblastine, or amsacrine. VP-16 uptake was enhanced 12-fold in the presence of IFN-alpha, but this did not appear to translate into an increase in topoisomerase-II (topo-II)-DNA complex formation as quantified by the sodium dodecyl sulfate-KCl precipitation assay. We also could not detect alterations in topo-II expression, topo-II protein production, or cell cycle kinetics that have been shown to correlate with increased VP-16 cell sensitivity. Therefore, at this time the mechanism of enhanced cell sensitivity to the combination treatment remains unclear.
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PMID:Interferon-alpha enhances the sensitivity of human osteosarcoma cells to etoposide. 1043 62

By using tissue miniunits, protein kinase modulators, and topoisomerase inhibitors in short-term incubation (0-90 min) we studied (1) the role of protein phosphorylation in the immediate control of DNA replication in the developing rat cerebral cortex and (2) the mechanism of action for genistein-mediated DNA synthesis inhibition. Genistein decreased the DNA synthesis within less than 30 min. None of the other protein kinase inhibitors examined (herbimycin A, staurosporine, calphostin-C) or the protein phosphatase inhibitor sodium orthovanadate inhibited DNA synthesis and they did not affect the genistein-mediated inhibition. The selective topoisomerase inhibitors camptothecin and etoposide decreased the DNA synthesis to an extent similar to that of genistein and within less than 30 min. In addition, the effects of these substances on topoisomerase I and II were studied. Etoposide and genistein but not herbimycin A, staurosporine, or calphostin-C strongly inhibited the activity of topoisomerase II. Our results (1) strongly suggest that the net rate of DNA replication during the S phase of the cell cycle is independent of protein phosphorylation and (2) indicate that the early inhibitory effect of genistein on DNA synthesis is mediated by topoisomerase II inhibition rather than protein tyrosine kinase inhibition.
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PMID:Early effects of protein kinase modulators on DNA synthesis in rat cerebral cortex. 1048 85

Determination of the clastogenic potential of new chemical entities, particularly pharmaceuticals, is an important part of the overall safety assessment of such drugs. It is appreciated that clastogenicity can arise from perturbation of many different cellular processes distinct from direct DNA/drug interactions. One such alternative clastogenic process is inhibition of DNA topoisomerase II, during which process the topoisomerase/DNA/drug ternary complex forms stable DNA double-strand breaks (cleavable complex), which become templates for recombinational, mutagenic, and chromosomal fragmentation events. Without extensive experimentation, it is generally not possible to distinguish clastogenicity arising from direct drug/DNA interaction from that arising from inhibition of topoisomerase II. In the present investigation, we demonstrate that specific catalytic inhibitors of DNA topoisomerase II reduce the clastogenicity of topoisomerase poisons but not that arising via non-topoisomerase-dependent mechanisms. In particular, it is shown that catalytic topoisomerase II inhibitors such as chloroquine, sodium azide, and A-74932, as well as certain intercalating agents such as 9-aminoacridine and ethidium bromide, strongly antagonize the formation of micronuclei induced by the DNA gyrase inhibitor clinafloxacin and the antitumor topoisomerase II poison etoposide. These catalytic inhibitors are also shown to antagonize the clastogenicity of experimental compounds and novel pharmaceuticals presumed to be DNA intercalating agents by virtue of their response in a cell-based bleomycin amplification assay. We extend our previous hypothesis, suggesting that the clastogenicity of some nonstructurally alerting drugs may be due to an as yet unappreciated propensity for DNA intercalation. It is further proposed that intercalation-dependent inhibition of DNA topoisomerase II may be responsible for this clastogenicity and that this may be detected in intact mammalian cells with the use of catalytic topoisomerase inhibitors.
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PMID:Use of catalytic topoisomerase II inhibitors to probe mechanisms of chemical-induced clastogenicity in Chinese hamster V79 cells. 1069 23

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.
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PMID:Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli. 1072 75

An Ehrlich ascites tumour cell line (EHR2) was selected for resistance to etoposide (VP16) by in vivo exposure to this agent. The resulting cell line (EHR2/VP16) was 114.3-, 5.7-, and 4.0-fold resistant to VP16, daunorubicin, and vincristine, respectively. The amount of salt-extractable immunoreactive topoisomerase IIalpha and beta in EHR2/VP16 was reduced by 30-40% relative to that in EHR2. The multidrug resistance-associated protein (MRP) mRNA was increased 20-fold in EHR2/VP16 as compared with EHR2, whereas the expression of P-glycoprotein was unchanged. In EHR2/VP16, the steady-state accumulation of [(3)H]VP16 and daunorubicin was reduced by 64% and 17%, respectively, as compared with EHR2. Deprivation of energy by addition of sodium azide increased the accumulation of both drugs to the level of sensitive cells. When glycolysis was restored by the addition of glucose to EHR2/VP16 cells loaded with drug in the presence of sodium azide, extrusion of [(3)H]VP16 and daunorubicin was induced. Addition of verapamil (25 microM) decreased the efflux of daunorubicin to the level of sensitive cells, but had only a moderate effect on the efflux of [(3)H]VP16. The resistant cells showed moderate sensitisation to VP16 on treatment with verapamil, whereas cyclosporin A had no effect. Compared with that of sensitive cells, the ATPase activity of plasma membrane vesicles prepared from EHR2/VP16 cells was very low. Vanadate inhibited the ATPase activity of EHR2/VP16 microsomes with a K(i) value of 30 microM. ATPase activity was slightly stimulated by daunorubicin, whereas vinblastine, verapamil, and cyclosporin A had no effect. In conclusion, development of resistance to VP16 in EHR2 is accompanied by a significant reduction in topoisomerase II (alpha and beta) and by increased expression of MRP mRNA (20-fold). MRP displays several points of resemblance to P-glycoprotein in its mode of action: 1) like P-glycoprotein, MRP causes resistance to a range of hydrophobic drugs; 2) MRP decreases drug accumulation in the cells and this decrease is abolished by omission of energy; and 3) MRP increases efflux of drug from cells. However, compared with that of P-glycoprotein-positive cells, the ATPase activity of MRP-positive cells is found to be low and not able to be stimulated by verapamil.
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PMID:Characterisation of multidrug-resistant Ehrlich ascites tumour cells selected in vivo for resistance to etoposide. 1085 30

A series of 4beta-arylamino-4'-O-demethylepipodophyllotoxins and 4beta-arylaminoepipodophyllotoxins have been synthesized with significant stereoselectivity and improved yields by employing the methanesulphonic acid/sodium iodide reagent system. Compounds NPF. W-68 and other DNA topoisomerase II inhibitors are prepared in good to excellent yields by this method and these are active or more active than etoposide in their inhibition of the human DNA topoisomerase II.
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PMID:Facile and efficient one-pot synthesis of 4beta-arylaminopodophyllotoxins: synthesis of DNA topoisomerase II inhibitors (NPF and W-68). 1099 70


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