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 number of studies in yeast have shown that DNA topoisomerase II is essential for chromosome condensation and disjunction during mitosis at the metaphase/anaphase transition and meiosis I. Accordingly, kinetic and mechanistic studies have implied a role for topoisomerase II in chromosome disjunction. As a step toward understanding the nature and role of topoisomerase II in a mammalian germline in vivo, we have purified topoisomerase II from rat testis to homogeneity and ascertained several of its catalytic activities in conjunction with that of the purified enzyme from liver. The purified enzymes appeared to be monomers under denaturing conditions; however, they differed in their relative molecular mass. Topoisomerase II from testis and liver have apparent molecular masses of 150 +/- 10 kDa and 160 +/- 10 kDa, respectively. The native molecular mass of testis topoisomerase II as assayed by immunoblot analysis of cell-free extracts, prepared in the presence of SDS and a number of protease inhibitors, corroborated with the size of the purified enzyme. Both enzymes are able to promote decatenation and relax supercoiled DNA substrates in an ATP and Mg(2+)-dependent manner. However, quantitative comparison of catalytic properties of topoisomerase II from testis with that of the enzyme from liver displayed significant differences in their efficiencies. Optimal pH values for testis enzyme are 6.5 to 8.5 while they are 6 to 7.5 for the liver enzyme. Intriguingly, the relaxation activity of liver topoisomerase II was inhibited by potassium glutamate at 1 M, whereas testis enzyme required about half its concentration. These findings argue that topoisomerase II from rat testis is structurally distinct from that of its somatic form and the functional differences between the two enzymes parallels with the physiological environment that is unique to these two tissues.
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PMID:Purification and functional characterization of type II DNA topoisomerase from rat testis and comparison with topoisomerase II from liver. 876 51

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a delta 32-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is > 10-fold more sensitive to heat treatment (52 degrees C) than the wild type. After brief treatment at 42 degrees C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52 degrees C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42 C (displayed on SDS-polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.
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PMID:Effect of the deletion of the sigma 32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance. 887 34

Using the technique of alkaline filter elution, we have evaluated the DNA damage induced by doxorubicin and etoposide in a rat glioblastoma cell line, C6, and its doxorubicin-selected resistant variant, C6 0.5. DNA damage paralleled drug-induced cytotoxicity, but it appeared that the same DNA damage generated much less cytotoxicity in resistant cells than in sensitive ones, resistant cells being able to tolerate more DNA damage than sensitive cells. We have then quantified the doxorubicin- and etoposide-induced complexes between topoisomerase II (topoII) DNA with the technique of SDS/KCl precipitation. Etoposide produced a concentration-dependent increase in topoII-DNA complexes, which was higher in resistant cells at equitoxicity, just as was DNA damage. In contrast, doxorubicin-induced topoII-DNA complexes, which were much less abundant than those induced by etoposide, were not differently produced in sensitive and resistant cells. This indicates that the DNA damage occurring in resistant cells at high doxorubicin concentrations might originate from source other than topoII-DNA complex formation. When verapamil was added during drug exposure, it restored doxorubicin intracellular accumulation to the level reached in sensitive cells, partially reversed both doxorubicin and etoposide resistance, increased the formation of etoposide-induced topoII-DNA complexes, but not those induced by doxorubicin. Immunoblot analysis of topoII as well as the measure of its catalytic activity in nuclear extracts revealed a quantitative defect of this enzyme in the resistant line. When inhibiting this activity by doxorubicin and etoposide, we observed that the concentrations of etoposide required for a given inhibition of kinetoplast DNA decatenation are much higher that those of doxorubicin. The topoII extracted from both cell lines is, therefore, much more sensitive to doxorubicin than to etoposide, but no difference in drug sensitivity was evident between sensitive and resistant cells, indicating that no qualitative alteration in topoII catalytic activity was likely to occur.
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PMID:Differential stabilization of topoisomerase-II-DNA cleavable complexes by doxorubicin and etoposide in doxorubicin-resistant rat glioblastoma cells. 915 58

Aclarubicin and doxorubicin are DNA binding anthracycline antibiotics of related chemical structure but differing cytotoxic action. Although doxorubicin mediates its cytotoxicity by poisoning the enzyme topoisomerase II, aclarubicin has been hypothesized to inhibit the catalytic action of topoisomerase II. We show here that aclarubicin, in contrast to doxorubicin, is highly effective in inhibiting the action of topoisomerase I. Aclarubicin not only inhibits this enzyme in a cell-free assay but also markedly inhibits DNA-protein cross-linking in H460 human lung adenocarcinoma cells as measured by the K(+)-SDS precipitation technique. It also displaces topoisomerase I from DNA as measured by Western blotting. Aclarubicin reverses the cytotoxicity of both amsacrine and camptothecin in clonogenic survival assays, consistent with the hypothesis that it is a dual topoisomerase I/II inhibitor. We suggest that the self-inhibition of topoisomerase I in short-term assays may mask the underlying activity of aclarubicin as a topoisomerase I poison. In short-term (1-H) drug exposure assays, aclarubicin kills both exponential and plateau phase cells by a non-cell cycle-selective mechanism apparently not involving G2 phase arrest. This may be a consequence of simultaneous inhibition of topoisomerases I and II.
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PMID:Differential actions of aclarubicin and doxorubicin: the role of topoisomerase I. 950 31

Since topoisomerase poisons allow the enzyme to cut and covalently bind to DNA but abort the subsequent rejoining of the molecule after relieving the torsional stress. To study their action we have made use of a supercoiled form of the pRYG plasmid that bears a specific topoisomerase recognition and binding region. The conversion of the supercoiled circular double-stranded DNA to the linear and open circle forms in the presence of a topoisomerase II poison and a denaturation step by proteinase K-SDS is indicative of the efficiency of our test agents to stabilize the cleavable complex. Using this system, three glucosylated isoflavones (6'-methoxy-pseudobaptigenin-7-O-beta-glucoside, genistin, and daidzin) isolated from cytotoxic chloroform and ethyl acetate extracts of Retama sphaerocarpa Boissier, were found to have the ability to stabilize the cleavage complex human DNA topoisomerase II.
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PMID:Glucosylated isoflavones as DNA topoisomerase II poisons. 1103 85

Chloroquinoxaline sulfonamide (chlorosulfaquinoxaline, CQS, NSC 339004) is active against murine and human solid tumors. On the basis of its structural similarity to the topoisomerase IIbeta-specific drug XK469, CQS was tested and found to be both a topoisomerase-IIalpha and a topoisomerase-IIbeta poison. Topoisomerase II poisoning by CQS is essentially undetectable in assays using the common protein denaturant SDS, but easily detectable with strong chaotropic protein denaturants. The finding that detection of topoisomerase poisoning can be so dependent on the protein denaturant used in the assay has implications for drug discovery efforts and for our understanding of topoisomerase poisons.
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PMID:Chloroquinoxaline sulfonamide (NSC 339004) is a topoisomerase IIalpha/beta poison. 1108 7

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.
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PMID:Topoisomerase II poisoning by ICRF-193. 1157 77

Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40 microM, with an IC50 of 5 microM. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC50=0.66 microM). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250 microM) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme-DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated 'cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS-K+-precipitable DNA-topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.
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PMID:Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I. 1202 7

The inhibition of cell proliferation by 1,4-bis (1-naphthyl)-2,3-dinitro-1,3-butadiene (Naph-DNB) was evaluated in vitro against 4 cell lines (L1210/DDP, A2780/DX3, HCT-8/FU7dR, A549-T12) selected for their resistance to cisplatin, doxorubicin, 5-fluorouracil and taxol, and their wild-type counterparts. Naph-DNB is a novel anti-cancer compound obtained years ago within a research project of Organic Chemistry aimed at synthesizing 2,3-dinitrobutadiene derivatives. Because of its chemical structure, Naph-DNB was suggested to interact with nucleic acids, in particular DNA, and the other cellular macromolecules. This hypothesis made us consider Naph-DNB as a candidate for studies concerning its antitumour activity. We used the MTT assay to test the inhibition of cell proliferation after incubation of the cell lines with Naph-DNB for 72 h. For comparison, resistant and wild-type cell lines were also tested against those anticancer drugs used in vitro for their selection. In these culture conditions Naph-DNB retained its inhibiting activity against all resistant cells with IC50 values similar to those obtained in corresponding wild-type cell lines. Moreover, Naph-DNB was twice as effective as 5-fluorouracil against wild-type HCT-8 cells. Our previous findings about the interaction of Naph-DNB with DNA through the formation of interstrand cross-links suggested a mechanism of action similar to that of platinum/alkylating agents or topoisomerase inhibitors (intercalating agents). Our present data obtained by the K-SDS precipitation assay in A2780 and A549 cells showed that Naph-DNB is not able to form a stable topoisomerase-DNA complex as is the case for topoisomerase inhibitors. In conclusion, our results indicate that Naph-DNB is able to overcome some of the classical mechanisms of resistance selected by some anticancer drugs mainly used in clinical setting.
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PMID:1,4-Bis(1-naphthyl)-2,3-dinitro-1,3-butadiene a novel anticancer compound effective against tumor cell lines characterized by different mechanisms of resistance. 1520 65

Reverse gyrase, a topoisomerase which introduces positive superhelical turns into DNA, has been purified from Sulfolobus to near homogeneity. It is a single polypeptide with a mol. wt. of 120 000 as determined by denaturing gel electrophoresis. Contrary to a previous report, it is a type I topoisomerase as judged by the linking-number change of closed circular DNA topoisomer. Unlike other known type I topoisomerases, ATP or dATP is required for introducing positive superhelical turns. In order to relax negatively supercoiled DNA, other nucleotide triphosphates (XTP) are also effective with low efficiency. In the absence of either XTP or divalent cations, the enzyme introduces nicks into closed circular DNA when the reaction is stopped by SDS. This suggests that reverse gyrase cuts one of the two strands of DNA in the course of its enzymatic reaction.
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PMID:Reverse gyrase; ATP-dependent type I topoisomerase from Sulfolobus. 1645 36


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