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)

The effect of high-level efflux activity on the MICs of fluoroquinolones against Streptococcus pneumoniae in the absence of topoisomerase mutations leading to fluoroquinolones resistance was investigated. A S. pneumoniae ATCC 46619-derived strain with high-level efflux activity was obtained (SP-25A). Both the parent and obtained strains were tested against efflux substrates acriflavine (Acr) and ethidium bromide (EtBr), and against norfloxacin (NFX), ciprofloxacin (CFX), levofloxacin (LFX), moxifloxacin (MFX), trovafloxacin (TVX) and sitafloxacin (SFX), in presence and absence of the efflux pump inhibitor reserpine. gyrA, gyrB, parC and parE QRDR genes were amplified by PCR and sequenced. MICs of NFX and CFX against SP-25A were 64-fold higher than parent strain MICs (256 mg/L versus 4 mg/L and 64 mg/L versus 1mg/L, respectively). MIC of LFX increased from 1 to 4 mg/L and MICs of MFX, TVX and SFX remained virtually unchanged (0.1-0.2 mg/L). MICs of Acr and EtBr against SP-25A were 8- and 16-fold higher than against parent strains. In both cases, reserpine reverted MICs to the parent strain values (1 and 0.2 mg/L). Only parE showed two mutations leading to a Pro(454) --> Ser and Glu(443) changes, which have previously been shown not to lead to significant fluoroquinolones MIC increases. SP-25A showed a significant increase of MICs of the hydrophilic fluoroquinolones, apparently derived only from efflux activity. Efflux activity, at these high levels, can lead to high-level resistance to older hydrophilic fluoroquinolones, but does affect newer fluoroquinolones such as moxifloxacin, trovafloxacin and sitafloxacin.
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PMID:In vitro activity of older and newer fluoroquinolones against efflux-mediated high-level ciprofloxacin-resistant Streptococcus pneumoniae. 1528 20

Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.
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PMID:Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy. 1538 64

The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIalpha inhibitor). We investigated the role of nuclear factor kappa B (NF-kappaB) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-kappaB associated with the transient loss of the inhibitory subunit IkappaB-alpha. Exposure to HGF/SF caused the activation of an NF-kappaB luciferase reporter that was blocked or attenuated by the expression of a mutant 'super-repressor' IkappaB-alpha. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-kappaB family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-kappaB-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the IkappaB-alpha super-repressor. The ability of HGF/SF to activate NF-kappaB signaling was dependent on c-Akt --> Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-kappaB, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-kappaB activity. HGF/SF induced the expression of several known NF-kappaB target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-kappaB-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-kappaB through a c-Akt --> Pak1 signaling pathway that is also dependent on Src, and that NF-kappaB contributes to HGF/SF-mediated protection against ADR.
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PMID:Role of NF-kappaB signaling in hepatocyte growth factor/scatter factor-mediated cell protection. 1568 34

Irinotecan is one of the most active drugs used in the treatment of small cell lung cancer (SCLC). 7-Ethyl-10-hydroxy-camptothecin (SN-38) is an active metabolite of irinotecan. We established an SN-38-resistant subline (SBC-3/SN-38) by continuous exposure of SN-38 to a human SCLC cell line, SBC-3. Using the 3-[4, 5-dimethyl-thiazol-2-yl] 2, 5-diphenyltetrazolium bromide assay, we evaluated the cytotoxicity of 17 anticancer agents. The SBC-3/SN-38 cells were 73-fold more resistant than the parental SBC-3 cells to SN-38 and showed cross-resistance not only to topoisomerase (topo) I inhibitors (irinotecan and topotecan), but also to topo II inhibitors (adriamycin and etoposide), antimicrotubule agents (vincristine, vindesine, vinorelbine and docetaxel), alkylating agents (cyclophosphamide and ifosfamide), platinum (cisplatin and carboplatin) and antifolate (methotrexate). Interestingly, the resistant subline reserved the sensitivity to bleomycin and 5-fluorouracil. The SBC-3/SN-38 cells had decreased topo I and II activity compared to the parent cells. The SN-38-resistant cell line, SBC-3/SN-38, will be useful to elucidate the mechanism of action of the topo I inhibitors.
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PMID:Establishment of a 7-ethyl-10-hydroxy-camptothecin-resistant small cell lung cancer cell line. 1573 31

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation.
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PMID:Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70. 1583 46

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.
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PMID:Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph. 1585 May 56

In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).
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PMID:Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. 1616 31

We evaluated the cytotoxicity and underlying mechanisms of cardiac glycosides, including digoxin, ouabain and proscillaridin A, on the proliferation of breast cancer MCF-7 cells. In terms of inhibition of cell proliferation of MCF-7 cells, the compounds rank in the order proscillaridin A>digoxin>ouabain. While both digoxin and ouabain inhibited topoisomerase II catalytic activity at nanomolar concentrations (100 nM), neither agent inhibited topoisomerase I catalytic activity even at concentrations as high as 100 microM. On the other hand, proscillaridin A was a potent poison of topoisomerase I and II activity at nanomolar drug concentrations (30 nM, 100 nM, respectively), suggesting that this agent may produce its cytotoxic activity by targeting both enzymes simultaneously. These studies suggest that the stabilization of DNA-topoisomerase II complexes is closely linked to the mechanism of digoxin, ouabain and proscillaridin A cytotoxicity. The potential DNA-binding properties of the cardiac glycosides have been assessed by measuring the displacement of ethidium bromide from calf thymus DNA. These results indicate that digoxin, ouabain and proscillaridin A neither intercalate nor interact with the minor groove of DNA.
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PMID:Inhibition of DNA topoisomerases I and II, and growth inhibition of breast cancer MCF-7 cells by ouabain, digoxin and proscillaridin A. 1681 97

The substituted phenazines XR11576 and XR5944 were originally described as dual topoisomerase-I/II poisons. Subsequent reports, however, indicated that the association of their cytotoxicity with cellular topoisomerases was not clear. We set out to study this further using human tumour cell lines, PEO1 ovarian cancer, MDA-MB-231 breast cancer and variants with acquired resistance to VP-16 and XR11576: PEO1VPR, MB-231VPR, MB-231-11576R and camptothecin: PEO1CamR. Cytotoxicity testing [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay], DNA-protein crosslink formation, cell cycle analysis (flow cytometry) for DNA content, apoptosis (flow cytometry) for Annexin V and Western blotting for apoptotic factors. Cytotoxicity testing showed potent cytotoxicity with no cross-resistance to XR11576 or XR5944 in VP-16 or camptothecin-resistant lines. Importantly, we have shown for the first time that the activities of XR5944 and XR11576 are similar as MB-231-11576R cells were resistant to both agents and to a similar extent. XR5944 showed the greatest, albeit slower, interaction with DNA with high levels of DNA-protein crosslinks. Levels of apoptosis in XR5944-treated cells were significantly less than those in VP-16 or XR11576 treatments, suggestive of a more cytostatic rather than cytotoxic mode of action. Interestingly, XR5944 failed to give rise to a G2/M blockade, in contrast to VP-16 or XR11576. XR5944 and XR11576, in line with a dual topoisomerase-I/II-directed mechanism of action, retain potent activity in tumour cells with acquired resistance to VP-16 and camptothecin. Although these agents appear to behave differently from each other according to experimental conditions, this study suggests a substantial overlap in their mechanism(s) of action.
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PMID:Mode of action of the novel phenazine anticancer agents XR11576 and XR5944. 1715

The aim of this work was to determine the functional activities of four different antioxidative enzymes (glutathione reductase, glutathione-S-transferase, glutathione peroxidase, thioredoxin reductase) and the protein expression of three ATP-binding cassette transporters (P-glycoprotein, multidrug resistance protein 1, multidrug resistance protein 2) in a panel of 14 human cancer cell lines. Enzyme activities and transporter expression were then correlated with the in-vitro cytotoxic activities (GI50 values) of 19 standard antitumor drugs. Analogous data from the National Cancer Institute were used for comparison. The GI50 values of the platinum complexes, alkylating agents, antimetabolites, topoisomerase inhibitors and antimitotic drugs were determined by crystal violet or 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. Standard enzymatic assays employed to measure the glutathione peroxidase, glutathione-S-transferase, glutathione reductase and thioredoxin reductase activities. The protein expression of the ATP-binding cassette transporter proteins was investigated by the Western-blot method. The delta method was used to normalize the data before bivariant correlation analysis. Only a few correlations between enzyme and cytotoxic activities of the antitumor agents were found. The GI50 values for melphalan and camptothecin correlated positively with the activity of glutathione-S-transferase, whereas GI50 values for methotrexate correlated positively with the cellular activities of both glutathione reductase and thioredoxin reductase. A significant correlation between glutathione reductase and thioredoxin reductase activities was found in our panel of cell lines. Neither P-glycoprotein nor multidrug resistance protein 2 expression could be detected by Western blot analysis in any cell lines investigated, but multidrug resistance protein 1 was consistently observed in all but four lines. Multidrug resistance protein 1 expression correlates positively with the GI50 values of several drugs, e.g. vinblastine and etoposide, and negatively with the GI50 values of 5-fluorouracil. The results confirm the complexity of resistance to antitumor agents and show that the GSH-thioredoxin system alone is not a good indication of intrinsic resistance for many of these anticancer drugs.
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PMID:Correlations between the activities of 19 standard anticancer agents, antioxidative enzyme activities and the expression of ATP-binding cassette transporters: comparison with the National Cancer Institute data. 1735 91


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