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)

Treatment of U-937 promonocytic cells with the DNA topoisomerase II inhibitor etoposide rapidly caused death by apoptosis, as determined by changes in chromatin structure, production of DNA breaks, nucleosome-sized DNA degradation, decrease in mitochondrial membrane potential and phosphatidyl serine translocation in the plasma membrane, and at the same time induced intracellular acidification. Both the execution of the apoptotic process and the intracellular acidification were reduced by the addition of forskolin plus theophylline or other cAMP increasing agents. These agents also attenuated the induction of apoptosis by camptothecin, heat-shock, cadmium chloride and X-radiation. Although etoposide slightly increased the production of reactive oxygen intermediates, this increase was not prevented by forskolin plus theophylline, and the addition of antioxidant agents failed to inhibit apoptosis. Etoposide caused a great increase in NF-(kappa)B binding activity, which was not prevented by forskolin plus theophylline, while AP-1 binding was little affected by the topoisomerase inhibitor. The treatments did not significantly alter the levels of Bcl-2 and Bax. By contrast, the expression of c-myc, which was very high in untreated U-937 cells and only partially inhibited by etoposide, was rapidly and almost totally abolished by the cAMP increasing agents. Finally, it was observed that etoposide caused a transient dephosphorylation of retinoblastoma (Rb), which was associated with cleavage of poly(ADP-ribose) polymerase (PARP). Both Rb dephosphorylation and PARP cleavage were inhibited by forskolin plus theophylline. The inhibition of Rb (type I) phosphatase and ICE/CED-3-like protease activities, and the abrogation of c-myc expression, are mechanisms which could explain the anti-apoptotic action of cAMP increasing agents in myeloid cells.
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PMID:cAMP increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells. 945 37

Fostriecin, a structurally unique phosphate ester, is presently under evaluation in clinical trials to determine its potential use as an antitumor drug in humans. Fostriecin has been reported as having inhibitory activity against DNA topoisomerase type II and protein phosphatases implicated in cell-cycle control. However, the relative contribution of these mechanisms to the antitumor activity of fostriecin has not yet been elucidated. In this study, after confirming that fostriecin is a potent inhibitor of serine/threonine protein phosphatase type 2A and a weak inhibitor of serine/threonine protein phosphatase type 1, we show that fostriecin inhibits approximately 50% of the divalent cation independent serine/threonine protein phosphatase (PPase) activity contained in whole cell homogenates of Chinese hamster ovary cells at concentrations associated with antitumor activity (1-20 microM). Investigations into the cellular effects produced by fostriecin treatment reveal that 1-20 microM fostriecin induces a dose-dependent arrest of cell growth during the G2-M phase of the cell cycle. Immunostaining of treated cells indicates that growth arrest occurs before the completion of mitosis and that fostriecin-induced growth arrest is associated with the aberrant amplification of centrosomes, which results in the formation of abnormal mitotic spindles. The "mitotic block" induced by fostriecin is reversible if treatment is discontinued in <24 h. However, after approximately 24-30 h of continuous treatment, growth arrest is not reversible, and treated cells die even when placed in fostriecin-free media. Correlative studies conducted with established PPase inhibitors reveal that, when applied at concentrations that inhibit PPase activity to a comparable extent, both okadaic acid and cantharidin also induce aberrant centrosome replication, the appearance of multiple aberrant mitotic spindles, and G2-M-phase growth arrest. These studies add additional support to the concept that PPase inhibition underlies the antitumor activity of fostriecin and suggest that other type-selective PPase inhibitors should be evaluated for potential antitumor activity.
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PMID:Fostriecin-mediated G2-M-phase growth arrest correlates with abnormal centrosome replication, the formation of aberrant mitotic spindles, and the inhibition of serine/threonine protein phosphatase activity. 972 69

We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parC genes for 30 clinical strains of Pseudomonas aeruginosa resistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248-2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC (codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrA allele. The remaining two strains harbored the first gyrB mutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e., gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 microg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6, P < 0. 01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrA mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.
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PMID:Type II topoisomerase mutations in ciprofloxacin-resistant strains of Pseudomonas aeruginosa. 986 66

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.
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PMID:Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. 1043 85

Fluoroquinolone resistance is mediated by target changes (DNA gyrase and/or topoisomerase IV) and/or decreased intracellular accumulation. The genes (gyrA/gyrB/parC/parE) and proteins of DNA topoisomerase IV show great similarity, both at the nucleotide and amino acid sequence level to those of DNA gyrase. It has been shown that there are hotspots, called the quinolone resistance determining region (QRDR), for mutations within gyrA and parC. Based on the Escherichia coli co-ordinates, the hotspots most favoured for giving rise to decreased susceptibility and/or full resistance to quinolones are at serine 83 and aspartate 87 of gyrA, and at serine 79 and aspartate 83 for parC. Few mutations in gyrB or parE/grlB of any bacteria have been described. Efflux of fluoroquinolones is the major cause of decreased accumulation of these agents; for Staphylococcus aureus, the efflux pump involved in norfloxacin resistance is NorA, and for Streptococcus pneumoniae, PmrA. By analysis of minimum inhibitory concentration (MIC) data derived in the presence and absence of the efflux inhibitor reserpine, it has been shown that up to 50% of ciprofloxacin-resistant clinical isolates of S. pneumoniae may possess enhanced efflux. This suggests that efflux may be an important mechanism of clinical resistance in this species. In Pseudomonas aeruginosa, several efflux operons have been demonstrated genetically and biochemically. These operons are encoded by mex (Multiple EffluX) genes: mexAmexB-oprM, mexCD-OprJ system and mexEF-oprN system. The E. coli efflux pump is the acrAB-tolC system. Both the mar operon and the sox operon can give rise to multiple antibiotic resistance. It has been shown that mutations giving rise to increased expression of the transcriptional activators marA and soxS affect the expression of a variety of different genes, including ompF and acrAB. The net result is that expression of OmpF is reduced and much less drug is able to enter the cell; expression of acrAB is increased, enhancing efflux from the cell.
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PMID:Mechanisms of fluoroquinolone resistance: an update 1994-1998. 1055 99

To detect if isolates susceptible to quinolones already carry mutations in the gyrA and parC genes, we selected 12 ciprofloxacin-susceptible Escherichia coli strains collected from patients with urinary tract infections in Latin America in 1998, as part of ongoing SENTRY Antimicrobial Surveillance Program. The isolates studied exhibited minimal inhibitory concentrations (MICs) for ciprofloxacin between < or = 0.015 microg/mL and 0.5 microg/mL. The molecular characterization of quinolone resistance was determinated by amplification of the gyrA and parC by PCR followed by sequencing of the respective amplicons. We observed that E. coli isolates exhibiting MIC, < or = 0.06 microg/mL for ciprofloxacin did not show mutations in either topoisomerase. On the other hand, all isolates with MIC between 0.12 microg/mL and 0.5 microg/mL demonstrated single mutation in the gyrA gene. The most frequent mutation occurred at position 83, where the amino acid serine was replaced by leucine. No mutations in the parC gene were observed. To preserve the potency and prevent the development of resistance, we suggest that quinolone usage should be rational, especially in the treatment of urinary tract infections, and in the prophylaxis of immunosupressed patient populations.
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PMID:Occurrence of single-point gyrA mutations among ciprofloxacin-susceptible Escherichia coli isolates causing urinary tract infections in Latin America. 1074 70

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.
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PMID:The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner. 1109 68

The activity of three new, 8-methoxy-nonfluorinated quinolones (NFQs) against multiple-drug-resistant staphylococci was investigated. First, using Staphylococcus aureus strains containing point mutations in the serine 84-80 hot spots of the target genes (gyrA and grlA), cell growth inhibition potencies of the NFQs as a result of DNA gyrase and topoisomerase IV inhibition were estimated and compared with those of known fluoroquinolones. The NFQs and clinafloxacin showed higher affinities toward both the targets than ciprofloxacin, trovafloxacin and gatifloxacin. Furthermore, the ratio of the calculated affinity parameter for DNA gyrase to that for topoisomerase IV was lower in the case of the NFQs, clinafloxacin, and gatifloxacin than in the case of ciprofloxacin and trovafloxacin. These results suggest that the former group of quinolones is better able to exploit both the targets. Next, using clinical isolates of methicillin-resistant S. aureus (MRSA; n = 34) and coagulase-negative staphylococci (CoNS; n = 24), the NFQs and clinafloxacin were shown to be more potent (MIC at which 90% of the isolates are inhibited [MIC90] = 2 microg/ml for MRSA and 0.5 microg/ml for CoNS) than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC90 = 16 to >64 microg/ml for MRSA and 4 to >32 microg/ml for CoNS). Bactericidal kinetics experiments, using two MRSA isolates, showed that exposure to the NFQs at four times the MIC reduced the bacterial counts (measured in CFU per milliliter) by > or =3 log units in 2 to 4 h. Overall, the NFQs and clinafloxacin were less susceptible than the other quinolones to existing mechanisms of quinolone resistance in staphylococci.
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PMID:Quinolone resistance in Staphylococci: activities of new nonfluorinated quinolones against molecular targets in whole cells and clinical isolates. 1125 24

Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.
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PMID:Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization. 1131 53

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.
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PMID:An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani. 1180 93


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