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)

By altering the accessibility of DNA sequences for alkylation or platination, and/or for subsequent repair, topoisomerase II can potentially affect the level of DNA interstrand cross-links induced in cells by bifunctional agents. In this study, we investigated the extent to which inhibition of topoisomerase II activity in a human glioblastoma multiforme cell line alters the kinetics of both the formation and the repair of total genomic DNA interstrand cross-links, as well as the sensitivity of the tumor cells to cis-diamminedichloroplatinum II (cis-DDP) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Cells were incubated with and without 200 microM novobiocin, a known topoisomerase II inhibitor, for 24 h, followed by exposure to 50 microM BCNU and 25 microM cis-DDP. DNA interstrand cross-linking was determined at various time points over 72 h, using a modified ethidium bromide-DNA binding assay. Sensitivity of the cells to cis-DDP and BCNU was also determined with and without novobiocin pretreatment with 200 microM novobiocin. This concentration of novobiocin showed no significant direct cytotoxicity, although it inhibited topoisomerase II activity in tumor cell nuclear extracts by 73%. A significant decrease in the rate of repair of both cis-DDP and BCNU induced DNA interstrand cross-links, with a corresponding decrease in the clonogenic survival of the cells, was observed following novobiocin exposure. Although the peak cross-link indices of novobiocin-treated cells relative to controls were not significantly increased, residual DNA cross-linking in the cells after 72 h was increased by 1.4-fold for BCNU and 3-fold for cells treated with cis-DDP, thus, indicating a greater effect of topoisomerase II on cross-link repair than on cross-link formation. These data suggest that inhibition of topoisomerase II may provide a potentially effective clinical strategy for sensitizing human brain tumors, and possibly other tumors as well, to DNA cross-linking anticancer agents.
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PMID:Topoisomerase II inhibition and altered kinetics of formation and repair of nitrosourea and cisplatin-induced DNA interstrand cross-links and cytotoxicity in human glioblastoma cells. 824 21

A function for topoisomerases I and II in DNA excision repair can be postulated from the organization of the mammalian chromosome, involving nucleosomal structures and matrix-attached DNA loops. To analyse this function we determined UV-induced DNA incision in confluent human fibroblasts in the presence of 16 inhibitors of topoisomerases I and II which belonged to at least five different drug categories, based on their mechanism of action. Dose-response experiments were performed, analysed by linear regression and the concentrations at which DNA-incising activity was reduced to 50% were calculated (K50 values). The majority of these values represent concentrations for which interfering cell toxicity could be excluded. K50 concentrations, which were determined by extrapolating dose-response data, may hit the toxicity range, nevertheless, we deem our K50 scale useful for making biochemical comparisons. With respect to topoisomerase I, camptothecin and topotecan diminished repair-specific DNA incision to a small extent, whereas distamycin, which binds to the minor groove of DNA, caused a stronger effect. With respect to topoisomerase II the results were as follows. (i) The DNA intercalator ethidium bromide decreased DNA-incising activity at rather low concentrations, which indicates marked inhibitory potency. Quinacrine was less effective. (ii) Inhibitors intercalating and binding to the 'cleavable' DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) strongly suppressed reparative DNA incision. (iii) Only small effects were observed using several drugs which act by trapping the 'cleavable' DNA-enzyme complex, namely nalidixic acid and oxolinic acid. In contrast, etoposide and teniposide inhibited post-UV DNA cleavage sizeably. (iv) Merbarone had to be applied at very high concentrations to reduce UV-induced DNA incision. (v) Novobiocin, an inhibitor of the ATPase subunit of topoisomerase II, markedly diminished repair-specific DNA cleavage. A comparison of the K50 values for DNA incision with those for DNA repair synthesis (1) shows that the majority of the investigated drugs inhibited both repair parameters. There were, however, differences in the concentrations required to achieve the 50% inhibition level. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kd form of topoisomerase II is a target enzyme for inhibitors which suppressed repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fibroblasts. 824 65

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.
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PMID:Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation. 857 84

A novel benzophenazine derivative, NC-190, is a potent antitumor compound. NC-190 has been shown to inhibit the DNA strand-passing activity of DNA topoisomerase II. We investigated further the mode of action of NC-190 against DNA topoisomerase II and DNA fragmentation. NC-190 inhibited the decatenation activity of purified topoisomerase II, but had only a weak inhibitory effect against topoisomerase I. A topoisomerase II-dependent DNA cleavage assay showed that NC-190 inhibited the enzyme activity by stabilizing a topoisomerase II-DNA cleavable complex. NC-190 induced growth inhibition, protein-linked DNA breaks, and DNA fragmentation in cultured HL-60 cells in a dose-dependent manner. These activities of NC-190 in HL-60 cells were comparable to those of etoposide (VP-16). These results demonstrate a good correlation among growth inhibition, topoisomerase II-dependent DNA cleavage, and DNA fragmentation induced by NC-190. A DNA unwinding assay showed that NC-190 had intercalating activity, but its activity appeared to be weaker than those of ethidium bromide and adriamycin. These results indicate that the mechanism by which NC-190 exhibits antitumor activity may be the inhibition of topoisomerase II.
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PMID:A novel antitumor compound, NC-190, induces topoisomerase II-dependent DNA cleavage and DNA fragmentation. 860 48

A single amino acid change transforms restriction enzyme NaeI to a topoisomerase and recombinase (NaeI-L43K) that shows no sequence similarity to these protein families. This transformation appears to result from coupled endonuclease and ligase domains. To further elucidate the relationship between NaeI-L43K and the topoisomerase protein family, we studied the effect of the topoisomerase inhibitors on NaeI-L43K activity. The intercalative drugs amsacrine, ellipticine, and daunorubicin inhibited NaeI-L43K, whereas the nonintercalating drugs camptothecin, VP-16, and oxolinic acid did not. Ethidium bromide also inhibited NaeI-L43K, implying that intercalation is responsible for its inhibition. The effects of the intercalative drugs on the DNA cleavage steps of NaeI and NaeI-L43K were compared. The drugs hardly inhibited DNA cleavage by wild type NaeI but completely inhibited DNA cleavage by NaeI-L43K. This difference in inhibition demonstrates that the L43K amino acid change sensitized NaeI to these drugs. Low concentrations of the intercalative drugs, except for ethidium bromide, enhance production of topoisomerase--DNA covalent intermediates but inhibited production of the NaeI-L43K--DNA covalent intermediate. These results imply some unique differences between DNA relaxation by NaeI-L43K and DNA topoisomerase. Concomitant with studying inhibition of the cleavage intermediate, NaeI-L43K was found to covalently bond with the 5' end of the cleaved DNA strand.
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PMID:Changing a leucine to a lysine residue makes NaeI endonuclease hypersensitive to DNA intercalative drugs. 875 63

This review summarises mutagenesis-related research on the major classes of DNA minor groove binding ligands. These compounds can bind to DNA covalently or non-covalently, and span a range of DNA sequence selectivities. Many of the non-covalent binders show effects on topoisomerase enzymes in mammalian cells, with the bisbenzimidazoles being the most active. Mutagenic effects consistent with topoisomerase inhibition are observed in vitro. Many of these compounds induce aneuploidy and polyploidy, properties which may also contribute to carcinogenic processes. Similarly, uvrA trapping by some minor groove binders may alter mutagenetic processes by inhibiting efficient repair. Distamycin has been shown to enhance the mutagenicity of ethidium bromide in bacteria by an undetermined mechanism. However, the inhibitory effects of minor groove binders on human DNA repair systems have not yet been reported. Hoechst 33258 and distamycin cause chromosome decondensation in both mouse and human cells particularly at heterochromatic regions which are rich in AT content. Various minor groove binders have been shown to induce fragile sites in cultured lymphocytes from susceptible individuals, which may have a propensity to develop particular cancers. Investigation of the relationship between fragile site inducing drugs and chromosomal rearrangements in fragile site carriers has not been investigated but may yield interesting results. Some DNA alkylating minor groove binders can generate lesions extremely toxic to mammalian cells (e.g., CC-1065 and analogues), and induce a range of DNA sequence changes in vivo, both at the site of covalent bonding as well as at surrounding sequences. This may be typical of alkylating minor groove binders which have a binding site size of several base pairs, and which stabilise helical structure. Minor groove binders have effects on gene expression in vitro by inhibiting the sequence selective binding of various transcription factors to DNA. These effects may result in expression or repression of downstream genes also. This class of ligand thus offers the possibility of mutations targeted to specific genes or genomic regions. It will be interesting to determine whether such examples of targeted mutagenesis, as has already been observed with CC-1065 and adozelesin, will result in an enhanced or in a lowered capacity to promote neoplastic disease. However it should be noted that pentamidine, a minor groove binder used in the treatment of AIDS-related PCP, has thus far shown no mutagenic effects in nuclear DNA and only a weak effect in mitochondrial DNA of yeast. These results suggest that minor groove binding does not necessarily lead to mutagenesis.
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PMID:The mutagenic properties of DNA minor-groove binding ligands. 878 82

The antitumor activity of pMC540 has been shown to be mediated via its interaction with topoisomerase (Topo) II eventually leading cells into apoptosis. This agent was also found to down regulate the expression of the c-myc oncogene in L1210 leukemia cells. To investigate the possibility that damage within select genomic regions may contribute to the antiproliferative activity of pMC540, differential damage in regions surrounding the c-myc locus as well as other select genes was determined. Southern blot hybridization experiments show that pMC540 treatment induces in situ DNA cleavage products in the 5' end of the c-myc oncogene of L1210 leukemia cells. In cells pre-treated with 50 microM ethidium bromide, an inhibitor of the Topo II-dependent DNA cleavage, a subsequent treatment with pMC540 failed to induce DNA cleavage, suggesting that the cleavage activity of pMC540 was Topo II dependent. pMC540-induced cleavage does not appear to correlate with the over-expression of the c-myc oncogene in these cells as another over-expressed gene c-myb was not affected. Thus, it is proposed that the c-myc gene may be a preferred target for pMC540 may mediated antiproliferative activity.
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PMID:In situ stimulation of topoisomerase II-induced cleavage sites in the c-myc protooncogene by antitumor agent pMC540 is associated with gene expression. 879 3

A wild type strain of Mycobacterium smegmatis mc2 155 was serially adapted to 64 fold of minimal inhibitory concentration of an antimycobacterial agent, ciprofloxacin. This clone (CIPr) exhibited cross resistance to ofloxacin and ethidium bromide. The rate of drug efflux was accelerated in CIPr compared to the wild type strain. Verapamil, a calcium channel blocker, enhanced the drug accumulation in CIPr by diminishing the efflux and thus reversed the resistant phenotype. Additionally, a missense mutation was detected in the quinolone resistance determining region of the DNA-gyrase A subunit of CIPr. Taken together, these results suggest that drug efflux plays a major role in conferring such a high level of resistance in CIPr, in addition to the mutation in the DNA-gyrase locus.
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PMID:Involvement of an efflux system in mediating high level of fluoroquinolone resistance in Mycobacterium smegmatis. 880 41

By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.
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PMID:Characterization of a protein recognizing minor groove binders-damaged DNA. 893 77

Treatment of leukemic cells with topoisomerase inhibitors can lead to growth arrest and subsequent apoptotic cell death. The relationships between cell cycle regulation and apoptosis triggering remain poorly understood. The gadd153 gene encodes the nuclear protein CHOP 10 that acts as a negative modulator of CCAAT/enhancer binding protein transcriptional factors and inhibits cell cycle progression. We have investigated the relationships between gadd153 gene expression and apoptosis induction in four human leukemic cell lines with different sensitivities to apoptosis induced by etoposide (VP-16), a topoisomerase II inhibitor. The gadd153 gene was constitutively expressed in the four studied cell lines. In U937 and HL-60 cells that were very sensitive to apoptosis induction by the drug, VP-16 induced a time- and dose-dependent increase of gadd153 gene mRNA expression. Using agarose gel electrophoresis and a quantitative filter elution assay, apoptotic DNA fragmentation was observed to begin when gadd153 gene expression increased. Equitoxic doses of VP-16 (as defined using a 96-h 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide assay) did not increase the gadd153 mRNA level in K562 and KCL22 cell lines that were more resistant to apoptosis induction by the drug. Nuclear run-on and mRNA stability experiments demonstrated that VP-16 treatment increased gadd153 gene transcription in the sensitive U937 cells. Cycloheximide did not prevent gadd153 expression increase. Both gadd153 mRNA level increase and internucleosomal DNA fragmentation were inhibited by N-tosyl-L-phenylalanine chloromethylketone, a serine threonine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal, an inhibitor of calpain, N-acetylcysteine, an inhibitor of oxidative metabolism, and overexpression of Bcl-2. Z-VAD and Z-DEVD peptides that inhibit interleukin 1beta-converting enzyme-like proteases suppressed DNA fragmentation without preventing gadd153 mRNA increase in VP-16-treated U937 cells. These results indicate that gadd153 gene expression increase occurs downstream of events sensitive to N-tosyl-L-phenylalanine chloromethylketone, calpain inhibitor I, and Bcl-2 and upstream of interleukin 1beta-converting enzyme-related proteases activation in leukemic cells in which treatment with VP-16 induces rapid apoptosis.
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PMID:Increased gadd153 messenger RNA level is associated with apoptosis in human leukemic cells treated with etoposide. 904 46


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