Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91-107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase alpha, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an 'adaptive' response in which the cell cycle continues to operate, perhaps in a 'repair' mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the 'adaptive' response until several hours after MBT, which is consistent with a slow transcriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.
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PMID:Effect of inhibitors of DNA replication on early zebrafish embryos: evidence for coordinate activation of multiple intrinsic cell-cycle checkpoints at the mid-blastula transition. 927 12

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.
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PMID:Identification of DNA replication and cell cycle proteins that interact with PCNA. 939 13

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is present in tobacco smoke and is hepatocarcinogenic in rats. Its bioactivation in rat hepatocytes leads to methylation and pyridyloxobutylation of DNA. Rat hepatocytes were cultured in serum-free William medium E on collagen-coated dishes. We demonstrated that some enzymes of the base and/or excision-repair pathways were involved in repair of NNK-induced DNA damage, measured by [methyl-3H] thymidine incorporation. Unscheduled DNA synthesis (UDS) induced by N-methyl-N-nitrosourea (MNU), NNK, N'-nitrosonornicotine (NNN) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) increased 2.9-, 2.8-, 1.5- and 3.5-fold, respectively, suggesting that methylated and/or pyridyloxobutylated-DNA by these four nitroso compounds is repaired by the excision pathway. Moreover, levels of NNK-induced UDS were dose (1-3 mM) and time (1-18 h) dependent. Enzymes involved in the excision repair pathways were selectively inhibited. Inhibitors of DNA topoisomerase I (camptothecin) and topoisomerase II (etoposide, nalidixic acid) did not decrease the induction of UDS, suggesting that topoisomerases are not involved in the repair of NNK-induced damage. While aphidicolin and arabinocytidine (DNA polymerase alpha, delta, epsilon inhibitors) totally inhibited NNK- and NNKOAc-induced UDS, dideoxythymidine (DNA polymerase beta inhibitor) inhibited NNK- and NNKOAc-induced UDS by 40 and 33%, respectively. We conclude that DNA polymerase alpha, delta or epsilon and to a lesser degree polymerase beta are involved in the repair of pyridyloxobutylated DNA. Previous studies showed that inhibition of poly(ADP-ribosyl) polymerase (PARP) by 3-aminobenzamide (3-ab) facilitated DNA ligation. Our results demonstrate that 3-ab increased NNK-induced UDS, but does not affect NNKOAc-induced UDS. These observations suggest that the ligation step is rate limiting in the repair of methylated DNA but not of pyridyloxobutylated DNA.
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PMID:Modulation of DNA repair by various inhibitors of DNA synthesis following 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced DNA damage. 956 22

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.
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PMID:Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism. 969 Dec 46

A series of 2-acetyl-pyridyl-4N-substituted thiosemicarbazones copper(II) complexes was evaluated for their cytotoxic mode of action in a variety of human and rodent tumor cell cultures. It was determined that these compounds may induce cytotoxicity by affecting several metabolic pathways including a reduction in de novo purine synthesis, and inhibition of IMP dehydrogenase, and DNA polymerase alpha activities. Selected compounds also demonstrated the ability to inhibit L1210 DNA topoisomerase II activity at micromolar concentrations. These agents were able to antagonize etoposide-induced formation of cleavable complexes as measured by K+/SDS precipitation and in vitro cleavage reactions.
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PMID:The cytotoxicity of copper(II) complexes of 2-acetyl-pyridyl-4N-substituted thiosemicarbazones. 989 58

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase alpha at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase alpha activity.
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PMID:Mode of action of thiocoraline, a natural marine compound with anti-tumour activity. 1036 4

We chemically synthesized epolactaene, a neuritogenic compound in human neuroblastoma cells, and investigated its biochemical action in vitro. Epolactaene and its derivatives selectively inhibited the activities of mammalian DNA polymerase alpha and beta and human DNA topoisomerase II, with IC(50) values of 25, 94, and 10 microM, respectively. By comparison with its structural derivatives, the long alkyl side chain in epolactaene seemed to have an important role in this inhibitory effect. The compound did not influence the activities of plant or prokaryotic DNA polymerases or of other DNA metabolic enzymes such as telomerase, RNA polymerase, and deoxyribonuclease I. Epolactaene did not intercalate into DNA. These results suggested that the neuritogenic compound epolactaene influences both DNA polymerases and topoisomerase II despite the dissimilarity in both structure and properties of these two enzymes and that inhibition of these enzymes could be related to the neuritogenic effect in human neuroblastoma cells. The relationship between the neuritogenic mechanism and cell cycle regulation by epolactaene was also discussed.
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PMID:Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, selectively inhibits the activities of mammalian DNA polymerases and human DNA topoisomerase II. 1087 81

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.
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PMID:Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats. 1096 99

(R)-(-)-Elenic acid (R-2,4-dimethyl-22-(p-hydroxyphenyl)-docos-3(E)-enoic acid) (EA) is a DNA topoisomerase II inhibitor found in an Indonesian sponge, Plakinastrella sp. We found and report here that it is a potent inhibitor of calf DNA polymerase alpha (IC(50)=7.7 microM) and rat DNA polymerase beta (IC(50)=12.9 microM). EA did not bind to DNA directly. EA did not influence the activities of DNA polymerases such as plant DNA polymerases I and II and prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I, or other DNA metabolic enzymes such as human immunodeficiency virus (HIV) reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. Interestingly, EA was also an inhibitor of DNA topoisomerases I and II, although the enzymatic characteristics including modes of action, amino acid sequences and three-dimensional structures were markedly different from those of DNA polymerases. EA could prevent the growth of NUGC-3 cancer cells, and the LD(50) value was 22.5 microM. The cells were halted at G1 and G2/M phase in the cell cycle. From these results, the action mode of EA is discussed.
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PMID:Selective inhibition of the activities of both eukaryotic DNA polymerases and DNA topoisomerases by elenic acid. 1185 91

Factors that reduce the intracellular concentration of triphosphorylated cytarabine (ara-CTP), the active form of cytarabine (ara-C), may induce chemoresistance in acute myeloid leukaemia (AML) patients. These factors include reduced influx of ara-C by the hENT1 transporter, reduced phosphorylation by deoxycytidine kinase (dCK), and increased degradation by high Km cytoplasmic 5'-nucleotidase (5NT) and/or cytidine deaminase (CDD). Increased levels of DNA polymerase alpha (DNA POL) and reduced levels of topoisomerase I (TOPO I) and topoisomerase II (TOPO II) have also been detected in ara-C-resistant cell lines. To determine whether these factors are implicated in clinical ara-C resistance, we analysed the expression of these parameters at diagnosis, using reverse transcription polymerase chain reaction, in the blast cells of 123 AML patients treated with ara-C. At diagnosis, hENT1, dCK, CDD, 5NT, TOPO I, TOPO II, DNA POL and MDR1 were expressed in 83%, 22%, 7%, 37%, 59%, 37%, 39% and 16% of patients respectively. In univariate analysis, patients with expression of 5NT or DNA POL at diagnosis had significantly shorter disease-free survival (DFS). In multivariate analysis, DNA POL positivity and hENT1 deficiency were related to a shorter DFS. In univariate analysis, patients with 5NT-positive blasts had significantly shorter overall survival (OS). In multivariate analysis, shorter OS was related to DNA POL positivity. These results suggest that expression of DNA POL, 5NT and hENT1 at diagnosis may be resistance mechanisms to ara-C in AML patients.
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PMID:In vivo mechanisms of resistance to cytarabine in acute myeloid leukaemia. 1206 Jan 21


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