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)

Three DNA damage-responsive cell cycle checkpoints can be shown to operate in diploid human fibroblasts. One checkpoint arrests growth in G1, another inhibits replicon initiation in S phase cells, and the third delays progression from G2 into mitosis. Progression from G2 into M is controlled in part by a cyclin-dependent kinase (cyclin B/Cdk1) that is regulated by tyrosine phosphorylation. Phosphorylation of Tyr15 on Cdk1 is inhibitory for kinase activity. Activation of cyclin B/Cdk1 at the onset of mitosis is accomplished by a phosphatase, Cdc25C, that interacts with cyclin B/Cdk1 in an autocatalytic feedback loop to remove the inhibitory phosphate at Tyr15 and activate kinase activity. DNA damage triggers G2 delay by inhibiting formation of the autocatalytic feedback loop so that dephosphorylation of Tyr15 does not occur. This suppression of activation of cyclin B/Cdk1 appears to account for the failure of damaged G2 cells to progress into mitosis. Once the damage to DNA is repaired, cells resume progression into mitosis as the cycle is re-engaged. The isoflavone genistein inhibits tyrosine kinases, including one that phosphorylates Cdk1 on Tyr15. This kinase, p56/p53lyn is rapidly induced by treatments that trigger cell cycle checkpoints (ionizing radiation, cytosine arabinoside), suggesting that this kinase may actively delay the onset of mitosis by phosphorylating Tyr15 on Cdk1. Genistein also inhibits type II DNA topoisomerase to produce a form of DNA damage that triggers all of the DNA damage-responsive cell cycle checkpoints. A brief 10 min incubation with the topoisomerase poison amsacrine was sufficient to trigger the S phase checkpoint response and inhibit replicon initiation. Inhibition of replicon initiation by 1 microM amsacrine was maximal 20-30 min after drug treatment and by 120 min, the checkpoint response had decayed to allow near control rates of replicon initiation. Topoisomerase II poisons also are powerful clastogens inducing lethal and carcinogenic chromosomal aberrations. Type II topoisomerase can break DNA in a region of chromosome 11q23 that contains the ataxia telangiectasia gene (ATM). The ATM gene controls all of the DNA damage-responsive cell cycle checkpoints. Chromosomal aberrations in 11q23 are frequently seen in acute myeloid leukemia that develops as a consequence of etoposide chemotherapy. Thus, topoisomerase poisons such as genistein may trigger chromatid breakage to inactivate AT gene function, disable cell cycle control, and induce genetic instability.
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PMID:Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints. 949 43

Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation.
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PMID:The ATM protein is required for sustained activation of NF-kappaB following DNA damage. 1032 72

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

Using a replica microwell method, four Chinese hamster lines which exhibit hypersensitivity to the topoisomerase I inhibitor camptothecin, designated CM1, CM2, CM3 and CM6, have been isolated. Their sensitivity towards camptothecin varied from 3.5- to 8.2-fold with relative sensitivity as follows: CM2 < CM3 < CM6 < CM1. Genetic analysis of the CM mutants has established that CM1, CM3 and CM6 fail to complement each other and can each be assigned to the irs2 (XRCC8) complementation group. The mutant CM2 could not be definitively assigned to a complementation group because it presented a semi-dominant phenotype. In contrast to their sensitivity to camptothecin, the four CM mutants were less sensitive (1.1- to 2.2-fold) to the topoisomerase II inhibitors etoposide and adriamycin, although CM1, CM3 and CM6 were more sensitive (2.5- to 3. 8-fold) to streptonigrin (a free radical generator and a topoisomerase II inhibitor). All four mutant lines displayed an increased sensitivity to the bifunctional alkylating agent mitomycin C (2.4- to 5.1-fold). Surprisingly, given their assignment to the irs2 (XRCC8) complementation group, CM1, CM3 and CM6 displayed only a minor increase in sensitivity to ionizing radiation (1.6-fold or less). Similar sensitivity of these CM mutants was observed for the radiomimetic compound bleomycin (1.7-fold sensitive or less). This study indicates that XRCC8 mutants are isolated at high frequency from the parent line V79 and that phenotypic heterogeneity amongst the irs2 (XRCC8) complementation group is greater than previously encountered. Mutations in different regions of the XRCC8 gene may be responsible for the differing cellular phenotypes. Hamster XRCC8 mutants show phenotypic similarities to cultured cells from ataxia telangiectasia and Nijmegen break syndrome (NBS) patients and are likely to be defective in the same pathway in which the ATM (ataxia telangiectasia-mutated) and the NBS genes operate.
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PMID:Isolation of camptothecin-sensitive chinese hamster cell mutants: phenotypic heterogeneity within the ataxia telangiectasia-like XRCC8 (irs2) complementation group. 1088 18

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

We report a novel MLL-associated chromosome translocation t(11;14)(q23;q24) in a child who showed signs of acute undifferentiated leukemia 3 years after intensive chemotherapy that included the topoisomerase-II inhibitor VP 16. Screening of a cDNA library of the patient's leukemic cells showed a novel fusion transcript between MLL and the Gephyrin (GPHN) gene on 14q24. The resulting MLL-GPHN fusion gene encodes MLL AT hook motifs and a DNA methyltransferase homology domain fused to the C-terminal half of Gephyrin, including a presumed tubulin binding site and a domain homologous to the Escherichia coli molybdenum cofactor biosynthesis protein MoeA. Genomic breakpoint analysis showed potential in vitro topoisomerase-II DNA-binding sites spanning the breakpoints in both MLL and GPHN but no flanking sequences that might mediate homologous recombination. This suggests that MLL-GPHN may have been generated by VP 16/topoisomerase-II-induced DNA double-strand breaks, followed by error-prone DNA repair via non-homologous end joining. Gephyrin was originally identified as a submembraneous scaffold protein that anchors and immobilizes postsynaptic membrane neurotransmitter receptors to underlying cytoskeletal elements. It also is reported to bind to phosphatidylinositol 3,4,5-triphosphate binding proteins involved in actin dynamics and downstream signaling and interacts with ATM-related family member RAFT1. Gephyrin domains in the chimeric protein therefore could contribute novel signal sequences or might modify MLL activity by oligomerization or intracellular redistribution.
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PMID:GPHN, a novel partner gene fused to MLL in a leukemia with t(11;14)(q23;q24). 1157 61

ATR, a human phosphatidylinositol 3-kinase-related kinase, is an important component of the cellular response to DNA damage. In the present study, we evaluated the role of ATR in modulating the response of cells to S phase-associated DNA double-stranded breaks induced by topoisomerase poisons. Prolonged exposure to low doses of the topoisomerase I poison topotecan (TPT) resulted in S phase slowing because of diminished DNA synthesis at late-firing replicons. In contrast, brief TPT exposure, as well as prolonged exposure to the topoisomerase II poison etoposide, resulted in subsequent G(2) arrest. These responses were associated with phosphorylation of the checkpoint kinase Chk1. The cell cycle responses and phosphorylation of Chk1 were markedly diminished by forced overexpression of a dominant negative, kinase-inactive allele of ATR. In contrast, deficiency of the related kinase ATM had no effect on these events. The loss of ATR-dependent checkpoint function sensitized GM847 human fibroblasts to the cytotoxic effects of the topoisomerase I poisons TPT and 7-ethyl-10-hydroxycamptothecin, as assessed by inhibition of colony formation, increased trypan blue uptake, and development of apoptotic morphological changes. Expression of kdATR also sensitized GM847 cells to the cytotoxic effects of prolonged low dose etoposide and doxorubicin, albeit to a smaller extent. Collectively, these results not only suggest that ATR is important in responding to the replication-associated DNA damage from topoisomerase poisons, but also support the view that ATM and ATR have unique roles in activating the downstream kinases that participate in cell cycle checkpoints.
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PMID:S phase and G2 arrests induced by topoisomerase I poisons are dependent on ATR kinase function. 1170 Mar 2

We have investigated the effects of three unrelated topoisomerase 2 inhibitors, genistein, adriamycin, and etoposide, on phosphorylation/activation of the checkpoint kinase Chk2 in normal or ATM-deficient (ATM-) human fibroblasts and in cells overexpressing a catalytically inactive ATR kinase. We demonstrate that genistein activates Chk2 in a strictly ATM-dependent manner, whereas etoposide and adriamycin can trigger Chk2 activation in long-term cultures of ATM- cells. Moreover, these two latter genotoxic compounds were found to activate Chk2 in fibroblasts expressing the dominant negative form of ATR. We also report a significant decrease in the accumulation in G2-phase of ATM- cells when genistein did not activate Chk2. In conclusion, our results strongly support that activation of Chk2 could be dependent on the type and/or extent of DNA damage and under the control of either an ATM-dependent or an ATM and, maybe, an ATR-independent pathway.
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PMID:Etoposide and adriamycin but not genistein can activate the checkpoint kinase Chk2 independently of ATM/ATR. 1174 20

The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes.
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PMID:Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80: analysis by cDNA microarray. 1195 61

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.
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PMID:Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization. 1242 35


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