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)

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

p53 protects mammals from neoplasia by inducing apoptosis, DNA repair and cell cycle arrest in response to a variety of stresses. p53-dependent arrest of cells in the G1 phase of the cell cycle is an important component of the cellular response to stress. Here we review recent evidence that implicates p53 in controlling entry into mitosis when cells enter G2 with damaged DNA or when they are arrested in S phase due to depletion of the substrates required for DNA synthesis. Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3 sigma. Binding of Cdc2 to Cyclin B1 is required for its activity, and repression of the cyclin B1 gene by p53 also contributes to blocking entry into mitosis. p53 also represses the cdc2 gene, to help ensure that cells do not escape the initial block. Genotoxic stress also activates p53-independent pathways that inhibit Cdc2 activity, activation of the protein kinases Chk1 and Chk2 by the protein kinases Atm and Atr. Chk1 and Chk2 inhibit Cdc2 by inactivating Cdc25, the phosphatase that normally activates Cdc2. Chk1, Chk2, Atm and Atr also contribute to the activation of p53 in response to genotoxic stress and therefore play multiple roles. p53 induces transcription of the reprimo, B99, and mcg10 genes, all of which contribute to the arrest of cells in G2, but the mechanisms of cell cycle arrest by these genes is not known. Repression of the topoisomerase II gene by p53 helps to block entry into mitosis and strengthens the G2 arrest. In summary, multiple overlapping p53-dependent and p53-independent pathways regulate the G2/M transition in response to genotoxic stress.
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PMID:Regulation of the G2/M transition by p53. 1131 28

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.
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PMID:The human decatenation checkpoint. 1159 14

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

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.
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PMID:Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents. 1267 25

The p21(WAF1/Cip1) gene plays a central role in cell cycle regulation. Here we show that topoisomerase II inhibitors, genistein and etoposide, induce p21(WAF1/Cip1) expression mainly in a p53-dependent manner in human lung cancer cell line A549. However, although p53 accumulated, p21(WAF1/Cip1) expression did not depend on the level of Ser15 phosphorylation of p53. Caffeine, an ataxia telangiectasia-mutated (ATM), and ATM- and Rad3-related kinase (ATR) inhibitor, abrogated genistein-induced p21(WAF1/Cip1) and largely blocked etoposide-induced p21(WAF1/Cip1) expression. Wortmannin, an ATM- and DNA-dependent protein kinase inhibitor, partially inhibited p21(WAF1/Cip1) expression induced by genistein and etoposide, whereas UCN-01, a Chk1 inhibitor, partially blocked etoposide, but not genistein-induced p21(WAF1/Cip1) expression. These data suggest that both genistein and etoposide induce p21(WAF1/Cip1) expression in a p53-dependent manner. Genistein appears to stimulate p21(WAF1/Cip1) expression through p53 via ATM, whereas etoposide may activate both ATM and ATR pathways. Our results suggest different mechanisms participate in genistein and etoposide induced p21(WAF1/Cip1) expression.
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PMID:P21 response to DNA damage induced by genistein and etoposide in human lung cancer cells. 1276 22

Previous studies have suggested two possible roles for Rad9 in mammalian cells subjected to replication stress or DNA damage. One model suggests that a Rad9-containing clamp is loaded onto damaged DNA, where it participates in Chk1 activation and subsequent events that contribute to cell survival. The other model suggests that Rad9 translocates to mitochondria, where it triggers apoptosis by binding to and inhibiting Bcl-2 and Bcl-x(L). To further study the role of Rad9, parental and Rad9(-/-) murine embryonic stem (ES) cells were treated with camptothecin, etoposide, or cytarabine, all prototypic examples of three classes of widely used anticancer agents. All three agents induced Rad9 chromatin binding. Each of these agents also triggered S-phase checkpoint activation in parental ES cells, as indicated by a caffeine-inhibitable decrease in [3H]thymidine incorporation into DNA and Cdc25A down-regulation. Interestingly, the ability of cytarabine to activate the S-phase checkpoint was severely compromised in Rad9(-/-) cells, whereas activation of this checkpoint by camptothecin and etoposide was unaltered, suggesting that the action of cytarabine is readily distinguished from that of classical topoisomerase poisons. Nonetheless, Rad9 deletion sensitized ES cells to the cytotoxic effects of all three agents, as evidenced by enhanced apoptosis and diminished colony formation. Collectively, these results suggest that the predominant role of Rad9 in ES cells is to promote survival after replicative stress and topoisomerase-mediated DNA damage.
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PMID:Rad9 protects cells from topoisomerase poison-induced cell death. 1498 9

In Schizosaccharomyces pombe, topoisomerase III is encoded by a single gene, top3(+), which is essential for cell viability and proper chromosome segregation. Deletion of rqh1(+), which encodes the sole RecQ family helicase in S. pombe, suppresses the lethality caused by loss of top3. Here, we provide evidence suggesting that the lethality in top3 mutants is due to accumulation of aberrant DNA structures that arise during S phase, as judged by pulsed-field gel electrophoresis. Using a top3 shut-off strain, we show here that depletion of Top3 activates the DNA damage checkpoint associated with phosphorylation of the checkpoint kinase Chk1. Despite activation of this checkpoint, top3 cells exit the arrest but fail to undergo faithful chromosome segregation. However, these mitotic defects are secondary to chromosomal abnormalities that lead to the lethality, because advance into mitosis did not adversely affect cell survival. Furthermore, top3 function is required for maintenance of nucleolar structure, possibly due to its ability to prevent recombination at the rDNA loci. Our data are consistent with the notion that Top3 has a key function in homologous recombinational repair during S phase that is essential for ensuring subsequent fidelity of chromosome segregation.
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PMID:Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity. 1534 8

The requirement for the serine/threonine protein kinase ATM in coordinating the cellular response to DNA damage induced by ionizing radiation has been studied extensively. Many of the anti-tumor chemotherapeutics in clinical use today cause DNA double strand breaks; however, few have been evaluated for their ability to modulate ATM-mediated pathways. We have investigated the requirement for ATM in the cellular response to doxorubicin, a topoisomerase II-stabilizing drug. Using several ATM-proficient and ATM-deficient cell lines, we have observed ATM-dependent nuclear accumulation of p53 and ATM-dependent phosphorylation of p53 on seven serine residues. This was accompanied by an increased binding of p53 to its cognate binding site, suggesting transcriptional competency of p53 to activate its downstream effectors. Treatment of cells with doxorubicin led to the phosphorylation of histone H2AX on serine 139 with dependence on ATM for the initial response. Doxorubicin treatment also stimulated ATM autophosphorylation on serine 1981 and the ATM-dependent phosphorylation of numerous effectors in the ATM-signaling pathway, including Nbs1 (Ser(343)), SMC1 (Ser(957)), Chk1 (Ser(317) and Ser(345)), and Chk2 (Ser(33/35) and Thr(68)). Although generally classified as a topoisomerase II-stabilizing drug that induces DNA double strand breaks, doxorubicin can intercalate DNA and generate reactive oxygen species. Pretreatment of cells with the superoxide scavenger ascorbic acid had no effect on the doxorubicin-induced phosphorylation and accumulation of p53. In contrast, preincubation of cells with the hydroxyl radical scavenger, N-acetylcysteine, significantly attenuated the doxorubicin-mediated phosphorylation and accumulation of p53, p53-DNA binding, and the phosphorylation of H2AX, Nbs1, SMC1, Chk1, and Chk2, suggesting that hydroxyl radicals contribute to the doxorubicin-induced activation of ATM-dependent pathways.
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PMID:Doxorubicin activates ATM-dependent phosphorylation of multiple downstream targets in part through the generation of reactive oxygen species. 1548 21


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