Chk1 Mediates S and G2 Arrests through Cdc25A Degradation in Response to DNA-damaging Agents

Abstract

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. 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. DNA damage caused by different agents induces cell cycle arrest at G1, S, or G2, thereby preventing replication of damaged DNA or aberrant mitosis until the damage is adequately repaired. These regulatory mechanisms are known as cell cycle checkpoints and involve an intricate network of protein kinase signaling pathways. They are central to the maintenance of genomic integrity and basic viability of the cells. Hence, defects in these pathways may result in either tumorigenesis or apoptosis depending on the severity and particular nature of the defects (3Walworth N. Curr. Opin. Cell Biol. 2000; 12: 697-704Crossref PubMed Scopus (115) Google Scholar, 4Zhou B.B. Elledge S. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar). Chk1 1The abbreviations used are: Chk1, checkpoint kinase 1; CDK, cyclin-dependent kinases; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; BrdUrd, bromodeoxyuridine; scrRNA, scrambled control RNA; siRNA, small interfering RNA; AT, ataxia telangiectasia; ATR, ataxia and Rad3 related.1The abbreviations used are: Chk1, checkpoint kinase 1; CDK, cyclin-dependent kinases; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; BrdUrd, bromodeoxyuridine; scrRNA, scrambled control RNA; siRNA, small interfering RNA; AT, ataxia telangiectasia; ATR, ataxia and Rad3 related. is a major checkpoint kinase and has been shown to be mainly responsible for the G2/M checkpoint (5Liu Q. Guntuku S. Cui X. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (191) Google Scholar). Upon DNA damage, Chk1 is activated by ATR-mediated phosphorylation. Active Chk1 phosphorylates the protein phosphatase Cdc25C at serine 216. This leads to the nuclear export of Cdc25C and its subsequent cytoplasmic sequestration by 14-3-3 protein, which prevents the activation of the downstream target of Cdc25C, the cyclin B/Cdc2 kinase that is responsible for G2/M transition (6Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1113) Google Scholar, 7Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1174) Google Scholar). In addition to the role of ATR/Chk1 in G2/M progression, recent experiments using dominant negative ATR showed that the ATR/Chk1 pathway may also play an important role in the S checkpoint induced by topoisomerase I inhibitors (8Cliby W.A. Lewis K.A. Lilly K.K. Kaufmann S.H. J. Biol. Chem. 2002; 277: 1599-1606Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Cdc25 phosphatases are essential cell cycle regulators. By removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases (CDKs), Cdc25 phosphatases activate the CDKs and promote cell cycle progression. In fission yeast, Cdc25 is a stimulator of the G2/M transition because of its activation of Cdc2. In mammalian cells, three Cdc25 analogues, Cdc25A, -B, and -C, have been identified (9Galaktionov K. Beach D. Cell. 1991; 67: 1181-1194Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 10Nagata A. Igarashi M. Jinno S. Suto K. Okayama H. New Biol. 1991; 3: 959-967PubMed Google Scholar) that rescue the growth defect of Cdc25-null Schizosaccharomyces pombe cells. In addition to the aforementioned Cdc25C, which promotes the G2/M transition through dephosphorylation of Cdc2, Cdc25B was proposed to act as an initiator of the same process (11Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar, 12Lammer C. Wagerer S. Saffrich R. Mertens D. Ansorge W. Hoffmann I. J. Cell Sci. 1998; 111: 2445-2453Crossref PubMed Google Scholar). In contrast, overexpression of Cdc25A activates cyclin E-Cdk2 and cyclin A-Cdk2 by inducing Cdk2 tyrosine dephosphorylation. This demonstrates that Cdc25A is involved in S phase (13Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (420) Google Scholar, 14Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (281) Google Scholar). Not surprisingly, Cdc25A overexpression causes a shortening of the G1 phase and faster S phase entry (15Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (252) Google Scholar, 16Sexl V. Diehl J.A. Sherr C.J. Ashmun R. Beach D. Roussel M.F. Oncogene. 1999; 18: 573-582Crossref PubMed Scopus (86) Google Scholar). Most interestingly, Cdc25A and -B, but not C have been found to be overexpressed in a subset of aggressive human cancers (17Galaktionov K. Lee A.K. Eckstein J. Draetta G. Meckler J. Loda M. Beach D. Science. 1995; 269: 1575-1577Crossref PubMed Scopus (498) Google Scholar, 18Dixon D. Moyana T. King M.J. Exp. Cell Res. 1998; 240: 236-243Crossref PubMed Scopus (52) Google Scholar, 19Wu W. Fan Y.H. Kemp B.L. Walsh G. Mao L. Cancer Res. 1998; 58: 4082-4085PubMed Google Scholar). The p53 tumor suppressor mediates DNA damage-induced G1 arrest, but has little or no effect on S or G2 arrest. Consequently, while normal cells mainly arrest in G1 in response to genotoxic stress, p53-null tumor cells are defective in G1 arrest. This leads to increased genetic instability in tumor cells, which further reinforces the tumorigenic process. On the other hand, tumor cells with p53-dependent G1 checkpoint deficiency rely more on the S or G2 checkpoint to repair their damaged DNA and preserve their essential genomic integrity for basic viability when treated with radiation or DNA-damaging drugs. Therefore, by specifically abrogating the G2 checkpoint, normal cells can still arrest in the G1 phase and repair the DNA damage; whereas, tumor cells that lack the G1 checkpoint will undergo mitotic catastrophe and eventually undergo apoptosis. These differences may confer a selective cytotoxicity in the tumor versus normal cells (20Powell S. DeFrank J. Connell P. Eogan M. Preffer F. Dombkowski D. Tang W. Friend S. Cancer Res. 1995; 55: 1643-1648PubMed Google Scholar, 21Koniaras K. Cuddihy A. Christopoulos H. Hogg A. O'Connell M. Oncogene. 2001; 20: 7453-7463Crossref PubMed Scopus (146) Google Scholar). Because of its prominent role in the DNA damage-induced G2 checkpoint, Chk1 has become an attractive target for this approach, and Chk1 inhibitors are being developed to potentiate the effects of genotoxic chemotherapeutics on p53-null tumors. Herein we demonstrate that camptothecin and doxorubicin, two DNA-damaging agents that cause S or G2 arrest, respectively, activate Chk1 and cause the rapid proteolysis of Cdc25A. Elimination of Chk1 expression through siRNA not only abrogated the S or G2 arrest, but also protected Cdc25A from degradation. These results suggest that Chk1 mediates the S or G2 phase checkpoints by targeting Cdc25A for proteolysis following chemically induced DNA damage. This study indicates that a Chk1 inhibitor may potentiate the cytotoxicity not only of DNA-damaging drugs causing G2 arrest, but also of agents conferring S arrest, which significantly broadens its applicability to cancer chemotherapy. Our results additionally demonstrate that Cdc25A plays functional roles not only in the G1/S transition but also in G2/M progression. This later conclusion is supported by our observation that both Cdc25A and Cdc25C overexpression can overcome the Chk1-mediated G2/M arrest induced by doxorubicin in a dose-dependent manner. Cell Culture—Human lung cancer cell line H1299 was obtained from ATCC (Manassas, VA). H1299 were grown in RPMI with and at in a was with Chk1 was with Chk1 from Cdc25C was by from was by from Cell Chk1 siRNA and scrambled control siRNA were to the of the Chk1 siRNA the following The scrambled siRNA from Chk1 siRNA siRNA H1299 cells were at a with of in The the cells were with siRNA with to the with of siRNA at was with of to A. In of was to of and at for and were and to for and to of the in a of Chk1 siRNA with of siRNA with of the were to of to A. In of was to of and at for and were to for and to Cdc25A degradation was with the following for either for for for doxorubicin, for cells were with and in with of activated and inhibitor were at for and on the and to using a The was with and with was using to the Cell in the cell was the cells were with to of the cells cells. The cells were and to the with the for at the cells were in and in The cells were with and treated with at for the cells were with and in the for or The were through using fluorescence-activated cell by of cells were for of cell and of the cells were using the by the Because cells in the more than of DNA of the cells were the cells at the the at the G2/M cells at the and the S phase cells and the of cell and of cells, also a to apoptosis and G2/M cells and a to cells in the S of DNA in the S H1299 cells were with siRNA on The cells were treated with camptothecin for by the cells with for the were for of and DNA were using the to the of Chk1 and Cdc25A in S was previously that Cdc25A proteolysis or UV N. J. Lukas C. M. J. Lukas J. Science. 2000; PubMed Scopus Google Scholar, J. N. J. Lukas J. Nature. 2001; PubMed Scopus Google which induce G2 or G1 arrest, this can be to DNA damage-induced arrest at other of the cell we the effect of a topoisomerase I inhibitor conferring S phase arrest. H1299 cells, a p53-null lung cancer cell were treated with or camptothecin and for of Cdc25A, Chk1 protein and cell cycle Cdc25A the and versus and its Cdc25C, not show in protein The of Cdc25A is because of because of cells with a specific abrogated this not the of Chk1 protein and the Chk1 only camptothecin addition and Chk1 activation in response to DNA damage. additionally that cells treated with camptothecin were in S phase and not further that Chk1 may S checkpoint by targeting Cdc25A for mediates both S and G2 Chk1 mediates doxorubicin-induced G2 was for H1299 cells with Chk1 or siRNA, treated with or doxorubicin for cell were and their in the cell cells phase S phase G2/M cells. Chk1 mediates S H1299 cells were with siRNA or control on The cells were treated with or camptothecin for by with for The cells were for cells in S phase were and the in the cell was of Chk1 and Cdc25A in G2/M also the effect of doxorubicin, a topoisomerase inhibitor conferring G2 arrest on different cancer cell H1299 cells and not to doxorubicin caused rapid degradation of Cdc25A while Cdc25C which can be to the and activation of Chk1 different DNA-damaging which cause at different of the cell to Chk1 activation and Cdc25A degradation. These are not specific to H1299 cells, because were also obtained from other cell and not Chk1 the of Cdc25A in to and the involvement of Chk1 in the DNA damage-induced proteolysis of Cdc25A, we an siRNA that specifically Chk1 Chk1 siRNA or the scrambled control RNA were H1299 cells that were to the same camptothecin scrRNA, we observed the proteolysis of Cdc25A in response to camptothecin at both and In contrast, with of the Chk1 siRNA, the proteolysis of Cdc25A was significantly The of the Cdc25A was by its with overexpressed Cdc25A protein not also The Chk1 the elimination of the Chk1 protein by the Chk1 siRNA but not the This result that Chk1 mediates the degradation of Cdc25A in response to camptothecin further this we Cdc25A in H1299 cells. Cdc25A is overexpressed camptothecin still induces the proteolysis of Cdc25A However, with Chk1 siRNA, this degradation was abrogated In the of Cdc25A was increased and that these Chk1 also the of Cdc25A in the of DNA damage. whether Chk1 also mediates doxorubicin-induced Cdc25A we treated H1299 cells with doxorubicin at to induce the proteolysis of Cdc25A. Chk1 this process and In contrast, the Chk1 siRNA this and These results suggest that Chk1 Cdc25A for degradation in response to both doxorubicin and Chk1 both S and whether Chk1 is involved in the doxorubicin-induced G2 checkpoint, we the cell cycle of H1299 cells in the of doxorubicin with or the elimination of Chk1 cells with were treated with doxorubicin for we observed a in the G2/M the of the G2 checkpoint and However, when Chk1 expression is the siRNA, the of G2/M arrest was significantly that Chk1 is responsible for the G2 Additionally, we observed a of cells in cells in versus in that of the checkpoint by Chk1 induced cell In to its role in the G2 checkpoint, the involvement of Chk1 in S phase arrest is not as widely demonstrate the role of Chk1 in S checkpoint, we a with H1299 cells to the S phase arrest. in the of were no differences in the cells, cells with Chk1 siRNA, and cells with camptothecin we a in of the S phase cells in either or cells to and to with the S phase arrest. However, Chk1 siRNA the DNA to in the of DNA damage, of the arrest and further that Chk1 also mediates S Chk1 was to the elimination of Chk1 protein by the siRNA not The of Cdc25A in G2/M we have shown the Chk1 mediates Cdc25A degradation and cells in either S or G2/M when induced by DNA-damaging further that Cdc25A plays a role in G2/M transition in addition to its more widely accepted in G1/S progression, we the cell cycle of H1299 cells with of expression either Cdc25A or Cdc25C The cells were treated with doxorubicin to induce G2 was to whether Cdc25 overexpression the DNA damage-induced G2 arrest. cells in the of doxorubicin showed a normal in G1 phase and a G2/M In contrast, cells a of the G2/M G2 arrest Cdc25C overexpression we observed a dose-dependent of the G2 arrest at the with its role in G2/M phase through dephosphorylation of C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1174) Google Scholar). In Cdc25A overexpression also a of the G2 arrest in a dose-dependent with the that Cdc25A mediates G2/M transition with as a of cells with of not confer not the overexpression of Cdc25A in the and further the of the observed of G2 arrest, we the same cell to for Cdc25A and Cdc2/Cdk1 Because is the kinase responsible for G2/M and its dephosphorylation at tyrosine by Cdc25C has been shown to cause its activation and phase (6Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1113) Google we that it also be the downstream target of Cdc25A. of of the Cdc25A a Cdc25A which doxorubicin and Chk1-mediated degradation in response to DNA damage. In contrast, with and of the significantly of Cdc25A, and more the overexpressed Cdc25A to proteolysis and but not the of Cdc25A the doxorubicin-induced of at tyrosine that Cdc25A activates through dephosphorylation of protein not show the different not The Cdc25A than the in more elimination of signal and in line with its more of the G2 arrest a negative of of a Cdc25A a (15Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (252) Google also in of Cdc25A to a that is than with same of and and and However, this Cdc25A is to and the thus that the phosphatase of Cdc25A is for protein was also for this and found to be the different not Cdc25C dephosphorylation of Cdc2. to Cdc25A, Cdc25C overexpression also to a in the signal in a dose-dependent that Cdc25C abrogates DNA damage-induced G2/M arrest of through its dephosphorylation. This that is a downstream target of Chk1 was proposed to play a role in the S or G2 checkpoints induced by DNA-damaging agents IR, and topoisomerase I inhibitors N. J. Lukas C. M. J. Lukas J. Science. 2000; PubMed Scopus Google Scholar, 4Zhou B.B. Elledge S. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar, W.A. Lewis K.A. Lilly K.K. Kaufmann S.H. J. Biol. Chem. 2002; 277: 1599-1606Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, X. J. G. Y. Cancer Res. 2002; Google Scholar). This is supported by the observation that in normal human and also in from with ataxia Chk1 is specifically at the S to phase of the cell cycle at both the RNA and protein and its is at the S to phase of the cell cycle N. H. H. A. K. M. A. K. M. Y. Oncogene. 1999; 18: PubMed Scopus Google Scholar). the involvement of Chk1 in these have not been and the mechanisms for these checkpoints still that Cdc25A protein Chk1-mediated degradation in response to DNA-damaging and this degradation with and the S or G2 Therefore, Chk1 elimination through siRNA leads to of both Cdc25A proteolysis and the S or G2 the of this a that Cdc25A is to Chk1-mediated proteolysis in response to DNA by H. Piwnica-Worms H. Sci. S. A. 2002; PubMed Scopus Google Scholar). This a study that that not Chk1, is responsible for Cdc25A degradation J. Lukas J. J. 2002; PubMed Scopus Google Scholar). In this we on two major topoisomerase inhibitors and the of cancer chemotherapy. is the commonly used and for doxorubicin is widely used to and cancers 2002; PubMed Scopus Google Scholar). Because and topoisomerase inhibitors confer DNA damage through different mechanisms may activate signal transduction pathways. The of cell cycle arrest by the topoisomerase inhibitors has not been This lack of has the of and of Our study this by that camptothecin and doxorubicin induce different cell cycle checkpoints and through the same process of targeting Cdc25A to degradation through Chk1, and Chk1 their Therefore, with we have shown that the checkpoint kinases to a of DNA by targeting Cdc25A to proteolysis and checkpoints at different of the cell Cdc25A G2/M in to G1/S is a of G1/S transition through its dephosphorylation and activation of Cdk2 kinase (9Galaktionov K. Beach D. Cell. 1991; 67: 1181-1194Abstract Full Text PDF PubMed Scopus (418) Google Scholar, I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (252) Google Scholar). However, we have that Cdc25A also rapid proteolysis in response to both S and G2 and of this through either elimination of Chk1 or Cdc25A overexpression abrogated the This that Cdc25A in both S phase and G2/M The is further supported by (a) a Cdc25A inhibitor cells at both G1 and and dephosphorylation of both Cdk2 and Cdc2/Cdk1 L. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google (b) of Cdc25A with mitosis (9Galaktionov K. Beach D. Cell. 1991; 67: 1181-1194Abstract Full Text PDF PubMed Scopus (418) Google and Cdc25A has been found to be of the cell cycle (13Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (420) Google Scholar, I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (252) Google Scholar). Cdc25C is a known mediator of G2/M transition through its activation of (6Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1113) Google Scholar, 7Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1174) Google Scholar). However, it may not be the only mediator of this process because are and not the of Cdc2, the of entry and the response to DNA damage were in Cdc25C, that Cdc25A Cdc25B may for of Cdc25C in the J. T. Piwnica-Worms H. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Our study that overexpression of Cdc25A overcome G2 arrest as as that of Cdc25C through dephosphorylation of Cdc2, the that is also a downstream target of Cdc25A Moreover, doxorubicin induces G2 arrest and Cdc25A degradation effect on Cdc25C and These suggest that Cdc25A may be a more and ubiquitous cell cycle than Cdc25C, and the pathway mediates both S and G2 Cdc25A and Cdc25C for G2/M cells is a of cyclin kinase which is for mitotic 1995; PubMed Scopus Google Scholar). this of kinase a of Cdc25 phosphatase may be the three Cdc25 Cdc25B is as an phosphatase that is responsible for the of cyclin kinase to in the mitotic process (11Gabrielli B.G. De Souza C.P. Tonks I.D. Clark J.M. Hayward N.K. Ellem K.A. J. Cell Sci. 1996; 109: 1081-1093Crossref PubMed Google Scholar, 12Lammer C. Wagerer S. Saffrich R. Mertens D. Ansorge W. Hoffmann I. J. Cell Sci. 1998; 111: 2445-2453Crossref PubMed Google Scholar). However, is an protein with a H. H. T. T. J. Cell Biol. 1997; PubMed Scopus Google and it is in a phase entry V. C. M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the cyclin kinase to the other of the Cdc25A and that the of protein is to the of kinase thus the of both are for phase progression. shown in this can be by the overexpression of either Cdc25A or Cdc25C cells show little mitotic J. T. Piwnica-Worms H. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). This not that Cdc25C plays no role in G2/M progression. only that Cdc25C is not essential for this process. other of the Cdc25 are to for its that in cells, Cdc25A may undergo of either at the or protein or at the phosphatase through to still a normal of Cdc25 phosphatase DNA-damaging Cdc25A it was that an S phase arrest, also Cdc25A degradation M. C. J. F. Draetta EMBO 2000; PubMed Scopus Google Scholar). showed that a topoisomerase I conferring S arrest, induces the same process specifically through Chk1. is not these two different of S will be to whether the DNA inhibitor causing S phase arrest through also leads to Cdc25A DNA damage through DNA this process not involve In contrast, camptothecin and doxorubicin induce DNA damage through of topoisomerase I and DNA by the DNA and DNA DNA I topoisomerase The cytotoxicity of their inhibitors from the of known as camptothecin and doxorubicin, other drugs in this and as as and 2002; PubMed Scopus Google Scholar). will be to whether we can our to the other topoisomerase this will further the of Chk1 inhibitors in the of cancer In our results showed that the pathway mediates both S and G2 This Chk1 as a more ubiquitous cell cycle checkpoint mediator and significantly broadens the of Chk1 inhibitor in the of cancer