The Radioresistance to Killing of A1–5 Cells Derives from Activation of the Chk1 Pathway

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G2 checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G2 checkpoint. Here we report that A1–5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G2 arrest response when compared with its counterpart, B4, although A1–5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1–5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1–5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G2 arrest and at the same time reduces the phosphorylation of Cdc2 in A1–5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1–5 cells to radiation-induced killing and reduces the G2 arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G2 arrest in A1–5 cells. Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G2 checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G2 checkpoint. Here we report that A1–5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G2 arrest response when compared with its counterpart, B4, although A1–5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1–5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1–5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G2 arrest and at the same time reduces the phosphorylation of Cdc2 in A1–5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1–5 cells to radiation-induced killing and reduces the G2 arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G2 arrest in A1–5 cells. ataxia telangiectasia-mutated ATM- and Rad3-related double strand breaks homologous recombination A1–5 and B4 are two independently isolated cell lines that were derived in the same laboratory from primary rat embryo fibroblasts transformed with activated Ras (T24) and mutant p53val-135 (1Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (494) Google Scholar). Both A1–5 and B4 cells show the typical phenotypes of the temperature-sensitive p53val-135(1Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (494) Google Scholar), but as reported here, only A1–5 cells show extreme radioresistance to killing accompanied by a strong G2checkpoint response. G2 checkpoint activation plays an important role in promoting cell survival following DNA damage (2Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1772) Google Scholar). The mechanism that regulates G2 arrest after DNA damage is conserved among species from yeast to human. The DNA damage checkpoint activated in G2 is believed to be mediated, at least in part, by an inhibition of the Cdc25C phosphatase that activates the Cdc2 kinase by removing inhibitory phosphates, thus allowing entry into mitosis (3Peng C.-Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1190) Google Scholar,4Dalal S.N. Schweitzer C.M. Gan J. DeCaprio J.A. Mol. Cell. Biol. 1999; 19: 4465-4479Crossref PubMed Scopus (241) Google Scholar). Cdc25C could be phosphorylated in vitro at serine 216 by either Chk1 or Chk2 (5Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1126) Google Scholar, 6Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (475) Google Scholar, 7Nurse P. Cell. 1997; 91: 865-867Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). This phosphorylation creates a binding site for the small acidic proteins 14-3-3 (3Peng C.-Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1190) Google Scholar) that cause the transport of Cdc25C to the cytoplasm and prevent Cdc2 activation. The Chk1 and Chk2 (Chk2 is the homologue of Rad53 in Saccharomyces cerevisiae and Cds1 in Schizosaccharomyces pombe) kinases, two important checkpoint regulators (3Peng C.-Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1190) Google Scholar, 5Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. 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Zhou B.-B.S. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar), were initially cloned in yeast, but homologues were subsequently identified in mammalian cells. Following DNA damage, both Chk1 and Chk2 are activated in human cells; however, it is not clear which pathway, Chk1/Cdc25C/Cdc2 or Chk2/Cdc25C/Cdc2, has a dominant role in G2 arrest. In this study we examine whether Chk1 or Chk2 plays the major role in the strong G2 arrest and the radioresistance to killing of A1–5 cells. UCN-01 and caffeine are two efficient inhibitors of G2checkpoint activation (14Lau C.C. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2942-2946Crossref PubMed Scopus (283) Google Scholar, 15Schlegel R. Pardee A.B. Science. 1986; 232: 1264-1266Crossref PubMed Scopus (268) Google Scholar, 16Powell S.N. DeFrank J.S. Connell P. Eogan M. Preffer F. Dombkowski D. Tang W. Friend S. Cancer Res. 1995; 55: 1643-1648PubMed Google Scholar, 17Yao S.-L. Akhtar A.J. McKenna K.A. Bedi G.C. Sidransky D. Mabry M. Ravi R. Collector M.I. Jones R.J. Sharkis S.J. Fuchs E.J. Bedi A. Nat. Med. 1996; 2: 1140-1143Crossref PubMed Scopus (159) Google Scholar, 18Wang Q. Fan S. Eastman A. Worland P.J. Sausville E.A. O'Connor P.M. J Natl. Cancer Inst. 1996; 88: 956-965Crossref PubMed Scopus (450) Google Scholar, 19Bunch R.T. Eastman A. Clin. Cancer Res. 1996; 2: 791-797PubMed Google Scholar, 20Bunch R.T. Eastman A. Cell Growth Differ. 1997; 8: 779-788PubMed Google Scholar) that act by targeting different proteins. UCN-01, a protein kinase inhibitor, potentiates the cytotoxicity of a variety of anticancer agents, including cisplatin, camptothecin, and ionizing radiation (18Wang Q. Fan S. Eastman A. Worland P.J. Sausville E.A. O'Connor P.M. J Natl. Cancer Inst. 1996; 88: 956-965Crossref PubMed Scopus (450) Google Scholar, 19Bunch R.T. Eastman A. Clin. Cancer Res. 1996; 2: 791-797PubMed Google Scholar, 20Bunch R.T. Eastman A. Cell Growth Differ. 1997; 8: 779-788PubMed Google Scholar, 21Shao R.-G. Cao C.-X. Shimizu T. O'Connor P.M. Kohn K.W. Pommier Y. Cancer Res. 1997; 57: 4029-4035PubMed Google Scholar). Therefore, it is currently undergoing testing in clinical trials for the treatment of human cancer. It is believed that UCN-01 sensitizes cells to DNA damage by abrogating the G2 checkpoint. Although UCN-01 inhibits multiple protein kinases (22Kawakami K. Futami H. Takahara J. Yamaguchi K. Biochem. Biophys. Res. Commun. 1996; 219: 778-783Crossref PubMed Scopus (127) Google Scholar), the way UCN-01 abrogates the G2 checkpoint is mainly by inhibiting Chk1 and affecting the Chk1-Cdc25C but not the Chk2-Cdc25C regulatory pathway (23Graves P.R., Yu, L. Schwarz J.K. Gales J. Sausville E.A. O'Connor P.M. Piwnica-Worms H. J. Biol. Chem. 2000; 275: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 24Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar). Caffeine, which sensitizes cells to ionizing radiation and other genotoxic agents by abrogating DNA damage checkpoints, has been shown to be an effective inhibitor of ATM1 and ATR (25Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar). ATM and ATR, both members of the phosphatidylinositol 3-kinase family, are upstream activators of Chk1 and Chk2 after DNA damage (8Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1089) Google Scholar, 12Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar, 13Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.-B.S. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar, 26Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar). Recently it has been reported that in mammalian cells, caffeine abolishes the G2 checkpoint by inhibiting the ATM-Chk2 pathway (27Zhou B.-B.S. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). To determine the effects of Chk1 and Chk2 on G2 checkpoint response and radioresistance to the killing of A1–5 cells, we used these and and Chk1 or Chk2 antisense in results to the Chk1 pathway as the major player for the extreme radioresistance and the strong G2 checkpoint response in A1–5 cells. The two cell A1–5 and B4 from A. (1Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (494) Google Scholar), were in with were at in an of and Caffeine in and UCN-01 from the and of in were to the cells were an at and with a The effective to radiation by the of In cells were with of were with or for and and to for were and at were for and for in the of to to were with cells were in with of were with either caffeine or UCN-01 for and to and to different cells were and in were in the and in at for The of cells in the cell cycle in a S.J. R. T. G. Cancer Res. 2000; 60: Google Scholar), cells were with the of DNA cells were and to the at the cells were were with an of to cells. were in and for the were in a and and for with in the same similar to determine the of DNA that in this cells were in to and after in in and at for were and DNA were as the of from the into the by of a The of DNA by this the capacity of the cells to nonhomologous end-joining repair. were with caffeine or UCN-01 for and to of were for at as and to cell this cell were in and and to of at for the and the protein to the ATR, Chk2, and which both and mutant were from The from The ATM from The from Cell The from The expression of these proteins were in a cell with of Chk1 or Chk2 in the of of a protein in of and in and for at were with and with and The kinase at for with of by (27Zhou B.-B.S. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar) in of and of were by and the kinase were by the into the Cdc25C protein the The antisense of Chk1 and Chk2 are to at the of the of Chk1 or Chk2 The used in this study are by The were to cells by to the antisense were to This to A1–5 cells in a with to the the cells were and to The cells were for the the and by the cell in A1–5 and B4 cells have a similar (1Martinez J. Georgoff I. Martinez J. Levine A.J. Genes Dev. 1991; 5: 151-159Crossref PubMed Scopus (494) Google Scholar). in the of DNA damage, no in phenotypes are after to A1–5 cells are to killing as compared with B4 cells The difference in is that in other transformed rat embryo fibroblast cell lines C.C. B. Res. PubMed Scopus Google Scholar, G. E.J. R.J. Res. 1991; PubMed Scopus Google Scholar, Y. G. Cancer Res. Google Scholar). The radioresistance of A1–5 could be by either caffeine or caffeine or UCN-01 the survival of A1–5 cells to similar to that of B4 cells. The that inhibitors of checkpoint activation A1–5 cells to radiation-induced killing that a strong checkpoint response the radioresistance of these cells. The results the between checkpoint response and cell to killing in A1–5 cells. shown in is a difference in the of cells between A1–5 and B4 cells. B4 cells show a in G2 after to but cells the arrest and after after to the same A1–5 cells a in the and an G2 The is not the This response is the we in a cell the cells were with either caffeine or UCN-01, the G2 response in A1–5 cells is The of A1–5 cells in the is similar to that of B4 cells, and the cells the arrest and after DNA are thought to be that or to cell G. 1991; PubMed Scopus Google Scholar, M. Biophys. PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar). Therefore, we whether the or of DNA the radioresistance of A1–5 cells. is no difference in the of DNA between A1–5 and B4 cells In addition, the of of DNA are similar in the two cell a similar capacity for nonhomologous end-joining whether the in the between A1–5 and B4 cells from in the of expression of either the human or the mutant Therefore, we the of expression of these proteins after is no difference in the of or expression between A1–5 and B4 cells. In addition, the and G2 response of A1–5 cells is not by a in from to or to not caffeine UCN-01 the expression of these proteins not transformed cell lines in by with the same have a similar to that of B4 cells not These results in suggest that and expression are not to the extreme radioresistance with the strong G2 checkpoint response of A1–5 cells. Chk1 and Chk2 are important regulators of the G2 we whether are in the strong G2 checkpoint response in A1–5 cells. shows that the of Chk1 were higher in A1–5 in B4 cells. The difference is much after a G2 arrest after DNA damage is by inhibitory on Cdc2 the of To determine whether the higher expression of Chk1 is to the pathway, we compared Cdc2 expression as as its phosphorylation in A1–5 and B4 cells. Although is no difference in Cdc2 expression between A1–5 and B4 cells, and is no in Cdc2 expression after not is a difference in the of Cdc2 phosphorylation between A1–5 and B4 cells The of phosphorylated Cdc2 is higher in A1–5 cells in B4 cells, which is with the higher Chk1 expression and the of cells in G2 and at for similar to the of Chk1 the of phosphorylated Cdc2 in A1–5 and B4 cells with a stronger in A1–5 cells Caffeine or UCN-01 reduces Chk1 expression in A1–5 cells at after and with the of Cdc2 which to the similar in A1–5 and B4 cells and results are when the kinase of Chk1 is but is by the in the of the protein not a is between Chk1 activation and G2 arrest in A1–5 cells. Chk2 could the of Cdc2 the of we whether Chk2 to the phosphorylation of Cdc2 in A1–5 cells. shows that is no difference in the of Chk2 expression between A1–5 and B4 cells, although Chk2 expression in both cell lines after In addition, caffeine UCN-01 Chk2 expression in A1–5 or B4 cells To determine whether this at the of kinase we the of Chk2 to phosphorylate The results are with at the of protein expression and suggest that is no difference in Chk2 between A1–5 and B4 cells In addition, caffeine UCN-01 the Chk2 kinase These suggest that Chk2 is not to the phosphorylation of Cdc2 in A1–5 cells. To that the higher expression of Chk1 is the major for the extreme radioresistance with the stronger G2 arrest response in A1–5 cells, we the effects of Chk1 or Chk2 antisense on the survival and G2 arrest of A1–5 cells. the Chk1 and Chk2 antisense show a different to A1–5 cells. with the cells, the cells with the Chk1 antisense show a of and of the at after the treatment On the other hand, Chk2 shows on the cell whether the antisense could Chk1 or Chk2 The are shown in the Chk1 or Chk2 antisense oligonucleotide reduces the protein as we the oligonucleotide the survival of A1–5 cells to the similar to that after UCN-01 treatment oligonucleotide reduces the strong G2 response in A1–5 cells The of the Chk1 A1–5 cells in the is much that of A1–5 cells, and cells the arrest and after radiation the same the Chk2 antisense oligonucleotide has on A1–5 cell survival and G2 arrest and These provide the that the Chk1 but not the Chk2 pathway plays the major role in the phenotypes of A1–5 cells. ATM and ATR are upstream of To determine whether the activation of these upstream the activation of the pathway, we expression in A1–5 cells. Although the expression of ATM and ATR increases after is no difference in the expression of these proteins between A1–5 and B4 cells not This that the higher expression of Chk1 in A1–5 cells is not to the expression of ATM or The activation of DNA damage the the cycle and which in increases the survival Although the mechanism of checkpoint response in its the have been A1–5 cells with extreme radioresistance and strong G2 checkpoint response provide a for the between to killing and checkpoint activation. The results that the radioresistance to the killing of A1–5 cells can be by the activation of the G2 checkpoint and suggest that G2 checkpoint activation cell Cdc2 is in an but can be activated by Therefore, the of Cdc25C could to an arrest of cells in G2 P. Cell. 1997; 91: 865-867Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Chk1 or Chk2 could phosphorylate Cdc25C after DNA damage and It is important to whether both of are in the strong G2 arrest in A1–5 cells. The of this the of the A1–5 phenotypes to DNA damage and of the G2 checkpoint. results suggest that A1–5 cells with higher of Cdc2 phosphorylation are accompanied by a stronger arrest in that this as caffeine and UCN-01, the of Cdc2 as the strong G2 arrest in A1–5 cells is associated with an in Cdc2 is the that the phosphorylation of Cdc2 with the higher expression of Chk1 in A1–5 cells and that caffeine or UCN-01 not only reduces the of Cdc2 phosphorylation but the expression of It has been reported that Chk1 is phosphorylated and activated by upstream following DNA damage (5Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1126) Google Scholar). results show phosphorylation of Chk1 in A1–5 cells, which be to the that the is not to the phosphorylation of Chk1 Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar). On the other hand, the expression of Chk2 are not with the of Cdc2 UCN-01 at could Chk1 but has no on Chk2 (23Graves P.R., Yu, L. Schwarz J.K. Gales J. Sausville E.A. O'Connor P.M. Piwnica-Worms H. J. Biol. Chem. 2000; 275: 5600-5605Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 24Busby E.C. Leistritz D.F. Abraham R.T. Karnitz L.M. Sarkaria J.N. Cancer Res. 2000; 60: 2108-2112PubMed Google Scholar). In this of UCN-01 abolishes G2 arrest with radioresistance in A1–5 cells, which with the of Cdc2 These results suggest that Chk1 but not Chk2 is for Cdc2 phosphorylation in A1–5 cells. Caffeine is an inhibitor of checkpoint activation to cells to radiation (14Lau C.C. Pardee A.B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2942-2946Crossref PubMed Scopus (283) Google Scholar, S.P. Res. PubMed Scopus Google Scholar, R. J. Biol. 1982; Scholar, C. Res. 1982; PubMed Scopus (91) Google Scholar, T. C. J. Biol. PubMed Scopus Google Scholar, R. Res. PubMed Scopus Google Scholar). the mechanism by which caffeine abolishes the G2 checkpoint and cell to killing The that caffeine inhibits the kinase of ATM and ATR (25Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar) a mechanism for caffeine Following DNA damage, the activation of Chk2 ATM (8Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1089) Google Scholar, 12Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar, 13Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.-B.S. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (360) Google Scholar), the activation of Chk1 ATR Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (199) Google Scholar). Caffeine both Chk1 expression and Cdc2 phosphorylation but has only a small on the Chk1 but not the Chk2 antisense oligonucleotide reduces the G2 and sensitizes A1–5 cells to killing by These results suggest by mainly affecting the Chk1 pathway, caffeine abolishes the strong G2 arrest in A1–5 cells. results from of an study (27Zhou B.-B.S. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), which that caffeine abolishes the G2checkpoint by inhibiting the The be to the different cell lines used and the different time in which Chk2 results not the that the Chk2 pathway plays a role in the strong G2 arrest A. Matsuoka S. A. J. H. D. Elledge S.J. Science. 2000; PubMed Scopus Google Scholar) in A1–5 cells. In although Chk1 expression in A1–5 cells is to the similar to that in B4 cells at after at the same the of G2 in A1–5 cells is higher that in B4 cells which Chk2 activation. is no difference in the expression of ATR between A1–5 and B4 cells not the higher Chk1 expression not be to ATR, the upstream of It A1–5 and B4 cells, similar a different DNA damage response. is that the of Chk1 is it is or by the recombination in A1–5 cells. This is in laboratory it is thought that checkpoint activation is by the cell to DNA and thus to cell Although DNA are homologous recombination in yeast and nonhomologous end-joining in mammalian cells, show that both are conserved from yeast to R. J.H. Genes 1997; PubMed Scopus Google Scholar). The results that A1–5 cells show a stronger G2checkpoint response but no in either the or the nonhomologous end-joining of DNA It is not clear the G2 arrest DNA and cell It that ATM to activation C. N. A. S. J. 2000; 19: PubMed Scopus Google Scholar, S. L. Y. Y. Lee Lee Nature. 2000; PubMed Scopus Google Scholar), thus to the that checkpoint activation in cells. report shows that caffeine cell but can ataxia cells J. J. G. Oncogene. 2000; 19: PubMed Scopus (91) Google Scholar). These results suggest that an of caffeine be is in repair. The between checkpoint and DNA and effects on the cell to the role of checkpoint proteins the of the response to DNA damage B.-B.S. Elledge S.J. Nature. 2000; PubMed Scopus Google Scholar). Here we show that caffeine and UCN-01 have similar effects on A1–5 cells, which that both the same is the The pathway is for cell activation and be two by this pathway, and the pathway a role for the in to the other it To results with A1–5 cells, a this is shown in In this is the major pathway to DNA damage and arresting cells in This pathway not only by arresting cells in G2 but by repair. On the other hand, the pathway plays a role for the that these two to the and efficient of DNA damage and to The or the of this be and it for the different of DNA J. Levine for the A1–5 and B4 cells and Zhou for the to for in the of the