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Department of Radiation Oncology, Case Comprehensive Cancer Center, University Hospitals of Cleveland and Case Western Reserve University School of Medicine, Cleveland, Ohio

Requests for reprints: Timothy J. Kinsella, Department of Radiation Oncology, LTR 6068, University Hospitals of Cleveland/Ireland Cancer Center, 11100 Euclid Avenue, Cleveland, OH 44106-6068. Phone: 216-844-2530; Fax: 216-844-4799. E-mail: timothy.kinsella{at}uhhs.com

The DNA mismatch repair (MMR) system plays an important role in mediating a G₂-M checkpoint arrest and subsequent cell death following treatment with a variety of chemotherapeutic agents. In this study, using 6-thioguanine (6-TG) as a mismatch-inducing drug, we examine the role of ataxia telangiectasia mutated (ATM)/CHK2 and ATM and Rad-3 related (ATR)/CHK1 signaling pathways in MMR-mediated cell cycle responses in MMR-proficient human colorectal cancer RKO cells. We show that, in response to 6-TG (3 µmol/L x 24 hours), activating phosphorylation of CHK1 at Ser³¹⁷ [CHK1(pS317)] and CHK2 at Thr⁶⁸ [CHK2(pT68)] are induced differentially during a prolonged course (up to 6 days) of MMR-mediated cell cycle arrests following 6-TG treatment, with CHK1(pS317) being induced within 1 day and CHK2(pT68) being induced later. Using chemical inhibitors and small interfering RNA of the signaling kinases, we show that a MMR-mediated 6-TG-induced G₂ arrest is ATR/CHK1 dependent but ATM/CHK2 independent and that ATR/CHK1 signaling is responsible for both initiation and maintenance of the G₂ arrest. However, CHK2(pT68) seems to be involved in a subsequent tetraploid G₁ arrest, which blocks cells that escape from the G₂-M checkpoint following 6-TG treatment. Furthermore, we show that CHK2 is hyperphosphorylated at later times following 6-TG treatment and the phosphorylation of CHK2 seems to be ATM independent but up-regulated when ATR or CHK1 is reduced. Thus, our data suggest that CHK1(pS317) is involved in a MMR-mediated 6-TG-induced G₂ arrest, whereas CHK2(pT68) seems to be involved in a subsequent tetraploid G₁-S checkpoint. The two signaling kinases seem to work cooperatively to ensure that 6-TG damaged cells arrest at these cell cycle checkpoints.

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Received 2/ 9/04; revised 6/ 8/04; accepted 6/18/04.