ATR-Chk1 Axis Is Activated, but the Function of Chk1 Is Disrupted in BCR/ABL Leukemia Cells Responding to DNA Damage.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.