Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G₂ checkpoint activation by the DNA strand-breaking nucleoside analogue 2′-C-cyano-2′-deoxy-1-β-D-arabino- pentofuranosylcytosine

2′-C-Cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC), the prodrug (sapacitabine) of which is in clinical trials, has the novel mechanism of action of causing single-strand breaks after incorporating into DNA. Cells respond to this unique lesion by activating the G₂ checkpoint, affected by the Chk1-Cdc25C-cyclin-dependent kinase 1/cyclin B pathway. This study aims at defining DNA damage checkpoint sensors that activate this response to CNDAC, particularly focusing on the major phosphatidylinositol 3-kinase-like protein kinase family proteins. First, fibroblasts, deficient in ataxia-telangiectasia mutated (ATM), transfected with empty vector or repleted with ATM, were arrested in G₂ by CNDAC to similar extents, suggesting ATM is not required to activate the G₂ checkpoint. Second, chromatin associations of RPA70 and RPA32, subunits of the ssDNA-binding protein, and the ataxia-telangiectasia and Rad3-related (ATR) substrate Rad17 and its phosphorylated form were increased on CNDAC exposure, suggesting activation of ATR kinase. The G₂ checkpoint was abrogated due to depletion of ATR by small interfering RNA, and impaired in ATR-Seckel cells, indicating participation of ATR in this G₂ checkpoint pathway. Third, the G ₂ checkpoint was more stringent in glioma cells with wild-type DNA-dependent protein kinase catalytic subunit (DNA-PKcs) than those with mutant DNA-PKcs, as shown by mitotic index counting. CNDAC-induced G₂ arrest was abrogated by specific DNA-PKcs inhibitors or small interfering RNA knockdown in ML-1 and/or HeLa cells. Finally, two phosphatidylinositol 3-kinase-like protein kinase inhibitors, caffeine and wortmannin, abolished the CNDAC-induced G₂ checkpoint in a spectrum of cell lines. Together, our data showed that ATR and DNA-PK cooperate in CNDAC-induced activation of the G₂ checkpoint pathway.