Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G(2) checkpoint activation by the DNA strand-breaking nucleoside analogue 2 '-C-cyano-2 '-deoxy-1-beta-D-arabino-pentofuranosylcytosine

作者：Liu Xiaojun; Matsuda Akira; Plunkett William^*

来源：Molecular Cancer Therapeutics, 2008, 7(1): 133-142.

DOI：10.1158/1535-7163.MCT-07-0416

2'-C-Cyano-2'-deoxy-1-beta-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 G(2) 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 G(2) by CNDAC to similar extents, suggesting ATM is not required to activate the G(2) 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 G(2) checkpoint was abrogated due to depletion of ATR by small interfering RNA, and impaired in ATR-Seckel cells, indicating participation of ATR in this G(2) 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 G(2) 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 G(2) checkpoint in a spectrum of cell lines. Together, our data showed that ATR and DNA-PK cooperate in CNDAC-induced activation of the G(2) checkpoint pathway.

• 出版日期2008-1