Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination

摘要

Purpose We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-beta-d-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-beta-d-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-beta-d-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. Methods Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. Results The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. Conclusion CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.