8-Chloro-cAMP (8-Cl-cAMP) and its metabolite 8-chloro-adenosine (8-Cl-Ado) inhibit cell growth by 8-Cl-Ado-converted 8-Cl-ATP that targets cell-cycle control and RNA metabolism. However, the cell-cycle checkpoint pathways remain to be identified. Recent studies have shown that 8-Cl-cAMP administration and 8-Cl-Ado exposure may damage chromosomal DNA in vivo and in vitro. In this study, we demonstrate that 8-Cl-Ado-induced DNA damage activates G2/M phase checkpoint, which is associated with ATM-activated CHK1-CDC25C-CDC2 pathway joined by BRCA1-CHK1 branch in apoptosis-resistant human myelocytic leukemia K562 (p53-null) cells. Inhibition of CHK1 kinase by Gö6976, an inhibitor of CHK1 activity, can promote DNA damage and lead to the activation of CHK2, converting G2/M checkpoint into intra-S-phase checkpoint in which two parallel branches, the ATM-CHK2-CDC25A-CDK2 and the ATM-NBS1/SMC1 cascades, are involved. These observations may provide aid in better understanding of the mechanisms of 8-Cl-cAMP and 8-Cl-Ado actions and in potential design of the combined therapy.