G(2) DNA damage checkpoint inhibition and antimitotic activity of 13-hydroxy-15-oxozoapatlin

Checkpoints activated in response to DNA damage cause arrest in the G(1) and G(2) phases of the cell cycle. Inhibitors of the G(2) checkpoint may be used as tools to study this response and also to increase the effectiveness of DNA-damaging therapies against cancers lacking p53 function. Using a cell-based assay for G(2) check. point inhibitors, we have screened extracts from the NCI National Institutes of Health Natural Products Repository and have identified 13-hydroxy-15-oxozoapatlin (OZ) from the African tree Parinari curatellifolia. Flow cytometry with a mitosis-specific antibody showed that checkpoint inhibition by OZ was maximal at 10 muM, which released 20% of irradiated MCF-7 cells expressing defective p53 and 30% of irradiated HCT116p53(-/-) cells from G(2) arrest. OZ additively increased the response to the checkpoint inhibitors isogranulatimide and debromohymenialdisine, but it did not augment the effects of UCN-01 or caffeine. Unlike other checkpoint inhibitors, OZ did not inhibit ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), Chk1, Chk2, Plk1, or Ser/Thr protein phosphatases in vitro. Treatment with OZ also caused G(2)-arrested and cycling cells to arrest in mitosis in a state resembling prometaphase. In these cells, the chromosomes were condensed and scattered over disordered mitotic spindles. The results demonstrate that OZ is both a G(2) checkpoint inhibitor and an antimitotic agent.