G₂ DNA Damage Checkpoint Inhibition and Antimitotic Activity of 13-Hydroxy-15-oxozoapatlin*

Checkpoints activated in response to DNA damage cause arrest in the G₁ and G₂ phases of the cell cycle. Inhibitors of the G₂ 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 G₂ checkpoint 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 μm, which released 20% of irradiated MCF-7 cells expressing defective p53 and 30% of irradiated HCT116p53 ^−/− cells from G₂arrest. 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 G₂-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 G₂ checkpoint inhibitor and an antimitotic agent.