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

Abstract

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 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 M, 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.In response to DNA damage, cell cycle progression pauses to allow time for DNA repair. This checkpoint response helps to maintain the integrity of the genome, and loss of checkpoints may enable the accumulation of mutations during carcinogenesis (1, 2). Cell cycle arrest is achieved by inhibiting the activities of cyclin-dependent kinases that govern entry into S phase or mitosis. In G 1 phase, DNA damage leads to the stabilization and nuclear localization of the p53 transcription factor, resulting in up-regulation of p21Waf1/Cip1 , an inhibitor of G 1 cyclindependent kinase activities (3-6). The G 2 checkpoint operates through a different mechanism, and the details of this pathway are beginning to emerge (7). In the current model, regulation is exerted through a phosphorylation cascade and by control of the subcellular localization of enzymes and their substrates. ATM 1 and ATR kinases play a role in the early signaling of DNA damage and cause phosphorylation of Chk1 and Chk2 kinases (8 -12). Phosphorylation of the phosphatase Cdc25C by Chk1 and Chk2 creates a 14 -3-3 binding site and inhibits its phosphatase activity in vitro, preventing its activation of Cdc2 kinase, the master regulator of mitosis (13-15). Cdc2 activity is also regulated by inhibitory phosphorylation by Wee1 and Myt1 kinases (16,17).Inhibitors of the G 2 checkpoint, such as caffeine, 2-aminopurine, pentoxifylline, staurosporine, and UCN-01, have been studied extensively in tissue culture model systems (18 -22). More recently, we have identified isogranulatimide (IGR) and debromohymenialdisine (DBH) as checkpoint inhibitors from marine inv...

G2 Checkpoint Kinase Inhibitors Exert Their Radiosensitizing Effects Prior to the G2/M Transition

“…Checkpoint inhibition was assessed by two-parameter flow cytometry as described in ref. 37. Briefly, irradiated cells were treated with checkpoint G 2 Checkpoint Inhibition and Radiosensitization kinase inhibitors and with 100 ng/mL nocodazole (Sigma-Aldrich Co.) to block cells that have entered mitosis.…”

Effect of combined DNA repair inhibition and G2 checkpoint inhibition on cell cycle progression after DNA damage

“…Flow Cytometry and Checkpoint Inhibitor Assay Cells were prepared for flow cytometry analysis exactly as described in (15). Checkpoint inhibition was assessed as described in ref.…”

“…Checkpoint inhibition was assessed as described in ref. 15, except that CHO cells were irradiated with 6.5 Gy and incubated for 8 hours followed by checkpoint inhibitor plus nocodazole addition for an additional 4 hours.…”

“…The cells were then analyzed by two-dimensional flow cytometry using the mitosis-specific antibody GF-7 to distinguish M cells from G 2 cells (ref. 15; see Supplementary Fig. S1 3 for examples).…”

In vitro Combination Treatment with Perifosine and UCN-01 Demonstrates Synergism against Prostate (PC-3) and Lung (A549) Epithelial Adenocarcinoma Cell Lines

“…It currently is being evaluated in early-phase clinical trials (17). Additional cellular targets of UCN-01 include the cell cycle checkpoint kinases chk1 (18,19) and possibly chk2 (20,21) with sensitization of treated cells to DNA damage. Recently, Sato et al (22) documented that an additional target of UCN-01 is the phosphatidylinositide-dependent kinase 1 (PDK1).…”