The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1.

The investigational anticancer agent 7-hydroxystaurosporine (UCN-01) abrogates the G2 checkpoint in tumor cells and sensitizes them to the lethal effects of genotoxic anticancer agents. On the basis of the role of the Cdc25C phosphatase in maintenance of this damage-inducible checkpoint, we hypothesized that UCN-01 inhibits a component of the signal transduction pathway that modulates Cdc25C phosphorylation. Of the three kinases known to phosphorylate Cdc25C on Ser216, both checkpoint kinase 1 (hChk1) and Cdc25C-associated protein kinase 1 (cTAK1) were potently inhibited by UCN-01 with IC50s of 11 and 27 nM, respectively. Treatment of K562 erythroblastoid leukemia cells with similar drug concentrations resulted in decreased levels of Ser216 phosphorylation of Cdc25C and complete disruption of the y-radiation-induced G2 checkpoint. In contrast to hChk1, the hChk2 kinase was 100-fold more resistant to inhibition by UCN-01 (IC50, 1040 nM). These results suggest that disruption of the DNA damage-induced G2 checkpoint by UCN-01 is mediated through the inhibition of the Cdc25C kinases, hChk1 and cTAK1, and that hChk2 activity is not sufficient to enforce the G2 checkpoint in cells treated with a pharmacological inhibitor of hChk1.

Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine.

Caffeine exposure sensitizes tumor cells to ionizing radiation and other genotoxic agents. The radiosensitizing effects of caffeine are associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints. The similarity of these checkpoint defects to those seen in ataxia-telangiectasia (A-T) suggested that caffeine might inhibit one or more components in an A-T mutated (ATM)-dependent checkpoint pathway in DNA-damaged cells. We now show that caffeine inhibits the catalytic activity of both ATM and the related kinase, ATM and Rad3-related (ATR), at drug concentrations similar to those that induce radiosensitization. Moreover, like ATM-deficient cells, caffeine-treated A549 lung carcinoma cells irradiated in G2 fail to arrest progression into mitosis, and S-phase-irradiated cells exhibit radioresistant DNA synthesis. Similar concentrations of caffeine also inhibit gamma- and UV radiation-induced phosphorylation of p53 on Ser15, a modification that may be directly mediated by the ATM and ATR kinases. DNA-dependent protein kinase, another ATM-related protein involved in DNA damage repair, was resistant to the inhibitory effects of caffeine. Likewise, the catalytic activity of the G2 checkpoint kinase, hChk1, was only marginally suppressed by caffeine but was inhibited potently by the structurally distinct radiosensitizer, UCN-01. These data suggest that the radiosensitizing effects of caffeine are related to inhibition of the protein kinase activities of ATM and ATR and that both proteins are relevant targets for the development of novel anticancer agents.

Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin.

Members of the phosphatidylinositol-3 kinase related kinase (PIKK) family function in both cell cycle progression and DNA damage-induced cell cycle checkpoints. The fungal metabolite, wortmannin, is an effective radiosensitizer that irreversibly inhibits certain members of the PIKK family. Based on their roles in DNA damage responses, several PIKKs, DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and the ataxia- and Rad3-related protein (ATR), are potential targets for the radiosensitizing effect of wortmannin. In this report, we demonstrate that wortmannin is a relatively potent inhibitor of DNA-PK (IC50, 16 nM) and ATM (IC50, 150 nM) activities, whereas ATR activity is significantly less sensitive to this drug (IC50, 1.8 microM). In intact A549 lung adenocarcinoma cells, wortmannin inhibited both DNA-PK and ATM at concentrations that correlated closely with those required for radiosensitization. Furthermore, pretreatment of A549 cells with wortmannin resulted in radioresistant DNA synthesis, a characteristic abnormality of ATM-deficient cells. These results identify wortmannin as an inhibitor of ATM activity and suggest that ATM and DNA-PK are relevant targets for the radiosensitizing effect of this drug in cancer cells.