KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation

Abstract

KU70 −/− and DNA-PKcs −/−/− chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 μM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70 −/− and DNA-PKcs −/−/− cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis −/− cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4 −/− , XRCC4 − , and XLF −/− cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.