FIGURE 5.

ATM suppresses DNA-PK activation in response to CPT. A, effects of ATM, replication, and transcription inhibitors on CPT-induced DNA-PKcs autophosphorylation. HeLa cells were treated with CPT (2 μm, 1 h) following ATM inhibitor (KU-55933; 10 μm, 1 h) with HU (2 mm, 10 min) or DRB (100 μm, 2 h) treatment, and DNA-PKcs autophosphorylation and Chk2 phosphorylation were analyzed by Western blotting. B, DNA-PK activation in ATM-deficient cells. Control cells (GM00637H or SuSa/Tn) and ATM-deficient cells (GM05849C or AT1OS/Tn) were treated with CPT (2 μm, 1 h), and DNA-PKcs autophosphorylation were analyzed by Western blotting. C, comet assay for detection of CPT-induced DNA strand breaks. HeLa cells were treated with CPT (0.25 μm) for 1 h following ATM inhibitor (10 μm, 1 h) treatment in the absence or presence of DRB (100 μm, 2 h), and induced-DNA strand breaks were detected by neutral comet assay. The comet tail moments were averaged in triplicate experiments, where the median among 100 cells was calculated in each experiment. Error bars represent S.E. calculated from three independent experiments. D, inhibition of ATM leads to DNA damage carry over from G₁ to S phase. U2OS cells were synchronized in M phase with nocodazole and released into G₁ and S phase upon incubation with fresh medium. Flow cytometry histograms show cell cycle profiles after release from the nocodazole block in the absence or presence of KU-55933 (10 μm, 1 h). Where indicated, cells were treated with CPT (5 μm, 1 h). The status of DNA-PK activation in the different experimental samples was determined by Western blotting with anti-phospho-DNA-PK antibodies.