Figure 2.
Importance of ATR kinase in p53 activation and cell resistance to Ni(II). H460 cells were treated with Ni(II) for 6 h in the presence of various inhibitors to identify a p53-targeting kinase: ATRi1: 1 μM AZD6738, ATRi2: 10 μM VE821, ATMi1: 10 μM KU55933, ATMi2: 1 μM KU60019, DNAPKi: 30 μM NU7026. A, phosphorylation levels of CHK1 (S317), CHK2 (T68) and KAP1 (S824) in Ni(II)-treated H460 cells. B, S15-p53 phosphorylation and p53 protein levels in the presence of inhibitors of DNA damage-responsive kinases (ATR, ATM, DNAPK). C, effects of inhibition of ATR alone and (D) ATR and ATM kinases together on p53 upregulation and phosphorylation of ATM targets (ATM-S1981, CHK2-T68). E, viability of H460 cells treated with Ni(II) for 18 h and assayed 48 h later (ATRi1: 0.5 μM AZD6738, ATRi2: 10 μM VE821). Means ± SD, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001. F, Ni(II) accumulation in H460 cells in the absence and presence ATR inhibitors. Cells were treated as in panel E and collected immediately for metal measurements. Data are means ± SD, n = 3. G, colony formation by Ni(II)-treated H460 cells in the absence and presence of 0.3 μM AZD6738 (ATRi1). Means ± SD, n = 3, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. H, viability of IMR90 cells grown and treated with Ni(II) under 5 or 20% O2. Cells were treated with Ni(II) for 48 h and assayed 24 h later. ATRi1: 0.3 μM AZD6738, ATRi2: 5 μM VE821. Data are means ± SD, n = 3, ∗∗p < 0.01, ∗∗∗p < 0.001.