The ATM/TP53/p21 pathway mediates the effect of chronic γ-irradiation on cell proliferation.

<p>(A) Western blotting was used to determine levels of TP53, phosphorylated TP53 (Ser-15), MDM2, MDMX, and p21 in human fibroblasts (TIG-3 p27) cultured under chronic γ-irradiation conditions for 24 or 96 hours (dose rates are indicated). β-actin served as a loading control. (B) Knockdown of TP53 or p21 in BJ1/hT cells abolishes the growth inhibitory effect of chronic γ-irradiation. Western blot analysis of cells transfected with control siRNA (si-Ctrl), TP53-specific siRNA (si-P53), or P21-specific siRNA (si-P21) are shown (top). β-tubulin served as a loading control. Whole cell lysates were prepared 48 hours after the transfection. Forty hours after the transfection. Forty-eight hours after transfection, BJ1/hT cells were placed into culture and grown under unirradiated control conditions (white circles) or a γ-irradiation dose rate of 0.347 mGy mGy/min (grey circles). The number of cells per dish was counted after indicated periods of time. (C) ATM kinase is activated in response to chronic γ-irradiation. BJ1/hT cells were exposed to indicated dose rates of chronic γ-irradiation in the presence or absence of the ATM kinase inhibitor KU55933 (10 µM). ATM kinase activity was assessed by detecting phosphorylated TP53 (Ser15) or phosphorylated CHEK2 (Thr68) by Western blotting. TP53 activation and P21 induction by chronic γ-irradiation were examined by Western blotting. (D) Inhibition of ATM kinase, but not DNA-PKcs, attenuates the growth inhibitory effect of chronic γ-irradiation. BJ1/hT cells were cultured under chronic γ-irradiation conditions at indicated dose rates in the presence or absence of KU55933 (10 µM) or the DNA-PKcs inhibitor NU7026 (10 µM). Values indicate the mean ± SD of three independent wells.