cAMP does not affect the IR-induced activation of ATM or Chk2 but attenuates p53 accumulation by decreasing its stability. (A) Reh cells were treated with or without forskolin or 8-CPT-cAMP for 30 minutes before exposure to 10 Gy IR. Cells were harvested at the indicated times after IR and subjected to Western blot analysis with the indicated antibodies. The figure shows one representative blot of 3 experiments. (B) Reh cells were treated as described in the legend to Figure 1C. Four hours after IR treatment, a portion of cells was harvested and examined for the expression of p53 and actin by immunoblotting. The histogram shows the level of cell death, measured by analysis of PI uptake, in the remaining portion of cells 18 hours after IR treatment. The right panel shows the densitometric analysis of the p53 band with the value obtained for untreated cells set as 1 (n = 5). *P < .01 (relative to cells treated with IR only). (C) Reh cells were treated with or without forskolin for 30 minutes before exposure to 10 Gy IR. After 4 hours, cells were exposed to CHX (25 μg/mL) over a 60-minute time course. Cells were harvested at the indicated time points after addition of CHX and analyzed for the expression of p53 and actin proteins by immunoblotting. The immunoblot is representative of 4 independent experiments. (Bottom panel) The immunoblots were scanned and the p53 band intensity was normalized to that of actin. The obtained values were then plotted with the value for cells not treated with CHX set as 1 (n = 4).