Contribution of Caspase‐3 Differs by p53 Status in Apoptosis Induced by X‐Irradiation

We investigated the effect of p53 status on involvement of caspase‐3 activation in cell death induced by X‐irradiation, using rat embryonic fibroblasts (REFs) transduced with a temperature‐sensitive mutant (mt) p53 gene. Cells with wild‐type (wt) p53 showed greater resistance to X‐irradiation than cells with mt p53. In cells with wt p53, X‐irradiation‐induced apoptosis was not inhibited by the caspase‐3 inhibitor acetyl‐L‐aspartyl‐L‐methionyl‐L‐glutaminyl‐L‐aspartyl‐aldehyde (Ac‐DMQD‐CHO) and caspase‐3 activity was not elevated following X‐irradiation, although induction of p53 and p21/WAF‐l protein was observed. In contrast, irradiated cells with mt p53 showed 89% inhibition of cell death with Ac‐DMQD‐CHO and 98% inhibition with the antioxidant N‐acetyl‐L‐cysteine (NAC). In cells with mt p53, caspase‐3 activity was increased approximately 5 times beyond baseline activity at 24 h after irradiation. This increase was almost completely inhibited by NAC. However, inhibition of caspase‐3 by Ac‐DMQD‐CHO failed to decrease production of reactive oxygen species by cells with mt p53. Differential involvement of caspase‐3 is a reason for differences in sensitivity to X‐irradiation in cells with different p53 status. Caspase‐3 activation appears to occur downstream from generation of reactive oxygen species occurring independently of wt p53 during X‐irradiation‐induced cell death.