Threshold level of p53 required for the induction of apoptosis in X-irradiated MOLT-4 cells.

PURPOSE

To determine the threshold level for the initiation of apoptosis by studying the quantitative aspect of p53 response to DNA damage in individual cells, to better understand the process in X-ray-induced p53-dependent apoptosis.

METHODS AND MATERIALS

Time-sequential changes in p53 protein level were obtained for X-irradiated MOLT-4 cells using flow cytometry and analyzed.

RESULTS

The changes in the cellular frequency distribution pattern of p53 content could be divided into two parts at a certain p53 level. The p53 vs. side-scatter in flow cytometry showed the sequential changes of p53 increase followed by an increase in cell death. On the basis of these results we determined a threshold level of p53 for the initiation of apoptosis. The level was estimated to be (1.08 +/- 0.05) x 10(5) molecules per cell, which was approximately threefold higher than the mean content of control cells. The minimum times for p53 level to reach this threshold level were independent of X-ray dose and 1.4-1.6 h. The times for the signal transduction from the p53 accumulation to disruption of the mitochondrial membrane potential, caspase-3 activation, and cell death were 1.6, 2.1, and 2.8 h, respectively.

CONCLUSIONS

The threshold level of p53 for the initiation of apoptosis and the time sequence in the course of apoptotic events were determined in X-irradiated MOLT-4 cells.