PML contributes to p53-independent p21 up-regulation in gamma-irradiation induced DNA damage responses

The cyclin-dependent kinase inhibitor p21(WAF1/Cip1) is a critical cell cycle regulator which translocates into the nucleus to participate in DNA repair during DNA damage responses. In the present study, we showed that the tumor suppressor, promyelocytic leukemia protein (PML) contributes to the up-regulation of p21 in a p53-independent pathway. Knock-down of PML in p53-null H1299 and HCT 116 (p53(-/-)) tumor cells by specific siRNA resulted in down-regulation of p21 protein expression, inhibition of gamma-irradiation-induced p21 up-regulation, and a decrease in p21 protein half-life. In PML knockdown H1299 cells, the down-regulation of p21 protein expression was reversed by MG132 treatment indicating that the proteasomal degradation of p21 protein was increased. Thus, PML positively regulates p21 expression by inhibiting proteasome-mediated proteolysis. Knockdown of PML decreased the repair of gamma-irradiation-induced double strand breaks (DSBs) as indicated by the delayed disappearance of gamma-H2AX foci and a decreased association between p21 and proliferating cell nuclear antigen (PCNA). Over-expression of p21 significantly restored the delayed DSB repair function. Taken together, these data provide evidence for a p53-independent functional relationship between PML and p21 in gamma-irradiation-induced DNA damage responses, and identify PML as a positive post-translational regulator of p21 in p53-deficient tumor cells.