Ionizing radiation down-regulates p53 protein in primary Egr-1(-/-) mouse embryonic fibroblast cells causing enhanced resistance to apoptosis

In this study, we sought to investigate the mechanism of the proapoptotic f unction of Egr-1 in relation to p53 status in normal isogenic cell backgrou nds by using primary MEF cells established from homozygous (Egr-1(-/-)) and heterozygous (Egr-1(+/-)) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1(+/-) cells (22.8%; p < 0.0001) wh en compared with Egr-1(-/-) cells (3.5%). Radiation elevated p53 protein in Egr-1(+/-) cells in 3-6 h, However, in Egr-1(-/-) cells, the p53 protein w as down-regulated 1 h after radiation and was completely degraded at the la ter time points. Radiation elevated the p53-CAT activity in Egr-1(+/-) cell s but not in Egr-1(-/-) cells. Interestingly, transient overexpression of E GR-1 in p53(-/-) MEF cells caused marginal induction of radiation-induced a poptosis when compared with p53(+/+) MEF cells. Together, these results ind icate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions ar e essential to mediate radiation-induced apoptosis, Rb, an Egr-1 target gen e, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were obser ved in Egr-1(-/-) MEF cells before and after radiation when compared with t he levels observed in Egr-1(+/-) cells. Elevated amounts of the p53-MDM2 co mplex and low amounts of Rb-MDM-2 complex were observed in Egr-1(-/-) cells after radiation. Because of a reduction in Rb binding to MDM2 and an incre ase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this le ads to inactivation of the p53-mediated apoptotic pathway in Egr-1(-/-) MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential tot overcome the antiapoptotic function of MDM2 on p53.