P53 REGULATES CDC2 INDEPENDENTLY OF INHIBITORY PHOSPHORYLATION TO REINFORCE RADIATION-INDUCED G2 ARREST IN HUMAN-CELLS

We have investigated the influence of p53 on radiation-induced G2 cell cycle arrest using human H1299 cells expressing temperature-sensitive p53. Gamma-irradiated cells lacking p53 arrested transiently in G2 wi th Cdc2 extensively phosphorylated at the inhibitory sites Thr14 and T yr15, and with both Cdc2 and cyclin B1 restricted to the cytoplasm. Ac tivation of p53 by temperature shift resulted in a more protracted G2 arrest that could not be overridden by checkpoint-abrogating drugs. Su rprisingly, this enhancement of G2 arrest was associated with a marked lack of inhibitory phosphorylation of Cdc2 and with the nuclear local ization of both Cdc2 and cyclin B1. While transient expression of an A 14F15 mutant form of Cdc2 that is not subject to inhibitory phosphoryl ation induced mitotic catastrophe in cells lacking p53, the p53-expres sing cells were relatively refractory to this effect. Enforced express ion of p21(WAF1/CIP1) was sufficient to confer nuclear localization on Cdc2 in the p53 null cells, though immunodepletion experiments demons trated that only a small proportion of Cdc2 was stably associated with p21(WAF1/CIP1) in the p53-expressing cells. We conclude that a p53-de pendent pathway can operate after exposure of human cells to ionising radiation to promote G2 arrest accompanied by nuclear translocation ra ther than inhibitory phosphorylation of Cdc2.