Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G(2)-M arrest

Recent studies have suggested that p53 regulates the G(2) checkpoint in the cell cycle and that this function is required for the maintenance of genom ic integrity. In this study, we investigated a regulatory role of p53 speci fically in G(2)-M transition. Human bladder carcinoma cells lacking functio nal p53 were synchronized at G(1)-S, which is preceded by p53-mediated G(1) arrest. p53 expression in the synchronized cells was induced by infection with a recombinant adenovirus that encodes p53, After release from the G(1) -S arrest, the cells progressed to S-phase and G(2) but failed to enter mit osis. Biochemical analysis showed that p53 inhibits cell cycle-dependent ex pression of cdc2 and cyclin B1 and consequently inhibits cdc2 kinase. The r ole of cyclin B1-associated cdc2 kinase in p53-mediated G(2)-M arrest was f urther investigated by expression of both cyclin B1 and cdc2AF, in which in hibitory phosphorylation sites were substituted. The cells expressing both cdc2AF and cyclin B1 showed a constitutive activation of cdc2 kinase during cell cycle progression and passed through G(2)-M regardless of p53 express ion. Therefore, inactivation of cdc2 kinase through cdc2 and cyclin B1 repr ession is an essential step in p53-mediated G(2)-M arrest.