GADD45 induction of a G2/M cell cycle checkpoint

G(1)/S and G(2)/M cell cycle checkpoints main tain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and Functional evidence of a Gadd45-mediated G(2)/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G(2)/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G(2)/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G(2)/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G(2)/M checkpoint is at least in part through modulation of the activity of the G(2)-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G(2)/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G(2)/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G(2) arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G(2)/M checkpoint initiated by ionizing radiation and failed to arrest at G(2)/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G(2)/M checkpoints in response to specific types of DNA damage.