GADD45 induction of a G 2 /M cell cycle checkpoint

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Abstract
G 1 /S and G 2 /M cell cycle checkpoints maintain 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, 4 n 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.