GADD45 induction of a G-2/M cell cycle checkpoint

摘要

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, 4n DNA content and, in 15 of the cells, centrosome separatiou. The Gadd45-mediated Gz/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 p~53 mutants. Increased expression of cyclin BI 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-spccific kinase, cyclin BI/p~34cdc2. 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 impaireil G-2/M check point after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G-2 arrest after ionizing radiation. Lymphocytes from gadd45-kiiockout mice (gadd45 -/-) also retained a G-2/M checkpoint initiated by ionixing radiation and failed to arrest at G-2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is. protected by a multiplicity of