GADD45 induction of a G(2)/M cell cycle checkpoint

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 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.

真核生物体内，G(1)/S与G(2)/M细胞周期检验点（cell cycle checkpoints）可响应遗传毒性应激，维持基因组稳定性。本研究报道了人类与小鼠细胞中存在Gadd45介导的G(2)/M检验点的遗传与功能学证据。通过向原代人成纤维细胞显微注射表达载体以升高Gadd45的表达水平，可将细胞阻滞于G(2)/M交界，其表型包括MPM2免疫阳性、4N DNA含量，且15%的细胞出现中心体分离。Gadd45介导的G(2)/M阻滞依赖于野生型p53：在p53基因敲除的李-弗劳默尼（Li–Fraumeni）成纤维细胞，或是共表达p53突变体的正常成纤维细胞中，均未观察到细胞阻滞现象。升高细胞周期蛋白B1（cyclin B1）与细胞分裂周期蛋白25C（Cdc25C）的表达水平，可抑制人成纤维细胞中Gadd45介导的G(2)/M阻滞，这表明Gadd45介导的G(2)/M检验点机制至少部分通过调控G(2)特异性激酶——细胞周期蛋白B1/p34(cdc2)的活性实现。通过使用GADD45基因敲除的人类或小鼠细胞，我们获得了Gadd45介导的G(2)/M检验点的遗传学与生理学证据。通过反义GADD45表达降低内源性Gadd45表达的人类细胞，在暴露于紫外线辐射或甲磺酸甲酯后，其G(2)/M检验点功能受损，但在电离辐射后仍可正常发生G(2)期阻滞。gadd45基因敲除小鼠（gadd45 −/−）的淋巴细胞同样保留了由电离辐射激活的G(2)/M检验点，但在紫外线辐射暴露后无法发生G(2)/M期阻滞。因此，哺乳动物基因组通过多重G(2)/M检验点响应特定类型的DNA损伤，从而获得保护。