Mechanisms of G2/M cell cycle checkpoint controls

哺乳动物细胞已经进化出一个复杂的防御网络，通过防止内源性和外源性诱变剂对永久DNA损伤的固定来维持基因组的完整性。主要的基因组监测机制包括细胞周期检查点，它存在于G1-S和G2/M过渡阶段，并在DNA损伤的响应中受到调节。这些阶段的缺陷可能导致与肿瘤发生相关的突变表型。p53通过激活G1/S和G2/M细胞周期检查点来保护细胞应激期间的基因组。p53的两个下游靶点p21waf1和Gadd45似乎参与了这些途径。p53介导的G1/S检查点至少部分是由于p21waf1的激活。最近，我们发现Gadd45对于响应紫外线辐射或烷基化剂甲磺酸甲酯以p53依赖的方式激活的G2/M检查点之一至关重要。DNA损伤激活Gadd45，继而与g2特异性激酶Cdc2结合，并阻止其与调节亚基细胞周期蛋白B1的结合和其激酶活性的失活。阻断Gadd45的表达可以使肿瘤细胞对顺铂（一种破坏dna的癌症化疗药物）的杀伤更加敏感。这一发现可能提供一种新的策略来识别抑制剂，这将为癌症治疗提供新的手段。

Mammalian cells have evolved an intricate defense network to maintain genomic integrity by preventing the fixation of permanent DNA damage from endogenous and exogenous mutagens. A major genomic surveillance mechanism involves cell cycle checkpoints that exist at the G1-S and G2/M transitions and are regulated in response to DNA damage. Defects in these stages may result in a mutator phenotype that is associated with tumorigenesis. p53 safeguards the genome during cellular stress by activating both G1/S and G2/M cell cycle checkpoints. Two p53 downstream targets, p21waf1 and Gadd45, appear to be involved in these pathways. p53-mediated G1/S checkpoint is at least, in part, due to the activation of p21waf1. Recently, we discovered that Gadd45 is essential for one of the G2/M checkpoints activated in response to ultraviolet radiation or the alkylating agent methyl methanesulfonate in a p53-dependent manner. DNA damage activates Gadd45, which, in turn, binds to a G2-specific kinase Cdc2 and prevents the association with its regulatory subunit cyclin B1 and the inactivation of its kinase activity. Blocking Gadd45 expression can sensitize tumor cells to killing by cisplatin, a DNA-damaging cancer chemotherapy drug. This finding may offer a novel strategy to identify inhibitors that will provide new means of cancer treatment. In human cells, two additional Gadd45 family members, Gadd45b and Gadd45g, have been identified based on their extensive sequence homology. Although both Gadd45b and Gadd45g also bind to Cdc2 in vivo, they do not inhibit Cdc2 kinase and induce a G2/M arrest. To further define the functional domain, we have constructed a series of Gadd45 deletion or missense mutants. We have identified that the region between 50-76 is essential for its ability to bind to Cdc2, PCNA and p21waf1 in vivo, and to induce a G2/M arrest. The unique effect of Gadd45 on the G2/M arrest may be due to the presence of a region containing DEDDDR residues, which differs from the DEEEED residues in Gadd45b and the GEEDEG residues in Gadd45g. Therefore, the binding of Gadd45 to Cdc2 is insufficient to induce a G2/M arrest and additional activity contributed by the DEDDDR residues may be necessary to regulate the G2/M checkpoint. Interestingly, Ran, a small nuclear GTPase implicated in both cell cycle progression and nuclear export, also contains this motif. Forced expression of Ran also induces a G2/M arrest, whereas the deletion of this motif abolishes such activity. These data suggest that Gadd45 and Ran may utilize a similar pathway to regulate cell cycle progression from the G2 phase to mitosis, and this motif may serve as a common structural entity to activate the G2/M checkpoint. Although Gadd45-mediated G2/M arrest is dependent on p53, it does not require p21waf1 and 14-3-3s (two proteins that have been proposed to be involved in the ionizing radiation-induced G2/M checkpoint). While Cdc25C and cyclin B1 overexpression can override Gadd45-induced G2/M arrest, induction of p53 causes a downregulation of Cdc25C and cyclin B1 (two rate-limiting factors essential for transition from G2 to mitosis). Therefore, we propose that Gadd45 may activate the G2/M checkpoint through two mechanisms: a direct binding and inhibition of the Cdc2/cyclin B1 kinase, and a direct activation of p53 during the G2 phase to eliminate the abundance of Cdc25C and cyclin B1, thereby insuring a persistent G2/M arrest. Currently, we are constructing a normal human fibroblast cell line with a somatic "knock-out" of the Gadd45 gene. In addition, we have made an adenovirus expressing Gadd45. These reagents will be useful to test futher the role of Gadd45 in the G2/M cell cycle checkpoint.