Mechanisms of G2/M cell cycle checkpoint controls

哺乳动物细胞已经进化出一种复杂的防御网络，通过防止固定内源和外源诱变剂造成的永久性DNA损伤来维持基因组的完整性。一个主要的基因组监测机制涉及细胞周期检查点，这些检查点存在于G1-S和G2/M过渡阶段，并受到DNA损伤的调节。这些阶段的缺陷可能导致与肿瘤发生相关的突变子表型。在细胞应激中，P53通过激活G1/S和G2/M细胞周期检查点来保护基因组。两个p53下游靶点p21waf1和GADD45似乎参与了这些通路。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对于其中一个G2/M检查点是必不可少的，这些检查点是由紫外线或烷化剂甲基甲烷磺酸以P53依赖的方式激活的。DNA损伤激活GADD45，GADD45进而与G2特异性的激酶CDC2结合，阻止与其调节亚单位细胞周期蛋白B1的联系，并使其激酶活性失活。阻断GADD45的表达可以使肿瘤细胞对顺铂的杀伤敏感，顺铂是一种破坏DNA的癌症化疗药物。这一发现可能提供一种新的策略，以寻找将提供新的癌症治疗手段的抑制剂。 在人类细胞中，根据广泛的序列同源性，已鉴定出另外两个GADD45家族成员Gadd45b和Gadd45g。虽然Gadd45b和Gadd45g在体内也与CDc2结合，但它们不抑制CDc2激酶并诱导G2/M期停滞。为了进一步定义功能域，我们构建了一系列GADD45缺失或错义突变体。我们已经确定，50-76之间的区域对于它在体内与CDc2、增殖细胞核抗原和p21waf1结合并诱导G2/M期停滞是必不可少的。GADD45对G2/M期阻滞的独特作用可能是由于存在一个含有DEDDDR残基的区域，该区域不同于Gadd45b中的DEEEED残基和Gadd45G中的GEEDEG残基。因此，GADD45与CDc2的结合不足以诱导G2/M停滞，可能需要DEDDDR残基贡献的额外活性来调节G2/M检查点。有趣的是，RAN，一个与细胞周期进程和核输出有关的小的核GTP酶，也包含这个基序。强制表达RAN也会导致G2/M期停滞，而该基序的缺失则取消了这种活性。这些数据表明，GADD45和RAN可能利用相似的途径来调节细胞周期从G2期到有丝分裂的进程，该基序可能作为一个共同的结构实体来激活G2/M检查点。 虽然GADD45介导的G2/M停滞依赖于p53，但它不需要p21waf1和14-3-3S(已被认为参与电离辐射诱导的G2/M检查点的两种蛋白质)。虽然CDC25C和Cyclin B1的过表达可以推翻GADD45诱导的G2/M期停滞，但P53的诱导导致CDC25C和Cyclin B1的下调(这两个限速因子是从G2向有丝分裂过渡所必需的)。因此，我们认为GADD45可能通过两种机制激活G2/M检查点：直接结合和抑制CDC2/Cyclin B1激酶，以及在G2期直接激活P53，以消除CDc25C和Cyclin B1的丰度，从而确保持续的G2/M停滞。目前，我们正在构建一个正常的人类成纤维细胞系，该细胞系具有GADD45基因的体细胞“敲除”。此外，我们还制备了表达GADD45的腺病毒。这些试剂将有助于进一步测试GADD45在G2/M细胞周期检查点中的作用。

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.