Mechanisms of G2/M cell cycle checkpoint controls

G2/M 细胞周期检查点控制机制

• 批准号: 6763835
• 负责人: XIN WEI WANG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763835
• 关键词: DNA damage; antineoplastics; cell cycle; cell cycle proteins; cell growth regulation; cis platinum compound; cyclins; enzyme activity; enzyme inhibitors; gene deletion mutation; gene expression; gene induction /repression; genome; guanosinetriphosphatases; human tissue; mutant; neoplastic cell; neoplastic growth; oncoprotein p21; p53 gene /protein; phosphoprotein phosphatase; protein binding; protein kinase; tissue /cell culture; ultraviolet radiation

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.

哺乳动物细胞通过防止内源性和外源性诱变剂固定永久性DNA损伤来发展一个复杂的防御网络，以维持基因组完整性。主要的基因组监测机制涉及在G1-S和G2/M转变处存在的细胞周期检查点，并对DNA损伤进行调节。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在细胞应激期间的基因组。这些途径似乎涉及两个p53下游目标P21WAF1和GADD45。 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对于响应于紫外线辐射或烷基化剂甲基甲磺酸甲酯的G2/M检查点之一至关重要。 DNA损伤激活了GADD45，而GADD45又与G2特异性激酶CDC2结合，并防止与其调节亚基细胞周期蛋白B1及其激酶活性的失活。阻断GADD45表达可以使肿瘤细胞对杀死肿瘤细胞（一种破坏DNA的癌症化学疗法药物）的杀伤。这一发现可能提供了一种新型策略，以鉴定将提供新的癌症治疗方法的抑制剂。 在人类细胞中，基于广泛的序列同源性，已经确定了另外两个GADD45家族成员GADD45B和GADD45G。尽管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停滞。为了进一步定义功能域，我们构建了一系列的GADD45缺失或错义突变体。我们已经确定，在50-76之间的区域对于在体内与CDC2，PCNA和P21WAF1结合以及诱导G2/M停滞的能力至关重要。 GADD45对G2/M停滞的独特作用可能是由于存在包含DEDDDR残基的区域，该区域与GADD45B中的DEEED残基有所不同，而GADD45G中的GEEDEG残基有所不同。因此，GADD45与Cdc2的结合不足以诱导G2/M停滞，并且对于调节G2/M检查点可能需要促进DEWDDR残基的额外活动。有趣的是，RAN是一种与细胞周期进展和核输出有关的小核GTPase，也包含该基序。 RAN的强迫表达也会引起G2/m停滞，而这种图案的缺失消除了这种活性。这些数据表明，GADD45和RAN可以利用类似的途径来调节细胞周期从G2相到有丝分裂的进程，并且该基序可以用作激活G2/M检查点的共同结构实体。 尽管GADD45介导的G2/m停滞取决于p53，但它不需要p21WAF1和14-3-3S（已提议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过表达可以覆盖GADD45诱导的G2/m停滞，但p53的诱导会导致CDC25C和Cyclin B1的下调（从G2到有丝毫分裂的过渡至关重要的两个速率限制因素）。因此，我们提出GADD45可以通过两种机制激活G2/M检查点：直接结合和抑制Cdc2/Cyclin B1激酶，以及在G2期间直接激活p53，以消除CDC25C和Cyclin B1的丰度，从而消除持久的G2/M持久耐药。目前，我们正在构建具有GADD45基因的体细胞“敲除”的正常人成纤维细胞系。此外，我们制作了表达GADD45的腺病毒。这些试剂将有助于测试GADD45在G2/M细胞周期检查点中的作用。