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DNA的永久性损伤的固定来维持基因组的完整性。一个主要的基因组监视机制涉及存在于G1-S和G2/M转换的细胞周期检查点，并响应于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子表型。p53通过激活G1/S和G2/M细胞周期检查点在细胞应激期间保护基因组。两个p53下游靶点p21 waf 1和Gadd 45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p21 waf 1的激活。最近，我们发现，Gadd 45是必不可少的G2/M检查点之一，激活响应紫外线辐射或烷化剂甲基甲磺酸在p53依赖的方式。DNA损伤激活Gadd 45，Gadd 45反过来又与G2特异性激酶Cdc 2结合，并阻止与其调节亚基细胞周期蛋白B1的结合及其激酶活性的失活。阻断Gadd 45表达可以使肿瘤细胞对顺铂（一种DNA损伤性癌症化疗药物）的杀伤敏感。这一发现可能提供一种新的策略，以确定抑制剂，将提供新的癌症治疗手段。 在人类细胞中，已经基于它们广泛的序列同源性鉴定了另外两个Gadd 45家族成员Gadd 45 b和Gadd 45 g。虽然Gadd 45 b和Gadd 45 g在体内也与Cdc 2结合，但它们不抑制Cdc 2激酶并诱导G2/M期阻滞。为了进一步确定其功能结构域，我们构建了一系列Gadd 45缺失或错义突变体。我们已经确定，50-76之间的区域是必不可少的，其能够结合到Cdc 2，PCNA和p21 waf 1在体内，并诱导G2/M期阻滞。Gadd 45对G2/M期阻滞的独特作用可能是由于含有DEDDDR残基的区域的存在，该区域不同于Gadd 45 b中的DEEEED残基和Gadd 45 g中的GEEDEG残基。因此，Gadd 45与Cdc 2的结合不足以诱导G2/M停滞，并且DEDDDR残基贡献的额外活性可能是调节G2/M检查点所必需的。有趣的是，Ran，一个涉及细胞周期进程和核输出的小的核GTdR，也包含这个基序。Ran的强制表达也诱导G2/M期阻滞，而该基序的缺失废除了这种活性。这些数据表明，Gadd 45和Ran可能利用类似的途径来调节细胞周期从G2期到有丝分裂的进程，并且该基序可能作为共同的结构实体来激活G2/M检查点。 虽然Gadd 45介导的G2/M期阻滞依赖于p53，但它不需要p21 waf 1和14-3-3s（已提出参与电离辐射诱导的G2/M检查点的两种蛋白质）。虽然Cdc 25 C和细胞周期蛋白B1过表达可以推翻Gadd 45诱导的G2/M期阻滞，p53的诱导导致Cdc 25 C和细胞周期蛋白B1（两个限速因子从G2到有丝分裂的过渡必不可少）下调。因此，我们认为Gadd 45可能通过两种机制激活G2/M检查点：直接结合和抑制Cdc 2/细胞周期蛋白B1激酶，以及在G2期直接激活p53以消除Cdc 25 C和细胞周期蛋白B1的丰度，从而确保持续的G2/M期停滞。目前，我们正在构建一个正常的人成纤维细胞系与体细胞“敲除”的Gadd 45基因。此外，我们还制备了表达Gadd 45的腺病毒。这些试剂将有助于进一步研究Gadd 45在G2/M细胞周期关卡中的作用。