THE ROLE OF GADD45 IN G2 - M CHECKPOINT

GADD45 在 G2 - M 检查点中的作用

• 批准号: 6350408
• 负责人: QIMIN ZHAN
• 金额: $ 20.46万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-11 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350408
• 关键词: DNA damage; animal tissue; cell cycle; cell cycle proteins; cell growth regulation; cyclins; enzyme activity; enzyme inhibitors; fibroblasts; flow cytometry; gene expression; human tissue; microinjections; p53 gene /protein; protein kinase; protein protein interaction; protein structure function; recombinant proteins; western blottings

Mammalian cells have evolved an intricate defense network to maintain genomic fidelity by preventing the fixation of permanent damage from genotoxic stress. Cell cycle checkpoint, a major genomic surveillance mechanism, is governed by a series of control systems and their inactivation may result in dramatic consequences on genomic stability and therapeutic sensitivity. In contrast to G1 checkpoint, the control of mammalian G2-M cell cycle checkpoint after DNA damage is poorly understood. We have previously reported that expression of GADD45, a p53-regulated and stress-inducible gene, is able to suppress cell growth although the mechanism by which GADD45-induced growth suppression remains unclear. Recent findings have demonstrated that over-expression of GADD45 in normal fibroblast via a microinjection approach caused cells to arrest in an early mitotic phase. The evidence includes that following microinjection with GADD45 expression vectors, the cells displayed a completely rounded shape, positive staining with the mitotic- specific marker (MPM2, Ab), a 4N amount of DNA and a single centrosome Also in agreement with these results, an increased level of GADD45 protein expression after transient transfection was shown to result in a higher G2/M fraction in human cells. These results raise the possibility that GADD45 may participate in the control of G2-M arrest in response to DNA damage. Therefore, the long- term objective described in this application will focus on three key issues: (1). To determine the role of GADD45 in the G2-M cell cycle checkpoint after genotoxic stress. (2) To define the molecular and biochemical mechanism(s) by which GADD45 plays a role in the G2-M checkpoint. Our hypothesis are as follows: (1). Cells with disrupted endogenous GADD45 will exhibit a perturbed G2-M delay following DNA damage. (2). In order to activate the machinery of G2-M transition, Gadd45 protein may target the Cdc2/cyclin B1 and Cdc2/cyclin A complexes, which "drive" cells from G2 to mitosis. (3). The capability of GADD45 on the control of G2-M checkpoint will contribute to the GADD45-induced growth suppression. Experimental techniques will include flow cytometric analysis, mitotic index assay, Cdc2 kinase assay, immunoblotting, construction of recombinant Gadd45 deletion proteins, cell survival assay and microinjection. The studies proposed in this application would define a new pathway controlling G2-M cell cycle checkpoint after certain DNA damage as well as determine the mechanism(s) by which GADD45 exerts its role in the negative control of cell growth. In addition, these studies would be helpful in the elucidation of how G2-M checkpoint affects therapeutic sensitivity and might provide the insights into the development of novel anti-cancer agents.

哺乳动物细胞已经进化出复杂的防御网络，通过防止基因毒性应激造成的永久性损伤来维持基因组的保真度。细胞周期检查点是一种主要的基因组监测机制，由一系列控制系统控制，它们的失活可能会对基因组稳定性和治疗敏感性产生巨大影响。与 G1 检查点相反，哺乳动物 G2-M 细胞周期检查点在 DNA 损伤后的控制知之甚少。我们之前曾报道过，p53 调节的应激诱导基因 GADD45 的表达能够抑制细胞生长，尽管 GADD45 诱导生长抑制的机制尚不清楚。最近的研究结果表明，通过显微注射方法在正常成纤维细胞中过度表达 GADD45 会导致细胞停滞在早期有丝分裂阶段。证据包括显微注射 GADD45 表达载体后，细胞显示出完全圆形的形状、有丝分裂特异性标记物（MPM2、Ab）的阳性染色、4N 量的 DNA 和单个中心体。同样与这些结果一致的是，瞬时转染后 GADD45 蛋白表达水平的增加显示出导致人类细胞中更高的 G2/M 分数。这些结果提出了 GADD45 可能参与响应 DNA 损伤而控制 G2-M 停滞的可能性。因此，本申请中描述的长期目标将集中在三个关键问题上：（1）。确定 GADD45 在基因毒性应激后 G2-M 细胞周期检查点中的作用。 (2) 明确GADD45在G2-M检查点中发挥作用的分子和生化机制。我们的假设如下：（1）．内源性 GADD45 被破坏的细胞在 DNA 损伤后会表现出受干扰的 G2-M 延迟。 （2）。为了激活 G2-M 转变机制，Gadd45 蛋白可能会靶向 Cdc2/cyclin B1 和 Cdc2/cyclin A 复合物，从而“驱动”细胞从 G2 进入有丝分裂。 （3）。 GADD45 对 G2-M 检查点的控制能力将有助于 GADD45 诱导的生长抑制。实验技术包括流式细胞术分析、有丝分裂指数测定、Cdc2 激酶测定、免疫印迹、重组 Gadd45 缺失蛋白的构建、细胞存活测定和显微注射。本申请中提出的研究将定义在某些 DNA 损伤后控制 G2-M 细胞周期检查点的新途径，并确定 GADD45 在细胞生长负控制中发挥作用的机制。此外，这些研究将有助于阐明 G2-M 检查点如何影响治疗敏感性，并可能为新型抗癌药物的开发提供见解。