THE ROLE OF GADD45 IN G2 - M CHECKPOINT

GADD45 在 G2 - M 检查点中的作用

• 批准号: 6497944
• 负责人: QIMIN ZHAN
• 金额: $ 21.03万
• 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 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497944
• 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一个复杂的防御网络，通过防止固定来自遗传毒性应激的永久性损伤来维持基因组保真度。细胞周期检查点是一种重要的基因组监视机制，它受一系列控制系统的控制，其失活可能导致基因组稳定性和治疗敏感性的严重后果。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。我们以前曾报道，GADD 45，p53调节和应激诱导基因的表达，能够抑制细胞生长，但GADD 45诱导的生长抑制的机制尚不清楚。最近的研究表明，通过显微注射方法在正常成纤维细胞中过度表达GADD 45导致细胞在早期有丝分裂期停滞。证据包括用GADD 45表达载体显微注射后，细胞显示完全圆形，有丝分裂特异性标记物（MPM 2，Ab）阳性染色，4 N量的DNA和单个中心体。与这些结果一致，瞬时转染后GADD 45蛋白表达水平的增加显示在人细胞中产生更高的G2/M分数.这些结果提高了GADD 45可能参与控制响应DNA损伤的G2-M期阻滞的可能性。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）。探讨GADD 45在遗传毒性应激后G2-M期细胞周期检查点中的作用。(2)确定GADD 45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）.具有破坏的内源性GADD 45的细胞将在DNA损伤后表现出受干扰的G2-M延迟。（二）、为了激活G2-M转换的机制，Gadd 45蛋白可以靶向Cdc 2/cyclin B1和Cdc 2/cyclin A复合物，其“驱动”细胞从G2到有丝分裂。（三）、GADD 45对G2-M期检查点的控制能力可能是GADD 45抑制细胞生长的重要原因。实验技术将包括流式细胞术分析、有丝分裂指数测定、Cdc 2激酶测定、免疫印迹、重组Gadd 45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，以及确定GADD 45在细胞生长的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，并可能为开发新的抗癌药物提供见解。