THE ROLE OF GADD45 IN G2 - M CHECKPOINT

哺乳动物细胞已经进化出一个复杂的防御网络，通过防止固定遗传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，受一系列调控系统的控制，它们的失活可能会对基因组的稳定性和治疗敏感性造成严重影响。与G1检查点不同，哺乳动物G2-M细胞周期检查点在DNA损伤后的调控机制尚不清楚。我们以前曾报道过GADD45基因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。最近的研究表明，通过显微注射方法在正常成纤维细胞中过表达GADD45导致细胞停滞在有丝分裂的早期阶段。结果表明，GADD45表达载体显微注射后，细胞呈完全圆形，有丝分裂特异性标记(MPM2，Ab)染色阳性，4N量DNA和单一中心体也与此结果一致，瞬时转染人细胞后GADD45蛋白表达水平增加，导致G2/M期细胞比例增加。这些结果提示GADD45可能参与了DNA损伤引起的G2-M期阻滞的调控。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GADD45在基因毒性应激后G2-M期细胞周期检查点中的作用。(2)明确GADD45参与G2-M检查点的分子和生化机制(S)。我们的假设如下：(1)。内源性GADD45被破坏的细胞将在DNA损伤后表现出扰动的G2-M延迟。(2)。为了激活G2-M转换机制，GADD45蛋白可能靶向CDC2/Cyclin B1和CDC2/Cyclin A复合体，从而“驱动”细胞从G2到有丝分裂。(3)。GADD45控制G2-M检查点的能力将有助于GADD45诱导的生长抑制。实验技术将包括流式细胞仪分析、有丝分裂指数分析、CDC2激酶分析、免疫印迹、重组GADD45缺失蛋白的构建、细胞存活分析和显微注射。本申请中提出的研究将定义一条控制特定DNA损伤后G2-M细胞周期检查点的新途径，并确定GADD45在细胞生长负调控中发挥作用的机制(S)。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。

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.