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，通过防止基因毒性应激造成的永久性损伤的固定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，其失活可能导致基因组稳定性和治疗敏感性的显著后果。与G1检查点相比，哺乳动物G2-M细胞周期检查点在DNA损伤后的控制尚不清楚。我们之前报道过GADD45（一种p53调控的应激诱导基因）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会导致细胞在早期有丝分裂阶段停滞。证据包括，在微注射GADD45表达载体后，细胞呈现完全圆形，有丝分裂特异性标记物（MPM2, Ab）染色阳性，DNA量为4N，单个中心体。与这些结果一致的是，瞬时转染后GADD45蛋白表达水平的增加导致人类细胞中G2/M分数更高。这些结果提出了GADD45可能参与控制G2-M阻滞以应对DNA损伤的可能性。因此，本应用程序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损伤后会出现G2-M延迟。(2)。为了激活G2- m过渡机制，Gadd45蛋白可能靶向Cdc2/cyclin B1和Cdc2/cyclin A复合物，这些复合物“驱动”细胞从G2到有丝分裂。(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。实验技术将包括流式细胞分析、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。本申请提出的研究将在一定DNA损伤后，定义一条控制G2-M细胞周期检查点的新途径，并确定GADD45负调控细胞生长的作用机制。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，并可能为开发新型抗癌药物提供见解。

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.