Functional analysis of cell cycle regulators by in vitro gene targeting using DT40 cells

DNA损伤后基因组完整性的维持依赖于DNA修复和检查点系统的协调作用。检查点的失效导致基因组的不稳定，从而导致多个DNA序列的改变和染色体畸变，从而导致癌症的发生。对酵母菌和果蝇进行遗传分析，鉴定出各种调节检查点的分子。我们的实验室已经鉴定出这些分子的哺乳动物同源物。为了研究这些分子在脊椎动物细胞中的功能，我们使用高重组鸡b细胞系DT40进行了体外基因敲除。本项目主要发现如下：1)后期促进复合体（APC）由两种调节蛋白Cdc20和Cdh1激活。在酵母和果蝇中，从有丝分裂结束到G1期，Cdh1-APC活性靶向有丝分裂周期蛋白。为了研究Cdh1在脊椎动物细胞中的功能，我们对鸡DT40细胞的Cdh1位点进行了克隆。Cdh1对于细胞活力和细胞周期进展是不可或缺的。然而，Cdh1的缺失诱导G1中有丝分裂周期蛋白的非预定积累，从而消除了由雷帕霉素（一种p27^<kip1 bb0的诱诱剂）治疗引起的G1阻滞。此外，我们发现Cdh1^<-/->细胞不能维持DNA损伤诱导的G2阻滞，并且Cdh1- apc被x辐射诱导的DNA损伤激活。因此，Cdh1-APC的激活在cdk抑制剂依赖性G1阻滞和dna损伤诱导的G2阻滞中都起着至关重要的作用。2)疣基因是基于其在果蝇中作为肿瘤抑制因子的能力被鉴定出来的。我们先前发现了疣的人类同源物，称为疣，并表明人类疣激酶在有丝分裂过程中定位于有丝分裂装置。我们生成了疣（-/-）DT40克隆，发现这些克隆的晚期有丝分裂明显延长。此外，许多细胞不能进行胞质分裂。这些发现表明，疣参与脊椎动物细胞的有丝分裂晚期事件，其功能失活可能导致正常有丝分裂进程失败，导致染色体不稳定。少

Maintenance of genomic integrity after DNA damage depends on the coordinated action of the DNA repair and checkpoint systems. The failure of checkpoint leads to genomic instability, which causes multiple DNA sequence alterations and chromosomal aberrations, and disposition to cancer.Genetic analyses of yeast and Drosophila allowed identification of various molecules regulating checkpoints. Our laboratory has identified mammalian homologues of those molecules. To investigate the function of those molecules in vertebrate cells, we performed in vitro gene knock-out using the hyper-recombinogenic chicken B-cell line DT40. Followings are major findings we obtained in this project :1) Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-APC activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of the chicken DT40 cells disrupted i … More n their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1 resulting in abrogation of G1l arrest caused by treatment with rapamycin, an inducer of p27^<kip1>. Furthermore, we found that Cdh1^<-/-> cells fail to maintain DNA-damage induced G2 arrest and that Cdh1-APC is activated by X-radiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both Cdk-inhibitor-dependent G1 arrest and DNA-damage-induced G2 arrest.2) The warts gene was identified based on its ability to act as a tumor suppressor in Drosophila. We previously identified a human homologue of the warts, termed WARTS and showed that the human WARTS kinase is localized to mitotic apparatus during mitosis. We generated WARTS (-/-) DT40 clones and found that late mitosis is significantly prolonged in those clones. Furthermore, many of the cells fail to undergo cytokinesis. These findings suggest that WARTS is involved in late mitotic events in vertebrate cells and that inactivation of its function may result in failure in normal mitotic progression, leading to chromosomal instability. Less