Chk1 signaling in the G2 DNA damage checkpoint

描述（由申请人提供）：该提案侧重于监测G2中DNA损伤的检查点，并防止有丝分裂发生，直到DNA修复完成。在G1/S期和G2/M期有不同的检查点通路，在存在DNA损伤的情况下延迟进入S期或有丝分裂。G1/S检查点基因的突变，如p53，在肿瘤中很常见，促进基因组不稳定，肿瘤进化和消除凋亡反应，导致化疗耐药。G2检查点基因的突变极为罕见，表明它们对肿瘤细胞的生存能力很重要。本研究的长期目标是全面剖析G2检查点反应的生物学特性，并在此基础上设计和评估靶向抗癌疗法，特别是针对p53突变肿瘤的治疗。该建议的核心是一系列旨在研究该检查点关键成分Chk1蛋白激酶调控的实验。G2检查点及其调控的细胞周期调控因子在进化过程中是高度保守的。因此，这里描述的实验将在人类细胞、小鼠和分裂酵母（分裂酵母）中进行，分裂酵母长期以来一直被用作G2细胞周期控制的范例。为了了解Chk1的功能和调控机制，酵母系统的遗传学将在一系列基于大量功能缺失和功能获得Chk1等位基因的筛选中得到利用。这些实验还将包括克隆已经确定的改变Chk1功能的基因，以及对建立在这种遗传学基础上的检查点信号进行生化解剖。被检查点捕获的细胞重新进入细胞周期的机制也将被研究。与此同时，研究结果将在培养的人类细胞中进行概括和扩展，采用生化方法研究野生型和突变型Chk1，以及在S. pombe中被鉴定为调节Chk1功能的蛋白的同源物。最后，我们将建立在细胞系中已经获得的数据，以抑制小鼠肿瘤细胞中的Chk1信号，以研究G2检查点抑制作为靶向抗癌治疗的效用。这项研究将产生的数据对细胞周期的基本生物学和基因组的稳定性以及癌症治疗的新方法和新目标的测试都很重要。

DESCRIPTION (provided by applicant): This proposal focuses on the checkpoint that monitors DNA damage in G2 and prevents mitosis from occurring until DNA repair is completed. There are distinct checkpoint pathways functioning at the G1/S and G2/M transitions that delay entry into S-phase or mitosis in the presence of DNA damage. Mutations in G1/S check point genes, such as p53, are commonplace in tumors, promoting genomic instability, tumor evolution and abolish apoptotic responses leading to chemo resistance. Mutations in G2 check point genes are extremely infrequent, suggesting they are important for tumor cell viability. The long-term goal of this study is to fully dissect the biology of G2 checkpoint responses, and on the basis of this, to devise and assess targeted anti-cancer therapies, especially for the treatment of tumors bearing mutations in p53. Central to this proposal is a series of experiments that aim to investigate regulation of a key component of this checkpoint, the Chk1 protein kinase. The G2 checkpoints and the cell cycle regulators that they regulate are highly conserved in evolution. Therefore, the experiments described here will be carried out in both human cells, mice, and in the fission yeast Schizosaccharomyces pombe, the organism that has long been used as a paradigm of G2 cell cycle control. To understand mechanisms of Chk1 function and regulation, the genetics of the yeast system will be utilized in a series of screens based on a large collection of both loss- and gain-of- function Chk1 alleles. These experiments will also involve the cloning of genes already identified to alter Chk1 function and the biochemical dissection of checkpoint signaling that builds on this genetics. The mechanism(s) by which checkpoint arrested cells re-enter the cell cycle will also be investigated. In parallel, findings will be recapitulated and expanded upon in human cells in culture, taking biochemical approaches to study wildtype and mutant Chk1, and homologs of proteins identified as modulating Chk1 function in S. pombe. Finally, we will build on data already obtained in cell lines to inhibit Chk1 signaling in tumor cells in the mouse to investigate the utility of G2 checkpoint inhibition as a targeted anti-cancer therapy. The study will yield data that will be important in terms of both the basic biology of the cell cycle and stability of the genome and also in the testing of new approaches and new targets for cancer therapy.