Chk1 signaling in the G2 DNA damage checkpoint

G2 DNA 损伤检查点中的 Chk1 信号传导

• 批准号: 7014491
• 负责人: MATTHEW J O'CONNELL
• 金额: $ 33.6万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014491
• 关键词: DNA damage; DNA repair; DNA replication; SDS polyacrylamide gel electrophoresis; Schizosaccharomyces pombe; biological signal transduction; cell cycle; cytogenetics; fungal genetics; gene mutation; human tissue; laboratory mouse; mass spectrometry; molecular cloning; p53 gene /protein; protein kinase; protein structure function; tissue /cell culture; transcription factor; western blottings; yeast two hybrid system

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.

描述（由申请人提供）：该提案的重点是检查点，即监测G2中的DNA损伤并防止有丝分裂发生直到DNA修复完成。在存在DNA损伤的情况下，在G1/S和G2/M的转变处有不同的检查点途径，该途径延迟进入S期或有丝分裂。 G1/S检查点基因（例如p53）中的突变在肿瘤中很常见，促进基因组不稳定性，肿瘤进化和废除凋亡反应，从而导致化学抗性。 G2检查点基因中的突变极少，表明它们对于肿瘤细胞活力很重要。这项研究的长期目标是完全剖析G2检查点反应的生物学，并在此基础上设计和评估靶向的抗癌疗法，尤其是用于治疗p53中携带突变的肿瘤。该提案的核心是一系列实验，旨在研究该检查点CHK1蛋白激酶的关键组成部分的调节。它们调节的G2检查点和细胞周期调节剂在进化中是高度保守的。因此，此处描述的实验将在人类细胞，小鼠和裂变酵母菌schizosacachomyces pombe中进行，该生物长期以来一直用作G2细胞周期控制的范式。为了了解CHK1功能和调节的机制，将基于大量损失和功能获得CHK1等位基因的大量筛选中使用酵母系统的遗传学。这些实验还将涉及已经鉴定出可以改变CHK1功能的基因的克隆以及基于该遗传学的检查点信号传导的生化解剖。还将研究将检查点逮捕的细胞重新进入细胞周期的机制。同时，将在培养物中的人类细胞中概括和扩展发现，采用生化方法研究野生型和突变体CHK1，以及被鉴定为调节pombe中CHK1功能的蛋白质的同源物。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信号传导，以研究G2检查点抑制作用作为靶向抗癌治疗的实用性。这项研究将产生有关细胞周期的基本生物学和基因组的稳定性以及新方法的测试和癌症治疗的新靶标的数据。