A novel regulator of cell cycle and cell death

细胞周期和细胞死亡的新型调节剂

• 批准号: 7406757
• 负责人: HONGLIN LI
• 金额: $ 17.72万
• 依托单位: LURIE CHILDREN'S HOSPITAL OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2009-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7406757
• 关键词: Affect; Age; Animals; Apoptosis; Apoptotic; Attenuated; Binding Proteins; Biochemical; Biological Assay; CDC2 Protein Kinase; Cdc25B protein; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cell Death; Cell division; Cells; Centrosome; Checkpoint kinase 1; Chromosome Segregation; Complex; Cytokinesis; DNA Damage; DNA Repair; DNA biosynthesis; DNA damage checkpoint; Data; Defect; Development; Docking; Ensure; Event; Foundations; G2/M Transition; Genetic Materials; Genome Stability; Genomic Instability; Genotoxic Stress; Goals; Homeostasis; Immunofluorescence Immunologic; Lead; Localized; Malignant Neoplasms; Mediating; Microtubule-Organizing Center; Mitosis; Mitotic; Mutagens; Normal Cell; Normal tissue morphology; Organelles; Pathogenesis; Phase; Phosphotransferases; Play; Principal Investigator; Process; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Solid; Testing; Trimethoprim-Sulfamethoxazole; aurora-A kinase; base; cancer cell; cancer therapy; chemotherapeutic agent; cyclin B1; daughter cell; driving force; human PLK1 protein; human disease; insight; novel; novel therapeutics; prevent; programs; promoter; repaired; response; therapeutic target; transmission process; tumorigenesis; Affect; Age; Animals; Apoptosis; Apoptotic; Attenuated; Binding Proteins; Biochemical; Biological Assay; CDC2 Protein Kinase; Cdc25B protein; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cell Death; Cell division; Cells; Centrosome; Checkpoint kinase 1; Chromosome Segregation; Complex; Cytokinesis; DNA Damage; DNA Repair; DNA biosynthesis; DNA damage checkpoint; Data; Defect; Development; Docking; Ensure; Event; Foundations; G2/M Transition; Genetic Materials; Genome Stability; Genomic Instability; Genotoxic Stress; Goals; Homeostasis; Immunofluorescence Immunologic; Lead; Localized; Malignant Neoplasms; Mediating; Microtubule-Organizing Center; Mitosis; Mitotic; Mutagens; Normal Cell; Normal tissue morphology; Organelles; Pathogenesis; Phase; Phosphotransferases; Play; Principal Investigator; Process; Protein Overexpression; Protein-Serine-Threonine Kinases; Proteins; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Solid; Testing; Trimethoprim-Sulfamethoxazole; aurora-A kinase; base; cancer cell; cancer therapy; chemotherapeutic agent; cyclin B1; daughter cell; driving force; human PLK1 protein; human disease; insight; novel; novel therapeutics; prevent; programs; promoter; repaired; response; therapeutic target; transmission process; tumorigenesis

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the roles of a novel protein C53 in regulation of cell cycle progression and DNA damage response, and to explore potential exploitation of this protein as a novel therapeutic approach for cancer treatment. Regulation of cell cycle and cellular response to DNA damage is critical for animal development and ageing, and the pathogenesis of human diseases like cancer. Defects in cell cycle regulation and DNA damage response may lead to genome instability and oncogenesis. Cells utilize the so-called checkpoint mechanisms to ensure the completion of a particular phase before the next phase is initiated, thereby maintaining truthful transmission of genetic material. One of the key checkpoints is so-called the G2/M checkpoint that regulates mitotic entry and activation of cyclin-dependent kinase 1 (Cdk1), a driving force for progression of cell division. We have recently identified Cdk5 activator p35 binding protein C53 as a novel regulator of cell cycle and DNA damage response. Our study demonstrated that C53 depletion leads to defective cell cycle and resistance to apoptosis induced by chemotherapeutic agents. Additionally, C53 overexpression abolished the G2/M checkpoint-mediated Cdk1 inactivation, thereby sensitizing cancer cells to various DNA damage agents. We further found that C53 depletion results in inactivation of Cdk1 and delayed mitotic entry, while C53 overexpression antagonizes Chk1-mediated Cdk1 inactivation. Importantly, we found that a fraction of endogenous C53 protein localizes at the centrosome, an organelle that is emerging as a pivotal multi- functional platform that integrates many important signal pathways. Based upon our preliminary study, we hypothesize that C53 plays a crucial role in unperturbed cell cycle progression. By antagonizing the activity of centrosome-associated Chk1, C53 acts as a positive promoter of initial activation of cyclin B1/Cdk1 kinase at the centrosome. To test our hypothesis, we aim: 1) To Investigate the centrosomal association of C53. In this aim, by using GFP-centrin stable cells and immunofluorescence and biochemical analyses, we will investigate the centrosomal association of C53 and its docking mechanism. Moreover, We will make a centrosome-targeted C53 to test whether C53 centrosomal localization is important for its function. 2) To characterize the role of C53 in initial activation of Cdk1/cyclin B1 during the G2/M transition. In this aim, we will use synchronized cells and immunofluorescence assays to examine the effect of C53 on initial activation of Cdk1/cyclin B1 at the centrosome. Furthermore, we will test whether C53 exerts its function through affecting the activity of centrosomal Chk1 and other centrosome-associated mitotic regulators. This study will provide insight on a novel regulatory mechanism for cell cycle control and checkpoint response. It will lay a foundation for exploitation of this protein as a novel therapeutic target in cancer treatment.

描述（由申请人提供）：我们的长期目标是阐明新型蛋白C53在调节细胞周期进程和DNA损伤反应中的作用，并探索对这种蛋白质的潜在剥削，作为一种新的治疗方法。细胞周期和对DNA损伤的细胞反应的调节对于动物发育和衰老以及癌症等人类疾病的发病机理至关重要。细胞周期调节和DNA损伤反应的缺陷可能导致基因组不稳定性和肿瘤发生。细胞利用所谓的检查点机制来确保在启动下一阶段之前完成特定阶段，从而维持遗传物质的真实传播。关键检查点之一是所谓的G2/M检查点，该检查点调节有丝分裂进入和激活Cyclin依赖性激酶1（CDK1），这是细胞分裂进展的驱动力。我们最近将CDK5激活剂p35结合蛋白C53鉴定为细胞周期和DNA损伤反应的新调节剂。我们的研究表明，C53的耗竭会导致细胞周期和对化学治疗剂诱导的凋亡的抗性。此外，C53的过表达废除了G2/M检查点介导的CDK1失活，从而使癌细胞对各种DNA损伤剂进行了敏感性。我们进一步发现，C53耗竭会导致CDK1失活并延迟有丝分裂进入，而C53的过表达拮抗CHK1介导的CDK1失活。重要的是，我们发现内源性C53蛋白的一部分位于中心体，这是一种呈现的细胞器，它是一个集成了许多重要信号途径的关键多功能平台。根据我们的初步研究，我们假设C53在不受干扰的细胞周期进程中起着至关重要的作用。通过拮抗与中心体相关的CHK1的活性，C53充当了中心体在中心体的Cyclin B1/CDK1激酶初始激活的阳性启动子。为了检验我们的假设，我们的目的是：1）研究C53的中心体关联。在此目的中，通过使用GFP-中心蛋白稳定的细胞以及免疫荧光和生化分析，我们将研究C53及其对接机制的中心体关联。此外，我们将制作一个以中心体为靶向的C53来测试C53中心体定位是否对其功能很重要。 2）表征C53在G2/M转变期间CDK1/Cyclin B1初始激活中的作用。在此目标中，我们将使用同步细胞和免疫荧光测定来检查C53对中心体在CDK1/Cyclin B1初始激活的影响。此外，我们将通过影响中心体CHK1和其他与中心体相关的有丝分裂调节剂的活性来测试C53是否发挥其功能。这项研究将提供有关细胞周期控制和检查点响应的新型调节机制的见解。它将为剥削该蛋白作为癌症治疗中的新治疗靶点奠定基础。