Normal and Neoplastic Regulation of Cyclin E

细胞周期蛋白 E 的正常和肿瘤调节

• 批准号: 6916340
• 负责人: Bruce E Clurman
• 金额: $ 38.49万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6916340
• 关键词: adeno associated virus group; antibody; carcinogenesis; cell growth regulation; cyclins; disease /disorder model; fibroblasts; gene mutation; gene targeting; genetically modified animals; human tissue; immunologic substance development /preparation; laboratory mouse; model design /development; molecular oncology; neoplastic growth; p53 gene /protein; phosphorylation; protein protein interaction; protein structure function; transfection /expression vector; ubiquitin

DESCRIPTION (provided by applicant): The cyclin E protein controls the transition from the G1 to the S-phase of the cell cycle. Deregulated cyclin E activity causes abnormal cell division, and mutations leading to aberrant cyclin E-cdk2 regulation are found in most human cancers. The research described in this proposal seeks to understand the mechanisms that govern cyclin E in normal cells and tumors. We are particularly interested in the regulation and function of cyclin E phosphorylation in cell cycle control and tumorigenesis. Multiple site-specific phosphorylations control cyclin E, but the role of specific mitogenic and signal transduction pathways in regulating these phosphorylations is unknown. The goal of the first aim is to characterize the regulation of cyclin E phosphorylation in normal cells and in tumor cells. We will develop antibodies that recognize phosphorylated forms of cyclin E, and these reagents will be used to examine cyclin E phosphorylation in normal cells, and to determine if it is abnormal in tumor cells. We are particularly interested in the relationships between phosphorylated cyclin E and the Fbw7 ubiquitin ligase. Because most studies on cyclin E phosphorylation have utilized overexpressed cyclin E, it has been difficult to distinguish the role of phosphorylation from the consequences of overexpression. We will thus use "knock-in" models in which phosphorylation sites are mutated in the context of the endogenous cyclin E locus to study the function of specific phosphorylations. These studies will use homologous recombination techniques in mice, as well as develop new methods based on adeno-associated virus vectors in human cells. The latter method may be broadly applicable to studies of protein phosphorylation in systems where mouse models are unnecessary or undesirable. The mechanisms of cyclin E-associated tumorigenesis are largely unknown. The overall goal of this aim is to use cyclin E transgenic and knock-in strains to develop models of cyclin E-associated cancer. Because we have discovered a homeostatic p53-dependent response that restrains excess cyclin E activity, we will specifically test the hypothesis that loss of p53 function is an integral step in the development of tumors with deregulated cyclin E expression. These models will be used to study the mechanisms of cyclin E-associated tumorigenesis and may facilitate the development of new therapeutic strategies.

描述（申请人提供）：细胞周期蛋白E控制细胞周期从G1期到S期的转变。细胞周期蛋白 E 活性失调会导致细胞分裂异常，并且在大多数人类癌症中都发现了导致细胞周期蛋白 E-cdk2 调节异常的突变。该提案中描述的研究旨在了解正常细胞和肿瘤中控制细胞周期蛋白 E 的机制。我们对细胞周期控制和肿瘤发生中细胞周期蛋白 E 磷酸化的调节和功能特别感兴趣。多个位点特异性磷酸化控制细胞周期蛋白 E，但特定有丝分裂和信号转导途径在调节这些磷酸化中的作用尚不清楚。第一个目标是表征正常细胞和肿瘤细胞中细胞周期蛋白 E 磷酸化的调节。我们将开发识别细胞周期蛋白E磷酸化形式的抗体，这些试剂将用于检查正常细胞中细胞周期蛋白E的磷酸化，并确定肿瘤细胞中的细胞周期蛋白E磷酸化是否异常。我们对磷酸化细胞周期蛋白 E 和 Fbw7 泛素连接酶之间的关系特别感兴趣。 由于大多数关于细胞周期蛋白 E 磷酸化的研究都利用了过度表达的细胞周期蛋白 E，因此很难区分磷酸化的作用与过度表达的后果。因此，我们将使用“敲入”模型来研究特定磷酸化的功能，其中磷酸化位点在内源性细胞周期蛋白E基因座的背景下发生突变。这些研究将在小鼠中使用同源重组技术，并开发基于人类细胞中腺相关病毒载体的新方法。后一种方法可能广泛适用于不需要或不需要小鼠模型的系统中蛋白质磷酸化的研究。细胞周期蛋白 E 相关肿瘤发生的机制很大程度上未知。该目标的总体目标是利用细胞周期蛋白 E 转基因和敲入菌株来开发细胞周期蛋白 E 相关癌症模型。因为我们发现了一种稳态 p53 依赖性反应，可以抑制细胞周期蛋白 E 的过度活性，所以我们将专门检验这样的假设：p53 功能的丧失是细胞周期蛋白 E 表达失调的肿瘤发展中不可或缺的一步。这些模型将用于研究细胞周期蛋白 E 相关肿瘤发生的机制，并可能促进新治疗策略的开发。