Role of p53 in cell cycle checkpoints

说明（由申请人提供）：p53显然是人类癌症中的肿瘤抑制因子，p53基因突变是常见事件。p53在介导对DNA损伤的细胞反应中具有充分表征的作用。G1期的检查点已被证明是严格依赖于p53的。由于G2/M检查点的存在，在其不存在的情况下发生，p53在防止有丝分裂进入的确切作用一直难以捉摸。本申请中的研究将解决p53生物学的这一关键方面。提出了三个具体的目标，以确定p53功能的分子细节，在这个检查点在响应短暂或持续的DNA损伤。在第一个目标，p53依赖的转录调控机制，影响有丝分裂的进入和进展将得到阐明。编码有丝分裂调节因子的基因包括Cdc 25 C、Survivin、Cdc 20和Cyclin B1是转录下调的靶点。为了阐明分子基础，将研究cdc 25 C基因的调节作为代表。在Cdc 25 C抑制的详细研究中获得的见解，然后将检查p53依赖性的Survivin，Cdc 20和细胞周期蛋白B1的转录下调。在第二个目标，p53依赖的转录后调节机制，在DNA损伤的反应将进行研究。这些包括Mdm 2在影响p21 CIP 1抑制细胞周期蛋白依赖性激酶的能力和调节Cdc 25 C蛋白稳定性方面的新作用。初步数据显示，不仅Cdc 25 C，而且Survivin、Cdc 20和Cyclin B1的蛋白质稳定性受p53调节。这一有趣的发现将进一步探讨。在第三个目标，p53及其下调的目标在有丝分裂进入和发展后，短暂或持续的DNA损伤的作用将被检查。在用DNA损伤剂瞬时处理后，野生型p53细胞可逆地阻滞和修复损伤，而p53无效细胞不能这样做并死亡。这些影响的分子基础将被阐明。拟议的研究解决了理解p53功能的一个核心问题，即它如何介导细胞周期检查点以应对DNA损伤。这些计划中的实验的创新包括研究p53的新靶点，其涉及以DNA结合依赖的方式发生的转录抑制，新观察到p53调节特定有丝分裂调节因子作为检查点激活的一部分的蛋白质稳定性，以及Mdm 2作为p53介导的细胞反应的下游效应子发挥积极作用的有趣的可能性。这项研究的意义涉及选择性靶向具有缺陷p53通路的肿瘤细胞的临床意义，特别是考虑到癌症中p53突变的频率。总之，计划中的实验将详细了解p53如何介导DNA损伤检查点。这有望为人类癌症的预后和治疗提供新的途径。公共卫生相关性：p53蛋白在人类癌症中作为肿瘤抑制因子发挥着重要作用，p53基因突变是人类肿瘤中的常见事件。p53在介导对DNA损伤的细胞反应中具有充分表征的作用。由于许多癌症化疗药物通过对基因组DNA造成损伤来发挥作用，因此了解p53参与的分子基础将确定增强治疗效果的新途径。

DESCRIPTION (provided by applicant): p53 has clearly been implicated as a tumor suppressor in human cancer with mutation of the p53 gene being a common event. p53 has a well-characterized role in mediating the cellular response to DNA damage. The checkpoint in the G1 phase has been shown to be strictly p53-dependent. Due to the existence of a G2/M checkpoint that occurs in its absence, the precise role of p53 in preventing mitotic entry has been elusive. The studies in this application will address this key aspect of p53 biology. Three specific aims are proposed to determine the molecular details of p53 function in this checkpoint in response to transient or sustained DNA damage. In the first aim, p53-dependent mechanisms of transcriptional regulation that affect mitotic entry and progression will be elucidated. The genes encoding mitotic regulators including Cdc25C, Survivin, Cdc20, and Cyclin B1 are targets for transcriptional downregulation. To elucidate the molecular basis, the regulation of the cdc25C gene will be studied as being representative. Insights gained in the detailed study of Cdc25C repression will then be examined for p53-dependent transcriptional downregulation of Survivin, Cdc20, and Cyclin B1. In the second aim, p53-dependent mechanisms of post-transcriptional regulation in response to DNA damage will be studied. These include novel roles for Mdm2 in affecting the ability of p21CIP1 to inhibit cyclin-dependent kinases and in regulating Cdc25C protein stability. Preliminary data show that protein stability of not only Cdc25C, but also Survivin, Cdc20, and Cyclin B1 is regulated by p53. This intriguing finding will be further explored. In the third aim, the role of p53 and its downregulated targets in mitotic entry and progression after transient or sustained DNA damage will be examined. Upon transient treatment with DNA damaging agents wild-type p53 cells reversibly arrest and repair the damage, whereas p53 null cells fail to do so and die. The molecular basis for these effects will be elucidated. The proposed studies address a central issue in understanding p53 function, namely how it mediates cell cycle checkpoints in response to DNA damage. The innovation of these planned experiments include the study of a new target for p53 that involves transcriptional repression occurring in a DNA binding-dependent manner, the novel observation that p53 regulates protein stability of particular mitotic regulators as part of checkpoint activation, and the intriguing possibility that Mdm2 plays a positive role as a downstream effector of p53-mediated cellular responses. The significance of this research relates to the clinical implications of selective targeting of tumor cells with a defective p53 pathway, especially given the frequency of p53 mutation in cancer. Taken together, the planned experiments will elaborate a detailed understanding of how p53 mediates DNA damage checkpoints. This is expected to provide new avenues of pursuit that are relevant for prognosis and treatment of human cancer. PUBLIC HEALTH RELEVANCE: The p53 protein has clearly been implicated in playing an important role as a tumor suppressor in human cancer with mutation of the p53 gene being a common event in human tumors. p53 has a well-characterized role in mediating the cellular response to DNA damage. As many cancer chemotherapeutic agents exert effects by causing damage to genomic DNA, understanding the molecular basis for the involvement of p53 will identify new avenues for enhanced therapeutic efficacy.