Role of p53 in cell cycle checkpoints

描述（由申请人提供）：p53在人类癌症中作为肿瘤抑制因子明显存在，p53基因突变是一种常见事件。p53在介导细胞对DNA损伤的反应中起着很好的作用。G1期的检查点已被证明是严格依赖p53的。由于G2/M检查点的存在，p53在阻止有丝分裂进入中的确切作用一直是难以捉摸的。本应用程序的研究将解决p53生物学的这一关键方面。我们提出了三个特定的目标来确定p53在这个检查点中对短暂或持续DNA损伤的反应的分子细节。在第一个目标中，p53依赖的转录调控机制影响有丝分裂的进入和进展将被阐明。编码有丝分裂调节因子的基因包括Cdc25C、Survivin、Cdc20和Cyclin B1是转录下调的靶点。为阐明其分子基础，本文将以cdc25C基因的调控为代表进行研究。在Cdc25C抑制的详细研究中获得的见解，然后将检查p53依赖性的Survivin， Cdc20和Cyclin B1的转录下调。在第二个目标中，p53依赖的转录后调控机制响应DNA损伤将被研究。其中包括Mdm2在影响p21CIP1抑制细胞周期蛋白依赖性激酶的能力和调节Cdc25C蛋白稳定性方面的新作用。初步数据显示，p53不仅调控Cdc25C蛋白的稳定性，还调控Survivin、Cdc20、Cyclin B1蛋白的稳定性。这一有趣的发现将被进一步探索。在第三个目标中，p53及其下调靶点在短暂或持续DNA损伤后有丝分裂进入和进展中的作用将被研究。在DNA损伤剂的短暂处理下，野生型p53细胞可逆地抑制和修复损伤，而p53无细胞不能这样做并死亡。这些效应的分子基础将被阐明。提出的研究解决了理解p53功能的核心问题，即它如何介导细胞周期检查点以响应DNA损伤。这些计划中的实验的创新之处包括研究p53的新靶点，该靶点涉及以DNA结合依赖的方式发生的转录抑制，p53调节特定有丝分裂调节剂的蛋白质稳定性作为检查点激活的一部分的新观察，以及Mdm2作为p53介导的细胞反应的下游效应物发挥积极作用的有趣可能性。本研究的意义在于选择性靶向具有p53通路缺陷的肿瘤细胞的临床意义，特别是考虑到p53在癌症中的突变频率。总的来说，计划中的实验将详细阐述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研究途径。

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.