Mouse Models to Dissect p53 Tumor Suppressor Function

描述(由申请人提供)：p53蛋白在肿瘤抑制中的关键作用被突显出来的结果是，超过一半的人类癌症中P53基因发生突变，并且P53基因缺陷的小鼠普遍发展为癌症。虽然P53通过诱导细胞周期停滞、衰老或凋亡来抑制癌症，但其在体内的分子作用机制尚不清楚。P53是一种转录激活剂，可以诱导许多靶基因的表达，但它也具有其他多种生化活性，调节转录抑制、DNA重组和线粒体膜的完整性。为了阐明与肿瘤抑制最相关的P53分子活性，我们以前产生了表达P53突变体p5325，26的敲入小鼠，该突变体对大多数基因的反式激活作用严重受损，但仍具有强大的反式激活一小部分靶标的能力，如Bax。此外，p5325，26还具有抗衰老和抑制肿瘤的生物活性。这些观察结果表明，p5325，26抑癌基因的功能是由1)靶基因如bax的残留强烈反式激活，2)大多数其他p53靶点的极低水平表达，或3)p53的另一种活性所解释的。在这里，这些可能性将通过产生和分析新的p53敲入突变株来区分。通过检测P53反式激活结构域p5325、26、53、54的突变而表达完全缺陷反式激活的敲入小鼠，将评估反式激活对细胞周期停滞、衰老和凋亡的P53效应功能以及体内组织中肿瘤抑制的重要性。此外，反式激活对P53诱导的肿瘤消退在癌症治疗中的作用将被确定。这些利用转基因小鼠的研究，其中突变体在天然P53启动子的控制下表达，在其中正常细胞可以在体外和在生物体的生理环境中进行研究，应该可以对P53在肿瘤抑制和治疗反应中的作用机制提供明确的见解。了解P53靶基因的全部或部分激活对肿瘤抑制是否重要，以及其他P53生化活动是否对肿瘤抑制重要，将有助于开发更好的癌症治疗方法。 与公共卫生相关：在美国，大约每2-3个人中就有1人会患上癌症，这是一种细胞增殖不受控制的疾病，可能是致命的。因此，了解这种疾病的基础是非常重要的，以便能够更有效地治疗和治愈它。这项研究旨在阐明对预防癌症至关重要的基因p53是如何发挥作用的，最终目标是设计出更好的癌症预防和治疗策略。

DESCRIPTION (provided by applicant): The crucial role of the p53 protein in tumor suppression is underscored by the findings that p53 is mutated in over half of all human cancers and that p53-deficient mice universally develop cancer. While p53 suppresses cancer by inducing cell cycle arrest, senescence or apoptosis in response to stress signals, its molecular mechanism of action in vivo remains unclear. p53 displays activity as a transcriptional activator that can induce numerous target genes, but it also has a variety of other biochemical activities, in regulating transcriptional repression, DNA recombination, and mitochondrial membrane integrity. To clarify the p53 molecular activities most relevant for tumor suppression, we previously generated knock-in mice expressing a p53 mutant, p5325,26, that is severely impaired for the transactivation of most genes but retains the ability to robustly transactivate a small set of targets, such as Bax. Moreover, p5325,26 retains biological activity in senescence and tumor suppression. These observations suggest that p5325,26 tumor suppressor function is accounted for by 1) residual strong transactivation of target genes like Bax, 2) very low level expression of most other p53 targets, or 3) another activity of p53. Here, these possibilities will be distinguished by generating and analyzing novel p53 knock-in mutant strains. By examining knock-in mice expressing a mutant totally defective for transactivation through mutation of both p53 transactivation domains, p5325,26,53,54, the importance of transactivation for p53 effector functions of cell cycle arrest, senescence and apoptosis, as well as for tumor suppression in tissues in vivo, will be assessed. Additionally, the roles of transactivation for p53- induced tumor regression in response to cancer therapy will be determined. These studies using knock- in mice, in which mutants are expressed under the control of the native p53 promoter, and in which normal cells can be studied both ex vivo and in the physiological context of the organism, should provide definitive insight into the mechanism of p53 action in tumor suppression and therapeutic responses. Understanding whether activation of the full complement of p53 target genes or a subset of these genes is important for tumor suppression, as well as whether other p53 biochemical activities are important for tumor suppression, will lead to the development of better cancer therapies. PUBLIC HEALTH RELEVANCE: In the US, approximately 1 in every 2-3 individuals will develop cancer, a disease of uncontrolled cellular proliferation that can be fatal. It is therefore very important to understand the basis of this disease in order to be able to more effectively treat and cure it. This research aims to elucidate how p53, a gene important for preventing cancer, acts, with the ultimate goal of devising better prevention and treatment strategies for cancer.