Mouse Models to Dissect p53 Tumor Suppressor Function

描述（由申请人提供）：研究结果强调了p53蛋白在肿瘤抑制中的关键作用，即p53在超过一半的人类癌症中发生突变，并且p53缺陷小鼠普遍发生癌症。虽然p53在应激信号下通过诱导细胞周期阻滞、衰老或凋亡来抑制癌症，但其在体内作用的分子机制尚不清楚。p53作为一种转录激活因子显示出活性，可以诱导许多靶基因，但它也具有多种其他生化活性，如调节转录抑制、DNA重组和线粒体膜完整性。为了阐明与肿瘤抑制最相关的p53分子活性，我们之前产生了表达p53突变体p5325,26的敲入小鼠，该突变体在大多数基因的反激活方面受到严重损害，但保留了一小部分靶标（如Bax）的强大反激活能力。此外，p5325,26在衰老和肿瘤抑制中保持生物活性。这些观察结果表明，p5325,26的肿瘤抑制功能是由以下因素造成的：1)靶基因如Bax残留的强反激活，2)大多数其他p53靶基因的极低水平表达，或3)p53的另一种活性。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的两个转激活域p5325,26,53,54的突变，表达完全不具备转激活的突变体的敲入小鼠，将评估转激活对细胞周期阻滞、衰老和凋亡的p53效应功能以及体内组织肿瘤抑制的重要性。此外，在对癌症治疗的反应中，p53诱导的肿瘤消退的转激活的作用将被确定。这些研究使用敲入小鼠，其中突变体在天然p53启动子的控制下表达，并且可以在离体和生物体生理环境中研究正常细胞，应该为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靶基因的全补体激活或其中一部分基因的激活对肿瘤抑制是否重要，以及其他p53生化活性对肿瘤抑制是否重要，将有助于开发更好的癌症治疗方法。

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.