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以下发现所强调，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仍不清楚。p53作为一种转录激活因子，可以诱导许多靶基因，但它也具有多种其他生物化学活性，在调节转录抑制，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，我们先前产生了表达p53突变体p5325的敲入小鼠，该突变体对于大多数基因的反式激活严重受损，但保留了稳健地反式激活一小部分靶点（如Bax）的能力。此外，p5325，26在衰老和肿瘤抑制中保留生物活性。这些观察结果表明，p5325，26肿瘤抑制功能是由1）靶基因（如Bax）的残留强反式激活，2）大多数其他p53靶标的极低水平表达，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。通过检查表达突变体的敲入小鼠，所述突变体通过两个p53反式激活结构域p5325、26、53、54的突变而完全缺乏反式激活，将评估反式激活对于细胞周期停滞、衰老和凋亡的p53效应子功能以及对于体内组织中的肿瘤抑制的重要性。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。这些使用敲入小鼠的研究，其中突变体在天然p53启动子的控制下表达，并且其中正常细胞可以离体和在生物体的生理环境中进行研究，应该提供对p53在肿瘤抑制和治疗反应中作用机制的明确认识.了解p53靶基因的完整补体或这些基因的子集的激活是否对肿瘤抑制很重要，以及其他p53生物化学活性是否对肿瘤抑制很重要，将导致更好的癌症治疗的发展。

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.