BUBRI in Normal and Neoplastic Growth

描述（由申请人提供）：染色体数目异常是绝大多数人类癌症的标志。这些癌症的一个子集与编码有丝分裂检查点蛋白（如BUB1、BUBR1或MAD2）的基因突变有关。这些检查点蛋白是复杂的调节机制的一部分，通过延迟姐妹染色单体分离，直到所有染色体都正确地附着在有丝分裂纺锤体上并在中期板上排列，从而防止非整倍体。因此，当它们有缺陷时，可能会导致染色体不稳定。最初试图通过经典的基因敲除方法研究小鼠有丝分裂检查点功能障碍与疾病之间可能的联系，但由于缺乏BUB3或MAD2的小鼠早期胚胎死亡而受到阻碍。为了建立一个有丝分裂检查点功能障碍的小鼠模型，我们产生了具有亚形态BUBR1等位基因（BUBR1 hypo）的小鼠。半胚型BUBR1等位基因纯合的小鼠尽管其BUBR1蛋白水平大幅降低，但仍能存活。初步数据表明，在几个纯合突变小鼠中形成组织学证实的肿瘤。对来自13.5日龄纯合突变胚胎的早期传代成纤维细胞的详细分析表明，BUBR1表达减少导致姐妹染色单体过早分离和非整倍体。在这个项目中，我们提出了实验来阐明BUBR1的生物学作用，以及BUBR1功能障碍可能导致恶性细胞转化的机制。在具体目标一中，我们将使用可诱导的BUBR1敲除细胞系来确定BUBR1及其关键功能域在体内的作用。在具体目标二中，我们将确定低形态BUBR1小鼠自发和致癌物质诱导的肿瘤形成率，并在机体水平上研究BUBR1功能障碍对染色体不稳定性的影响。在具体目标3中，我们将确定能够与BUBR1合作形成肿瘤的致瘤途径。总之，这些研究将为BUBR1的正常功能提供有价值的见解，并有助于阐明控制有丝分裂纺锤体组装检查点的错误如何导致对肿瘤形成的易感性增加。

DESCRIPTION (provided by applicant): Abnormalities of chromosome number are a hallmark of the vast majority of human cancers. A subset of these cancers is associated with mutations in genes encoding for mitotic checkpoint proteins such as BUB1, BUBR1 or MAD2. These checkpoint proteins are part of an intricate regulatory mechanism that prevents aneuploidy by delaying sister chromatid separation until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Therefore, they may contribute to chromosomal instability when defective. Initial attempts to study the possible connection between mitotic checkpoint dysfunction and disease by classical gene knockout approaches in the mouse have been hampered by the early embryonic death of mice lacking BUB3 or MAD2. In an effort to generate a mouse model for mitotic checkpoint dysfunction, we generated mice with a hypomorphic BUBR1 allele (BUBR1 hypo). Mice homozygous for the hypomorphic BUBR1 allele are viable despite a strong reduction of their BUBR 1 protein levels. Preliminary data demonstrate the formation of histological confirmed tumors in several homozygous mutant mice. Detailed analyses of early-passage fibroblasts derived from 13.5-day-old homozygous mutant embryos show that the diminished BUBR1 expression causes premature sister chromatid separation and aneuploidy. In this project we have proposed experiments to elucidate the biological role of BUBR1, and the mechanisms by which BUBR1 dysfunction may lead to malignant cell transformation. In specific aim one, we will use inducible BUBR1 knockout cell lines to establish the in vivo role of BUBR1 and its critical functional domains. In specific aim two, we will determine the rates of spontaneous and carcinogen-induced tumor formation in hypomorphic BUBR1 mice, and investigate the effect of BUBR1 dysfunction on chromosomal instability at the organismal level. In specific aim three, we will identify the tumorigenic pathways able to cooperate with BUBR1 in the formation of tumors. Together these studies will provide valuable insights into the normal function of BUBR1 and help to elucidate how errors in the control of the mitotic spindle assembly checkpoint lead to increased susceptibility to tumor formation.