Nucleoporins in Chromosomal Instability and Cancer

摘要 绝大多数人类癌症都有异常数量的染色体，称为非整倍体。 然而，非整倍体的分子基础及其在肿瘤发生中的作用仍然知之甚少。 最近的研究导致了一类新的有丝分裂调节因子的鉴定，其由介导细胞分裂的蛋白质组成。 间期核质运输。我们广泛的长期目标是提供对生物学的深入了解， 这些核转运因子的相关性及其在防止染色体不稳定性中的可能作用， 肿瘤发生目前建议的具体目标是解剖核孔的有丝分裂功能 复杂蛋白RanBP 2在分子，细胞和有机体水平，并确定如何RanBP 2 下调促进肿瘤发生。我们已经产生了一系列的小鼠，其中核孔蛋白 RanBP 2蛋白通过使用野生型、敲除和表达载体以从正常到零的分级方式降低。 亚型等位基因缺乏RanBP 2的小鼠是胚胎致死的，但是具有非常低量的RanBP 2的小鼠是胚胎致死的。 蛋白质是可行的和明显正常的。与RanBP 2在有丝分裂中的作用一致，我们发现这些小鼠 发展成严重的非整倍体我们观察到的主要有丝分裂缺陷是染色质桥的形成， 分裂后期，一种让人联想到拓扑异构酶II功能受损的表型。在具体目标一中，我们将使用 遗传和生物化学方法来建立RanBP 2精确调节的机制 后期姐妹染色单体分离。此外，使用条件性敲除细胞，我们将确定 RanBP 2的关键功能结构域。初步研究表明，RanBP 2水平低的小鼠 增加对自发性和致癌物诱导的肿瘤的易感性，尤其是肺肿瘤。重要的是， 使用原发性人类肿瘤和人类癌细胞系的定量RT-PCR分析，我们发现， RanBP 2在许多肺腺癌中的表达显著降低，表明RanBP 2在肺腺癌中具有一定的生物学活性。 在小鼠和人类中的肿瘤抑制功能。在具体目标二中，我们将利用已经建立的和 新设计的RanBP 2突变小鼠模型，以解决RanBP 2不足的机制 促进肿瘤发生。RanBP 2突变小鼠对致癌物DMBA的高度敏感性表明， RanBP 2不足与肿瘤发生中的其他基因突变强烈协同作用。具体目标 第三，我们将通过使用“睡美人”转座子系统来识别这些癌症基因突变。在 除了这种无偏的方法，我们将使用候选基因的方法来确定是否K-ras 在肺癌发生中与RanBP 2缺陷协同作用。在基础研究层面，完成这些 aims将提供对一个重要的核输运因子维持的机制的深入了解。 染色体稳定性和预防癌症。在临床层面，这些研究可能为以下方面提供依据： 改善人类癌症的检测、预防和治疗。

ABSTRACT The vast majority of human cancers have abnormal numbers of chromosomes, known as aneuploidy. However, the molecular basis of aneuploidy and its role in tumor development remain poorly understood. Recent studies led to the identification of a new class of mitotic regulators consisting of proteins that mediate nucleocytoplasmic transport in interphase. Our broad long-term goal is to provide insight into the biological relevance of these nuclear transport factors and their possible role in preventing chromosomal instability and tumorigenesis. The specific goal of the current proposal is to dissect the mitotic functions of the nuclear pore complex protein RanBP2 at the molecular, cellular and organismal levels, and to determine how RanBP2 downregulation promotes tumorigenesis. We have generated a series of mice in which the nucleoporin RanBP2 protein is reduced in a graded fashion from normal to zero by the use of wild-type, knockout and hypomorphic alleles. Mice lacking RanBP2 are embryonically lethal, but mice with very low amounts of the protein are viable and overtly normal. Consistent with a role for RanBP2 in mitosis, we find that these mice develop severe aneuploidy. The main mitotic defect that we observe is chromatin-bridge formation in anaphase, a phenotype reminiscent of impaired topoisomerase II¿ function. In specific aim one, we will use both genetic and biochemical approaches to establish the mechanism by which RanBP2 regulates accurate sister chromatid segregation in anaphase. Furthermore, using conditional knockout cells we will determine the critical functional domain(s) of RanBP2. Preliminary studies show that mice with low levels of RanBP2 have increased susceptibility to spontaneous and carcinogen-induced tumors, especially lung tumors. Importantly, using quantitative RT-PCR analysis of primary human tumors and human cancer cell lines we found that RanBP2 expression is dramatically reduced in many lung adenocarcinomas, suggesting that RanBP2 has a tumor suppressive function in both mice and humans. In specific aim two, we will use already established and newly designed RanBP2 mutant mouse models to resolve the mechanism by which RanBP2 insufficiency promotes tumorigenesis. The profound sensitivity of RanBP2 mutant mice to the carcinogen DMBA indicates that RanBP2 insufficiency strongly synergizes with other gene mutations in tumorigenesis. In specific aim three, we will identify these cancer gene mutations by the use of the "Sleeping Beauty" transposon system. In addition to this unbiased approach, we will use a candidate gene approach to determine whether K-ras synergizes with RanBP2 deficiency in lung carcinogenesis. At the basic research level, completion of these aims will provide insight into the mechanism by which a prominent nuclear transport factor maintains chromosomal stability and prevents cancer. At the clinical level, these studies may provide the basis for improved detection, prevention and treatment of cancer in humans.