Analysis of Loss of Function of MAD2 in Mammals

描述(申请人提供)：癌症中的遗传不稳定性 通常发生在整个染色体水平，称为染色体 不稳定(CIN)。有丝分裂检查点将中期有丝分裂延迟到 微管与运动中枢的连接完成，允许 将染色体平等地分离到两个子细胞。我们最近做了 在原代小鼠胚胎成纤维细胞和人类肿瘤细胞中均显示 MAD2是一种有丝分裂检查点基因，丢失一个等位基因就足以导致 染色体不稳定。我们假设这个检查站的中断 随之而来的非整倍体将导致细胞生长失控，并可能 促进肿瘤的形成。因此，这项建议的目标是 进一步阐明MAD2在基因组维持中的作用 稳定性、肿瘤启动和/或进展，以及对有丝分裂的反应 通过使用各种遗传方法来挑战检查点。 我们的具体目标是：(L)确定单倍体不足在 MAD2基因座对小鼠细胞生长和肿瘤发生的影响。MAD2+/-小鼠 完全可行，尽管MEF的非整倍体程度很高。 对于这种单倍体不足的表型，我们将研究 自发的肿瘤形成，细胞活力和转化的改变， 以及对纺锤体断裂和化学致癌的反应。(2)至 确定非整倍体是否足以改变肿瘤的侵袭性和 它是否能促进人类细胞的转化。我们已经使用了体细胞 细胞基因打靶技术灭活一个MAD2等位基因并诱导 染色体稳定的人类肿瘤细胞系中的非整倍体。这个 这些细胞的转移行为及其对临床的敏感性 相关的纺锤体抑制剂将在裸鼠模型中进行研究。同样， 在未转化的成纤维细胞中将产生杂合子MAD2缺失 和上皮细胞，以及非整倍体对细胞生长、活力的影响， 并将对转型进行调查。(3)确定 小鼠完全失去有丝分裂检查点控制。考虑到胚胎 MAD2-/-小鼠的致命性，这个问题将使用CRE- 后续组织特异性LOX条件性基因打靶方法 乳腺上皮和B细胞系中MAD2的缺失。对以下方面的影响 组织特异性MAD2的发育、染色体稳定性与肿瘤发生 零突变将被研究。

DESCRIPTION (provided by applicant): Genetic instability in cancers frequently occurs at the whole chromosome level, referred to as chromosome instability (CIN). The mitotic checkpoint delays mitosis at metaphase until the attachment of microtubules to the kinetochores is complete, allowing for equal segregation of chromosomes to the two daughter cells. We have recently shown in both primary murine embryonic fibroblasts and human tumor cells that loss of one allele of MAD2, a mitotic checkpoint gene, is sufficient to cause chromosome instability. We hypothesize that disruption of this checkpoint with its attendant aneuploidy will result in deregulated cell growth and may facilitate tumor formation. Therefore, the objective of this proposal is to further elucidate the role that MAD2 plays in the maintenance of genomic stability, tumor initiation and/or progression, and responses to mitotic checkpoint challenges by the use of various genetic approaches. Our specific aims are: (l) To determine the role of haploinsufficiency at the Mad2 locus on cell growth and tumorigenesis in mice. Mad2 +/- mice are completely viable despite the high degree of aneuploidy in MEF's. The effects o f this haploinsufficient phenotype will be studied with respect to spontaneous tumor formation, alterations in cell viability and transformation, and response to spindle disruption and chemical carcinogenesis. (2) To determine if aneuploidy alone is sufficient to alter tumor aggressiveness and whether it can facilitate transformation in human cells. We have used somatic cell gene targeting techniques to inactivate one MAD2 allele and induce aneuploidy in an otherwise chromosomally stable human tumor cell line. The metastatic behavior of these cells and their sensitivity to clinically relevant spindle inhibitors will be studied in a nude mouse model. Similarly, a heterozygous MAD2 deletion will be generated in non-transformed fibroblasts and epithelial cells, and the effect of aneuploidy on cell growth, viability, and transformation will be investigated. (3) To identify the consequences of complete loss of mitotic checkpoint control in mice. Given the embryonic lethality of the Mad2-/- mice, this question will be addressed using the Cre- lox conditional gene targeting approach with subsequent tissues specific deletion of Mad2 in mammary epithelium and B cell lineages. The effects on development, chromosome stability, and tumorigenesis of tissue specific Mad2 null mutations will be studied.