Analysis of Loss of Function of MAD2 in Mammals

哺乳动物 MAD2 功能丧失分析

• 批准号: 6693841
• 负责人: LOREN Scott MICHEL
• 金额: $ 13.08万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-09 至 2004-10-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693841
• 关键词: aneuploidy; athymic mouse; carcinogenesis; cell growth regulation; fibroblasts; gene deletion mutation; lung neoplasms; lymphocyte; mammary epithelium; metastasis; neoplasm /cancer genetics; oncoproteins; p53 gene /protein

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.

描述（由申请人提供）： 癌症的遗传不稳定性 通常发生在整个染色体水平，称为染色体 不稳定性（CIN）。 有丝分裂检查点延迟中期有丝分裂， 微管与动粒的连接完成， 两个子细胞的染色体平均分离。 我们最近 在原代鼠胚胎成纤维细胞和人肿瘤细胞中显示， 有丝分裂检查点基因MAD 2的一个等位基因的缺失足以引起 染色体不稳定性 我们假设破坏这个检查点 伴随着非整倍性将导致细胞生长失调， 促进肿瘤形成。 因此，本建议的目的是 进一步阐明MAD 2在维持基因组中的作用， 稳定性、肿瘤发生和/或进展以及对有丝分裂的反应 检查点的挑战，通过使用各种遗传方法。 我们的具体目标是：（1）确定单倍不足在 Mad 2基因座对小鼠细胞生长和肿瘤发生的影响。 Mad 2 +/-小鼠 尽管MEF中的非整倍性程度很高，但它是完全可行的。 的影响 外来资产 将研究这种单倍不足表型， 自发肿瘤形成，细胞活力和转化的改变， 以及对纺锤体破坏和化学致癌作用的反应。 (2)到 确定单独的非整倍性是否足以改变肿瘤的侵袭性， 是否能促进人类细胞的转化。 我们使用了体细胞 细胞基因靶向技术灭活一个MAD 2等位基因并诱导 在其他染色体稳定的人肿瘤细胞系中的非整倍性。 的 这些细胞的转移行为和它们对临床上 将在裸鼠模型中研究相关的纺锤体抑制剂。 与此类似， 在非转化的成纤维细胞中将产生杂合的MAD 2缺失 以及非整倍性对细胞生长，活力， 将对转型进行研究。 (3)为了确定的后果 小鼠中完全丧失有丝分裂检查点控制。 鉴于胚胎 Mad 2-/-小鼠的致死率，这个问题将使用Cre- lox条件性基因靶向方法，随后组织特异性 在乳腺上皮和B细胞谱系中缺失Mad 2。 的效果 组织特异性Mad 2的发育、染色体稳定性和肿瘤发生 将研究无效突变。