MAMMALIAN GENOME ORGANIZATION AND GENE AMPLIFICATION

癌基因扩增在人类肿瘤中是非常常见的现象， 而药物外排转运体的放大是导致 癌症中的多药耐药。我们已经通过原位荧光显示 杂交(FISH)在CHO细胞中扩增dhfr基因是 通常是由染色体断裂引起的。另外两个重要的 最近有报道说：1)肿瘤抑制基因p53， 显然参与了损伤感知信号转导通路 这阻止了复制，直到损伤可以修复为止，以及2)带有 野生型P53不能进行扩增。这些数据调用了一个统一的 没有活性P53的肿瘤细胞通过 单链损伤，导致染色体断裂，然后 启动扩音器。然而，我们的数据并没有涉及放大问题 由双微小染色体介导的，如在人类和小鼠身上所发生的 细胞。此外，相对于后来的情况，信息仍然缺乏。 导致较高扩增拷贝数的扩增事件。 此外，上面概述的简单模型与 观察到，可以扩增DNA的CHO细胞(意味着缺乏 野生型P53)表现出损伤阻止表型 (暗示存在P53活性)。建议的具体目标 项目有：1)利用双色FISH分析获得详细的 在初始染色体之后发生的后续重排的图片 2)确定DHFR基因的扩增是否也 在自发扩增过程中和之后由染色体断裂引发的 DNA损伤处理，提高扩增频率；3) 为了测试扩增是否在其他细胞系中扩增出 也是由染色体断裂引起的； 4)确定中国仓鼠细胞中P53的表达状况 并确定P53活性的急剧变化是否可以调节 细胞扩增dhfr基因的能力。

Amplification of oncogenes is a very frequent phenomenon in human tumors, and the amplification of drug efflux transporters is a major cause of multi-drug resistance in cancer. We have shown by fluorescence in situ hybridization (FISH) that amplification of the DHFR gene in CHO cells is usually initiated by chromosome breaks. Two additional important observations have recently been reported: 1) the tumor suppressor, p53, is apparently involved in a damage-sensing signal transduction pathway that prevents replication until damage can be repaired, and 2) cells with wild-type p53 cannot undergo amplification. These data invoke a unifying model in which tumor cells that have no active p53 replicate through single-strand lesions, leading to chromosome breaks which can then initiate amplification. However, our data does not address amplification mediated by double minute chromosomes, as occurs in human and murine cells. In addition, information is still lacking relative to subsequent amplification events that lead to higher amplicon copy numbers. Furthermore, the simple model outlined above is not consistent with the observation that CHO cells, which can amplify their DNA (implying a lack of wild-type p53) nevertheless exhibit the damage-arrest phenotype (implying the presence of p53 activity). Specific aims of the proposed project are: 1) to use two-color FISH analysis to obtain a detailed picture of subsequent rearrangements that occur after initial chromosome breaks; 2) to determine whether amplification of the DHFR gene is also initiated by chromosome breaks during spontaneous amplification and after DNA damaging treatments that increase the frequency of amplification; 3) to test whether amplification in other cell lines in which the amplicons are carried on double minutes is also initiated by chromosome breaks; and 4) to determine the status of p53 in appropriate Chinese hamster cells and to determine whether an acute change in p53 activity can regulate a cell's ability to amplify the DHFR gene.