MAMMALIAN GENOME ORGANIZATION AND GENE AMPLIFICATION

癌基因扩增是人类肿瘤中非常常见的现象， 而药物外排转运蛋白的扩增是 癌症的多药耐药性。 我们已经通过荧光原位 杂交（FISH）在CHO细胞中扩增DHFR基因， 通常由染色体断裂引发。 另外两个重要 最近已经报道了观察结果：1）肿瘤抑制因子，p53， 显然参与了损伤感应信号转导途径 防止复制，直到损伤可以修复，和2）细胞与 野生型p53不能进行扩增。 这些数据引发了一个统一的 模型中没有活性p53的肿瘤细胞通过 单链病变，导致染色体断裂，然后可以 启动扩增。 然而，我们的数据并不涉及放大 由双微小染色体介导的，如在人类和小鼠中发生的 细胞 此外，与后续行动有关的信息仍然缺乏。 扩增事件导致更高的扩增子拷贝数。 此外，上面概述的简单模型与 观察到CHO细胞，可以扩增它们的DNA（这意味着缺乏 野生型p53）仍然表现出损伤-停滞表型 （暗示存在p53活性）。 拟议方案的具体目标 项目有：1）使用双色FISH分析，以获得详细的 在初始染色体之后发生的随后重排的图片 2）确定DHFR基因的扩增是否也是 在自发扩增期间和之后由染色体断裂引发 增加扩增频率的DNA损伤处理; 3） 为了测试在扩增子 进行双分钟也是由染色体断裂启动;和 4)以确定p53在适当的中国仓鼠细胞中的状态 并确定p53活性的急性变化是否可以调节 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.