AMPLIFICATION--MODEL FOR GENETIC INSTABILITY IN CANCER

癌基因的扩增是人类肿瘤进展的重要决定因素。我们已经通过荧光原位杂交（FISH）获得了大量证据，证明在CHO和人类细胞系中，模型二氢叶酸还原酶（DHFR）基因的扩增是由姐妹染色单体融合引起的染色体断裂引起的。主要的未知因素包括断裂的潜在原因，以及它们是否与端粒维持有关，染色单体融合的机制，以及在扩增过程中修剪和均匀重复单位以获得更高拷贝数的过程。这些步骤中的任何一个都是化疗的潜在目标，我们的长期目标是了解每一步的分子机制。本提案的具体目的是：1)通过分离和表征第一步扩增子中明确定义的断裂染色体末端的融合产物，来确定初始染色体断裂后染色单体融合的机制；一种有效的同源重组方法将在DHFR基因下游引入一盒稀有切割的限制性位点，并通过在反式中引入相关的限制性内切酶来诱导断裂；2)确定端粒的短暂揭膜是否会导致基因扩增；端粒结合的显性阴性TRF2蛋白将在具有dhfr -近端端粒的细胞中过度表达，从而诱导端到端染色体融合；然后确定扩增频率，并通过荧光原位杂交（FISH）和限制性位图分析重排；3)确定断裂诱导复制在CHO细胞中发生的程度，作为扩增子均匀化和修剪的可能模型；我们将确定截断的DHFR基因的结构是否通过广泛的基因转换事件恢复到野生型，同时保留了原始的缺失连接；4)验证断裂诱导复制可以启动扩增或可以缩短、均匀化和扩增最初较大的异质扩增子的假设；FISH分析将用于将Aim 2中分离的细胞系分为那些明显经历了初始桥接-断裂-融合周期的细胞系和那些似乎在原位扩增了DHFR基因的细胞系；限制映射将揭示在没有初始桥接-断裂-融合循环的情况下，那些在loco中扩增的细胞是否通过滚动圈复制来实现。

The amplification of oncogenes is an important determinant of tumor progression in humans. We have obtained substantial evidence by fluorescence in situ hybridization (FISH) that amplification of the model dihydrofolate reductase (DHFR) gene in both CHO and human cell lines is initiated by chromosome breaks resulting from sister chromatid fusions. Major unknowns include the underlying causes of the breaks and whether they relate to telomere maintenance, the mechanism of chromatid fusion, and the processes that trim and homogenize repeating units during amplification to higher copy number. Any of these steps is a potential target for chemotherapy, and our long-range goals are to understand the molecular mechanisms operating at each step. Specific aims of this proposal are: l) to define the mechanism that fuses chromatids after an initial chromosome break by isolating and characterizing the fusion product(s) of well-defined broken chromosome ends in first-step amplificants; an efficient homologous recombination approach will be used to introduce a cassette of rare-cutting restriction sites just downstream from the DHFR gene, and breaks will be elicited by introduction of the relevant restriction enzyme in trans; 2) to determine whether transient unmasking of telomeres can lead to gene amplification; a dominant-negative telomere-binding TRF2 protein will be over- expressed in cells with a DHFR-proximal telomere to induce end-to-end chromosome fusions; the frequency of amplification will then be determined, and rearrangements will be analyzed by fluorescence in situ hybridization (FISH) and restriction mapping; 3) to determine the extent to which break-induced replication can occur in CHO cells, as a possible model for homogenization and trimming of amplicons; we will determine whether the structures of truncated DHFR genes that have been restored to wild-type while retaining the original deletion junction have done so by an extensive gene conversion event; and 4) to test the hypothesis that break-induced replication can initiate amplification or can shorten, homogenize, and amplify initially large, heterogenous amplicons; FISH analysis will be used to divide the cell lines isolated in Aim 2 into those that obviously underwent initiating bridge-breakage-fusion cycles from those that appear' to have amplified the DHFR gene in loco; restriction mapping will reveal whether those amplifying in loco did so by rolling circle replication in the absence of an initiating bridge-breakage-fusion cycle.