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。我们已经获得了大量的证据，通过荧光原位杂交（FISH）的模型二氢叶酸还原酶（DHFR）基因在CHO和人类细胞系的扩增是由姐妹染色单体融合导致的染色体断裂。主要的未知因素包括断裂的根本原因，以及它们是否与端粒维持、染色单体融合的机制以及在扩增过程中修剪和均质化重复单元以获得更高拷贝数的过程有关。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个步骤的分子机制。1）通过分离和表征第一步扩增物中明确断裂的染色体末端的融合产物，确定在初始染色体断裂后融合染色单体的机制;将使用有效的同源重组方法在DHFR基因的下游引入稀有切割限制性位点的盒，并通过反式引入相关限制性内切酶来引发断裂; 2）确定端粒的瞬时暴露是否可导致基因扩增;显性负性端粒结合TRF 2蛋白将在具有DHFR近端端粒的细胞中过表达以诱导端对端染色体融合;然后确定扩增频率，并通过荧光原位杂交（FISH）和限制性作图分析重排; 3）确定断裂诱导的复制在CHO细胞中可发生的程度，作为扩增子均质化和修剪的可能模型;我们将确定已恢复为野生型同时保留原始缺失连接的截短DHFR基因的结构是否是通过广泛的基因转换事件实现的;和4）检验断裂诱导的复制可以启动扩增或可以缩短、均质化和扩增最初大的异源扩增子的假设; FISH分析将用于将Aim 2中分离的细胞系分成明显经历起始桥断裂的那些细胞系。从那些看起来已经扩增DHFR基因的片段中分离出融合循环;限制性作图将揭示那些原位扩增的片段是否在没有起始桥断裂融合循环的情况下通过滚环复制而这样做。

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.