AMPLIFICATION--MODEL FOR GENETIC INSTABILITY IN CANCER

扩增——癌症遗传不稳定性模型

• 批准号: 6845723
• 负责人: JOYCE L HAMLIN
• 金额: $ 31.17万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-02 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845723
• 关键词: CHO cells; DNA damage; DNA repair; DNA replication; Herpesviridae; dihydrofolate reductase; fluorescent in situ hybridization; ganciclovir; gene redundancy; gene targeting; genetic models; methotrexate; natural gene amplification; neoplasm /cancer genetics; restriction mapping; telomere; thymidine kinase; virus genetics

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.

癌基因的扩增是人类肿瘤进展的重要决定因素。我们通过荧光原位杂交 (FISH) 获得了大量证据，表明 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导致的染色体断裂启动的。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有关、染色单体融合的机制以及在扩增到更高拷贝数期间修剪和均质化重复单元的过程。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个步骤的分子机制。该提案的具体目标是： l) 通过分离和表征第一步扩增中明确断裂染色体末端的融合产物，定义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 基因下游引入稀有切割限制性位点盒，并通过反式引入相关限制性酶来引发断裂； 2) 确定端粒的短暂暴露是否会导致基因扩增；显性失活端粒结合 TRF2 蛋白将在具有 DHFR 近端粒的细胞中过表达，以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) 和限制性图谱分析重排； 3) 确定 CHO 细胞中断裂诱导的复制发生的程度，作为扩增子均质化和修剪的可能模型；我们将确定截短的 DHFR 基因的结构是否已通过广泛的基因转换事件恢复为野生型，同时保留原始的缺失连接； 4) 检验断裂诱导的复制可以启动扩增或可以缩短、均质化和扩增最初较大的异质扩增子的假设； FISH 分析将用于将目标 2 中分离的细胞系分为明显经历了起始桥断裂融合循环的细胞系和似乎在局部扩增 DHFR 基因的细胞系；限制性图谱将揭示在没有启动桥-断裂-融合循环的情况下，那些局部扩增是否是通过滚环复制来实现的。