AMPLIFICATION--MODEL FOR GENETIC INSTABILITY IN CANCER

扩增——癌症遗传不稳定性模型

• 批准号: 6693857
• 负责人: JOYCE L HAMLIN
• 金额: $ 28.04万
• 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 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693857
• 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。我们已经通过荧光原位杂交(FISH)获得了大量证据，表明模型二氢叶酸还原酶(DHFR)基因在CHO和人类细胞系中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，以及在扩增过程中为了获得更高的拷贝数而修剪和均质重复单位的过程。这些步骤中的任何一个都是潜在的化疗靶点，我们的长期目标是了解每一步的分子机制。这一建议的具体目的是：L)通过分离和鉴定第一步扩增中明确定义的染色体末端的融合产物(S)来确定在最初的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基因下游引入一盒稀有切割限制位点，并通过在反式中引入相关的限制性酶来引发断裂；2)确定瞬时暴露端粒是否可以导致基因放大；在具有DHFR-近端端粒的细胞中过表达显性负端粒结合的TRF2蛋白，以诱导染色体端到端的融合；然后将确定扩增的频率，并将通过荧光原位杂交(FISH)和限制性内切酶图谱分析重排；3)确定断裂诱导的复制在CHO细胞中发生的程度，作为均质和修剪扩增的可能模型；我们将确定已恢复到野生型并保留原始缺失连接的截断的DHFR基因的结构是否通过广泛的基因转换事件来实现；以及4)检验断裂诱导的复制可以启动扩增或可以缩短、均质和扩增最初大的、异源的扩增片段的假设；FISH分析将用于将在Aim 2中分离的细胞系分为明显经历了启动断桥-融合循环的细胞系，而那些似乎在LOCO中扩增了DHFR基因的细胞株；限制性内切酶图谱将揭示在没有启动断桥-融合循环的情况下，在LOCO中扩增的那些细胞系是否通过滚环复制做到了这一点。