MECHANISMS OF GENOMIC REARRANGEMENT IN CARCINOGENESIS

基因组重排致癌机制

• 批准号: 2102814
• 负责人: FREDERIC G BARR
• 金额: $ 11.42万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-02-04 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2102814
• 关键词: DNA damage; DNA footprinting; DNA methylation; alternatives to animals in research; carcinogenesis; carcinogens; chromatin; chromosome translocation; computer assisted sequence analysis; human genetic material tag; human tissue; in situ hybridization; method development; neoplasm /cancer genetics; nuclear matrix; nucleic acid sequence; pediatric neoplasm /cancer; polymerase chain reaction; pulsed field gel electrophoresis; restriction mapping; rhabdomyosarcoma; southern blotting; thymidine kinase; tissue /cell culture

The combined work of many investigators has demonstrated the importance of genomic rearrangements in neoplastic development and has established that environmental carcinogens can induce their formation. To analyze the mechanism by which carcinogens induce genomic rearrangement, we have developed a cell culture system in which the inactive endogenous thymidine kinase gene in a hamster cell line is activated and rearranged by carcinogen treatment. Our initial analysis of carcinogen-induced rearrangements in the model cell culture system has suggested a potential role for nuclear organization as well as DNA sequence in the rearrangement process. To investigate the role of these mechanistic features in a human cancer, we have identified the genes involved in the t(2;13) translocation of the pediatric soft tissue tumor alveolar rhabdomyosarcoma. Furthermore, we have developed PCR-based methodologies for isolating rearrangement breakpoints and the associated wild-type rearrangement partners. In the proposed project, we will employ several carcinogenic agents in our cell culture system to isolate multiple independent clones with rearrangements in the vicinity of the hamster thymidine kinase gene. The distribution of breakpoints will be characterized by Southern blot, PCR, and sequencing analyses. These experiments will thereby explore the hypothesis that cancer-causing agents differ in their ability to cause rearrangements. Following isolation of the rearrangement partners, the DNA sequence, chromatin structure, methylation status, nuclear matrix association, and genomic location of breakpoint regions will be examined to test the hypothesis that both genetic and epigenetic features influence the propensity of a region to rearrange. The findings from the cell culture system will be compared to the structural features of the t(2; 13) translocation breakpoints in the human cancer alveolar rhabdomyosarcoma. Finally, PCR methodology will be applied to develop assays for quantitation of the rearrangement frequency of a sequence in a population. Such assays will permit exploration of rearrangement events at a variety of genomic loci as well as evaluation of the role of specific environmental and parental cell features in the rearrangement process by directly manipulating components of the inducible system.

许多调查人员的合并工作表明了 肿瘤发育中的基因组重排，并确定 环境致癌物可以诱导其形成。分析 致癌物诱导基因组重排的机制，我们有 开发了一种细胞培养系统，其中无活性内源性胸苷 仓鼠细胞系中的激酶基因被激活并重新排列 致癌物治疗。我们对致癌诱导的初步分析 模型细胞培养系统中的重排已经提出了潜力 核组织的作用以及重排中的DNA序列 过程。研究这些机械特征在人类中的作用 癌症，我们已经确定了t（2; 13）易位涉及的基因 小儿软组织肿瘤肺泡横纹肌肉瘤。此外， 我们已经开发了基于PCR的方法来隔离重排 断点和相关的野生型重排伙伴。 在拟议的项目中，我们将在我们的 细胞培养系统将多个独立克隆与 仓鼠胸苷激酶基因附近的重排。这 断点的分布将以Southern印迹，PCR为特征 和测序分析。这些实验将探索 假设引起癌症的药物的能力有所不同 重排。隔离重排伙伴后，DNA 序列，染色质结构，甲基化状态，核基质 将检查关联和断点区域的基因组位置 测试遗传和表观遗传特征的假设 区域重新排列的倾向。细胞的发现 将将培养系统与t（2; 13）的结构特征进行比较 人类癌肺泡横纹肌肉瘤中的易位断点。 最后，将应用PCR方法来开发 对人群中序列的重排频率的定量。 这样的测定将允许在各种各样的重新安排事件探索 基因组基因局以及对特定的作用的评估 重排过程中的环境和父母细胞特征 直接操纵诱导系统的组件。