MECHANISMS OF GENOMIC REARRANGEMENT IN CARCINOGENESIS

许多调查人员的共同工作表明了以下方面的重要性： 基因组重排的肿瘤发展，并已确定， 环境致癌物质可诱发其形成。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，其中无活性的内源性胸苷 仓鼠细胞系中的激酶基因被激活并重排， 致癌物治疗我们对致癌物诱导的 模型细胞培养系统中的重排表明了一种潜在的 核组织以及DNA序列在重排中的作用 过程为了研究这些机械特征在人类 癌症，我们已经确定了参与t（2;13）易位的基因 小儿软组织肿瘤腺泡状横纹肌肉瘤此外，委员会认为， 我们开发了基于PCR的方法来分离重排， 断裂点和相关的野生型重排伴侣。 在建议的项目中，我们将在我们的 分离多个独立克隆的细胞培养系统， 在仓鼠胸苷激酶基因附近的重排。的 断裂点的分布将通过Southern印迹，PCR， 和测序分析。这些实验将探索 致癌物质在引起癌症的能力上不同的假设 重新安排在分离重排伴侣后，DNA 序列，染色质结构，甲基化状态，核基质 相关性和断裂点区域的基因组位置将被检查 为了验证遗传和表观遗传特征都影响 一个地区重新排列的倾向。从细胞中的发现 文化系统将比较的结构特征的t（2; 13） 人癌腺泡状横纹肌肉瘤中的易位断裂点。 最后，PCR方法将被应用于开发以下检测方法： 群体中序列重排频率的定量。 这样的测定将允许在多种细胞中探索重排事件。 基因组位点以及评估的作用， 环境和亲本细胞的特点，在重排过程中， 直接操纵诱导系统的组件。

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.