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MECHANISMS OF GENOMIC REARRANGEMENT IN CARCINOGENESIS

基因组重排致癌机制

基本信息

批准号：
2654124
负责人：
FREDERIC G BARR
金额：
$ 11.45万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-02-04 至 2000-01-31
项目状态：
已结题

项目摘要

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.

许多调查人员的共同工作证明了在肿瘤发育中的基因组重排，并建立了环境致癌物可以诱导它们的形成。要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，在该系统中，非活性内源性胸腺嘧啶核苷仓鼠细胞系中的激酶基因被激活和重排致癌物治疗。我们对致癌物诱发的初步分析模型细胞培养系统中的重排表明了一种潜在的核组织和DNA序列在重排中的作用进程。为了研究这些机械特征在人类中的作用癌症，我们已经确定了与t(2；13)易位有关的基因儿童软组织肿瘤的泡状横纹肌肉瘤。此外，我们已经开发了基于聚合酶链式反应的分离重排方法。断点和相关的野生型重排伙伴。在拟议的项目中，我们将在我们的用于分离多个独立克隆的细胞培养系统仓鼠胸苷激酶基因附近的重排。这个断点的分布将通过Southern印迹、聚合酶链式反应、和测序分析。因此，这些实验将探索认为致癌物质的致癌能力不同的假说重新安排。在分离重排伙伴之后，DNA 序列，染色质结构，甲基化状态，核基质将研究断裂点区域的关联性和基因组位置检验遗传特征和表观遗传特征共同影响的假设一个地区重新排列的倾向。来自细胞的发现文化系统将与t(2；13)的结构特征进行比较人癌泡状横纹肌肉瘤中的易位断裂点。最后，将应用聚合酶链式反应方法开发检测方法。对种群中序列重排频率的量化。这样的分析将允许探索各种不同的重排事件以及对特定基因座作用的评估重排过程中的环境和亲本细胞特征直接操纵感应系统的组件。