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.
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