Abstract 3888: Ionizing radiation destabilizes inverted ALU repeats linked to LOH

Abstract

Abstract Ionizing radiation is a potent inducer of Loss of Heterozygosity (LOH), driven primarily by DNA double strand breaks. Such a process impacts both tumorigenesis through deletion of linked tumor suppressor genes and, similarly, the potential development of radiation resistant tumor clones during radiation therapy. The human mucoepidermoid cell line, H292, was exposed to either 4 or 8 Gy and surviving clones developed from two rounds of cloning analyzed for LOH using polymorphic microsatellite markers. A high level of LOH was observed within individual clones extending from 11q21 to 11q24 (6/49 after 4 Gy and 8/50 after 8Gy). More detailed analysis using a SNP screen on selected LOH positive clones indicated a sharp telomeric termination of LOH adjacent to a common SNP location. The telomeric extent of LOH observed co-mapped to a region of elevated recombination instability documented previously using linkage analysis. Further, SNP analysis showed a region of approximately 1 Mbp LOH that included the region of recombination instability identified through linkage analysis, in some clones isolated either with or without irradiation. The consistency of the telomeric boundary of LOH observed in the irradiated clones permitted a targeted analysis of DNA double strand breaks at this location, using LM-PCR, both before and after 4Gy irradiation of the same cell line. Using a six primer set screen covering a 9 kbp region at the telomeric extent of radiation-induced LOH, a single primer set showed an elevation in DNA breaks from 2-8h after irradiation. The timing and discrete distribution of such breaks indicated that these were not directly induced by the radiation itself. Cells that were unirradiated were not affected. Further inspection of the location demonstrating selective radiation-linked fragmentation showed the presence of an ALU inverted repeat, separated by approximately 1kbp, where the DNA breaks determined by LM-PCR were clustered within the 5’ ALU. Others have shown previously that inverted ALU repeats separated by <20bp are recombinationally unstable and are underrepresented within the human genome. It is proposed that, within the H292 cell line, otherwise stable ALU inverted repeats are capable of forming a transient hairpin conformation, perhaps facilitated by their own transcription via RNA polymerase III, which is unstable within a genome undergoing DNA damage and repair. The combination of such inverted ALU repeats and genotoxic exposure, such as during radiation therapy, may therefore destabilize specific regions of the genome and contribute to the development of aberrant cell clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2011-3888