DNA Hypomethylation in Radiosensitivity and Genomic Stability

Exposure to ionising radiation can result in genome destabilisation. Genomic instability is observed in many cancers and is thought to lead to tumourigenesis. A correlation between genomic instability and global hypomethylation, another common feature of tumour cells, has been demonstrated and attributed to dysregulated gene expression, retroviral activation and increased homologous recombination. Furthermore, global hypomethylation has been correlated with increased radiosensitivity, and ionising radiation has been demonstrated to reduce methylation levels. Using mouse embryonic stem cell lines containing wild type or catalytically inactive DNA methyltransferase enzymes (DNMT1, DNMT3A and DNMT3B), this thesis investigated the effect of radiation on global methylation, and the effect of global hypomethylation on radiosensitivity and genomic stability. Global hypomethylation was not found to increase radiation sensitivity in these cells, and did not correlate with genomic instability on a genome wide scale, or at specific gene loci. However, the DNMT3A and DNMT3B enzymes specifically appeared to influence radiosensitivity, whilst absence of catalytically active DNMT1 resulted in a 10-fold increase of instability at the Hprt gene locus, indicating the importance of this enzyme for maintaining genomic stability. Ionising radiation did not induce hypomethylation or delayed chromosomal instability in these cells. However, the wild type cell line displayed radiation induced delayed genomic instability at Hprt, indicated by a 5-fold increase of mutation rate. Furthermore, disruption of the normal methylation pattern, or absence of the DNMTs, resulted in failure to manifest radiation-induced delayed genomic instability. This is the first direct evidence that normal DNA methylation levels, or the presence of functional DNMTs, are required for the propagation of radiation induced delayed genomic instability in murine embryonic stem cells.