Artemis over-expression and radiosensitivity in human cell lines

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The cellular radiosensitivity of two fibroblast cell lines derived from a breast cancer patient that “over-reacted” to radiotherapy (84BR) and a patient with multiple independent tumours (175BR) was examined. Both patients had not been previously diagnosed with a mutation in any known DNA repair gene. The clonogenic assay revealed the 84BR and 175BR cell lines were hypersensitive to gamma radiation when compared to repair normal NB1 and 1BR.3 fibroblast cells. In addition, DNA DSB repair was found to be defective in both patient cell lines due to the abnormal persistence of -H2AX foci over a 24 hour time point in the nuclei of gamma irradiated cells when compared to normal fibroblasts. Also normal response to the cross-linking agent nitrogen mustard in a clonogenic assay was observed in 84BR and 175BR cell lines indicating a normal homologous recombination (HR) DNA repair pathway (since HR is essential for DNA crosslink repair). From these data it was concluded that these cells were defective in one or more components of the Non Homologous End Joining (NHEJ) pathway. The Artemis gene which has an endonuclease activity in the NHEJ repair pathway trims the ends of the double strand breaks before the two ends are ligated. Quantitative real time PCR analysis detected approximately 1.5 to 2 fold over-expression in Artemis gene in 84BR and 175BR cell lines compared to normal cells. Also an increase in the level of apoptosis before and following radiation exposure and a failure to efficiently repair DNA DSB were observed in the patient cell lines. Consequently, it was demonstrated in the cell lines described in this study that increased expression of Artemis endonuclease leads to abnormal and illegitimate DNA DSBs due to unregulated action of the protein thus, contributing to increased radiosensitivity and elevated apoptosis