Studies To Validate The Comet Assay In Measuring Differential Radiation-Induced DNA Damage In Cancer Cells.

DNA damage, induced by both exogenous and exogenous sources, is a key cause of cancer. However, further induced DNA damage, in the form of radiotherapy and genotoxic-chemotherapy, are key means of treating cancer. Consequently, methods to assess DNA damage are important in studies of both cancer causation and treatment. The alkaline comet assay (ACA) has become a standard method for detection of differential DNA damage formation and repair, both in vitro and in vivo. In this study two different theories were investigated to determine the reason for the differential measures of comet formation noted between bladder cancer cell lines. First, that differential comet formation reflects actual differences in DNA damage formation between the radioresistant and radiosensitive cell lines. Second, that differential comet formation is simply due to the additional release of DNA sequences/loops from the comet head to the tail, for the same level of DNA damage. To determine which scenario occurs, cell lines, which exhibited differential ACA responses, were examined using alternate DNA damage assays (including LC-MS/MS, γ-H2AX & PFGE). An equivalent differential response noted between the assays would indicate that the comet assay does indeed measure differences in IR- induced DNA damage between cell lines; whilst the absence of a differential effect for the alternate methods could indicate that the differential effect noted by the comet assay is possibly due to the additional release of the adjacent/contiguous loops of DNA from the comet head. The key finding of this project is that an equivalent differential response noted between ACA and both LC-MS/MS and PFGE reveals that the comet assay does indeed measure differences in induced DNA damage between cells lines. This project also suggests further new insight regarding γ-H2AX foci formation, with foci formation being marker of DSBs that are ‘marked’ for repair, and therefore possibly considered as markers indicative of resistance.