Fig. 6.

Radiosensitisation in p53 mutant cells is due to production of reactive oxygen species (ROS). A. Data from a clonogenic assay demonstrating that the radiosensitisation induced by inhibiting glycolysis can be rescued by adding N-acetylcysteine which acts to increase the levels of reduced glutathione. Results represent mean values obtained from at least three separate experiments and error bars represent the standard error of the mean. P values were calculated according to the method described by Franken [31]. B. Flow cytometry analysis of cells either untreated, or exposed to hydrogen peroxide, ionising radiation (IR), 2-DG or the indicated combinations and harvested 24 h later. Percentages indicate the percent of events (cells) within the gated region, indicating the majority of the normal ROS level. ROS is measured in the FL1-H channel and is quantitated using 2′,7′-dichloro-dihydro-fluorescein diacetate. See also Supplementary Data Tables 4 and 5 for additional flow cytometry data. In all cell lines with abrogation of wtp53 function (either through mutation or knock-down) addition of 2-DG to IR resulted in a significant shift in fluorescence indicative of increased ROS (measured by the proportion of cells inside/outside the gated parameter) (*P < 0.05 in all cases), while in cell lines harbouring functional wtp53 no significant increase was observed. Results presented are typical results from an independent experiment that has been repeated on at least three occasions.