Live cell observation of expression of X-irradiated EGFP-plasmid DNA in non-irradiated human cells

Whole-cell irradiation in radiobiological studies inevitably causes damage not only in genomic DNA but also intracellular organelles. To investigate separately the repairability of DNA damage from the effects on organelles, the solutions (in TE buffer) or hydrated films of the EGFP-expressing plasmids were exposed to X-rays and then transfected into non-irradiated human MCF7 breast cancer cells. Live-cell imaging of EGFP fluorescence of the cells was performed to measure repair efficiency of the plasmids in cells. The kinetics of the fluorescet expression aftrer irradiation of several doses were compared with those following treatment with nicking or restriction enzymes used as positive controls for SSB or DSB, respectively. Assuming a Poisson distribution of the strand breaks in the plasmid, expected kinetic curves were calculated. The numbers of cells expressing EGFP in the experimental results were considerably fewer than expected, particularly for the hydrated plasmid film sample. These results suggest that the lower EGFP expression efficiencies were not only due to complex chemical structures of termini created by SSBs compared with those created by enzyme treatments, but also that localization of non-DSB type lesions might be facilitated by irradiation and thus compromise DNA repair efficiency. This trend becomes evident particularly for the hydrated sample, indicating that more complex damage could be induced through direct effect of radiation rather than indirect effect of diffusible water radicals.The 16th International Congress of Radiation Research(ICRR2019