Expression of an X-Ray Irradiated EGFP-Expressing Plasmid Transfected into Nonirradiated Human Cells.

To investigate the repairability of X-ray induced DNA damage, particularly non-double-strand breaks in living cells, enhanced green fluorescent protein (EGFP)-expressing plasmids X-ray irradiated and then transfected into nonirradiated human cells, MCF7 and MCF10A. Live-cell imaging of EGFP fluorescence was performed to measure the efficiency of plasmid repair in cells. The number of EGFP-expressing cells significantly decreased with increasing X-ray dose for both cell lines. The obtained kinetic curves of EGFP expression indicating plasmid repair were quantitatively compared against algebraically calculated ones based on the values of the transfected plasmids that had been treated with nicking or restriction enzymes. Then, assuming a Poisson distribution of single-strand breaks (SSBs), the number of cells carrying these nicked plasmids that could express EGFP were estimated. Our experimental results revealed considerably fewer cells expressing EGFP compared to the expected values we had calculated. These results suggest that the lower proportion of cells expressing EGFP as a measure of plasmid repair was due not only to the complex chemical structures of termini created by SSBs compared to those created by enzyme treatments, but also that base lesions or AP sites proximately arising at the strand-break termini might compromise EGFP expression. These results emphasize that radiation-induced DNA breaks are less repairable than enzymatically induced DNA breaks, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA.

VISUALIZATION OF THE DNA REPAIR PROCESS IN MAMMALIAN CELLS TRANSFECTED WITH EGFP-EXPRESSING PLASMID DNA AFTER EXPOSURE TO X-RAYS IN VITRO.

To investigate the repair process of DNA damage induced by ionizing radiation in isolation from various types of cytoplasmic damage, we transfected X-irradiated enhanced green fluorescent protein (EGFP)-expressing plasmid DNA into non-irradiated mammalian cells using lipofectamine. The repair kinetics of the irradiated plasmids in the cells were visualized under microscopy as the EGFP fluorescence emitted by transfected cells. Using an agarose gel electrophoresis method, the yields of single- and double-strand breaks of the plasmids were also quantified. As positive control experiments, plasmid DNA with single- or double-strand breaks induced by a nicking or restriction enzyme were also transfected into the cells. The DNA repair rates for X-ray-irradiated plasmids were significantly lower than those of the enzymatically digested positive control samples. These results indicate that X-rays could induce less repairable damage than that induced by enzymes.