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 were exposed to X-rays and then transfected into non-irradiated 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 with 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 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 with 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 single-strand breaks compared with 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 ones, which is not apparent when using conventional gel electrophoresis assays of plasmid DNA

Repair susceptibility of X-irradiated EGFP plasmid DNA transfected into non-irradiated cells

Ionizing radiation is known to cause various chemical damage to cells. Whole-cell irradiation inevitably causes damage not only in genomic DNA but also intracellular organelles. To investigate the repairability of DNA damage separately from the effects on organelles, EGFP-expressing plasmids were exposed to X-rays in solution and then transfected into non-irradiated human MCF-7 breast cancer cells, which induce a low expression of DNA damage response protein BRCA1. Live-cell imaging of EGFP fluorescence of the cells was performed to measure repair efficiency of the plasmids in the cells. The kinetics of the fluorescent expression after irradiation of several doses were compared with those treated with a nicking or restriction enzyme used as positive controls to induce single- (SSB) or double-strand breaks (DSB), respectively. Assuming a Poisson distribution of the strand breaks in the plasmid, expected kinetic curves were also calculated. The numbers of EGFP-expressing cells observed were considerably fewer than the calculated values. That is, the difficulty of DNA repair is peculiar to irradiation. These results suggest that the lower EGFP expression efficiencies were not only due to complex chemical structures of DNA-strand-break termini 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. In the future, we test MCF-10A (BRCA1 positive) to reveal the role of DNA damage responses in the repair dynamics. We also conduct high-LET irradiation to cells to produce more dense ionizations in the plasmid.第3回QST国際シンポジウム「Quantum Life Science

ヒト細胞中に移入されたX 線照射水和EGFP 発現プラスミド DNA の修復感受性

Clustered DNA damage is defined as two or morelesions (base lesions, single strand breaks or abasic(AP) sites ) localized within one or two helical turn(s).Although repair susceptibility of a synthesized baselesion (or AP) cluster in oligonucleotides have beeninvestigated, those induced actually by irradiation is notclarified yet. To elucidate the repair susceptibility of radiation-induced clustered damage, in the present study,fully hydrated DNA films containing 35 water moleculesper nucleotide, as well as DNA solutions (1×TE, 1μg/μl) were used as samples. In the hydrated DNA films,diffusible water radicals are hardly generated by ionizingradiation. Thus, the major process of radiation actionis direct ionization or impact of secondary electrons toDNA. The EGFP-expressing plasmids were exposedto X-rays with a 1/e dose for each sample, and thentransfected into non-irradiated human cells (MCF-7).Those cells were observed for 48h with fluorescence microscope.DNA repair efficiencies were obtained as thenumber of fluorescence expression cells. The efficiencyfor the hydrated DNA film was significantly lower thanthat for the solution sample. These results indicate thatDNA damage induced in the hydrated DNA would beclustered lesions and difficult to be repaired.日本放射線影響学会第62回大

Live cell observation of DNA repair using EGFP plasmid exposed to X-rays in various conditions

Clustered DNA damage, which is defined as two or more lesions (base lesions, single strand breaks or apurinic/apyrimidinic(AP) sites) localized within one or two helical turn(s), is mainly induced by the direct deposition of radiation energy to DNA . Repair susceptibility of clustered DNA damage has not been clarified yet. To elucidate its repair susceptibility, in the present study, fully hydrated DNA films containing 35 water molecules per nucleotide, as well as DNA solutions were used as samples. In the hydrated DNA films, the major process of radiation action is direct ionization or impact of secondary electrons to DNA, because diffusible water radicals are hardly generated. Thus, clustered DNA damage yield in hydrated DNA films is much higher than that produced in DNA solution. The EGFP-expressing plasmids were exposed to X-rays with a 1/e dose that causes loss of the closed circular form resulting 37% of residual intact plasmids, and then transfected into non-irradiated human cells (MCF-7). Those cells were observed for 48h with a fluorescence microscope. DNA repair efficiency was obtained as the number of fluorescence expression cells. The efficiency for the irradiated DNA film was approximately 20% lower than that for the solution sample. These results indicate that DNA damage induced in the hydrated DNA films would be less repaired because of complexity of the damage, namely clustered damage, produced by direct effect.第3回QST国際シンポジウム「Quantum Life Science

非照射ヒト細胞におけるin vitro X 線照射プラスミドDNA の EGFP 発現のライブセル観察

Whole cell irradiation inevitably causes variousdamage in not only genomic DNA but also intracellularorganelles. In order to separately examine repairabilityof DNA damage from damage responses of organelles,EGFP-expressing plasmid DNA (in TE buffer) wasexposed to X-rays in vitro, and then transfected into "NON-irradiated " human breast cancer cells (MCF7). Thedynamics of EGFP fluorescence of cells was observedby a live cell imaging technique to analyze DNA repairefficiency in the cells. The EGFP expression kinetics asDNA repair efficiency was analyzed for the cells transfectedwith the plasmids exposed to several X-ray doses.The similar experiments were also performed usingplasmids treated with nicking or restriction enzyme toobtain positive control data of SSB or DSB induction,respectively. Based on those positive control values,theoretically expected efficiencies were also calculatedassuming that strand breakage of plasmid DNA by irradiationfollowed Poisson distribution. The numbers ofcells expressing EGFP in the experimental results wereconsiderably fewer than the theoretical values, indicatingthat the irradiated plasmids were less repaired in thecells. These results strongly suggest that X-irradiationcauses complex types of strand break termini ratherthan those created by enzymatic treatments, presumablyso called non-DSB type clustered DNA damage.These particular types of radiation induced damagewould compromise DNA repair efficiency, consequentlyinduce down-regulation of EGFP expression.日本放射線影響学会第62回大