TY - JOUR
AU - Yokoya,, Akinari
AB - Abstract 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. INTRODUCTION Radiation damage to DNA and its repair mechanism have been studied at both molecular (in vitro) and cellular (in vivo) levels. To date, chemical or biochemical analyses of DNA or its related molecules exposed to ionizing radiation reveal aspects of the chemical processes by which damage is induced(1). In cellular level studies based on whole-cell irradiation, however, damage is inevitably caused in not only DNA but also organelles that may impact DNA damage responses. To clarify the situation, a pioneering study conducted by Rainbow(2) infected non-irradiated human cells with γ-ray-irradiated human adenovirus. The repair efficiency of the single-strand DNA breaks (SSBs) to the viral genome were analyzed in terms of the recovered molecular weight. Recombinant plasmids carrying the bacterial gpt or neo genes were exposed to ultraviolet light(3, 4) or γ-rays(5) and transferred into unirradiated cells. The repair efficiency of the damage was monitored using a colony formation assay in selective media. The significance of these researches was emphasized by Thacker (see his review(6) and references therein). In the present study, we established a novel experimental method using green fluorescent protein (GFP)-expressing plasmid DNA irradiated with X-rays and transfected into non-irradiated human cancer cells. The time-dependence of the numbers of cells expressing fluorescence, as an indicator of DNA repair rate, were observed using a live-cell imaging technique. MATERIALS AND METHODS Cell Culture The experimental procedure of this study is schematically shown in Figure 1. Human breast cancer cells (MCF7) were kindly provided by Dr Imai and Dr Fukunaga of the Institute of Development, Aging and Cancer, Tohoku University, Japan. Figure 1. View largeDownload slide Experimental procedure of plasmid irradiation and transfection into mammalian cells. Figure 1. View largeDownload slide Experimental procedure of plasmid irradiation and transfection into mammalian cells. The cells were cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemicals, Osaka, Japan) containing 10% fetal bovine serum (Biological Industries, Beit-HaEmek, Israel) and 1% Antibiotic-Antimycotic (Life Technologies, Carlsbad, CA) in a humidified incubator maintained at 37°C in an atmosphere consisting of 95% ambient air and 5% CO2. Cells were harvested, and 1.0 × 104 cells were seeded into each well of a 96-well plate filled with the above medium 24 h prior to plasmid transfection. MEASUREMENT OF TRANSFECTION EFFICIENCY DEPENDING ON THE PLASMID CONFORMATION The cells were transfected with an enhanced green fluorescent protein (EGFP) expressing plasmid DNA (pEGFP-C1) exposed to X-rays. Before performing X-irradiation experiments, we determined the transfection efficiencies for closed circular, open circular and linear forms of the plasmid DNA as positive control data. The plasmids (1 mg/1 ml) in a buffer of 10 mM Tris and 1 mM EDTA (TE buffer, pH 8.0) were treated with a nicking (Nb.BsmI) or restriction enzyme (HindIII) solution at 65°C or 37°C, respectively, for 1 h to induce an SSB or double-strand break (DSB) in the plasmid. The sites cleaved by Nb.BsmI and HindIII are outside of the EGFP coding sequence (Figure 2). Using an agarose gel analysis method, we confirmed that the number of undigested plasmids was negligible in the incubation conditions of this study (data not shown). Figure 2. View largeDownload slide (a) Restriction sites of the pEGFP-C1 plasmid and (b) obtained fluorescent images of the EGFP-expressing cells transfected with the pEGFP-C1 plasmid, shown at 4× magnification 48 h after transfection. Figure 2. View largeDownload slide (a) Restriction sites of the pEGFP-C1 plasmid and (b) obtained fluorescent images of the EGFP-expressing cells transfected with the pEGFP-C1 plasmid, shown at 4× magnification 48 h after transfection. The treated plasmids were transfected into the cultured cells using a lipofection reagent kit (Invitrogen Lipofectamine 3000 Transfection Reagent, Thermo Fisher Scientific, Yokohama, Japan) with 0.3 μl of Lipofectamine 3000 Reagent, 0.4 μl of P3000 Reagent, and 0.2 μg of the plasmid DNA in 10 μl of Opti MEM Medium at 37°C. X-RAY IRRADIATION OF THE PLASMID DNA The plasmid solution (1 mg/1 ml) in the TE buffer was exposed to X-rays in 1.5-ml microtubes at 4°C using an X-ray generator with a W-target operated at a tube voltage of 150 kVp (Softex, Kanagawa, Japan). The dose rate was 11.8 Gy/min. The doses of X-rays absorbed by the samples were 0.4 and 1.5 kGy. After X-ray irradiation, the plasmids were transfected into MCF7 cells using the procedure described above. TIME-LAPSE IMAGING FOR CELL CYCLE ANALYSIS The EGFP fluorescence expression in the transfected cells were investigated as live-cell images. To visualize green fluorescence emission from EGFP in the cells, a fluorescence microscope (BZ-X710, Keyence, Osaka, Japan) equipped with a filter set for an excitation wavelength of 485 nm and emission wavelength of 535 nm was used. The observation was started 1 h after transfection. Fluorescent cell images of irradiated and non-irradiated (control) cells were acquired every 30 min for 33 h. The captured images were finally reconstituted as time-lapse recordings. The number of green cells were counted using fluorescent microscope software (Hybrid Cell Count, Keyence). To measure the total number of cells, phase contrast images were also taken. RESULTS The fluorescent images of the cells expressing EGFP 48 h after the transfections are shown in Figure 2. Transfection rates were obtained as ratios of the number of green cells to the observed total of 1000 cells. When the plasmids were treated with the nicking enzyme, Nb.BsmI, the number of green cells was significantly lower than that for the control plasmids. When treated with the restriction enzyme, HindIII, the green cells were scarce. The results are summarized in Table 1. The transfection efficiencies were 61% for the nicking enzyme and 8% for the restriction enzyme. Table 1. Transfection efficiency of the pEGFP-C1 plasmid DNA treated with nicking (Nb.BsmI) or restriction (HindIII) enzymes. Treatments The number of EGFP-expressing cells The number of total cells observed Fraction of EGFP-expressing cells Ratio to control (%) Control 478 1000 0.48 100 Nb. BsmI 293 1000 0.29 61 Hind III 42 1000 0.04 9 Treatments The number of EGFP-expressing cells The number of total cells observed Fraction of EGFP-expressing cells Ratio to control (%) Control 478 1000 0.48 100 Nb. BsmI 293 1000 0.29 61 Hind III 42 1000 0.04 9 Table 1. Transfection efficiency of the pEGFP-C1 plasmid DNA treated with nicking (Nb.BsmI) or restriction (HindIII) enzymes. Treatments The number of EGFP-expressing cells The number of total cells observed Fraction of EGFP-expressing cells Ratio to control (%) Control 478 1000 0.48 100 Nb. BsmI 293 1000 0.29 61 Hind III 42 1000 0.04 9 Treatments The number of EGFP-expressing cells The number of total cells observed Fraction of EGFP-expressing cells Ratio to control (%) Control 478 1000 0.48 100 Nb. BsmI 293 1000 0.29 61 Hind III 42 1000 0.04 9 The conformational changes of the plasmids exposed to X-rays are shown in the agarose gel images in Figure 3. Figure 3. View largeDownload slide Image of the agarose gel electrophoresis of X-ray-irradiated pEGFP-C1 plasmid DNA. Figure 3. View largeDownload slide Image of the agarose gel electrophoresis of X-ray-irradiated pEGFP-C1 plasmid DNA. At the 0.4 kGy dose, about half of the intact closed circular form plasmids were converted into open circular forms by SSB induction. At the higher 1.5 kGy dose, the open circular form was dominant, and linear form plasmids were also observed at a lower frequency. These results were consistent with our previous report using pUC18 plasmids irradiated in the TE buffer(7). The dose inducing an average of one SSB (D37) in pUC18 was 0.34 kGy. The fluorescent images of the cells transfected by the X-ray-irradiated pEGFP-C1 plasmid DNA are shown in Figure 4. The green cells increased with culture time for both doses. Although the numbers of cells under 0.4 kGy exposure were seemingly higher than those under the control (0 Gy), these numbers were nearly identical when the cells expressing slight fluorescence were counted. To investigate the EGFP expression kinetics, the numbers of green cells were plotted against incubation times for both doses, with data from doses of 0.8 and 1.0 kGy as well (Figure 5). Figure 4. View largeDownload slide Fluorescent images of the EGFP-expressing cells transfected with pEGFP-C1 plasmid DNA after X-ray exposure under 4× magnification. Because there were no EGFP-expressing cells observed before 5 h after lipofection, the images taken for this period are not shown. Figure 4. View largeDownload slide Fluorescent images of the EGFP-expressing cells transfected with pEGFP-C1 plasmid DNA after X-ray exposure under 4× magnification. Because there were no EGFP-expressing cells observed before 5 h after lipofection, the images taken for this period are not shown. Figure 5. View largeDownload slide Dependence of the number of EGFP-expressing cells on incubation time. The error bars indicate the standard variation among four replicates. Because there were no EGFP-expressing cells observed before 5 h after lipofection, the data for this period were omitted from the graph. Figure 5. View largeDownload slide Dependence of the number of EGFP-expressing cells on incubation time. The error bars indicate the standard variation among four replicates. Because there were no EGFP-expressing cells observed before 5 h after lipofection, the data for this period were omitted from the graph. We considered that the number of EGFP-expressing cells was proportional to incubation time after 9 h. Using the slopes of the straight section, we calculated the EGFP expression rates as the increasing number of green cells per hour and plotted them against dose (Figure 6). Figure 6. View largeDownload slide Dependence of the EGFP expression rate on dose. The error bars indicate the standard variation obtained by the four samples. Figure 6. View largeDownload slide Dependence of the EGFP expression rate on dose. The error bars indicate the standard variation obtained by the four samples. DISCUSSION This study revealed the EGFP expression of transfected mammalian cells with plasmid DNA that had been exposed to X-rays in vitro. Because of the experimental design, the EGFP expression observed reflects only DNA damage repair in cells without any confounding factors such as cytoplasmic damage caused by irradiation. As even DSBs induced by HindIII in the plasmids exhibited a very low transfection efficiency, as shown in Table 1, the linear form plasmids were likely to have been transfected. The plasmids treated with the nicking enzyme (Nb.BsmI), on the other hand, showed a higher transfection efficiency (61%, Table 1), indicating that the strand breaks introduced enzymatically can be readily religated because they contain 5′-phosphate and 3′-hydroxyl termini. The EGFP expression rate decreased as irradiation dose increased (Figure 6). At the maximum dose of 1.5 kGy, almost all plasmids were converted to an open circular form, and a small proportion of linear form plasmids were created, as shown by the gel image (Figure 3). This condition was comparable with that of the nicking enzyme treatment. Interestingly, the EGFP expression rate under 1.5 kGy irradiation was 38% of that in the control, and it was considerably less than that expected from the results of the nicking enzyme treatment. These results strongly suggest that the SSBs induced by X-rays are different from an enzymatically induced SSB (i.e. nick). The strand break terminus generated by ionizing irradiation are known to often produce 3′-phosphate or 3′-phosphoglycolate groups at their termini(1) and need to be processed to remove these 3′-phosphate (and sugar residual) groups and restore 5′-phosphate groups for completion of religation. More complex termini could be generated by higher linear energy transfer (LET) irradiation, accompanied by base damage or loss of bases, i.e. clustered lesions(8, 9). To date, many studies have attempted to reveal their chemical structure (see review(10)). Biochemical techniques, such as molecular weight analysis by gel electrophoresis, are poorly suited to detect clustered lesions. Base excision repair enzymes were also used to visualize the base lesions converted to SSBs(11, 12). Some artificial combinations of the base lesions or AP sites, however, have been reported to strongly retard enzymatic activity(13, 14), indicating that this biochemical technique is unable to detect very proximally arising lesions(7, 15, 16). The new assay introduced by the present study will be a promising approach for monitoring the repair of high LET radiation-induced non-DSB clustered DNA damage in future studies. The fluorescence intensity varied among cells, indicating that the transfection efficiency was not constant among cells. Further investigation will be needed to reveal the efficiency of plasmid uptake independent of the repair efficiency. ACKNOWLEDGEMENTS The authors deeply appreciate Dr Hiroo Imai and Dr Hisanori Fukunaga at the Institute of Development, Aging and Cancer, Tohoku University, for kindly providing the MCF7 cells. FUNDING This work was supported by JSPS KAKENHI [Grant no. 15H02823]. REFERENCES 1 von Sonntag , C. and Free-Radical-Induced , D. N. A. Damage and Its Repair: A Chemical Perspective ( Berlin, Germany : Springer-Verlag Berlin Heidelberg ) ( 2006 ). 2 Rainbow , A. J. Repair of radiation-induced DNA breaks in human adenovirus . Radiat. Res. 60 , 155 – 164 ( 1974 ). Google Scholar Crossref Search ADS PubMed 3 Spivak. , G. , Ganesan , A. K. and Hanawalt , P. C. Enhanced transformation of human cells by UV-irradiated pSV2 plasmids . Mol. Cell. 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Google Scholar Crossref Search ADS 16 Urushibara , A. , Shikazono , N. , O’Neill , P. , Fujii , K. , Wada , S. and Yokoya , A. LET dependence of the yield of single-, double-strand breaks and base lesions in fully hydrated plasmid DNA films by 4He(2+) ion irradiation . Int. J. Radiat. Biol. 84 , 23 – 33 ( 2008 ). Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - VISUALIZATION OF THE DNA REPAIR PROCESS IN MAMMALIAN CELLS TRANSFECTED WITH EGFP-EXPRESSING PLASMID DNA AFTER EXPOSURE TO X-RAYS IN VITRO
JF - Radiation Protection Dosimetry
DO - 10.1093/rpd/ncy241
DA - 2019-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/visualization-of-the-dna-repair-process-in-mammalian-cells-transfected-eyTIXjHwCs
SP - 79
VL - 183
IS - 1-2
DP - DeepDyve
ER -