International Journal of Radiation Biology, May 2015; 91(5): 383–388 © 2015 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2015.1021960
The bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality Yuichiro Yokota1, Tomoo Funayama1, Yasuko Mutou-Yoshihara1, Hiroko Ikeda1,2 & Yasuhiko Kobayashi1,2 1Microbeam Radiation Biology Group, Japan Atomic Energy Agency, Takasaki, Gunma, and 2Gunma University Graduate
School of Medicine, Maebashi, Gunma, Japan
Abstract Purpose: To investigate the dependence of the bystander cellkilling effect on radiation dose and quality, and to elucidate related molecular mechanisms. Materials and methods: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method. Results: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ⱕ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations. Conclusion: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.
of cell population but also medium transfer from and noncontact co-cultures with irradiated cells can induce effects on non-irradiated bystander cells (Hamada et al. 2011). Bystander effects induced by various types of radiation may contribute to the health risks of low-dose irradiation during manned space flight, and the therapeutic outcomes of localized irradiation in advanced radiotherapy. Considering these situations, it is necessary to elucidate the dependency of bystander effects on radiation quality. Anzenberg et al. (2008) demonstrated that X-rays but not α-particles increased phosphorylated histone H2AX (γ-H2AX) foci formation and decreased survival fraction in bystander cells. Shao et al. (2001, 2002, 2003a, 2004) also reported that X-rays and carbon ions enhanced the plating efficiency, proliferation, apoptosis, necrosis and micronucleus (MN) formation rates of bystander cells in radiation qualitydependent manner. These reports support radiation qualitydependent bystander effect. In contrast, Fournier et al. (2007) showed that carbon ions and uranium ions induced the cyclin-dependent kinase inhibitor 1A in bystander cells in radiation quality-independent manner. Kanasugi et al. (2007) observed that increased frequency of chromosomal aberrations in bystander cells was independent of radiation types used when cells were irradiated with X-rays or neon ions. Shao et al. (2003b, 2006) also reported that enhanced MN formation rate in bystander cells was independent of radiation quality when a part of cell population was targeted with argon- or neon-ion microbeam. Furthermore, Yang et al. (2007) found that X-rays and iron ions increased the MN and γ-H2AX-foci formation rates of bystander cells in radiation quality-independent manner. Although the bystander effects induced by different types of radiation have been observed in a number of reports, it remains unclear how radiation quality relates to these effects. Increasing evidence indicates that bystander effects are mediated by various molecules, including reactive oxygen species (Shao et al. 2003b, Yang et al. 2007), cytokines
Keywords: Bystander effect, cell killing, co-culture, nitric oxide, radiation quality, carbon ion
Introduction The radiation-induced bystander effect was reported by Nagasawa and Little (1992) as an excessive increase in sister chromatid exchanges in cells exposed to low fluences of α-particles. It is now accepted that the bystander effect manifests various influences including reduced survival rates of non-irradiated bystander cells (Mothersill and Seymour 2001, Morgan 2003), and that not only low fluence irradiation
Correspondence: Dr Yuichiro Yokota, PhD, Microbeam Radiation Biology Group, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan. Tel: ⫹ 81 27 346 9113. Fax: ⫹ 81 27 346 9688. E-mail: [email protected] (Received 2 April 2014; revised 10 December 2014; accepted 11 February 2015)
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(Rastogi et al. 2012), and growth factors (Gow et al. 2010), as well as by gap junction intercellular communication (Azzam et al. 2001, Shao et al. 2003b, 2006, Autsavapromporn et al. 2013). In addition, many studies have shown that nitric oxide (NO) radicals are key initiators or mediators of the bystander effect, and that they participate in signal transduction between irradiated cells and non-irradiated bystander cells (Matsumoto et al. 2000, 2001, Yang et al. 2007, Maeda et al. 2010, Tomita et al. 2010, 2012). In the present study, normal human fibroblast WI-38 cells were thus irradiated with the same dose ranges of γ-rays or carbon ions, and were co-cultured with non-irradiated cells for 6 - 24 h to determine whether the bystander cell-killing effect is dependent on radiation dose or quality, and to investigate the role of NO radicals.
Materials and methods Cell culture Normal human lung fibroblast WI-38 cell line was purchased from American Type Cell Culture (ATCC; Manassas, VA, USA). Cells were regularly subcultured in minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% glutamine, penicillin and streptomycin mixture (Life Technologies, Carlsbad, CA, USA), and 10% fetal bovine serum (ATCC). Subcultured cells with population doubling levels of ⬍ 35 were seeded onto membrane-based inserts with 1.0-μmϕ pores (Corning, Corning, NY, USA) and companion plates (Corning) at a density of 2 ⫻ 104 cells/cm2. Cells were cultured for a week and the culture medium was replaced every 3 or 4 days. Confluent cell monolayers were used in irradiation and co-culture experiments.
Scavenger treatment The specific NO radical scavenger 2-(4-Carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; Wako, Osaka, Japan) was dissolved in water and was used to investigate the role of NO radicals in the bystander cellkilling effect. Culture medium was replaced with fresh media containing 20-μM carboxy-PTIO at least 2 h before irradiation.
Irradiation and co-culture Monoenergetic 18.3-MeV/n carbon ions were accelerated using an AVF930 cyclotron (Sumitomo Heavy Industries, Tokyo, Japan) installed at the Japan Atomic Energy Agency (JAEA), Takasaki. The linear energy transfer (LET) value and dose rates of carbon ions on cell surface were estimated as previously described (Yokota et al. 2003), and were 108 keV/μm and 0.01 - 0.2 Gy/sec, respectively. γ-rays were emitted from a Cobalt-60 radiation source installed at the JAEA-Takasaki. The LET value and dose rates of γ-rays were 0.2 keV/μm (Kiefer 1990) and 0.8 - 24 Gy/h, respectively. Culture medium was temporarily removed from irradiation vessels just before irradiation, and the vessels were capped with a thin sheet of polyimide film (Du Pont-Toray, Tokyo, Japan) to prevent dryness and contamination during irradiation at room temperature. After irradiation, inserts
containing irradiated cells were placed into companion plates with non-irradiated cell cultures. Irradiated and nonirradiated cells were then co-cultured for 6 - 24 h.
Colony formation assay Clonogenic abilities of non-irradiated bystander cells were investigated using traditional colony formation assays. In brief, cells were trypsinized, counted using a Muse cell analyzer (Merck, Darmstadt, Germany), and seeded in 100-mmϕ tissue culture dishes (AGC Techno Glass, Shizuoka, Japan) at about 600 cells per dish. Formed colonies were fixed with formaldehyde (Wako) 14 days after seeding, stained with 0.002% crystal violet (Merck), and observed under a stereoscopic microscope (Olympus, Tokyo, Japan). Colonies of ⱖ 50 cells were counted as having been derived from surviving single cells. Survival rates were calculated by dividing colony formation rates of bystander cells with those of sham-treated control cells.
Analysis of cell cycle phase distributions Cell cycle phase distributions of confluent cell monolayers were analyzed as previously described (Yokota et al. 2005). In brief, trypsinized cells were suspended in phosphate buffered saline (-) (Life Technologies) containing 0.2% Triton X-100 (Nacalai Tesque, Kyoto, Japan), and the suspension was filtered through a 30-μm-mesh nylon sieve (Partec, Münster, Germany) to isolate cell nuclei. Fluorescence intensities of cell nuclei stained with 4′6-diamidino-2-phenylindole (DAPI) solution (Partec) were measured using a flow cytometer (Partec), and cell cycle phase distributions were analyzed using a DPAC computer program (Partec).
Quantification of nitrite concentrations Concentrations of nitrite were quantified using the Saltzman method with some modifications (Saltzman 1954, Matsumoto et al. 2002). In brief, culture medium was collected from inserts 24 h after co-culture and was centrifuged at 15,000 g force for 5 min to remove cells and debris. Subsequently, 100 μl of supernatant was mixed with 150 μl of the Saltzman reagent containing 0.5% sulfanilic acid (Wako), 0.002% N-1-naphthylethylendiamine dihydrochloride (Wako), and 14% acetic acid (Kanto Chemical, Tokyo, Japan). After 15-min incubation at room temperature, absorbance of the mixture was measured at 550 nm using an absorptiometer (Shimadzu, Kyoto, Japan). Serial dilutions of sodium nitrite (Wako) in medium were prepared to test the performance of the experimental system and to obtain the standard curve (Figure 1). Their absorbance values increased linearly with nitrite concentrations in the range of 0.1 - 10 μM, indicating accurate quantification of a broad concentration range of nitrite. Excess nitrite production was calculated by subtracting concentrations of control samples from those of test samples.
Statistical analysis Differences in a couple of data sets were identified using the Student’s t-test.
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Figure 1. Calibration curve for nitrite determinations using the modified Saltzman method. Absorbance of the mixture of Saltzman reagents and nitrite was measured at 550 nm. Data are presented as the mean ⫾ standard error of the mean (SEM) from 3 or more independent experiments. The error bars are so small that they are hidden behind the data points. A straight-line approximation was performed using the leastsquares method. The correlation coefficient was over 0.999, indicating accurate determinations over a broad range of nitrite concentrations.
Results Confluent cell populations were predominantly in the G0/G1 phase Cell cycle phase distributions of confluent cell populations were assessed because it can modulate the irradiation effect (Pawlik and Keyomarsi 2004). Flow cytometry assays showed that 87.7 ⫾ 4.2%, 2.7 ⫾ 0.7%, and 9.7 ⫾ 3.5% [mean ⫾ standard error of the mean (SEM) of 3 or more independent experiments] of confluent cell populations were in G0/G1, S, and G2/M phases, respectively. Thus, bystander cell-killing effects mainly occurred in G0/G1-phase cells were investigated in this study.
Reduced survival rates in non-irradiated bystander cells co-cultured for 6 h or longer with γ-irradiated cells
Co-cultures of γ-irradiated and non-irradiated cells were incubated in the upper and lower sides of porous membranes, respectively, and time courses of the bystander cell-killing effect were monitored. Survival rates of nonirradiated bystander cells were reduced after co-culture for 6 h or longer with irradiated cells (Figure 2), clearly indicating partial loss of clonogenic ability following intercellular communication with irradiated cells. Reductions in survival rates increased with time and were maximal after 24 h. In subsequent experiments, irradiated cells and non-irradiated cells were co-cultured for 24 h to compare the bystander cellkilling effects of γ-rays and carbon ions.
Bystander cell-killing effect induced by various doses of γ-rays and carbon ions Survival rates of non-irradiated bystander cells co-cultured with cells irradiated with varying doses of γ-rays or carbon ions for 24 h were investigated to elucidate the influence of radiation dose and quality on the bystander cell-killing effect. Survival rates of bystander cells decreased with increasing doses, and plateaued at 0.5 Gy (Figure 3). In addition, the survival rates did not significantly differ between γ-rays and carbon ions at the same irradiation doses (p ⱖ 0.05).
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Figure 2. Time course analysis of intercellular communication between irradiated and non-irradiated cells. Cells were irradiated with 0.5 Gy of γ-rays, and were co-cultured with non-irradiated cells for 6 - 24 h in the upper and lower sides of porous membranes, respectively. Survival rates of non-irradiated bystander cells (closed bar) and of sham-treated cells (open bar) were measured using colony formation assays. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Survival rates of non-irradiated cells co-cultured with irradiated cells for 6 h or longer decreased according to a medium-mediated bystander cell-killing effect. Differences between non-irradiated bystander cells and sham-treated cells were considered significant when *p ⬍ 0.05, **p ⬍ 0.01.
The specific NO radical scavenger carboxy-PTIO prevented reductions in survival rates of bystander cells To investigate the molecular mechanisms and intercellular communications involved in the bystander cell-killing effect, 20-μM of carboxy-PTIO was added to the media of irradiated
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Dose (Gy) Figure 3. Dose responses of the bystander cell-killing effect and nitrite concentrations in the medium. Cells were irradiated with various doses of γ-rays (A) or carbon ions (B), and were co-cultured with nonirradiated cells for 24 h. Survival rates of non-irradiated bystander cells were measured using colony formation assays (solid line) and nitrite concentrations in the medium were determined using the modified Saltzman method (dotted line). Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Decrease in survival rates and increase in nitrite concentrations depended in part on irradiation dose, but not on radiation quality.
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and non-irradiated cells during irradiation and co-culture. Carboxy-PTIO molecules accelerate conversion of NO radicals to nitrogen dioxide (NO2) radicals (Akaike et al. 1993). Carboxy-PTIO treatment prevented the reduction in survival rates of bystander cells co-cultured with cells that had been irradiated at 0.5 Gy with γ-rays or carbon ions (Figure 4).
Nitrite concentrations in the co-culture media of irradiated cells and non-irradiated bystander cells Nitrite concentrations were measured in the medium after co-culture of irradiated cells and non-irradiated cells for 24 h using a modified Saltzman method. Nitrite concentrations tended to increase with increasing γ-ray and carbon-ion doses of less than 0.25 Gy and 1 Gy, respectively (Figure 3), and then fluctuated at around 0.1 μM at higher doses. In addition, nitrite concentrations did not differ significantly between co-cultures exposed to γ-rays and carbon ions at the same irradiation doses (p ⱖ 0.05), excepting with 0.25 Gy (p ⫽ 0.048). The difference at 0.25 Gy may be caused by a low fluence of carbon ions (an average of 2.2-ions hit in 150-μm2 cell nucleus; based on the Poisson distribution, 11.4% of nuclei were not hit and 24.8% of them were hit by only one ion), although further study is necessary to clarify this issue.
Discussion In the present study, survival rates of bystander cells cocultured with cells irradiated with various doses of γ-rays or carbon ions were investigated to evaluate the dependencies of medium-mediated bystander cell-killing effects on irradiation dose and radiation quality. Concerning irradiation dose dependency, reductions in survival rates of non-irradiated bystander cells were more pronounced with increasing doses of up to 0.5 Gy (Figure 3), indicating that the bystander cell-killing effect depends on
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Figure 4. Effects of the specific nitric oxide (NO) radical scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) on the bystander cell-killing effect. Cells were treated with 20-μM carboxy-PTIO or water in the culture medium for 2 h before irradiation. After irradiated with 0.5 Gy of γ-rays (A) or carbon ions (B), irradiated cells and non-irradiated cells were co-cultured for 24 h with or without carboxy-PTIO. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Carboxy-PTIO prevented the reduction in survival rates of bystander cells. Differences between non-irradiated bystander cells and sham-treated cells were considered significant when *p ⬍ 0.05, **p ⬍ 0.01 (N.S., not significant).
the energy absorbed into irradiated cells in the lower dose range. Schettino et al. (2003, 2005) and Maeda et al. (2010) also showed that the bystander cell-killing effect in hamster V79 cells after exposure to ultrasoft X-ray and synchrotron X-ray microbeams, respectively, depends on the energy absorbed into irradiated cells in the lower dose range. However, the molecular mechanisms underlying these phenomena remained unclear. A typical NO scavenger carboxy-PTIO was thus supplemented to irradiated cells and non-irradiated cells to investigate molecular mechanism of the bystander cellkilling effect. Treatment with carboxy-PTIO during periods of irradiation and co-culture suppressed the reduction in survival rates of bystander cells (Figure 4), indicating that NO radicals play an important role in the bystander cell-killing effect in this non-contact co-culture system. In addition, Sokolov et al. (2005) found that carboxy-PTIO and a selective iNOS inhibitor aminoguanidine (AG) prevented DNA double-strand break induction in bystander WI-38 cells following medium transfer from and co-culture with irradiated cells. Tomita et al. (2010, 2012) also reported that either of them suppressed bystander cell-killing effect following X-ray microbeam irradiation in WI-38 cells. However, activation of p53 tumor suppressor gene product following irradiation can inhibit transcription of inducible NO synthase (iNOS, also known as NOS2) in irradiated cells through the interaction between p53 and TATA binding protein and/or nuclear factor κB which are essential for iNOS expression (Forrester et al. 1996, Ambs et al. 1998, Matsumoto et al. 2000, 2001). Collectively, NO radicals might be produced by iNOS in bystander WI-38 cells as the second mediator or effector to lead bystander effects. NO radicals are rapidly oxidized into NO2 radicals, and NO2 radicals are finally transformed into nitrite and nitrate. Thus, we measured concentrations of nitrite to suppose NO radical production in the co-culture system. As a result, positive relationships between irradiation dose and nitrite concentration were found, but only in the lower dose range (Figure 3). To suppose the molecular mechanism of the dose dependency of bystander cell-killing effect in more detail, the relationships between the bystander cell-killing effect and the nitrite concentration in the medium were analyzed (Figure 5). Because the dose responses of the nitrite concentration in the medium as well as the bystander cell-killing effect were quite similar between γ-rays and carbon ions (Figure 3), the data sets of γ-rays and carbon ions were taken together to perform a straight-line approximation. The data showed a negative correlation between survival rates of bystander cells and concentrations of nitrite in the medium (slope, ⫺ 1.95; correlation coefficient, 0.701; Figure 5). From this negative correlation, it is suggested that the amount of NO radicals produced is a determinant of survival rates of bystander cells. In contrast, Matsumoto et al. (2000, 2001) reported that functional p53 expression was enhanced and radioresistance was given in human glioblastoma A172 cells through NO radical-mediated bystander effect. Su et al. (2010) also described that the lower concentrations of NO-radical donor reagent gave radioresistance in human lung cancer H1299 cells having the
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on irradiation dose but not on radiation quality, and negative relationships were observed between them. Therefore, it is suggested that the amounts of NO radicals produced are one of the most important determinants of γ-ray- and carbon ion-induced bystander effects. In future studies, we will elucidate the molecular mechanisms of NO radical-mediated bystander cell death.
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Figure 5. Negative relationships between nitrite concentrations in culture medium and survival rates of bystander cells. Survival rates of bystander cells were plotted as a function of nitrite concentrations using the data from Figure 3, and a straight-line approximation was performed using the least-squares method. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. The slope and correlation coefficient were ⫺ 1.95 and 0.701, respectively. Survival rate of bystander cells decreased with increasing nitrite concentrations in the medium.
This work was financially supported by JAEA and Japan Society for the Promotion of Science KAKENHI [grant number 25740019 to Y. Y. and 24310073 to T. F.]. The authors thank Drs T. Sakashita and M. Suzuki for their useful comments. The authors are also grateful to the operators of the Takasaki Ion Accelerators for Advanced Radiation Application, and to the operators of the Cobalt-60 irradiation facility in JAEA-Takasaki for their technical cooperation.
Declaration of interest functional p53. Collectively, it is supposed that the influence of NO radical-mediated bystander effects depends on the differences in functional signaling pathways resulting from the gene expression status of the cells used. Next plan is to investigate where NO radicals are produced in and how they lead cell death to non-irradiated bystander cells. Concerning radiation quality dependency, the dose responses of survival curves in bystander cells and of nitrite concentrations in medium were quite similar between γ-rays and carbon ions in the dose range investigated (Figure 3). From this, it can be concluded that the bystander cell-killing effect and the NO production mechanism are independent of radiation quality in normal human fibroblasts. This conclusion is partly supported by some reports employed human normal fibroblasts as donors and recipients of bystander signaling (Shao et al. 2003b, 2006, Fournier et al. 2007, Kanasugi et al. 2007, Yang et al. 2007), but neither by Anzenberg et al. (2008) and Shao et al. (2004) using human cancer cells as donors, nor by Shao et al. (2001, 2002, 2003a) using human cancer cells as donors and recipients. Furthermore, Autsavapromporn et al. (2013) recently reported a clastogenic bystander effect that depended on radiation quality in confluent fibroblast monolayers irradiated with microbeams of X-rays and heavy ions. However, this dependency looked like being abolished by the specific inhibitor of gap junction intercellular communication pathways 18-α-glycyrrhetinic acid. Taken with the present data, it can be supposed that the dependency of radiation-induced bystander effects on radiation quality varies with donor cell type and intercellular communication pathways. In summary, bystander cell-killing effects of γ-rays and carbon ions were investigated in the non-contact co-culture system of normal human fibroblast WI-38 cells. The specific NO radical scavenger carboxy-PTIO treatment clearly indicated that the bystander cell-killing effect was mediated by NO radicals. Importantly, survival rates of bystander cells and nitrite concentrations of the co-culture medium depended
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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The bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality Yuichiro Yokota1, Tomoo Funayama1, Yasuko Mutou-Yoshihara1, Hiroko Ikeda1,2 & Yasuhiko Kobayashi1,2 1Microbeam Radiation Biology Group, Japan Atomic Energy Agency, Takasaki, Gunma, and 2Gunma University Graduate
School of Medicine, Maebashi, Gunma, Japan
Abstract Purpose: To investigate the dependence of the bystander cellkilling effect on radiation dose and quality, and to elucidate related molecular mechanisms. Materials and methods: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method. Results: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ⱕ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations. Conclusion: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.
of cell population but also medium transfer from and noncontact co-cultures with irradiated cells can induce effects on non-irradiated bystander cells (Hamada et al. 2011). Bystander effects induced by various types of radiation may contribute to the health risks of low-dose irradiation during manned space flight, and the therapeutic outcomes of localized irradiation in advanced radiotherapy. Considering these situations, it is necessary to elucidate the dependency of bystander effects on radiation quality. Anzenberg et al. (2008) demonstrated that X-rays but not α-particles increased phosphorylated histone H2AX (γ-H2AX) foci formation and decreased survival fraction in bystander cells. Shao et al. (2001, 2002, 2003a, 2004) also reported that X-rays and carbon ions enhanced the plating efficiency, proliferation, apoptosis, necrosis and micronucleus (MN) formation rates of bystander cells in radiation qualitydependent manner. These reports support radiation qualitydependent bystander effect. In contrast, Fournier et al. (2007) showed that carbon ions and uranium ions induced the cyclin-dependent kinase inhibitor 1A in bystander cells in radiation quality-independent manner. Kanasugi et al. (2007) observed that increased frequency of chromosomal aberrations in bystander cells was independent of radiation types used when cells were irradiated with X-rays or neon ions. Shao et al. (2003b, 2006) also reported that enhanced MN formation rate in bystander cells was independent of radiation quality when a part of cell population was targeted with argon- or neon-ion microbeam. Furthermore, Yang et al. (2007) found that X-rays and iron ions increased the MN and γ-H2AX-foci formation rates of bystander cells in radiation quality-independent manner. Although the bystander effects induced by different types of radiation have been observed in a number of reports, it remains unclear how radiation quality relates to these effects. Increasing evidence indicates that bystander effects are mediated by various molecules, including reactive oxygen species (Shao et al. 2003b, Yang et al. 2007), cytokines
Keywords: Bystander effect, cell killing, co-culture, nitric oxide, radiation quality, carbon ion
Introduction The radiation-induced bystander effect was reported by Nagasawa and Little (1992) as an excessive increase in sister chromatid exchanges in cells exposed to low fluences of α-particles. It is now accepted that the bystander effect manifests various influences including reduced survival rates of non-irradiated bystander cells (Mothersill and Seymour 2001, Morgan 2003), and that not only low fluence irradiation
Correspondence: Dr Yuichiro Yokota, PhD, Microbeam Radiation Biology Group, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan. Tel: ⫹ 81 27 346 9113. Fax: ⫹ 81 27 346 9688. E-mail: [email protected] (Received 2 April 2014; revised 10 December 2014; accepted 11 February 2015)
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(Rastogi et al. 2012), and growth factors (Gow et al. 2010), as well as by gap junction intercellular communication (Azzam et al. 2001, Shao et al. 2003b, 2006, Autsavapromporn et al. 2013). In addition, many studies have shown that nitric oxide (NO) radicals are key initiators or mediators of the bystander effect, and that they participate in signal transduction between irradiated cells and non-irradiated bystander cells (Matsumoto et al. 2000, 2001, Yang et al. 2007, Maeda et al. 2010, Tomita et al. 2010, 2012). In the present study, normal human fibroblast WI-38 cells were thus irradiated with the same dose ranges of γ-rays or carbon ions, and were co-cultured with non-irradiated cells for 6 - 24 h to determine whether the bystander cell-killing effect is dependent on radiation dose or quality, and to investigate the role of NO radicals.
Materials and methods Cell culture Normal human lung fibroblast WI-38 cell line was purchased from American Type Cell Culture (ATCC; Manassas, VA, USA). Cells were regularly subcultured in minimum essential medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% glutamine, penicillin and streptomycin mixture (Life Technologies, Carlsbad, CA, USA), and 10% fetal bovine serum (ATCC). Subcultured cells with population doubling levels of ⬍ 35 were seeded onto membrane-based inserts with 1.0-μmϕ pores (Corning, Corning, NY, USA) and companion plates (Corning) at a density of 2 ⫻ 104 cells/cm2. Cells were cultured for a week and the culture medium was replaced every 3 or 4 days. Confluent cell monolayers were used in irradiation and co-culture experiments.
Scavenger treatment The specific NO radical scavenger 2-(4-Carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; Wako, Osaka, Japan) was dissolved in water and was used to investigate the role of NO radicals in the bystander cellkilling effect. Culture medium was replaced with fresh media containing 20-μM carboxy-PTIO at least 2 h before irradiation.
Irradiation and co-culture Monoenergetic 18.3-MeV/n carbon ions were accelerated using an AVF930 cyclotron (Sumitomo Heavy Industries, Tokyo, Japan) installed at the Japan Atomic Energy Agency (JAEA), Takasaki. The linear energy transfer (LET) value and dose rates of carbon ions on cell surface were estimated as previously described (Yokota et al. 2003), and were 108 keV/μm and 0.01 - 0.2 Gy/sec, respectively. γ-rays were emitted from a Cobalt-60 radiation source installed at the JAEA-Takasaki. The LET value and dose rates of γ-rays were 0.2 keV/μm (Kiefer 1990) and 0.8 - 24 Gy/h, respectively. Culture medium was temporarily removed from irradiation vessels just before irradiation, and the vessels were capped with a thin sheet of polyimide film (Du Pont-Toray, Tokyo, Japan) to prevent dryness and contamination during irradiation at room temperature. After irradiation, inserts
containing irradiated cells were placed into companion plates with non-irradiated cell cultures. Irradiated and nonirradiated cells were then co-cultured for 6 - 24 h.
Colony formation assay Clonogenic abilities of non-irradiated bystander cells were investigated using traditional colony formation assays. In brief, cells were trypsinized, counted using a Muse cell analyzer (Merck, Darmstadt, Germany), and seeded in 100-mmϕ tissue culture dishes (AGC Techno Glass, Shizuoka, Japan) at about 600 cells per dish. Formed colonies were fixed with formaldehyde (Wako) 14 days after seeding, stained with 0.002% crystal violet (Merck), and observed under a stereoscopic microscope (Olympus, Tokyo, Japan). Colonies of ⱖ 50 cells were counted as having been derived from surviving single cells. Survival rates were calculated by dividing colony formation rates of bystander cells with those of sham-treated control cells.
Analysis of cell cycle phase distributions Cell cycle phase distributions of confluent cell monolayers were analyzed as previously described (Yokota et al. 2005). In brief, trypsinized cells were suspended in phosphate buffered saline (-) (Life Technologies) containing 0.2% Triton X-100 (Nacalai Tesque, Kyoto, Japan), and the suspension was filtered through a 30-μm-mesh nylon sieve (Partec, Münster, Germany) to isolate cell nuclei. Fluorescence intensities of cell nuclei stained with 4′6-diamidino-2-phenylindole (DAPI) solution (Partec) were measured using a flow cytometer (Partec), and cell cycle phase distributions were analyzed using a DPAC computer program (Partec).
Quantification of nitrite concentrations Concentrations of nitrite were quantified using the Saltzman method with some modifications (Saltzman 1954, Matsumoto et al. 2002). In brief, culture medium was collected from inserts 24 h after co-culture and was centrifuged at 15,000 g force for 5 min to remove cells and debris. Subsequently, 100 μl of supernatant was mixed with 150 μl of the Saltzman reagent containing 0.5% sulfanilic acid (Wako), 0.002% N-1-naphthylethylendiamine dihydrochloride (Wako), and 14% acetic acid (Kanto Chemical, Tokyo, Japan). After 15-min incubation at room temperature, absorbance of the mixture was measured at 550 nm using an absorptiometer (Shimadzu, Kyoto, Japan). Serial dilutions of sodium nitrite (Wako) in medium were prepared to test the performance of the experimental system and to obtain the standard curve (Figure 1). Their absorbance values increased linearly with nitrite concentrations in the range of 0.1 - 10 μM, indicating accurate quantification of a broad concentration range of nitrite. Excess nitrite production was calculated by subtracting concentrations of control samples from those of test samples.
Statistical analysis Differences in a couple of data sets were identified using the Student’s t-test.
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Figure 1. Calibration curve for nitrite determinations using the modified Saltzman method. Absorbance of the mixture of Saltzman reagents and nitrite was measured at 550 nm. Data are presented as the mean ⫾ standard error of the mean (SEM) from 3 or more independent experiments. The error bars are so small that they are hidden behind the data points. A straight-line approximation was performed using the leastsquares method. The correlation coefficient was over 0.999, indicating accurate determinations over a broad range of nitrite concentrations.
Results Confluent cell populations were predominantly in the G0/G1 phase Cell cycle phase distributions of confluent cell populations were assessed because it can modulate the irradiation effect (Pawlik and Keyomarsi 2004). Flow cytometry assays showed that 87.7 ⫾ 4.2%, 2.7 ⫾ 0.7%, and 9.7 ⫾ 3.5% [mean ⫾ standard error of the mean (SEM) of 3 or more independent experiments] of confluent cell populations were in G0/G1, S, and G2/M phases, respectively. Thus, bystander cell-killing effects mainly occurred in G0/G1-phase cells were investigated in this study.
Reduced survival rates in non-irradiated bystander cells co-cultured for 6 h or longer with γ-irradiated cells
Co-cultures of γ-irradiated and non-irradiated cells were incubated in the upper and lower sides of porous membranes, respectively, and time courses of the bystander cell-killing effect were monitored. Survival rates of nonirradiated bystander cells were reduced after co-culture for 6 h or longer with irradiated cells (Figure 2), clearly indicating partial loss of clonogenic ability following intercellular communication with irradiated cells. Reductions in survival rates increased with time and were maximal after 24 h. In subsequent experiments, irradiated cells and non-irradiated cells were co-cultured for 24 h to compare the bystander cellkilling effects of γ-rays and carbon ions.
Bystander cell-killing effect induced by various doses of γ-rays and carbon ions Survival rates of non-irradiated bystander cells co-cultured with cells irradiated with varying doses of γ-rays or carbon ions for 24 h were investigated to elucidate the influence of radiation dose and quality on the bystander cell-killing effect. Survival rates of bystander cells decreased with increasing doses, and plateaued at 0.5 Gy (Figure 3). In addition, the survival rates did not significantly differ between γ-rays and carbon ions at the same irradiation doses (p ⱖ 0.05).
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Figure 2. Time course analysis of intercellular communication between irradiated and non-irradiated cells. Cells were irradiated with 0.5 Gy of γ-rays, and were co-cultured with non-irradiated cells for 6 - 24 h in the upper and lower sides of porous membranes, respectively. Survival rates of non-irradiated bystander cells (closed bar) and of sham-treated cells (open bar) were measured using colony formation assays. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Survival rates of non-irradiated cells co-cultured with irradiated cells for 6 h or longer decreased according to a medium-mediated bystander cell-killing effect. Differences between non-irradiated bystander cells and sham-treated cells were considered significant when *p ⬍ 0.05, **p ⬍ 0.01.
The specific NO radical scavenger carboxy-PTIO prevented reductions in survival rates of bystander cells To investigate the molecular mechanisms and intercellular communications involved in the bystander cell-killing effect, 20-μM of carboxy-PTIO was added to the media of irradiated
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Dose (Gy) Figure 3. Dose responses of the bystander cell-killing effect and nitrite concentrations in the medium. Cells were irradiated with various doses of γ-rays (A) or carbon ions (B), and were co-cultured with nonirradiated cells for 24 h. Survival rates of non-irradiated bystander cells were measured using colony formation assays (solid line) and nitrite concentrations in the medium were determined using the modified Saltzman method (dotted line). Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Decrease in survival rates and increase in nitrite concentrations depended in part on irradiation dose, but not on radiation quality.
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and non-irradiated cells during irradiation and co-culture. Carboxy-PTIO molecules accelerate conversion of NO radicals to nitrogen dioxide (NO2) radicals (Akaike et al. 1993). Carboxy-PTIO treatment prevented the reduction in survival rates of bystander cells co-cultured with cells that had been irradiated at 0.5 Gy with γ-rays or carbon ions (Figure 4).
Nitrite concentrations in the co-culture media of irradiated cells and non-irradiated bystander cells Nitrite concentrations were measured in the medium after co-culture of irradiated cells and non-irradiated cells for 24 h using a modified Saltzman method. Nitrite concentrations tended to increase with increasing γ-ray and carbon-ion doses of less than 0.25 Gy and 1 Gy, respectively (Figure 3), and then fluctuated at around 0.1 μM at higher doses. In addition, nitrite concentrations did not differ significantly between co-cultures exposed to γ-rays and carbon ions at the same irradiation doses (p ⱖ 0.05), excepting with 0.25 Gy (p ⫽ 0.048). The difference at 0.25 Gy may be caused by a low fluence of carbon ions (an average of 2.2-ions hit in 150-μm2 cell nucleus; based on the Poisson distribution, 11.4% of nuclei were not hit and 24.8% of them were hit by only one ion), although further study is necessary to clarify this issue.
Discussion In the present study, survival rates of bystander cells cocultured with cells irradiated with various doses of γ-rays or carbon ions were investigated to evaluate the dependencies of medium-mediated bystander cell-killing effects on irradiation dose and radiation quality. Concerning irradiation dose dependency, reductions in survival rates of non-irradiated bystander cells were more pronounced with increasing doses of up to 0.5 Gy (Figure 3), indicating that the bystander cell-killing effect depends on
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Figure 4. Effects of the specific nitric oxide (NO) radical scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) on the bystander cell-killing effect. Cells were treated with 20-μM carboxy-PTIO or water in the culture medium for 2 h before irradiation. After irradiated with 0.5 Gy of γ-rays (A) or carbon ions (B), irradiated cells and non-irradiated cells were co-cultured for 24 h with or without carboxy-PTIO. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. Carboxy-PTIO prevented the reduction in survival rates of bystander cells. Differences between non-irradiated bystander cells and sham-treated cells were considered significant when *p ⬍ 0.05, **p ⬍ 0.01 (N.S., not significant).
the energy absorbed into irradiated cells in the lower dose range. Schettino et al. (2003, 2005) and Maeda et al. (2010) also showed that the bystander cell-killing effect in hamster V79 cells after exposure to ultrasoft X-ray and synchrotron X-ray microbeams, respectively, depends on the energy absorbed into irradiated cells in the lower dose range. However, the molecular mechanisms underlying these phenomena remained unclear. A typical NO scavenger carboxy-PTIO was thus supplemented to irradiated cells and non-irradiated cells to investigate molecular mechanism of the bystander cellkilling effect. Treatment with carboxy-PTIO during periods of irradiation and co-culture suppressed the reduction in survival rates of bystander cells (Figure 4), indicating that NO radicals play an important role in the bystander cell-killing effect in this non-contact co-culture system. In addition, Sokolov et al. (2005) found that carboxy-PTIO and a selective iNOS inhibitor aminoguanidine (AG) prevented DNA double-strand break induction in bystander WI-38 cells following medium transfer from and co-culture with irradiated cells. Tomita et al. (2010, 2012) also reported that either of them suppressed bystander cell-killing effect following X-ray microbeam irradiation in WI-38 cells. However, activation of p53 tumor suppressor gene product following irradiation can inhibit transcription of inducible NO synthase (iNOS, also known as NOS2) in irradiated cells through the interaction between p53 and TATA binding protein and/or nuclear factor κB which are essential for iNOS expression (Forrester et al. 1996, Ambs et al. 1998, Matsumoto et al. 2000, 2001). Collectively, NO radicals might be produced by iNOS in bystander WI-38 cells as the second mediator or effector to lead bystander effects. NO radicals are rapidly oxidized into NO2 radicals, and NO2 radicals are finally transformed into nitrite and nitrate. Thus, we measured concentrations of nitrite to suppose NO radical production in the co-culture system. As a result, positive relationships between irradiation dose and nitrite concentration were found, but only in the lower dose range (Figure 3). To suppose the molecular mechanism of the dose dependency of bystander cell-killing effect in more detail, the relationships between the bystander cell-killing effect and the nitrite concentration in the medium were analyzed (Figure 5). Because the dose responses of the nitrite concentration in the medium as well as the bystander cell-killing effect were quite similar between γ-rays and carbon ions (Figure 3), the data sets of γ-rays and carbon ions were taken together to perform a straight-line approximation. The data showed a negative correlation between survival rates of bystander cells and concentrations of nitrite in the medium (slope, ⫺ 1.95; correlation coefficient, 0.701; Figure 5). From this negative correlation, it is suggested that the amount of NO radicals produced is a determinant of survival rates of bystander cells. In contrast, Matsumoto et al. (2000, 2001) reported that functional p53 expression was enhanced and radioresistance was given in human glioblastoma A172 cells through NO radical-mediated bystander effect. Su et al. (2010) also described that the lower concentrations of NO-radical donor reagent gave radioresistance in human lung cancer H1299 cells having the
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on irradiation dose but not on radiation quality, and negative relationships were observed between them. Therefore, it is suggested that the amounts of NO radicals produced are one of the most important determinants of γ-ray- and carbon ion-induced bystander effects. In future studies, we will elucidate the molecular mechanisms of NO radical-mediated bystander cell death.
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Figure 5. Negative relationships between nitrite concentrations in culture medium and survival rates of bystander cells. Survival rates of bystander cells were plotted as a function of nitrite concentrations using the data from Figure 3, and a straight-line approximation was performed using the least-squares method. Data are presented as the mean ⫾ SEM of 3 or more independent beam times. The slope and correlation coefficient were ⫺ 1.95 and 0.701, respectively. Survival rate of bystander cells decreased with increasing nitrite concentrations in the medium.
This work was financially supported by JAEA and Japan Society for the Promotion of Science KAKENHI [grant number 25740019 to Y. Y. and 24310073 to T. F.]. The authors thank Drs T. Sakashita and M. Suzuki for their useful comments. The authors are also grateful to the operators of the Takasaki Ion Accelerators for Advanced Radiation Application, and to the operators of the Cobalt-60 irradiation facility in JAEA-Takasaki for their technical cooperation.
Declaration of interest functional p53. Collectively, it is supposed that the influence of NO radical-mediated bystander effects depends on the differences in functional signaling pathways resulting from the gene expression status of the cells used. Next plan is to investigate where NO radicals are produced in and how they lead cell death to non-irradiated bystander cells. Concerning radiation quality dependency, the dose responses of survival curves in bystander cells and of nitrite concentrations in medium were quite similar between γ-rays and carbon ions in the dose range investigated (Figure 3). From this, it can be concluded that the bystander cell-killing effect and the NO production mechanism are independent of radiation quality in normal human fibroblasts. This conclusion is partly supported by some reports employed human normal fibroblasts as donors and recipients of bystander signaling (Shao et al. 2003b, 2006, Fournier et al. 2007, Kanasugi et al. 2007, Yang et al. 2007), but neither by Anzenberg et al. (2008) and Shao et al. (2004) using human cancer cells as donors, nor by Shao et al. (2001, 2002, 2003a) using human cancer cells as donors and recipients. Furthermore, Autsavapromporn et al. (2013) recently reported a clastogenic bystander effect that depended on radiation quality in confluent fibroblast monolayers irradiated with microbeams of X-rays and heavy ions. However, this dependency looked like being abolished by the specific inhibitor of gap junction intercellular communication pathways 18-α-glycyrrhetinic acid. Taken with the present data, it can be supposed that the dependency of radiation-induced bystander effects on radiation quality varies with donor cell type and intercellular communication pathways. In summary, bystander cell-killing effects of γ-rays and carbon ions were investigated in the non-contact co-culture system of normal human fibroblast WI-38 cells. The specific NO radical scavenger carboxy-PTIO treatment clearly indicated that the bystander cell-killing effect was mediated by NO radicals. Importantly, survival rates of bystander cells and nitrite concentrations of the co-culture medium depended
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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