Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

Life Sciences in Space Research 6 (2015) 36–43

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Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation Masanori Tomita a,∗ , Hideki Matsumoto b , Tomoo Funayama c , Yuichiro Yokota c , Kensuke Otsuka a , Munetoshi Maeda a,d , Yasuhiko Kobayashi c a

Radiation Safety Research Center, Central Research Institute of Electric Power Industry, 2-11-1 Iwado Kita, Komae, Tokyo 201-8511, Japan Division of Oncology, Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuoka-Shimoaitsuki, Eiheiji-cho, Fukui 910-1193, Japan c Microbeam Radiation Biology Group, Radiation Biology Research Division, Quantum Beam Science Center, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan d Proton Medical Research Group, Research and Development Department, The Wakasa Wan Energy Research Center, 64-52-1 Nagatani, Tsuruga-shi, Fukui 914-0192, Japan b

a r t i c l e

i n f o

Article history: Received 23 April 2015 Received in revised form 17 June 2015 Accepted 22 June 2015 Keywords: Bystander response Heavy ion Microbeam Nitric oxide Non-targeted effect

a b s t r a c t In general, a radiation-induced bystander response is known to be a cellular response induced in nonirradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell–cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by highLET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κ B and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κ B, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κ B-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time after irradiation. © 2015 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

1. Introduction Astronauts in space are affected constantly by radiation and microgravity (Yatagai and Ishioka, 2014). Space radiation includes protons, heavy ions with high charge and energy, and secondary radiation, including neutrons and the recoil nuclei generated from reactions with spacecraft walls or within tissues (Cucinotta and Durante, 2006; Cucinotta et al., 2008). High-linear energy transfer (LET) heavy ions are major contributors to the total dose equivalent

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Corresponding author. Tel.: +81 3 3480 2111; fax: +81 3 3480 3113. E-mail address: [email protected] (M. Tomita).

(Held, 2009). Previously, high-LET heavy ions have been demonstrated to have higher relative biological effectiveness (RBE) than low-LET photons (γ -rays and X-rays) for a variety of biological endpoints (Held, 2009; Asaithamby and Chen, 2011). If these highLET heavy ions hit cell nuclei, they induce multiple local DNA lesions. Complex clustered DNA damage exhibits strong LET dependence (Goodhead, 1999), and thus it is a good candidate as a prime determinant of the LET-RBE relationship. The complex clustered DNA damage induced by high-LET heavy ions is more difficult to repair by cells than simple individual damage (Asaithamby and Chen, 2011). Recently, it was suggested that the determining factor for a high RBE value in the presence of high-LET heavy ions

http://dx.doi.org/10.1016/j.lssr.2015.06.004 2214-5524/© 2015 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.

M. Tomita et al. / Life Sciences in Space Research 6 (2015) 36–43

may be the inefficacy or inefficiency of DNA double-strand break (DSB) repair via non-homologous end-joining (NHEJ) (Takahashi et al., 2014). In addition, it is considered that mitochondria are a potential cytoplasmic target of high-LET α particles that mediate cellular damage (Zhang et al., 2014). In the space environment, it is also important to evaluate the biological effects induced by low fluence in low fluence-rate irradiation conditions to accurately estimate the human health risk (Held, 2009; Yatagai and Ishioka, 2014). The heterogeneity of the absorbed dose within the irradiated tissues or cells is more relevant for high-LET heavy ions than for low-LET photons because the absorbed dose per single hit (one nucleus traversal) in the former case is much greater than that in the latter. Therefore, directly irradiated and non-irradiated cells co-exist within tissues exposed to the low fluence of high-LET heavy ions. Non-targeted effects include the direct consequences of radiationinduced initial lesions produced in cellular DNA, as well as intraand inter-cellular communication involving both targeted and nontargeted cells (Matsumoto et al., 2011; Tomita and Maeda, 2015), which mainly comprise radiation-induced adaptive responses, lowdose hypersensitivity, genomic instability, and radiation-induced bystander response (RIBR). In general, RIBR is defined as a cellular response induced in a non-irradiated cell that receives bystander signals from directly irradiated cells within an irradiated cell population (Matsumoto et al., 2011; Tomita and Maeda, 2015). RIBRs are mediated mainly by physical cell–cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium during cell culture. The connexin 43 (Cx43)-mediated GJIC is involved in α -particleinduced bystander signaling in confluent cell cultures (Azzam et al., 2001) and the induction of Cx43 has been observed after mean α -particle doses as low as 1.6 mGy (Azzam et al., 2003). The mean propagation distance of the bystander signal ranges from 20 to 40 μm around the intranuclear α -particle impact point (Gaillard et al., 2009). In the bystander response elicited via cell culture medium, nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling molecules (Matsumoto et al., 2011; Tomita and Maeda, 2015). Therefore, the modes of action of reactive nitrogen species (RNS) in bystander signaling could help to elucidate the mechanism of RIBRs (Matsumoto et al., 2011). The heavy-ion microbeam system at the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) in the Japan Atomic Energy Agency (JAEA, Gunma, Japan) is a pioneering collimated heavy-ion microbeam system, which can provide targeted irradiation of several types to biological materials, where heavycharged particles are accelerated using an azimuthally varying field (AVF) cyclotron with a minimum beam diameter of 5 μm (Funayama et al., 2008). An additional excellent feature of the TIARA facility is that the same particles with similar energy in the broadbeams and microbeams can be used to expose biological materials, thereby contributing significantly to the understanding of heavy-ion-induced bystander responses (Shao et al., 2003; Funayama et al., 2005; Kanasugi et al., 2007; Hamada et al., 2008; Iwakawa et al., 2008; Harada et al., 2009; Fournier et al., 2009; Hino et al., 2010; Mutou-Yoshihara et al., 2012; Autsavapromporn et al., 2013 and so on). The significant role of NO in the bystander response was determined in studies using heavy-ion microbeams (Kanasugi et al., 2007; Mutou-Yoshihara et al., 2012). The contribution of NO to the high-LET heavy-ion-induced bystander response and radioadaptive response was also identified using other broadbeam heavy-ion irradiation facilities (Matsumoto et al., 2000; Shao et al., 2001, 2002, 2004; Yang et al., 2007). In our previous studies using X-ray microbeams, bystander cell killing was mainly initiated/mediated by NO in normal human lung fibroblast WI-38 cells, cultured confluent, exponentially growing human non-small-cell lung cancer H1299 cells expressing wild-

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type or mutated p53, and Chinese hamster V79 cells (Tomita et al., 2010, 2012, 2013; Maeda et al., 2010, 2013). However, the biological effects and signal transduction events induced by NO-mediated high-LET heavy-ion-induced bystander responses are still unclear. In this study, we determined the contribution of NO to foci formation by DSB repair-related proteins and the induction of reproductive cell death in bystander cells of normal human fibroblasts using a 13 MeV/u Ar14+ -ion microbeam. We also demonstrated the activation of NF-κ B, Akt, and cyclooxygenase-2 (COX-2, also known as prostaglandin endoperoxide synthase-2) by bystander signaling. The activation of these proteins depended on the incubation time after irradiation and the presence of NO. 2. Materials and methods 2.1. Cell culture Normal human lung fibroblast WI-38 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in D-MEM/F-12 medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin, and they were maintained at 37 ◦ C in a humidified incubator with a 95% air/5% CO2 atmosphere. 2.2. Heavy-ion broadbeam irradiation WI-38 cells were cultured on a 25-mm cover glass in a six-well culture plate for 1 week until they formed confluent monolayers. The cover glass was then placed in a 60-mm dish and 2–3 ml fresh medium was added at least 2 h before irradiation. Immediately before irradiation, the cell culture medium was removed and the dish was covered with 8-μm thickness polyimide film (Du Pont-Toray, Tokyo, Japan) to maintain hydration during irradiation (approximately 10 min). The cells were irradiated with 5 Gy of 13 MeV/u Ar14+ ions delivered from the AVF cyclotron at TIARA, JAEA (Gunma, Japan), at room temperature. The LET value at the cell surface was 1370 keV/μm, which was calculated according to the kinetic energy loss, assuming water equivalence. The absorbed dose (Gy) was calculated as the fluence (number of ion particles/cm2 ) × LET (keV/μm) × (1.6 × 10−9 ). 2.3. Heavy-ion microbeam irradiation To study the formation of 53BP1 and γ -H2AX foci, the cell suspension concentration was adjusted to 1 × 106 cells/ml. The cell suspension (5 μl) was spotted onto the 25-mm cover glass in the 60-mm dish where five spots (one at the center and four satellites) were placed on the cover glass, as shown in Figs. 1A and 1B. The distance between the midpoint of the central and satellite colonies was 5 mm. After incubation for 1 h, 3 ml of fresh medium was added to the dish and the cells were incubated for 1 day to form colonies. Immediately before irradiation, the medium was removed and the cover glass was covered with 8-μm thickness polyimide film. A single cell nucleus at the midpoint of the central colony was irradiated with an average of five particles of collimated Ar14+ -ion microbeam (13 MeV/u) delivered from the AVF cyclotron at the TIARA. The set-up and irradiation procedures were described previously (Funayama et al., 2008). The LET value on the cell surface was 1130 keV/μm. Irradiation was performed at room temperature. When the WI-38 cell nucleus was irradiated with five Ar ions, the estimated absorbed dose was 5 Gy. In the cell survival and western blot analyses, WI-38 cells were cultured on a 25-mm cover glass for 1 week, as described above. On average, five particles of the Ar-ion microbeam were irradiated on five points every 400 μm along a straight line in the center of the cover glass, as shown in Fig. 2A.

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Fig. 1. Induction of DNA double-strand breaks (DSBs) in bystander cells. A. Schematic showing how five colonies of WI-38 cells (one central and four satellite colonies) were produced on a 25-mm cover glass. Cells on the cover glass were cultured and treated in a 60-mm dish. B. Image showing five colonies on the cover glass. The colonies were stained with crystal violet. C. Numbers of 53BP1 and γ -H2AX co-localized foci per cell in the center of the satellite colonies. A targeted cell nucleus at the midpoint of the central colony was irradiated with an average of five particles of Ar-ion microbeam. c-PTIO (20 μM) was added to the culture medium 2 h before irradiation. Cells were fixed for 6 h after irradiation followed by immunostaining. The numbers of 53BP1 and γ -H2AX co-localized foci in the bystander cells were counted in the centers of the satellite colonies. The error bars represent standard errors of the mean (SEM) based on three independent experiments. ∗ P < 0.05. D. Distribution of the 53BP1 and γ -H2AX co-localized foci in the bystander cells counted in Fig. 1C.

then rinsed three times with T-PBS and incubated at room temperature for 1 h with PBS containing Alexa-488-conjugated anti-rabbit IgG and Alexa-568-conjugated anti-mouse IgG (Molecular Probes, Life Technologies, Carlsbad, CA, USA), which were diluted to 1:200. The cover glass was washed twice with T-PBS and once with PBS, mounted with SlowFade Gold Antifade Reagent with DAPI (Molecular Probes, Life Technologies), and then analyzed using a laser scanning microscope (C1si, Nikon, Tokyo, Japan). The numbers of 53BP1 and γ -H2AX co-localizing foci were counted in a minimum of 94 cells per cover glass. 2.5. Cell survival assays Clonogenic survival was determined in a colony formation assay. At 16–24 h after irradiation, all of the cells on the cover glass were harvested by trypsinization and resuspended in fresh medium. Cells were counted, diluted, and plated in 100-mm tissue culture dishes in medium containing 20% FBS. After a 2-week incubation period, the dishes were stained with crystal violet and colonies comprising more than 50 cells were counted. 2.6. Reagents Fig. 2. Suppression of the bystander cell killing effect by pretreatment with c-PTIO. A. Schematic showing the irradiation of cells with a microbeam. WI-38 cells were cultured on a 25-mm cover glass for one week until they formed a confluent monolayer. On average, five particles of Ar-ion microbeams were irradiated at five points every 400 μm along a straight line in the center of the cover glass. B. Surviving fractions of WI-38 cells. Cells were irradiated with a microbeam, as shown in Fig. 2A. c-PTIO (20 μM) was added to the culture medium at 2 h before irradiation. Cells were harvested at 16–24 h after irradiation and plated on tissue culture dishes to allow colony formation. The error bars represent standard errors of the mean (SEM) based on five independent experiments. ∗ P < 0.05.

2.4. Immunofluorescence After irradiation, cells were washed three times with phosphatebuffered saline (PBS), fixed with 4% paraformaldehyde-PBS (Nacalai Tesque, Kyoto, Japan) at room temperature for 20 min, and rinsed with PBS. After treatment with PBS supplemented with 0.1% Triton X-100 at room temperature for 20 min, the cells were rinsed with PBS supplemented with 0.01% Tween-20 (T-PBS) and blocked with 10% bovine serum albumin (BSA, Wako, Japan) in PBS at room temperature for 20 min. After washing once with T-PBS, the cells were incubated overnight at 4 ◦ C in 1% BSA in T-PBS containing 1:500 diluted antibodies, i.e., anti-53BP1 (Merck, Darmstadt, Germany) and anti-γ -H2AX (Merck Millipore, Billerica, MA, USA). The cells were

2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3oxide (c-PTIO; Dojindo, Kumamoto, Japan) was dissolved in PBS and diluted before use to obtain the desired final concentration (20 μM) in the culture medium. Cells were treated with c-PTIO for 2 h before irradiation. 2.7. Immunoblotting Whole cell extracts (2 × 106 cells) were prepared as described previously (Tomita et al., 2003) and proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using 10% (NF-κ B, Akt), 7.5% (COX-2), or 15% (γ -H2AX and β -actin) SDS polyacrylamide gel, and then transferred onto polyvinylidene difluoride membranes (Immobilon, Merck Millipore). After blocking with 5% nonfat dried milk in Tris-buffered saline (TBS) supplemented with 0.05% Tween-20 (T-TBS) or Blocking One-P (Nakalai Tesque), each membrane was incubated in 5% milk in T-TBS or Blocking One-P containing primary antibodies: anti-COX-2 (1/500) (Santa Cruz Biotechnology, Dallas, TX, USA), anti-β -actin (1/100 000), anti- phospho-Akt1 (Ser473) (1/1000), anti-Akt1 (1/500) (Abcam, Cambridge, UK), anti-phospho-NF-κ B

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p65 (Ser536) (1/1000), anti-phospho-NF-κ B p65 (1/1000) (Cell Signaling Technology, Beverly, MA, USA), or anti-γ -H2AX (1/10 000) (Merck Millipore). After rinsing once with T-TBS, the membrane was incubated with horseradish peroxidase-conjugated antibodies (1/1000) (DAKO, Glostrup, Denmark) and developed with an ECL system (GE Healthcare, Buckinghamshire, UK). 2.8. Statistical analysis Significant differences were determined using the Student’s t-test. P < 0.05 was considered statistically significant. 3. Results 3.1. Induction of DNA damage in bystander cells First, we investigated the NO-mediated induction of DSBs in bystander cells. We produced five colonies (one central and four satellites) of normal human fibroblast WI-38 cells on a cover glass by spotting 5 μl condensed cell suspension (1 × 106 cells/ml) and incubating for 1 day, as shown in Figs. 1A and 1B (Matsumoto et al., unpublished). The distance between the midpoint of the central colony and each satellite was 5 mm. Therefore, GJIC did not contribute directly to the induction of DSBs in the cells in the satellite colonies. To determine the importance of NO in the induction of DSBs by bystander signaling, cells were pretreated with or without an NO scavenger, c-PTIO. A single cell nucleus at the midpoint of the central colony was targeted for irradiation with an average of five particles (approximately 5 Gy) from a high-LET (1130 keV/μm) Ar-ion microbeam. 53BP1 (p53 binding protein 1) responds to DSBs and co-localizes with phosphorylated histone H2AX at Ser139 (γ -H2AX), and this is used widely as a surrogate marker for DSBs (Asaithamby and Chen, 2011). Fig. 1C shows the number of 53BP1 and γ -H2AX co-localized foci per cell in the centers of satellite colonies at 6 h after irradiation. The incubation period after irradiation was determined according to our previous study (Matsumoto et al., unpublished). The number of foci in the bystander cells was 1.4 times higher than that in the control cells. Foci formation was suppressed significantly by pretreatment with c-PTIO. The ratio of cells with two or more co-localized foci in the bystander cells was higher than that in the control cells and bystander cells pretreated with c-PTIO (Fig. 2D). These results show that DSBs were induced by NO-mediated bystander signaling after single cell nuclear irradiation with high-LET Ar ions. 3.2. Bystander cell killing Next, we examined the induction of NO-mediated bystander cell killing. In this study, WI-38 cells were cultured for 1 week on a cover glass until they formed confluent monolayers. On average, five particles of Ar ions were irradiated at five points every 400 μm along a straight line at the center of the cover glass because a large number of cells were required to count the exact cell number in the colony formation assay (Fig. 2A). There were approximately 6 × 105 cells on the cover glass. Thus, the influence of the directly irradiated cells was negligible in this assay. Cells were harvested at 16–24 h after microbeam irradiation, and the surviving fraction was 0.88 ± 0.04 (Fig. 2B). By contrast, the surviving fraction was 1.01 ± 0.05 in the cells treated with c-PTIO before microbeam irradiation. These results suggest that the effect of high-LET heavy-ion-induced bystander cell killing was mediated mainly by NO when only a few cells within the cell population were irradiated.

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3.3. Activation of NF-κ B, Akt, and COX-2 mediated bystander signaling The NF-κ B and COX-2 signaling pathways have important roles during the α -particle-induced bystander response (Zhou et al., 2005, 2008). In addition, a pronounced and prolonged increase in the Akt activity has been detected in α -particle-induced bystander cells (Ivanov et al., 2010). These results were previously summarized by Hei et al. (2011). To demonstrate the induction of bystander signaling by high-LET heavy ions, confluent WI-38 cells on a cover glass were irradiated with an average of 5 Gy Ar-ion broadbeam (1370 keV/μm) or microbeam (1130 keV/μm) (Fig. 3). The difference in LET between the broadbeam and microbeam was due to the use of different types of vacuum window. Ar-ion microbeam irradiation was performed as shown in Fig. 2A. The phosphorylation of Akt at Ser473 and of NF-κ B p65 at Ser536, as well as the accumulation of COX-2 in the cells at 6 h after broadbeam or microbeam irradiation are shown in Fig. 3A. Phosphorylated Akt was observed in both the bystander and directly irradiated cells. Phosphorylation was also suppressed by pretreatment with c-PTIO. The phosphorylation of NF-κ B p65 was detected in the directly irradiated cells and suppressed by c-PTIO. However, in the bystander cells, the phosphorylation of NF-κ B p65 was not detected. Furthermore, the accumulation of COX-2 was induced in the bystander cells but not in the directly irradiated cells and this was partly suppressed by c-PTIO. The accumulation of COX-2 depends on the activation of NF-κ B during α -particle-induced bystander signaling (Zhou et al., 2008). However, the phosphorylation of NF-κ B p65 was not detected in the bystander cells at 6 h after irradiation (Fig. 3A). To clarify the possibility that the activation of NF-κ B was induced earlier, we also investigated the phosphorylation of Akt and NF-κ B p65, as well as the accumulation of COX-2 in the bystander cells at 3 h post-irradiation (Fig. 3B). The phosphorylation of both Akt and NF-κ B was detected in the bystander cells, which was suppressed by pretreatment with c-PTIO, but the accumulation of COX-2 was not observed. These results suggest that the transient activation of NF-κ B may be a prerequisite for the accumulation of COX-2 in bystander cells. Next, we investigated the activation of those proteins in directly irradiated cells at 1 or 3 h after irradiation (Fig. 3C). The phosphorylation of Akt was observed at 3 h and it was suppressed by c-PTIO. In addition, the phosphorylation of NF-κ B p65 was observed at 1 and 3 h after irradiation, which was not affected by pretreatment with c-PTIO. It has been reported that the activation of NF-κ B depends on the activation of ATM after the induction of DSBs (Wu et al., 2006; Ivanov et al., 2010). Thus, to clarify whether the induction of DSBs was affected by NO, we also analyzed γ -H2AX. We observed γ -H2AX at 1 and 3 h after irradiation, but it was not suppressed by c-PTIO. The level of γ -H2AX did not decrease at 3 h after irradiation, thereby suggesting that complex DNA damage was induced in the cells irradiated with high-LET Ar ions. The accumulation of COX-2 was not detected at 3 h after irradiation. 4. Discussion In this study, we showed that DSBs and reproductive cell death were induced by a NO-mediated high-LET heavy-ion-induced bystander response in normal human fibroblasts. In addition, the activation of NF-κ B, Akt, and COX-2 by bystander signaling depended on the incubation time after irradiation and the presence of NO. According to our results, the numbers of 53BP1 and γ -H2AX co-localized foci in bystander cells were 1.4 times higher than those in control cells. In addition, foci formation was suppressed by pretreatment with c-PTIO (Figs. 1C and 1D). In our method, the distance was 5 mm between the midpoint of the central colony

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Fig. 3. Phosphorylation and accumulation of bystander signaling-related molecules in directly irradiated and bystander cells. WI-38 cells were cultured on a 25-mm cover glass for one week until they formed a confluent monolayer. c-PTIO (20 μM) was added to the culture medium at 2 h before irradiation. Microbeam irradiation was performed as shown in Fig. 2A. Cells on the cover glass were also directly irradiated with 5 Gy of Ar-ion broadbeam. A. Phosphorylation or accumulation of bystander signaling-related molecules in the cells at 6 h after microbeam or broaedbeam irradiation. B. Phosphorylation or accumulation of signaling molecules in the cells at 3 h after microbeam irradiation. C. Phosphorylation or accumulation of signaling molecules in the cells at 1 or 3 h after broadbeam irradiation.

where a single targeted was cell irradiated with Ar-ion microbeam and the midpoint of each satellite colony. Our results suggest that the NO-mediated high-LET heavy-ion-induced bystander response causes DSBs via the cell culture medium in non-irradiated cells, which are at least 5 mm away. Our results are consistent with previous studies, which showed that foci formation by γ -H2AX or 53BP1 in bystander cells was induced in normal human fibroblast AG01522 cells irradiated with 1 GeV/n iron (Fe) ions with 151 keV/μm (Yang et al., 2007, 2011). By contrast, foci formation by γ -H2AX in bystander AG01522 cells did not increase significantly after high-LET carbon- or nickel-ion microbeam irradiation (Fournier et al., 2009). The process of DNA damage induction mediated by NO is complex and it occurs via multiple pathways (reviewed in Tomita and Maeda, 2015). Therefore, slight differences in the cell culture or irradiation conditions might affect the induction of DSBs in bystander cells. In our previous studies using X-ray microbeams, we observed a bystander cell killing effect in confluent cultured WI-38 cells at 24 h after irradiation (Tomita et al., 2010, 2012). The minimum surviving fractions of cells determined using a colony formation assay were 0.85 and 0.88 after irradiation with 5.35 keV of synchrotron X-ray microbeam at 1.4 Gy and 1.49 keV Al-K X-ray microbeam at 1.2 Gy, respectively. The X-ray-induced bystander cell killing effect also depended mainly on NO. In the present study, the surviving fraction of bystander cells decreased to 0.88 ± 0.04 after irradiation. The decrease in cell survival was also suppressed significantly by pretreatment with c-PTIO. Funayama et al. (2005) reported that the number of non-irradiated CHOK1 cells in colonies decreased at 60 h after exposure to a highLET Ar-ion microbeam (11.5 MeV/n, 1260 keV/μm) via the by-

stander response, using heavy-ion microbeams at the TIARA. The bystander cell killing effect was also reported in A549 (human lung cancer cell line) cells and AG01522D cells after exposure to carbon ions (18.3 MeV/n, 103 keV/μm) or neon ions (13.0 MeV/n, 375 keV/μm) (Hamada et al., 2008; Harada et al., 2009). In these studies, clonogenic survival decreased by about 10%. However, enhancements of the plating (colony forming) efficiency by the medium-mediated bystander response have also been reported in other cell lines after exposure to high-LET α particles (Baskar et al., 2007) or carbon ions (Shao et al., 2001), but pretreatment with c-PTIO decreased the enhanced plating efficiency. The differences among these findings may be attributable to differences in the number of directly irradiated cells, the microbeam irradiation method, co-culture techniques (Shao et al., 2001), or the irradiated conditioning medium treatment (Baskar et al., 2007). Therefore, further studies are warranted, including dose response investigations, to elucidate the bystander cell killing effect using different methods. Our microbeam study showed that the high-LET heavyion-induced bystander cell killing effect in normal confluent human fibroblasts cultures was mediated by NO. NF-κ B-, COX-2-, and Akt-mediated signaling pathways may be involved in the α -particle-induced bystander response, as reviewed by Hei et al. (2011). However, the biological effectiveness of highLET heavy ions where LET >1000 keV/μm was quantitatively and qualitatively different from that of α particles where LET was approximately 100 keV/μm, at least in the directly irradiated cells (Asaithamby and Chen, 2011; Takahashi et al., 2014). To elucidate the mechanism of high-LET heavy-ion-induced bystander signaling mediated by NO, we examined the phosphorylation of NF-κ B and Akt, as well as the accumulation of COX-2, using both a high-

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Fig. 4. A possible model for the NO-mediated high-LET heavy-ion-induced bystander response. In the irradiated cells, ATM is activated by the induction of DNA damage. NF-κ B is activated downstream of ATM signaling, which may then activate cNOS. Akt is phosphorylated in a NO-dependent manner, although Akt may be a downstream target of activated ATM. Activated Akt is known to activate NF-κ B. Bystander signaling may be amplified via this feedback system. In the bystander cells that receive NO, NF-κ B and Akt are activated within 3 h after irradiation. iNOS may be activated in the bystander cells followed by NF-κ B activation. COX-2 is accumulated only in the bystander cells at 6 h after irradiation. COX-PGE2-dependent ROS may react with NO to generate ONOO− following the induction of DNA damage and the suppression of GJIC. Reproductive cell death is induced as a result of these signaling pathways.

LET Ar-ion broadbeam and microbeam (Fig. 3). In the directly irradiated cells, NF-κ B p65 was phosphorylated at Ser536 1 h after irradiation, but it was not affected by treatment with c-PTIO (Fig. 3C). NF-κ B p65 was phosphorylated at Ser536 by ATM in vitro (Sabatel et al., 2012). In addition, a previous study showed that NF-κ B essential modulator (NEMO), the regulatory subunit of Iκ B kinase (IKK), was associated with activated ATM after the induction of DSBs (Wu et al., 2006). The activation of ATM was also confirmed by the phosphorylation of histone H2AX at Ser139 (γ -H2AX), which was not affected by c-PTIO (Fig. 3C). Thus, NF-κ B is activated by the active form of ATM after the induction of DSBs in Ar-ion-irradiated cells. Although the mechanism is unclear, calcium-dependent constitutive nitric oxide synthase (cNOS, also known as nNOS or eNOS) might be activated immediately after irradiation to release NO (Leach et al., 2002; Han et al., 2007; Matsumoto et al., 2011; Matsumoto, 2013). The phosphorylation of Akt at Ser473 in the directly irradiated cells was observed at 3 and 6 h after irradiation, which was suppressed by pretreatment with c-PTIO (Figs. 3A and 3C), although the ATM-dependent phosphorylation of Akt was suggested previously (Hawkins et al., 2011). Recently, it was reported that the production of NO in cells irradiated with high-LET carbon ions activated the PI3K-Akt signaling pathway (Fujita et al., 2014). Akt regulates signaling pathways that lead to the activation of NF-κ B (Kane et al., 1999); thus, NO-mediated bystander signaling may be amplified via this positive-feedback loop. At 3 h after irradiation, Akt and NF-κ B p65 were also phosphorylated in the bystander cells and this effect was suppressed by pretreatment with c-PTIO (Fig. 3B). Thus, the initial activation of Akt and NF-κ B in the bystander cells was mediated mainly by NO. The continuous phosphorylation of Akt in bystander cells was observed at 6 h after irradiation (Fig. 3A), and this effect was also reported after α -particle irradiation (Ivanov et al., 2010). However, the phosphorylation of NF-κ B p65 in bystander cells was observed transiently at 3 h and it diminished at 6 h after irradiation (Figs. 3A and 3B), whereas in irradiated cells, the phosphorylation of NF-κ B p65 continued until at least 6 h after irradiation (Figs. 3A and 3C). Therefore, the transient activation of NF-κ B by the high-LET Ar-ion-induced bystander response differs from the prolonged activation of NF-κ B by the α -particle-induced bystander response (Ivanov et al., 2010). However, it is possible that NF-κ B is re-activated later during the high-LET Ar-ion-induced bystander

response because Akt was phosphorylated continuously (Figs. 3A and 3B). The active form of NF-κ B may activate inducible NOS (iNOS) in bystander cells (Zhou et al., 2008). In addition, the accumulation of COX-2, which is a downstream target of NF-κ B, was induced in the bystander cells at 6 h after irradiation (Fig. 3A). The accumulation of COX-2 was also partially suppressed by pretreatment with c-PTIO. After α -particle irradiation, the induction of COX-2 was observed in both irradiated and bystander cells (Zhou et al., 2005, 2008; Ivanov et al., 2010), but COX-2 induction was not detected at least 6 h after irradiation in the high-LET Ar-ion irradiated cells (Figs. 3A and 3C). The suppression (or delay) of COX-2 induction in the irradiated cells may have been caused by the induction of complex DNA damage, which may contribute to the high cell killing effect of high-LET heavy ions. It has been reported that the COX-2 mRNA contains an AU-rich RNA element, which targets it for rapid decay and translational inhibition (Dixon, 2003). Moreover, multiple signaling pathways are involved in the regulation of COX-2 expression. Further studies are needed to elucidate the factors or signaling pathways involved in the suppression of COX-2 induction in cells exposed to high-LET heavy ions. Based on the results obtained in this study, we proposed a possible model for NO-mediated high-LET heavy-ion-induced bystander signaling, as shown in Fig. 4. After the induction of DSBs in high-LET heavy-ion-irradiated target cells, activated ATM induces NF-κ B activation, which then activates cNOS, thereby releasing NO. NO then activates Akt following the activation of NF-κ B and this positive-feedback loop may amplify bystander signaling. In the bystander cells, both NF-κ B and Akt are activated via NO. NF-κ B may induce iNOS and COX-2. Prostaglandin E2 (PGE2), which is produced in a COX-2-dependent manner, could be involved in the regulation of ROS production (Hei et al., 2011). Peroxynitrite, ONOO− , which is formed by the reaction between NO and ROS (superoxide anions), can induce DNA damage. Even at low levels (1 μM), ONOO− can increase the proportion of γ -H2AX-positive cells (Han et al., 2007). In addition, ONOO− may be responsible for the inhibition of GJIC (reviewed in Matsumoto et al., 2011; Matsumoto, 2013). The damage accumulated throughout these processes may lead to bystander cell death. In summary, our study showed that high-LET (>1000 keV/μm) heavy-ion-induced bystander signaling mediated by NO leads to the induction of DSBs and reproductive cell death in nonirradiated normal human fibroblasts. NF-κ B, Akt, and COX-2 are

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key molecules in the NO-mediated bystander signaling pathway, where their activation depends on the incubation time after irradiation. In addition, we found that COX-2 only accumulated in the bystander cells at least 6 h after high-LET heavy-ion irradiation. Therefore, COX-2 can be used as a molecular marker of high-LET heavy-ion-induced bystander cells, although this may depend on the time after irradiation. In space environments, astronauts are affected by both space radiation and microgravity, and thus it is possible that the low fluence of high-LET heavy ions may modify the effect of microgravity, as discussed by Yatagai and Ishioka (2014). Therefore, it will be necessary to obtain a better understanding of the biological effects associated with both high-LET heavy ions and microgravity to evaluate the human health risks of space environments. Conflict of interest The authors declare that there are no conflicts of interests. Acknowledgements The authors are grateful to Drs Katsumi Kobayashi, Noriko Usami, Atsushi Ito, Hiroshi Maezawa, Yoshiya Furusawa, Ryoichi Hirayama, and Yoshihisa Matsumoto for helpful comments and discussions. We also thank members of the Microbeam Radiation Biology Group (Drs Tetsuya Sakashita and Michiyo Suzuki, and Miss Hiroko Ikeda) and the staff at TIARA, JAEA for their assistance with heavy-ion irradiation. References Autsavapromporn, N., Suzuki, M., Funayama, T., Usami, N., Plante, I., Yokota, Y., et al., 2013. Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures: the impact of radiation quality. Radiat. Res. 180, 367–375. Asaithamby, A., Chen, D.J., 2011. Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation. Mutat. Res. 711, 87–99. Azzam, E.I., de Toledo, S.M., Little, J.B., 2001. Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha-particle irradiated to nonirradiated cells. Proc. Natl. Acad. Sci. USA 98, 473–478. Azzam, E.I., de Toledo, S.M., Little, J.B., 2003. Expression of CONNEXIN43 is highly sensitive to ionizing radiation and other environmental stresses. Cancer Res. 63, 7128–7135. Baskar, R., Balajee, A.S., Geard, C.R., 2007. Effects of low and high LET radiations on bystander human lung fibroblast cell survival. Int. J. Radiat. Biol. 83, 551–559. Cucinotta, F.A., Durante, M., 2006. Cancer risk from exposure to galactic cosmic rays: implications for space exploration by human beings. Lancet Oncol. 7, 431–435. Cucinotta, F.A., Kim, M.H., Willingham, V., George, K.A., 2008. Physical and biological organ dosimetry analysis for international space station astronauts. Radiat. Res. 170, 127–138. Dixon, D.A., 2003. Regulation of COX-2 expression in human cancers. In: Dannenberg, A.J., DuBois, R.N. (Eds.), Progress in Experimental Tumour Research. Karger, Basel, Switzerland, pp. 52–71. Fournier, C., Barberet, P., Pouthier, T., Ritter, S., Fischer, B., Voss, K.O., et al., 2009. No evidence for DNA and early cytogenetic damage in bystander cells after heavyion microirradiation at two facilities. Radiat. Res. 171, 530–540. Fujita, M., Imadome, K., Endo, S., Shoji, Y., Yamada, S., Imai, T., 2014. Nitric oxide increases the invasion of pancreatic cancer cells via activation of the PI3K-AKT and RhoA pathways after carbon ion irradiation. FEBS Lett. 588, 3240–3250. Funayama, T., Wada, S., Kobayashi, Y., Watanabe, H., 2005. Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam. Radiat. Res. 163, 241–246. Funayama, T., Wada, S., Yokota, Y., Fukamoto, K., Sakashita, T., Taguchi, M., et al., 2008. Heavy-ion microbeam system at JAEA-Takasaki for microbeam biology. J. Radiat. Res. 49, 71–82. Gaillard, S., Pusset, D., de Toledo, S.M., Fromm, M., Azzam, E.I., 2009. Propagation distance of the alpha-particle-induced bystander effect: the role of nuclear traversal and gap junction communication. Radiat. Res. 171, 513–520. Goodhead, D.T., 1999. Mechanisms for the biological effectiveness of high-LET radiations. J. Radiat. Res. 40 (Suppl), 1–13. Hamada, N., Ni, M., Funayama, T., Sakashita, T., Kobayashi, Y., 2008. Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions. Mutat. Res. 639, 35–44.

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