Original Article | Published:

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells

Oncogene volume 26, pages 23302339 (05 April 2007) | Download Citation

Subjects

Abstract

The initiation and propagation of the early processes of bystander signaling induced by low-dose α-particle irradiation are very important for understanding the underlying mechanism of the bystander process. Our previous investigation showed that the medium collected from cell culture exposed to low-dose α-particle rapidly induced phosphorylated form of H2AX protein foci formation among the non-irradiated medium receptor cells in a time-dependent manner. Using NG-methyl-L-arginine, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate and Nω-nitro-L-arginine (L-NNA) treatment before exposure to 1 cGy α-particle, we showed in the present study that nitric oxide (NO) produced in the irradiated cells was important and necessary for the DNA double strand break inducing activity (DIA) of conditioned medium and the generation of NO in irradiated confluent AG1522 cells is in a time-dependent manner and that almost all NO was generated within 15 min post-irradiation. Concurrently, the kinetics of NO production in the medium of irradiated cells after irradiation was rapid and in a time-dependent manner as well, with a maximum yield observed at 10 min after irradiation with electron spin resonance analysis. Furthermore, our results that 7-Nitroindazole and L-NNA, but not aminoguanidine hemisulfate, treatment before exposure to 1 cGy α-particle significantly decrease the DIA of the conditioned medium suggested that constitutive NO from the irradiated cells possibly acted as an intercellular signaling molecule to initiate and activate the early process (30 min) of bystander response after low-dose irradiation.

Introduction

Nitric oxide (NO), generated from arginine by the activation of nitric oxide synthase (NOS), is a major signaling molecule in the immune, cardiovascular and nervous systems (reviewed by Stefano et al., 2000), either by acting within the cell in which it is produced or by penetrating cell membranes to affect adjacent cells (Murad, 1998). The uniqueness of NO as a redox signaling molecule resides in part in its relative stability and hydrophobic properties that permit its diffusion through the cytoplasm and plasma membranes over several cell diameter distances (Beckman and Koppenol, 1996). The toxicity of NO is linked to its ability to combine with superoxide anions (O2) to form peroxynitrite (ONOO), an oxidizing free radical that can cause DNA fragmentation and lipid oxidation (Beckman et al., 1990; Jourd'heuil et al., 1997). Recent findings show that ONOO may act as a signaling molecule capable of upregulating protein tyrosine phosphorylation, which plays an important role in the regulation of cell communication, proliferation, migration, differentiation and survival (Murad, 1998; Minetti et al. 2002). The activation of NOS is involved in the cellular response to such stimulants as chemical or irradiation. Stefano et al. (1995) and Stefano (1998) demonstrated that activation of human endothelial cells with morphine or anandamide stimulated NO release.

Recent studies have demonstrated the important role of NO in mediating the bystander response induced by low-dose irradiation. For instance, Matsumoto has shown that X-ray irradiation activates the activity of inducible nitric oxide synthase (iNOS) as early as 3 h post-irradiation and the activity of iNOS keeps increasing by 24 h post-irradiation (Matsumoto et al., 2001). The release of NO from irradiated cells, measured to be in the micromolar level to the medium, induced an increase in radioresistance among the non-irradiated cells which were co-cultured with the irradiated ones (Matsumoto et al., 2001). Shao et al. (2003a, 2004) presented evidence for a significant increase in the incidence of micronuclei in the non-irradiated bystander cells that were in the vicinity of ones irradiated through either the nuclei or the cytoplasm with a microbeam facility. Furthermore, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) (a scavenger of NO) abolished excess micronuclei formation. Using the fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM), and targeted with a precision microbeam, Shao et al. (2003a) detected an increase in the number of fluorescent-positive cells than the actual number of directly irradiated cells. The author suggested that NO mediated the bystander effect and attributed the induced NO to the activation of iNOS. In our previous studies, based on the immunochemistry of phosphorylated form of H2AX (γ-H2AX), a reliable marker to reflect the induction of DNA double strand breaks (DSBs) in cellular nucleus (Rothkamm and Lobrich, 2003; Sedelnikova et al., 2002, 2004), we demonstrated that in situ visualization of DSBs could be used to assess the early events of radiation-induced extranuclear/extracellular (bystander) effects (Hu et al., 2005). In addition, the bystander DSBs were induced in a time-dependent manner and could be detected as early as 2 min post-irradiation (Hu et al., 2006). However, the nature of these intercellular signaling molecule(s) and how they participate in initiating radiation-induced bystander effects (RIBE) are not clear. As NO generated by iNOS has been reported to be important for induction of late event of RIBE, such as MN, it is not clear if NO also plays a functional role in the initiation of early effects and, if it does, by which pathway. Leach et al. have revealed that after 2 Gy X-ray irradiation, the activity of constitutive nitric oxide synthase (cNOS) is transiently enhanced at 5 min post-irradiation and by 30 min the activity has returned to basal levels. These results indicated that activation of cNOS is an early signaling event induced by ionizing radiation (Leach et al., 2002).

In the present study, we investigated the nature of the intercellular signaling molecule(s) in the initiation and early process of low-dose α-particle-induced bystander response. We hypothesized that NO acted as a signaling molecule in the early process of radiation-induced DSBs in non-irradiated bystander cells. To address these goals, a medium transfer approach was adopted. By treating the cells with NG-methyl-L-arginine (L-NMMA), NG-methyl-D-arginine (D-NMMA), 7-Nitroindazole (7-Ni), Nω-nitro-L-arginine (L-NNA), aminoguanidine hemisulfate (AG), DAF-FM diacetate or by electron spin resonance (ESR) analysis, we studied the kinetics in the generation of NO in irradiated cells, as well as the reactive nitrogen species (RNS) stress to the non-irradiated, medium receptor cells. Our results suggested that NO, more likely constitutive NO, act as an intercellular signaling molecule which initiates and activates the early process (30 min) of radiation-induced bystander response.

Results

NO mediates the DSBs induction in bystander normal human skin fibroblasts

Using the medium transfer approach, our previous study showed that irradiated cells secreted the DSB-inducing factor(s) into the medium rapidly after irradiation in a time-dependent manner and this factor(s) contributed to the induction of excessive DSB-positive cells in the non-irradiated (medium receptor) cells (Han et al., accepted). It was shown that the incidence of DSBs induction was maximal in non-irradiated cells (medium receptor) that received medium from cells irradiated for 10 min, or in other word, the DSB-inducing factor(s) released from the irradiated cells, reached a maximum by 10 min post-irradiation. Furthermore, the DSB-inducing activity (DIA) of conditioned medium was dose-independent (Figure 2–3 in Supplementary data). Therefore, in the present study, 10 min post 1 cGy irradiation was chosen as the time point to study the characteristic of the DSB-inducing factor(s). To investigate whether NO mediated the early process of bystander effect based on the incidence of DSBs in the medium receptor cells, an inhibitor of NOS, L-NMMA (1 mM), was used in the incubation of donor cells for 1 h immediately before irradiation. The conditioned medium collected at 10 min post 1 cGy α-particle irradiation was transferred to the medium receptor cells. L-NMMA (Molecular Probes, Eugene, Oregon, USA) has been shown to be an effective NOS inhibitor (Leach et al., 2002; Liu et al., 2005). However, in the presence L-NMMA, the conditioned medium collected at 10 min post-irradiation showed no distinct DIA (15.7–15.1%, P=0.83) (Figure 1), whereas only a marginal decrease (29.6–26.0%) in DIA was found with D-NMMA (Molecular Probes, USA), a negative control of L-NMMA (Figure 1). These results suggested that NO contributed to the formation of excessive DSBs in the receptor cells.

Figure 1
Figure 1

Effect of the NOS inhibition, L-NMMA (a NOS inhibitor, 1 mM), D-NMMA (non-reactive D-enantiomer, 1 mM), 7-Ni (a selective cNOS inhibitor, 4 μM) or AG (a selective iNOS inhibitor, 0.1 mM) on the induction of bystander DSBs. Media from either sham-irradiated donor culture or donor culture irradiated with 1 cGy dose of α-particles were treated with or without drug treatment and DSBs were quantified by γ-H2AX immunochemical staining. Results show that L-NMMA or 7-Ni treatment 1 h before irradiation effectively blocked the DSBs induction in receptor bystander cells treated with the conditioned medium collected at a period after 10 min post-irradiation. However, treatment with D-NMMA or AG had no distinct effect. Data are pooled from three independent experiments and the results represent mean±s.d. P-value <0.05 is considered significant between groups.

To distinguish the functional role of cNOS or iNOS in DSBs induction, 4 μM 7-Ni (Sigma, St Louis, MO, USA), a selective inhibitor of cNOS (Qu et al., 2001), or 0.1 mM AG (Sigma), a selective inhibitor of iNOS (Rao et al., 2002; Matsumoto et al., 2000, 2001), was used to treat the medium donor culture 1 or 2 h before irradiation, respectively. The results with chemicals treatment were shown in Figure 1. In the presence of 7-Ni, the conditioned medium collected at 10 min post-irradiation had no distinct DIA (16.1–16.2%, P=0.94), but AG treatment showed no inhibitory effect on the DIA of conditioned medium (30.8–19.5%, P<0.01). These results indicated that NO, derived from the constitutive pool of cNOS, mediated the early process of DSBs induction.

Generation of NO, most likely constitutive NO, is derived from the irradiated cells

To distinguish whether the NO modifier acts on the donor or the recipient cells, the medium donor culture was treated with 20 μM DAF-FM diacetate, a fluorescent dye for detecting NO and/or nitrite (NO2) (Kojima et al., 1998, 1999; Lacza et al., 2003), for 30 min. The medium with excessive dye was removed and the cultures were rinsed and replaced with fresh culture medium. After irradiation, the cells were cultured for 10 min and the medium was transferred to the recipient cells to detect the DIA. DAF-FM diacetate can permeate well into cells and is quickly transformed into water-soluble DAF-FM (non-membrane permeable) by esterases in the cytosol, where the dye can remain for a long time. DAF-FM will react with NO (or NO2) to yield highly fluorescent DAF-FM T, which is also non-membrane permeable. DAF-FM T is not formed in the absence of NO (or NO2). In some experiments, the medium donor culture was incubated with 1 μM L-NNA (Sigma), an irreversible selective inhibitor of cNOS (Dawson et al., 1991; Kiang et al., 2003), for 30 min, then the excess inhibitor was removed and the culture was rinsed with fresh culture medium three times before irradiation. Our results indicated that treatment of the donor culture with DAF-FM diacetate or L-NNA before irradiation prevented the conditioned medium, harvested at 10 min post-irradiation, from inducing DSB in receptor cells (14.5–15.6%, P=0.69 for DAF-FM diacetate; 15.8–15.2%, P=0.67 for L-NNA) (Figure 2). These results clearly indicated that the generation of NO, most likely constitutive NO, in the irradiated cells was very important and necessary to the DSBs induction in the medium receptor cells.

Figure 2
Figure 2

Effect of DAF-FM diacetate (20 μM), a NO/NO2 radical quencher or L-NNA (1 μM), an irreversible inhibitor of cNOS, on the induction of bystander DSBs. Medium from either sham-irradiated donor cultures or donor cultures irradiated with 1 cGy dose of α-particles were used to treat the medium receptor culture. DSBs were quantified by γ-H2AX immunochemical staining. Incubation of medium from irradiated donor cells with DAF-FM diacetate or L-NNA before irradiation inhibited radiation-induced damage signal being released into the medium. Data are pooled from three independent experiments and the results represent mean±s.d. P-value <0.05 is considered significant between groups.

Radiation stimulates cellular NO/NO2 production in irradiated cells with time

With the fluorescent probe DAF-FM diacetate, the production of NO/NO2 in confluent AG1522 cells was monitored as a function of time post-irradiation. Figure 3 shows that 1 cGy dose of α-particles stimulated NO/NO2 production in AG1522 cells in a time-dependent manner with most of the NO production reaching a peak in about 10–15 min post-irradiation. After 10–15 min, no distinct increase in fluorescence intensity is observed, which indicates no NO was produced in cells (Kojima et al., 1998, 1999).

Figure 3
Figure 3

Induction of NO/NO2 in confluent AG1522 human fibroblasts as a function of time post-irradiation in cultures that were either sham-irradiated or exposed to a dose of 1 cGy of α-particles. The results shown are a representative time-dependent response generated using the fluorescent probe DAF-FM diacetate.

Total NO/ONOO generation in the medium of irradiated cells within 30 min post-irradiation and the kinetics of NO/ONOO in the medium of irradiated cells

To investigate the total amount of NO in the medium of irradiated cells within 30 min post-irradiation, 20 μM c-PTIO was added to the medium for 30 min before irradiation and NO level was monitored by ESR. As c-PTIO selectively reacts with NO/ONOO, and no other oxidants (O2, NO2 or OH) react with c-PTIO to form c-PTI (Pfeiffer et al., 1997; Janssen et al., 1998), the decreased intensity of c-PTIO signal implied the total NO/ONOO production (Janssen et al., 1998) within 30 min post-irradiation. The spectrum of PTIO consists of a five-line signal with an intensity ratio of 1:2:3:2:1 (Janssen et al., 1998). In the presence of NO/ONOO, the intensity of the signal decreases in a dose-dependent manner (Janssen et al., 1998). In the irradiated donor culture, the measured signal intensity was only about one-half comparing with the sham-irradiated culture (Figure 4a). The loss in signal intensity could be attributed to the reduction of PTIO, which reacts with NO/ONOO. In the absence of cells, irradiated medium alone had no measurable effect on the signal intensity (Figure 4b). Treatment of the cells in culture with an NOS inhibitor, L-NMMA, resulted in no distinct difference of signal intensity between sham-irradiated culture and the 1 cGy dose of α-particle-irradiated culture (Figure 4c). The results suggest that the irradiated cells and not the medium produces NO/ONOO.

Figure 4
Figure 4

Representative ESR spectra of c-PTIO of control or irradiated cells. The trap regent, c-PTIO, was added before irradiation or sham-irradiation at a final concentration of 20 μM. The treatment groups included (a) sham-irradiated donor cultures and donor cultures irradiated with 1 cGy dose of α-particles; (b) sham-irradiated medium without cells and medium irradiated with 1 cGy dose of α-particles; (c) condition A+L-NMMA, 1 mM. The results indicate that it is the irradiated cells but not the ionization of medium that mediate the production of NO/ONOO.

We further investigated the kinetics of NO/ONOO production in the medium of irradiated cells at different time points post-irradiation. At time points of 5, 10 and 15 min post-irradiation, 95 μl conditioned medium was collected immediately, mixed with 5 μl 20 μM c-PTIO and then frozen in liquid nitrogen for ESR analysis. The time function of the c-PTIO remnant is shown in Figure 5. The decrease in c-PTIO signal intensity indicates that the amount of NO/ONOO in the medium is a time-dependent phenomena with the maximal release observed at 10 min after irradiation. Furthermore, and NO was produced essentially within the first 15 min post-irradiation.

Figure 5
Figure 5

The time function of the c-PTIO remnant in the medium after 1 cGy α-particle irradiation monitored by ESR (monitored at −20°C) and the decrease in c-PTIO signal intensity indicates the amount of NO/ONOO into the medium. The results indicated that the amount of NO/ONOO in the medium was in a time-dependent manner with the maximum at 10 min after irradiation. Data are pooled from three independent experiments and the results represent mean±s.d.

Enhanced nitrosated stress from NO/NO2 in the receptor cell culture after medium transfer

The enhanced nitrosated stress from NO/NO2 in receptor culture was monitored with the fluorescent probe, DAF-FM diacetate. The increase in relative fluorescence intensity in bystander cells treated with the medium collected from donor cells at 10 min post-irradiation for 30 min is shown in Figure 6. The relative fluorescence intensity of NO/NO2 in receptor culture was about threefolds higher than that of cells treated with medium from sham-irradiated donor culture. The increased fluorescence indicated an enhanced RNS stress in the receptor cells.

Figure 6
Figure 6

Enhanced fluorescent signal of fluorescent probe DAF-FM diacetate in receptor AG1522 fibroblasts after treated with medium transferred from either sham-irradiated or cultures irradiated with 1 cGy dose of α-particles. The conditioned medium was collected at 10 min post-irradiation and fluorescent signals in receptor cells were measured at 30 min post medium transfer. Data are pooled from three independent experiments and the results are represented as mean±s.d. P-value <0.05 is considered significant between groups.

Induction of DSBs with low-level ONOO anions and the activation of iNOS might not be involved in the early process of DSB inductions

The toxicity of NO is linked to its ability to combine with O2 to form ONOO, an oxidizing free radical that can cause DNA fragmentation and lipid oxidation (Beckman et al., 1990; Jourd'heuil et al., 1997). To investigate whether low level of NO (several hundred nM to 1 μM) could induce DSBs in the cultures, 1 μM ONOO, synthesized as described (Fernandes et al., 2005), was used to treat the confluent AG1522 for 30 min and the cells were fixed for DSBs assessment. The results shown in Figure 7 implied that even low level of ONOO (1 μM) could induce DSBs effectively (28.7–14.6%, P=0.005) in the cultures.

Figure 7
Figure 7

Induction of DSBs with 1 μM ONOO treatment. Results show that low-level ONOO can induce DSBs effectively. DSBs were quantified by γ-H2AX immunochemical staining. Data are pooled from three independent experiments and the results represent mean±s.d. P-value <0.05 is considered significant between groups.

Discussion

DSBs are considered to be the most relevant lesion for the deleterious effects of ionizing radiation (Dahm-Daphi et al., 2000; Hoeijmakers, 2001; Van Gent et al., 2001). Using a fluorescent antibody specific for γ-H2AX, discrete nuclear foci can be visualized at sites of DSBs, induced either by exogenous agents such as ionizing radiation or generated endogenously during programmed DNA rearrangements (Rogakou et al., 1998; Rogakou and Pilch, 1999; Chen et al., 2000; Burma et al., 2001; Mahadevaiah et al., 2001; Petersen et al., 2001; Rothkamm and Lobrich, 2003). Several studies have shown consistently that a γ-H2AX focus represents one DSB and that γ-H2AX foci formation can be used to measure the repair of individual DSBs in human cells (Rothkamm and Lobrich, 2003; Sedelnikova et al., 2002, 2004). For the fast phosphorylation of H2AX, γ-H2AX can be monitored even at 1 min post-damage and this protein becomes fully phosphorylated at 10 min and will then remain in a phosphorylated state for 20 min (Rogakou and Pilch, 1999). In our previous study, we demonstrated that in situ visualization of DSBs in confluent AG1522 cultures could be used to assess the early or initiation events of radiation-induced extranuclear/extracellular (bystander) effects (Hu et al., 2005; Han et al., accepted).

The role of soluble transmissible factor(s) generated by irradiated cells that in turn induces toxic effects in non-irradiated cells has been demonstrated by many medium transfer experiments (reviewed by Azzam and Little, 2003). Mothersill and Seymour (1997) first demonstrated a highly significant reduction in cloning efficiency in both non-irradiated normal as well as malignant epithelial cell lines that had received media from 60Co-γ-ray-irradiated human epithelial cell cultures. Further studies found that transferring media from low or high linear energy transfer-irradiated cultures to non-irradiated cells led to increased levels of various bystander effects, such as cell killing (Mothersill and Seymour, 1998; Lyng et al., 2000; Nagar et al., 2003), neoplastic transformation (Lewis et al., 2001), proliferation (Iyer and Lehnert, 2000) and genomic instability (Seymour and Mothersill, 1997). These studies suggested that irradiated cells secreted a cytotoxic factor such as reactive oxygen species (ROS), transforming growth factor-β1 (TGF-β1) or interleukin-8 (IL-8), or proteins (reviewed by Azzam et al., 2004) into the culture medium which was capable of causing damage in non-irradiated cells. Consistent with these studies, by using the γ-H2AX foci as a surrogate marker of DSBs, we found that, as early as 2.5 min post-irradiation, the irradiated cells secreted certain DSB-inducing factors into the medium and resulted in the excessive DSBs in non-irradiated medium receptor cells (Han et al., accepted). In the present studies, we investigated the nature of the DSB-inducing factor(s) and how it was generated and transferred. By treating the cells with L-NMMA to inhibit the NOS activity (Leach et al., 2002; Liu et al., 2005) (D-NMMA), it was found that L-NMMA, could effectively block the DIA of conditioned medium in the receptor cells. In contrast, D-NMMA, the inactive enantiomer had no effect. These are suggested that NO was a possible signal molecular in this process (Figure 1).

There are two principal pathways for the generation of NO after stimulation of the cells with chemicals or radiation, that is, by activation of iNOS or cNOS. Distinguishing the two forms of NOS are based on the activation time after the stimuli, the quantity of the NO generated and whether it is Ca2+ dependent or not (reviewed by Stefano et al., 2000). For constitutive NO, although the release level is only in the nM range, its transient release can have profound physiological activations for a longer period of time after NO has returned to its basal level (reviewed by Stefano et al., 2000). Stefano et al. demonstrated that the release of NO from human endothelial cells treated with morphine or anandamide peaked within 5 min and returned to basal levels within 10 min. They attributed this to the activation of c- form of NOS, as iNOS can be activated only after prolonged (2 h) exposure to bacterial lipopolysaccharide and interferon-γ, but remained significantly elevated over basal levels for 24–48 h (Stefano et al., 1995, 1998). Leach et al. (2002) showed that after 2 Gy X-ray irradiation, the activity of cNOS was transiently enhanced at 5 min post-irradiation and by 30 min NOS activity had returned to basal levels, implying that constitutive NO or its derivatives were the effectors activating the signal transduction pathways after irradiation. Shao et al. (2002, 2003a, 2003b) indicated the importance of NO in the induction of micronuclei in bystander cells and suggested that the transmissible signaling molecule(s), such as TGF-β1 secreted from the irradiated cells activated iNOS in the non-irradiated cells. To investigate which form of NOS is involved in the initiation process of RIBE, herein the donor culture was treated with 7-Ni and L-NNA, the inhibitors of cNOS, before irradiation. The results showed that the DIA of conditioned medium decreased significantly when compared with the control without drug treatment, which implied NO might be derived from the activation of cNOS (Figure 1). On the other hand, treatment with AG did not decrease the DIA of conditioned medium, suggesting that the activation of iNOS might not be involved in the early process of RIBE. Moreover, our results show that even low-level (1 μM) ONOO can induce the increase of distinct DSB-positive cells (Figure 7).

The next critical question is whether NO was produced inside the irradiated cells or presented in the medium of irradiated cells. Based on the treatment of cells with DAF-FM diacetate or L-NNA as well as c-PTIO with ESR analysis, we found that NO was generated in the irradiated cells (Figure 2), and presented in the medium post-irradiation (Figure 4), which contributed to the induction of nitrosated stress signal in the medium receptor cells (Figure 6). Moreover, by treating the irradiated cells with the fluorescent probe DAF-FM diacetate before 1 cGy α-particle irradiation, the generation of NO in confluent AG1522 cells was found to be time-dependent. Furthermore, almost all NO was generated in about 15 min post-irradiation and no distinct fluorescence increase was observed after 10–15 min (Figure 3). Similarly, the kinetics of the NO in the medium after irradiation was rapid and in the time-dependent manner with maximum levels observed 10 min after irradiation (Figure 5). The time dependence of NO concentration presented in the medium was similar to that of DIA of conditioned medium (Han et al., accepted), that is, transiently increased post-irradiation and then reached a maximum level at 10 min. As DSBs induction in cells is very sensitive and can be detected rapidly after irradiation, our findings on NO-mediated DSBs induction in non-irradiated bystander cells may reflect the initiation and early process of RIBE.

Finally, it should be pointed out that although our results indicate that NO, more likely constitutive NO, is generated in the irradiated cells, present in the medium of irradiated cell and formed the RNS stress in the medium receptor cells, the pathway of NO signaling and the contribution, if any, of other kinds of molecules such as ROS is not clear. Many studies have demonstrated that ROS is very important for the induction of RIBE (reviewed by Azzam et al., 2004). Our previous studies (Hu et al., 2005, 2006) also indicate that ROS is an important mediator for the induction of DSBs in non-irradiated cells. Furthermore, by treating medium donor cells with dimethyl sulfoxide, a quencher of ROS, before irradiation could effectively reduce the DIA of conditioned medium (Han et al., accepted). As a result, it is probably that both ROS and NOS are involved in the initiation of RIBE and ROS may be the upstream or downstream mediator of NO induction. Moreover, NO is also known to regulate the expression of TGF-β1 and IL-8 in some cells and the enhancement of NO might trigger the release of these cytokines to the medium (Vodovotz et al., 1999; Xiong et al., 2001).

Materials and methods

Cell culture

AG1522 normal human diploid skin fibroblasts, received as a kind gift from Dr Barry Michael (Gray Cancer Institute, UK), were maintained in α-Eagle's minimum essential medium (Gibco) supplemented with 2.0 mM L-glutamine and 20% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) plus 100 μg/ml streptomycin and 100 U/ml penicillin (Gibco, Grand Island, NY, USA) at 37°C in a humidified 5% CO2 incubator. For irradiation, approximately 1 × 104 exponentially growing AG1522 cells in passages 12–15 were seeded into each specially designed rectangular dish (internal area: 10 × 6 mm2) consisting of a 3.5 μm thick mylar film bottom. The culture medium was replaced every 2 days and the cells were irradiated at day 3 under full confluent condition. At that time, about 92% of the cells were in G0–G1 as determined by flow cytometry (Azzam et al., 2001).

α-Particle irradiation and medium transfer experiments

The average energy of α-particles derived from the 241Am irradiation source was 3.5 MeV measured at the cell layer and the particles were delivered at a dose rate of 1.0 cGy s−1. To confirm whether the irradiated cells secreted the soluble transmissible factor(s) to the medium or whether these factor(s) resulted in DSBs induction in the non-irradiated cells, the medium transfer experiment was used in the present study. AG1522 cells cultured in a rectangular dish under confluent conditions were irradiated 100% with 1 or 20c Gy α-particles. After incubation for 10 min, the medium from the irradiated population was immediately collected and then transferred to a rectangular dish full of non-irradiated, confluent AG1522 cells. Thereafter, these receptor cells were incubated for 30 min with the conditioned medium and then were fixed for DSBs immuno-labeling or NO/NO2 fluorescence detection with fluorescent probe. Medium from 100% sham-irradiated dish was transferred to the receptor cells to sever as control. In some experiments, only medium without cells was irradiated and then the medium was transferred as described above. To test for the amount of NO/ONOO production, medium was collected from irradiated cells and subjected to ESR measurement at the indicated time points.

Immunochemical staining of cells (γ-H2AX) and DSBs measurement

Immunochemical staining of cells was performed as described (Aten et al., 2004). Briefly, after incubation, cells in the dish were washed with phosphate-buffered saline (PBS) three times, fixed in a 2% paraformaldehyde solution in PBS for 15 min at room temperature and then rinsed three times with PBS again. Before immunochemical staining, cells were incubated for 30 min in TNBS solution (PBS supplemented with 0.1% Triton-X 100 and 1% FBS) to improve their permeability and then incubated with anti-γ-H2AX antibody (Upstate Biotechnology, Lake Placid, NY, USA) in PBS+ (PBS supplemented with 1% FBS) for 90 min, washed in TNBS (3 × , 5 min) and incubated in PBS+ containing the fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (Haoyang Biological Manufacture Company, Tianjin, China) for 60 min. After washing with TNBS (3 × , 5 min), cells were counter-stained with Hoechst 33342 at a concentration of 5 μg/ml for 20 min at room temperature. After a final wash with TNBS, the stained cells on the mylar film were mounted using 50% glycerol-carbonate buffer (pH 9.5).

The stained rectangular dishes were loaded on a 35-mm diameter glass bottom dish (glass thickness: 0.17 mm, Netherlands), which was used as a holder. Immunofluorescent images were captured by Confocal Laser Scanning Microscope (Leica, TCS SP2, Bensheim, Germany). For quantitative analysis, the cells with γ-H2AX foci were regarded as the positive cells and the fraction of positive cells was calculated (cells with DSBs/total cells) as described previously (Limoli et al., 2002; Fagagna et al., 2003). At least 1000 cells in each sample were counted and statistical analysis was performed on the means of the data obtained from at least three independent experiments.

DSBs induction in receptor cells with treatment of NOS inhibitors or DAF-FM diacetate

To test whether NO is involved in the rapid release of signal and the rapid induction of DSBs among the receptor cells, the medium in the donor cells was replaced with the fresh medium containing either L-NMMA (1 mM), D-NMMA (1 mM) or 7-Ni (4 μM) 1 h, or AG (0.1 mM) 2 h before irradiation. After irradiation, the donor cell culture was incubated for 10 min and then the medium was immediately collected and transferred to the receptor cells to quantify its DIA as described above.

To trap the possibly induced intracellular NO, DAF-FM diacetate at a working concentration of 20 μM or L-NNA (1 μM) was added to the irradiated culture 30 min before irradiation. The additional dye or drug in the medium was removed by rinsing the cells in culture three times with fresh culture medium. After irradiation, the irradiated cultures were incubated for 10 min and then the medium was immediately collected and transferred to the receptor cells to quantify its DIA as described.

Direct detection of NO/NO2 both in the donor and receptor cells with the fluorescence probe

To monitor the kinetics of NO/NO2 production and the RNS stress from NO/NO2 in receptor cells, DAF-FM diacetate was used as the fluorescent probe, which shows a stable and intense fluorescence in a wide range of pH after reacting with NO/NO2 (Kojima et al., 1998, 1999). The cells in culture were incubated with DAF-FM diacetate (10 μM in saline buffer containing the following: NaCl 140 mM, KCl 5 mM, MgCl2 2 mM, 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid 10 mM, glucose 10 mM, sucrose 6 mM, CaCl2 2 mM (Li et al., 2003) for 40 min at 35°C. After the excess probe was removed, the cells were rinsed with the buffer (3 ×). Fluorescence was measured with a Spetra Max M2 (Molecular Devices, Synnyvale, CA, USA) fluorescence reader and the excitation/emission were set at 495/515 nm. To follow the production of NO/NO2 in the irradiated cells, the confluent culture grown in the 8.2 mm diameter dish with mylar film bottom was irradiated with a 1 cGy dose of α-particles and the kinetics in NO/NO2 induction was followed up to 30 min post-irradiation.

To investigate the NO/NO2 stress in the receptor culture, confluent cells growing in 96-well cell culture plates treated with the dye before medium transfer were mixed with the medium collected from donor cells at 10 min post-irradiation. After incubation with conditioned medium for 30 min, the intensity of the fluorescent signals, which served as surrogate marker of NO/NO2 was measured using a micro-plate fluorescence reader.

Measurement of NO/ONOO in the medium of irradiated cells with ESR

To monitor the total amount of α-particle-induced NO/ONOO in the irradiated cell culture among the period of 30 min post-irradiation, c-PTIO was added to the medium (20 μM) before irradiation. After irradiation, the tissue culture dishes were immediately incubated for 30 min. Hundred microliters cell culture medium was collected and quickly frozen in liquid nitrogen for ESR analysis. Cells in culture treated with NOS inhibitor (1 mM L-NMMA) 1 h before irradiation and with the medium without cells were used as control.

To examine time dependence of NO/ONOO concentration in the culture medium, 95 μl medium was collected immediately at predetermined time point and then mixed with 5 μl c-PTIO (20 μM). The mixture was quickly frozen in liquid nitrogen for ESR analysis. ESR spectra were recorded at −20°C. Samples in the 100 μl capillaries inserted into 4 mm quartz tubes were used for ESR analysis. ESR spectra were recorded at room temperature or −20°C on a JES-FA 200 ESR spectrometer operating at 9.43 gHZ (room temperature) or 9.05 gHZ (−20°C) and with a 100-kHz field modulation. Spectrometery conditions were as follows: microwave power, 2 mW; scan range, 20 mT; modulation amplitude, 0.1 mT; time constant, 0.1 s; scan time, 1 min. The relative signal intensity of c-PTIO is represented by dividing the ratio of the c-PTIO signal intensity of conditioned medium (collected from 1 cGy α-particle irradiated culture) by that of sham-irradiated culture. The decrease in c-PTIO signal intensity indicates that the increase of NO/ONOO production and release.

Statistics

Data are presented as means±s.d. Significance levels are assessed using Student's t-test. A P-value of 0.05 or less between groups is considered to be significant.

Supplementary information is available at Oncogene's website.

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Acknowledgements

We thank Drs Mohammad Athar, Hari Bhat and Vladimir Ivanov for critical reading of the paper and their helpful discussions, and Dr Haiying Hang for his technical assistance with DSB detection. This work was funded by National Nature Science Foundation of China under Grant nos. 10225526 and 30570435, Grant KSCX2-SW-324 of Chinese Academy of Sciences, and US National Institutes of Health Grants ES 012888 and Environmental Center Grant ES09089.

Author information

Affiliations

  1. Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, People's Republic of China

    • W Han
    • , L Wu
    • , S Chen
    • , L Bao
    • , L Zhang
    • , E Jiang
    • , Y Zhao
    • , A Xu
    •  & Z Yu
  2. Center for Radiological Research, College of Physicians and Surgeons,Columbia University, New York, NY, USA

    • T K Hei

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Supplementary information

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    Supplement Data

Glossary

AG

aminoguanidine hemisulfate

c-PTIO

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

DAF-FM diacetate

4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate

DIA

DSB-inducing activity (of conditioned medium collected from the irradiated culture)

D-NMMA

NG-methyl-D-arginine

DSBs

DNA double strand breaks

ESR

electron spin resonance

γ-H2AX

phosphorylated form of H2AX protein

L-NMMA

NG-methyl-L-arginine

L-NNA

Nω-nitro-L-arginine

7-Ni

7-Nitroindazole

NO

nitric oxide

NO2

nitrite

NOS

nitric oxide synthase

ONOO

peroxynitrite

RIBE

radiation-induced bystander effects

RNS

reactive nitrogen species

ROS

reactive oxygen species

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DOI

https://doi.org/10.1038/sj.onc.1210024

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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