Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of ∼1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 ± 0.02-fold (P < 0.005) relative to control with ∼40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.

The response of cells and tissues to radiation exposure has been assumed to be a direct consequence of energy deposition in DNA within the cell nucleus. The observation of epigenetic or nontargeted responses in a range of studies has questioned this basic assumption (1, 2, 3, 4, 5). Bystander responses are an important example of these responses where cells, which have not been directly exposed to radiation, respond to radiation when their neighbors are exposed. Recent evidence has shown bystander responses manifested as increased chromosomal damage (6, 7), genomic instability (8, 9), mutations (7, 10, 11), and malignant transformation (12, 13). Many of the studies of bystander responses have measured either the ability of factors to be transferred from irradiated cells to unirradiated cells by medium transfer, or the response of cells to low fluences of α-particles where only a few percent of cells have been randomly exposed. Using these approaches, several factors have been identified as playing a role in bystander responses. These have included cytokines (14), ROS1(15, 16), and membrane-mediated responses (17). It has been reported recently that NO, an important signaling molecule, produces multiple bystander effects of enhancing cell growth, inducing micronuclei, and radioprotection (18, 19, 20).

The development of microbeams has allowed individual cells to be targeted at specific subcellular locations with precise doses of radiation (21, 22). This is a major advance from conventional broad beam irradiations where, although very low-dose studies allow only a fraction of cells to be irradiated, it is impossible to know which part of a cell is actually targeted. Moreover, when using conventional α-particle irradiations as a tool to study processes such as the bystander effect, only the targeted nuclei but not the targeted cytoplasm have been considered in calculations of the fraction of targeted cells (23, 24). In fact, evidence already suggests that cytoplasmic traversal could induce cell killing and genetic mutation (25). Hence, microbeams allow more rigorous studies of mechanisms underpinning bystander responses as the dose delivered to the target cells and the number of target cells exposed can be carefully controlled. The Gray Cancer Institute Charged Particle Microbeam allows individual charged particles to be delivered to cells with high reproducibility. Using this approach we have shown direct evidence for bystander responses in primary human fibroblasts when only a single cell within a population is targeted with a single helium ion used as a surrogate for an α-particle (26).

Most studies to date on bystander responses have used normal human or mouse cells. Recent studies in vivo have suggested a role for bystander responses in tumors (27). In this study we report responses in a radioresistant glioma line, T98G, to localized radiation and show direct evidence for a significant bystander response mediated by NO.

Cell Culture.

Human glioblastoma T98G cells bearing a mutant p53 gene (28) were obtained from the European Collection of Animal Cell Cultures. Cells were cultured in RPMI 1640 supplemented with 10% (v/v) FCS, and 0.01% sodium pyruvate, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. All of the cultures were maintained at 37°C in an atmosphere of 95% air and 5% CO2. For microbeam experiments, plateau phase cells were seeded 2–7 h before irradiation in a φ5-mm central area of the specially designed microbeam dish consisting of a 3-μm thick Mylar film base. The region prepared for cell seeding had been pretreated with 1.7 μg/cm2 Cell-Tak adhesive (Collaborative Biomedical Products). The full-attached cells were stained with 0.2 μg/ml Hoechst 33342 for 1 h before irradiation, enabling individual nuclei to be identified by the microbeam imaging system. Excess stain was removed by washing the cells with serum-free medium containing 10 mm HEPES before irradiation, and cells were maintained in this medium during microbeam irradiation. Typically, 1270 ± 32 (mean ± SE) individual cells in total could be scanned in the microbeam dish just before irradiation. Immediately after irradiation, the culture medium was replaced with 2 ml of complete medium, and incubation continued until additional MN analysis or medium transfer experiments. In some experiments, 20 μm c-PTIO (Molecular Probes Inc.) or 20 μm AG was present in the medium during and after irradiation. c-PTIO is a NO-specific scavenger. AG inhibits the activity of NO synthase.

Microbeam Irradiation.

The Gray Cancer Institute microbeam system was used for this study. Details of the experimental set-up have been described previously elsewhere (21, 22). The coordinates of each stained cell nucleus was found and stored using a computerized imaging system so that the cells could be revisited and irradiated automatically. A fraction of cells, from 1 cell to all of the cells in the population, were individually irradiated at their nuclear center by a precise number of 3He2+ with a linear energy transfer (LET) of 100 keV/μm. Using these ions, 99% of cell nuclei could be precisely targeted with an accuracy of ±2 μm.

Medium Transfer Experiments.

After irradiation, cells were incubated for 1 h, and then the medium of the irradiated population was collected and filtered through a 0.22-μm filter to ensure that no cells were still present. Then the conditioned medium was transferred to a 60-mm Petri dish where 2 × 105 T98G cells had been seeded 8–9 h previously. Cells were treated with the conditioned medium for 20 h and then analyzed using the MN assay.

MN Scoring.

The cytokinesis block technique was used to assay for MN in situ. 1 h after irradiation, the culture medium in the microbeam dishes was replaced with one containing 1 μg/ml cytochalasin-B. With respect to the medium transfer experiments, the transferred medium was replaced with one containing 1.5 μg/ml cytochalasin-B. The cells were incubated for an additional 24–26 h then fixed with Carnoys solution [3:1 (v/v) methanol: acetic acid] for 20 min. After air-drying, cells were stained with 10 μg/ml acridine orange for 5 min. MN were scored in BN cells and classified according to the criteria method described previously (29). The MN yield, YMN, was calculated as the ratio of the number of MN to the scored number of BN cells.

NO Measurement.

The NO level in the T98G cells was measured in situ by using DAF-FM diacetate (Molecular Probes Inc.), which is cell-permeant and essentially nonfluorescent until it reacts with NO and other NO derivatives such as N2O3 produced by auto-oxidation of NO to yield highly fluorescent benzotriazole. Briefly, 1 h after irradiation, cells were washed twice with serum-free medium containing 10 mm HEPES and then treated with 3.5 μm DAF-FM diacetate for 45 min at 37°C. After the excess probe was removed, cells were incubated for an additional 20 min to allow complete de-esterification of the intracellular diacetates. The fluorescence images of at least 100 randomly selected cells per dish was captured using a 3CCD Color Coolview HR Camera (Photonic Science Ltd., East Sussex, United Kingdom) attached to a fluorescent microscope (Zeiss Axioskop) with manual UV-light shutter and filters. Each CCD in this camera provided a resolution of 1392 × 1040 pixels, and the total color image was thus composed of >4.3 million pixels. The gray scale dynamic range of cell fluorescence intensities was between 0 and 255 for each color. The exposure conditions including the intensity of the lamp, the exposure time of the CCD camera, and the gain of the amplifier were standardized to allow quantitative comparisons of the relative fluorescence intensity of the cells between groups.

Predicted Micronuclei Yields.

Assuming no bystander effect occurred in the irradiated cell population, the MN yield in the population, where a known fraction of the cells was irradiated through the nucleus with a precise number of 3He2+ ions, was mathematically predicted as follows. Assuming N, F, PE, and Q are the total number of cells in the population, the fraction of irradiated cells, the plating efficiency, and the BN cell formation frequency of which 100% of cells have been irradiated with exact number of 3He2+ ions, respectively. Then the number of BN cells formed from the cells that were irradiated and survived is given as: F × N × PE × Q. Similarly, the number of BN cells formed from unirradiated cells is given as: (1−F) × N × PEc × Qc, where PEc and Qc are the plating efficiency, and the BN cell formation frequency of the unirradiated control.

If YMNc and YMN are the MN yields of the unirradiated control and the cell population where 100% of cells have been irradiated with a precise number of 3He2+ ions, respectively. Then, the predicted MN yield in population where a known fraction of cells was irradiated is therefore:

\[\frac{\mathrm{F}\ {\times}\ \mathrm{N}\ {\times}\ \mathrm{PE}\ {\times}\ \mathrm{Q}\ {\times}\ \mathrm{Y}_{\mathrm{MN}}\ {+}\ (1\ {-}\ \mathrm{F})\ {\times}\ \mathrm{N}\ {\times}\ \mathrm{PE}_{\mathrm{c}}\ {\times}\ \mathrm{Q}_{\mathrm{c}}\ {\times}\ \mathrm{Y}_{\mathrm{MNc}}}{\mathrm{F}\ {\times}\ \mathrm{N}\ {\times}\ \mathrm{PE}\ {\times}\ \mathrm{Q}\ {+}\ (1\ {-}\ \mathrm{F})\ {\times}\ \mathrm{N}\ {\times}\ \mathrm{PE}_{\mathrm{c}}\ {\times}\ \mathrm{Q}_{\mathrm{c}}}\]

It was found that 1 h after irradiation the cells still attached on the microbeam dish and, thus, their plating efficiency for the MN assay was the same as the control. Therefore, the predicted MN yield becomes

\[\frac{\mathrm{F}\ {\times}\ \mathrm{Q}\ {\times}\ \mathrm{Y}_{\mathrm{MN}}\ {+}\ (1\ {-}\ \mathrm{F})\ {\times}\ \mathrm{Q}_{\mathrm{c}}\ {\times}\ \mathrm{Y}_{\mathrm{MNc}}}{\mathrm{F}\ {\times}\ \mathrm{Q}\ {+}\ (1\ {-}\ \mathrm{F})\ {\times}\ \mathrm{Q}_{\mathrm{c}}}\]

Experiments showed that when 100% of cells in the population were irradiated with one 3He2+ ion, the frequency of BN cell formation in the irradiated population was 0.35 ± 0.06, the same as that of unirradiated controls. Whereas when 100% of cells in the population were irradiated with five 3He2+ ions, the frequency of BN cell formation was reduced to 0.29 ± 0.06.

Statistical Analysis.

Statistical analysis was done on the means of the data obtained from at least three independent experiments. Two replicates were counted for each experimental point in each experiment to determine the MN yield. All of the results are presented as means ± SE. Significance was assessed using Student’s t test at P < 0.01.

Induction of Micronuclei Exceeds the Predicted Value.

It was found that when only one cell in the T98G population (∼1200 cells) was irradiated with one or five 3He2+ ions and subsequently incubated for 1 h, the MN induction in the population was increased by 20% so that MN appeared on average in an additional ∼16 BN cells. Additional study showed that the MN yield increased very quickly when the fraction of irradiated cells increased from 0 to 20%. An example of this MN formation induced by one 3He2+ irradiation is illustrated in Fig. 1. When the fraction of irradiated cells was >20%, the MN yield of the irradiated populations approached a saturation value and was near to that measured when 100% of the cells were irradiated.

According to the calculation method described above, we estimated the MN yields of the population where 10% or 20% of cells were individually targeted by one 3He2+ ion by assuming no bystander effect occurred in the irradiated population. Fig. 1 illustrates that the predicted MN yields are significantly less than the measured MN yields. These results give direct evidence of a bystander effect for the production of micronucleated cells. The difference in the MN yields between observed and predicted values should result from the bystander response. Because there is no GJIC in this low density T98G population during irradiation and subsequent incubation, a radiation-induced signaling factor is probably involved in this bystander response.

c-PTIO Reduced the Micronucleus Induction to the Predicted Value.

To investigate what kind of signaling factors are involved in the bystander effect, we treated the cell population with c-PTIO, a NO-specific scavenger, during and after irradiation until cells were assayed for MN. Fig. 2 illustrates the influence of c-PTIO treatment on the MN yields of the population where a fraction of cells are individually irradiated with either one or five 3He2+ ions. It was found that the c-PTIO treatment reduced the MN yields to the values predicted if only direct effects were being produced, indicating that the radiation-induced bystander response was abolished by the c-PTIO. Similar elimination of the bystander MN induction was observed with cells treated with 20 μm AG, the inhibitor of NO synthase (data not shown). Accordingly, NO contributes to the radiation-induced bystander MN induction in the T98G population when these are exposed to 3He2+.

NO Production in the Irradiated Cell Population.

To strengthen the above finding and get more direct evidence of radiation-induced NO production, we measured the NO level in situ in the T98G population 1 h after microbeam irradiation by using DAF-FM. Fig. 3 illustrates a typical response of the percentage of DAF-FM stained cells to the cell fluorescence intensity in the population where 1% of cell nuclei have been individually irradiated with a single 3He2+ ion. It is seen that most of the cells in the unirradiated control have fluorescence intensities <155, but in the irradiated population, the percentage of cells with a fluorescence intensity >155 was obviously increased from 36.1 ± 5.3% of the control cells to 72.9 ± 0.9% in the population where 1% were irradiated. Taking the population of cells as a whole this led to the mean relative cell fluorescence intensity of the irradiated population being increased to 1.13 ± 0.02-fold (P < 0.005) of that of the control population. However, the treatment of cells with 20 μm AG diminished the cell fluorescence intensity of the irradiated population to the background level, and this AG treatment itself had no significant influence on the cell fluorescence intensity.

Signal Transfer from Cell Culture Medium.

To investigate whether there is any long-lived signaling factor besides the short-lived NO involved in the bystander effect, the medium from the population where 100% of cells were individually irradiated by a precise number of 3He2+ ions was harvested 1 h after irradiation and then transferred to another population of unirradiated cells. Fig. 4 illustrates the MN yields of the unirradiated population after treatment with the conditioned medium. It was found that the MN yield in the T98G cell population was increased by ∼25% on average due to this conditioned medium treatment, but this increase had no significant relationship to irradiation dose. Moreover, when c-PTIO was present in the medium during irradiation and subsequent cell culture, the conditioned medium did not show a cytotoxic effect of MN induction in the unirradiated cells, which indicates that some NO-downstream long-lived biological active factors may also contribute to the radiation-induced bystander effect.

Interaction of the Bystander Signal Molecules and Direct Irradiation.

Fig. 5 illustrates the dose response for the induction of MN in the population where all of the cells were individually irradiated with a precise number of 3He2+ ions. With increasing particle number per cell, the MN yield increased and then started to saturate at high doses. An interesting finding was that when the population was treated with c-PTIO, the MN induction in the population where cells were irradiated with 1 or 2 particles was decreased significantly, but it was not influenced when the cells were irradiated with 5 or 10 particles. This result indicates that with low-dose irradiation the induction of MN could be generated from the complex interaction of direct irradiation and NO-related bystander signal molecules. In contrast, however, with high-dose irradiation, the MN mainly results from the direct irradiation effect. The complex cellular effect of direct irradiation and bystander signaling factors will reduce slightly the predicted MN yield in Fig. 1 assuming no bystander effect occurred in the low-dose irradiation. However, this decrease increases slightly the gap between the measured MN yield and the predicted value, and, therefore, does not influence the deduction of the generation of a bystander response.

In the present study, it is found that when a fraction of cells, including even only 1 cell, in a low density T98G cell population are individually targeted by a precise number of 3He2+ ions, a significant bystander induction of MN can be produced in the neighboring unirradiated cells. Using c-PTIO as a NO-specific scavenger, we find that NO is involved in this radiation-induced bystander effect within a cell population in the absence of GJIC. Moreover, 1 h after irradiation, NO derivatives generated from the oxidation of NO were indeed detected in situ by DAF-FM in the irradiated cell population. This early NO response is in agreement with a report that the NO level detected by DAF-FM was increased by about 20–25% in six different human hepatocellular carcinoma cell lines 1 h after irradiation (30). Nakagawa et al.(31) also detected a significant electron paramagnetic resonance (EPR) signal from NO in mice livers just 1 h after whole-body X-ray irradiation. But Matsumoto et al.(32) found that the accumulation of iNOS in T98G cells was increased significantly only 3 h after irradiation. Thus, detecting NO by a direct chemical capture method may be more sensitive than using Western blot analysis to assay iNOS expression.

As an important messenger molecule, NO is known to be generated endogenously from l-arginine by inducible NO synthase (33, 34) that can be activated in p53-mutated mammalian cells after exposure to radiation (32). The NO-mediated bystander effect reported here is detected as induction of MN in the unirradiated cells, which is in contrast to a NO-initiated bystander effect measured as radioresistance, which has been reported previously (32). These contrasting observations may result from the multiple biofunctions of NO, which depend on NO concentration and the fact that a microbeam was used in the studies here where individual cells were targeted with low doses. Basically, NO and its derivatives have a number of different molecules serving as potential targets for their chemical reactions (35). They can induce cellular damage by acting on DNA, or alternatively, they can protect from radiation-induced cell death by scavenging peroxyl radicals, inducing protective proteins, inhibiting caspase activity and other interactions (36). We have observed previously a NO-initiated bifunctional bystander response of enhanced cell proliferation and induced MN simultaneously in unirradiated human salivary gland tumor cells when they are cocultured with irradiated cells (20). In fact, these NO-induced multiple responses have been demonstrated directly by using the NO-donor: sper/NO (37).

How does NO initiate MN formation in unirradiated bystander cells? It is believed that NO itself does not induce DNA strand breaks (38). Therefore, the bystander MN induction might result from NO-downstream products and/or NO derivatives. The result of medium-mediated bystander response in Fig. 4 indicates that some NO-related long-lived reactive signaling factors should be present in the conditioned medium harvested from the irradiated T98G cells. It has been reported that cytokines such as TGF-β can be activated by NO stress in a time- and dose-dependent fashion (39, 40). With a low dose of α-particle irradiation, TGF-β1 can be secreted from HFL1 cells into the supernatant and additionally induces a bystander effect by enhancing cell growth (14). Others have found that cells treated with TGF-β1 undergo a time-dependent increase in DNA cleavage (41) and the induction of apoptosis (42). TGF-β1 may activate cell surface membrane-associated NADH oxidase, which, in turn, increases the intracellular production of superoxide anions and hydrogen peroxide, and it may also activate the release of extracellular and cell-permeating H2O2(14). These ROS molecules attack at DNA bases and induce DNA strand breaks. Thus, the cytokines including TGF-β1 are likely to be a candidate for the long-lived signaling factors involved in the bystander responses measured here.

On the other hand, the data in Figs. 2 and 4 show that when only 10% of cells are individually targeted with one 3He2+ ion, the MN yield of the whole population is increased by 31% compared with the predicted value assuming no bystander effect, whereas the MN yield of the conditioned medium-treated cells only increased by 17% even when 100% of cells were individually irradiated with one 3He2+ ion. Therefore, the bystander response induced by in situ coculture of the unirradiated cells with the irradiated cells is greater than that induced by the conditioned-medium, which indicates that, besides long-lived cytokines, some other factors with relatively short lives may also contribute to the bystander response during cell coculture. However, the detailed mechanism including the direct effectors of this NO-mediated bystander response in MN formation is still unknown.

The bystander cellular damage described here, for instance, of ∼16 additional micronucleated BN cells produced after 1 h of cell culture after a single cell irradiation, is smaller than that of our previous reports. We found that when only one cell within a population of human fibroblasts at low density was targeted with one 3He2+ ion, after 3 days of cell incubation after irradiation, an additional 80–100 damaged cells were observed (26). On the other hand, when a single cell within a confluent culture processing good GJIC was targeted, after 15 h of cell incubation after irradiation, MN was produced in 3000 BN cells (43). It has also been reported that the induction of broad-beam irradiation induced signaling molecules, such as ROS and NO, increased with the cell incubation time after irradiation (16, 18). Thus, the bystander response might be related to the status of cell population and the incubation time subsequent to irradiation.

A signal amplification process might participate in the bystander response. When a few cells in the population are individually irradiated, signaling factors including NO and perhaps cytokines are released from damaged cells and then react with some vicinal unirradiated cells simultaneously; the newly damaged cells will release additional reactive species and cause other cells to be damaged again. With such a series of cascade reactions, the original signaling factors generated from the directly irradiated cells can be magnified so that a measurable bystander cellular damage can be produced. The observation of bystander responses not only indicates that low-dose radiation-induced cancer risk might be greater than we thought but also is of significance in terms of radiotherapy. It has been reported that the expression of iNOS can be enhanced in irradiated T98G cells so that the medium from these irradiated cells can transfer a radioprotective effect to other glioblastoma cell lines such as A-172 that have a wild-type p53gene (18, 32). In contrast, Kurimoto et al.(44) showed that NO generating agent S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) are potential radiosensitizers for T98G cells, and Mitchell et al.(45) have also shown that treatment with NO yields a marked increase in the radiosensitization of hypoxic cell. Moreover, it was found that radiation-induced NO regulated tumor microenvironments leading to angiogenesis and vasorelaxation (46, 47), changes that will increase the sensitivity of radiotherapy. Accordingly, if radiation-induced signaling factors that transfer to bystander tumor cells have a cell-killing effect, potential new approaches may be developed to improve the efficiency of radiation treatment, if the mechanisms underpinning these responses can be controlled.

Grant support: Cancer Research UK and the Gray Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Kevin M. Prise, Gray Cancer Institute, P. O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, United Kingdom. Phone: 44-1923-828611; Fax: 44-1923-835210; E-mail: prise@gci.ac.uk

1

The abbreviations used are: ROS, reactive oxygen species; NO, nitric oxide; c-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; MN, micronucleus; BN, binucleated; GJIC, gap junctional intercellular communication; AG, aminoguanidine; 3He2+, helium-3 ions; TGF, tumor growth factor; iNOS, inducible nitric oxide synthase.

Fig. 1.

MN yield of the population where a fraction of cells were individually irradiated with one 3He2+ ion. The predicted MN yield is calculated by the method that has been described in the text.

Fig. 1.

MN yield of the population where a fraction of cells were individually irradiated with one 3He2+ ion. The predicted MN yield is calculated by the method that has been described in the text.

Close modal
Fig. 2.

MN yields of the population where a fraction of cells were individually irradiated with one 3He2+ ion (A) and five 3He2+ ions (B), respectively. In some experiments, 20 μm c-PTIO were present in the medium during irradiation and subsequently 1 h after irradiation until MN assay. The predicted MN yield is calculated by the method that has been described in the text. ∗ indicates that the MN yield of the irradiated population is significantly larger than that of unirradiated control, c-PTIO treated population, and predicted values assuming no bystander effect (P < 0.01).

Fig. 2.

MN yields of the population where a fraction of cells were individually irradiated with one 3He2+ ion (A) and five 3He2+ ions (B), respectively. In some experiments, 20 μm c-PTIO were present in the medium during irradiation and subsequently 1 h after irradiation until MN assay. The predicted MN yield is calculated by the method that has been described in the text. ∗ indicates that the MN yield of the irradiated population is significantly larger than that of unirradiated control, c-PTIO treated population, and predicted values assuming no bystander effect (P < 0.01).

Close modal
Fig. 3.

Typical relationship of the frequency of DAF-FM stained cells to the cell fluorescence intensities in irradiated or control populations treated with or without 20 μm AG. For the irradiated populations, 1% of the cell nuclei were individually irradiated with a single 3He2+ ion.

Fig. 3.

Typical relationship of the frequency of DAF-FM stained cells to the cell fluorescence intensities in irradiated or control populations treated with or without 20 μm AG. For the irradiated populations, 1% of the cell nuclei were individually irradiated with a single 3He2+ ion.

Close modal
Fig. 4.

MN yields of the conditioned medium-treated cell populations. The conditioned medium was harvested from the cell population where 100% of cells were individually irradiated by a precise number of 3He2+ ions. In some experiments, 20 μm c-PTIO was present in the conditioned medium during and subsequently 1 h after irradiation until MN assay.

Fig. 4.

MN yields of the conditioned medium-treated cell populations. The conditioned medium was harvested from the cell population where 100% of cells were individually irradiated by a precise number of 3He2+ ions. In some experiments, 20 μm c-PTIO was present in the conditioned medium during and subsequently 1 h after irradiation until MN assay.

Close modal
Fig. 5.

MN induction in the population where 100% of cells were individually irradiated with a precise number of 3He2+ ions. In some experiments, 20 μm c-PTIO was present in the medium during irradiation and subsequently 1 h after irradiation until MN assay. ∗ indicates that the MN yield of the irradiated population is significantly larger than that of the c-PTIO-treated population (P < 0.01).

Fig. 5.

MN induction in the population where 100% of cells were individually irradiated with a precise number of 3He2+ ions. In some experiments, 20 μm c-PTIO was present in the medium during irradiation and subsequently 1 h after irradiation until MN assay. ∗ indicates that the MN yield of the irradiated population is significantly larger than that of the c-PTIO-treated population (P < 0.01).

Close modal

We thank Stuart Gilchrist and Bob Sunderland for assistance with the irradiations using the Gray Cancer Institute Charged Particle Microbeam and Dr. Steven Everett for helpful comments on the manuscript.

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