Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions

Available online at www.sciencedirect.com

Mutation Research 639 (2008) 35–44

Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions Nobuyuki Hamada a,b,c,∗ , Meinan Ni c , Tomoo Funayama c , Tetsuya Sakashita c , Yasuhiko Kobayashi a,b,c a

b

Department of Quantum Biology, Division of Bioregulatory Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan The 21st Century Center of Excellence (COE) Program for Biomedical Research Using Accelerator Technology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan c Microbeam Radiation Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Takasaki, Gunma 370-1292, Japan Received 29 August 2007; received in revised form 19 October 2007; accepted 2 November 2007 Available online 12 November 2007

Abstract Ionizing radiation-induced bystander effects have been documented for a multitude of endpoints such as mutations, chromosome aberrations and cell death, which arise in nonirradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. To address this, we employed precise microbeams of carbon and neon ions for targeting only a very small fraction of cells in confluent fibroblast cultures. Conventional broadfield irradiation was conducted in parallel to see the effects in irradiated cells. Exposure of 0.00026% of cells led to nearly 10% reductions in the clonogenic survival and twofold rises in the apoptotic incidence regardless of ion species. Whilst apoptotic frequency increased with time up to 72 h postirradiation in irradiated cells, its frequency escalated up to 24 h postirradiation but declined at 48 h postirradiation in bystander cells, indicating that bystander cells exhibit transient commitment to apoptosis. Carbon- and neon-ion microbeam irradiation similarly caused almost twofold increments in the levels of serine 15-phosphorylated p53 proteins, irrespective of whether 0.00026, 0.0013 or 0.0066% of cells were targeted. Whereas the levels of phosphorylated p53 were elevated and remained unchanged at 2 h and 6 h postirradiation in irradiated cells, its levels rose at 6 h postirradiation but not at 2 h postirradiation in bystander cells, suggesting that bystander cells manifest delayed p53 phosphorylation. Collectively, our results indicate that heavy ions inactivate clonogenic potential of bystander cells, and that the time course of the response to heavy ions differs between irradiated and bystander cells. These induced bystander responses could be a defensive mechanism that minimizes further expansion of aberrant cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Ionizing radiation; Heavy ions; Bystander effect; Broadbeam; Microbeam; Normal human diploid fibroblasts

1. Introduction ∗

Corresponding author at: Department of Quantum Biology, Division of Bioregulatory Medicine, Gunma University Graduate School of Medicine, 1233 Watanuki-machi, Takasaki, Gunma 370-1292, Japan. Tel.: +81 2734 69542; fax: +81 2734 69688. E-mail address: [email protected] (N. Hamada). 0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2007.11.001

Convincing evidence has accumulated that ionizing radiation induces biological response in nonirradiated bystander cells that have received signals from directly irradiated cells [1–4]. Bystander cells manifest a range of biological consequences, such as chromosome

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aberrations, mutagenesis, micronucleation, apoptosis, inactivation of cellular clonogenic potential, activation of signaling pathways, and delayed effects in their progeny [5–13]. Proposed mechanisms mediating radiation-induced bystander effects involve gap junctional intercellular communication, reactive oxygen/nitrogen species, secreted soluble factors, lipid rafts, and calcium fluxes [14–22]. There is plentiful evidence that biological effectiveness varies with the linear energy transfer (LET) of radiation. Higher-LET heavy ions produce more dense ionization along their trajectories, thereby causing more complex clustered DNA damage [23,24]. Whereas extensive studies have established that heavy ions are more cytotoxic and genotoxic to irradiated cells than low-LET photons like X-rays and ␥-rays [24–26], less information is available hitherto as to the potential impact of heavy ions on bystander effect. Considering that less irradiated cells coexist with more nonirradiated counterparts in a population exposed to a lower dose of higher-LET heavy ions [27], a clarification of the effects occurring not only in irradiated cells but in their bystander cells would be crucial to comprehend the mechanism of action of heavy ions. In the current study, we set out to examine the response of heavy ion-irradiated and bystander cells in confluent cultures of normal human diploid fibroblasts. To address this, we employed two different broadbeams that randomly deliver particles, and corresponding microbeams that selectively target a preset fraction of cells each with the precise number of particle(s). We demonstrate that heavy ion-induced bystander responses are manifested as inactivated clonogenic potential, a transient apoptotic response, and delayed p53 phosphorylation.

2. Materials and methods 2.1. Cell culture AG01522D primary normal human diploid fibroblasts were obtained and routinely sub-cultured as described elsewhere [28,29]. Cells at passages 7–9 were seeded at 1 × 104 cells/cm2 , re-fed on days 4, 7 and 9, and used for irradiation experiments on day 11. This procedure for preparing confluent densityinhibited cultures has been shown to synchronize 95–98% of the cells in G0 and G1 phases and also to maximize intercellular interactions [13–15]. All cell cultures were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 in air unless otherwise stated. The size of the cell nucleus and the whole cell was determined to be 165 ± 7 ␮m2 and 1370 ± 50 ␮m2 , respectively, for confluent AG01522 cells as described [16,30,31].

Fig. 1. Diagrammatic presentation of the two different conditions used for heavy ion irradiation in this study. We carried out conventional broadfield irradiation using broadbeams (left panel), and targeted irradiation using microbeams (right panel). Arrows, ovals, and bold lines depict the particle fluence, the cells, and the dish, respectively.

2.2. ␥-ray irradiation Cell monolayers cultured on 25-cm2 tissue culture flasks were exposed to 0–8 Gy of ␥-rays from 60 Co sources at a dose rate of 2 Gy/min at room temperature, followed by incubation at 37 ◦ C in 95% air/5% CO2 . 2.3. Heavy ion irradiation Two different conditions were used for heavy ion irradiation in this study (a schematic diagram depicted in Fig. 1). Targeted microbeam irradiation of only a very small fraction of cells within the whole cell population (referred hereinafter to as microirradiation) was conducted using collimated microbeams installed at TIARA (Takasaki Ion Accelerators For Advanced Radiation Application) facilities of Japan Atomic Energy Agency (JAEA), for which the setup and irradiation procedure have been described [16,27,32–34]. Taking into consideration that roughly 80% of the particle fluence collimated via a 20-␮m-diameter microaperture was confined to the area of central 20-␮m circle around which the remaining 20% was scattered to the area of 15-␮m-wide annulus [35], we refer to the unit of the target number herein as the site, rather than the cell. We selectively targeted neither cell nucleus nor cytoplasm on account of the followings: (a) broadbeam randomly irradiates nuclei or cytoplasms; (b) growing evidence indicates that both cytoplasmic and nuclear irradiation induces bystander responses to a comparable extent [17,18]; (c) microbeam scatters as aforementioned. For all the experiments, 10 particles were delivered to each site. In a 35-mm microbeam dish, 1.1 × 106 ± 8.2 × 104 cells populated at the time of irradiation, so that the fraction of hit cells among the whole population was estimated to be 0.00026, 0.0013 and 0.0066% when 10 particles of two different types of heavy ions (physical properties listed in Table 1) were delivered to each of 1, 5 and 25 sites with a matrix distribution of 3 mm × 3 mm area in the center of the dish, respectively. Therefore, under these conditions, the vast majority of the cells could be considered as bystander cells, and contribution of the effects induced in hit cells to the overall response should be negligible. Absorbed dose (cGy) given by a traversal of maximal 10 particles through a single

N. Hamada et al. / Mutation Research 639 (2008) 35–44 Table 1 Physical properties of heavy-ion microbeams Ion species

Energy (MeV/nucleon)

LET (keV/␮m)

Dose (cGy) to a target cella

Carbon ions Neon ions

18.3 13.0

103 375

12.0 43.9

37

the dishes were then covered with 8-␮m-thick Kapton film (DuPont-Toray, Tokyo, Japan) to avoid drying, followed by irradiation at room temperature. Immediately after irradiation, the conditioned medium was added back to the dishes, and the cultures were thereafter maintained at 37 ◦ C in 95% air/5% CO2 . Control cells were sham-irradiated and handled in parallel with the test cells for all the experiments.

a

The dose (cGy) given by a traversal of a single target cell with 10 particles was calculated as 160.2 × LET (keV/␮m) × fluence (number of ion particles/␮m2 ), which was further divided by the average size of the whole cell (1370 ␮m2 ).

target cell was calculated as 160.2 × LET (keV/␮m) × fluence (number of ion particles/␮m2 ), which was further divided by the average size of the whole cell (1370 ␮m2 ). Conventional broadfield irradiation was performed using broadbeams as previously described [28,29]. Cell monolayers grown on 60-mm dishes were exposed to graded dose of two different types of heavy ions (properties listed in Table 2). The LET at the cell surface was calculated according to the kinetic energy loss, assuming water equivalence. Dose (Gy) was calculated as fluence (number of ion particles/ cm2 ) × LET (keV/␮m) × 1.602 × 10−9 . The Poissondistributed mean number of particles that traversed each nucleus and whole cell was calculated as Dose (Gy)/[0.1602 × LET (keV/␮m)], which was further multiplied by the average size of the nucleus (165 ␮m2 ) and the whole cell (1370 ␮m2 ), respectively. Irradiation with 1–3 Gy should give particle traversal to all cells: exposure to 1, 2 and 3 Gy was estimated to give traversal by a Poisson-distributed mean of 79, 158 and 238 particles/cell (9.5, 19 and 29 particles/nucleus) for carbon ions, and 20, 39 and 59 particles/cell (2.4, 4.7 and 7.1 particles/nucleus) for neon ions, respectively. Conditioned medium from cell monolayers was collected just prior to microbeam and broadbeam irradiation, and

2.4. Cell survival Cell survival was determined by the clonogenic survival assay as previously mentioned [28,29]. In brief, confluent cultures were reseeded within 1 h postirradiation into 100-mm dishes in quadruplicate at numbers estimated to produce 50–80 clonogenic colonies per dish. After incubation for 12–13 days, cultures were fixed in methanol and stained with crystal violet. Only colonies comprising more than 50 cells were scored as survivors. Survival curves illustrated in Fig. 2A were fitted against the means for three independent experiments to the exponential equation y = exp (−ax) where y, x and a are surviving fraction, the dose and slope, respectively. The 10% survival dose (D10 ) presented in Table 2 was calculated as [ln (1/0.1)]/a. 2.5. TUNEL assay Apoptosis was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method [36], using the ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (S7101, Millipore, Billerica, MA) as previously described [37]. Briefly, cell monolayers cultivated on coverslips were fixed in 1% paraformaldehyde and methanol, and permeabilized in 0.5% Triton X-100. After endogenous peroxidase was quenched with 3% hydrogen peroxide, the coverslips were reacted with TdT enzyme and peroxidase-conjugated anti-digoxigenin antibody. Positive TUNEL staining was developed with diaminobenzidine and

Table 2 Physical properties of heavy-ion broadbeams Ion species

Energy (MeV/nucleon)

LET (keV/␮m)

Dosea

Carbon ions

18.3

108

1.3 Gy (D10 ) 13 cGy (0.1 D10 ) 13 mGy (0.01 D10 )

Neon ions

13.0

437

1.9 Gy (D10 ) 19 cGy (0.1 D10 ) 19 mGy (0.01 D10 )

No. of particles traversed throughb A nucleus

A whole cell

12.4 1.24 0.12

103 10.3 1.03

4.48 0.45 0.04c

37.2 3.72 0.37c

a Survival curves presented in Fig. 2A were fitted against the means of three or four independent experiments to the exponential equation y = exp (−hx) where y, x and h are surviving fraction, dose and slope, respectively. D10 was calculated as [ln (1/0.1)]/h. Cultures were exposed to above-indicated doses of broadbeams for the experiments demonstrated in Figs. 3A and 4. b The Poisson-distributed mean number of particles that traversed each nucleus and whole cell was calculated as Dose (Gy)/ [0.1602 × LET (keV/␮m)], which was further multiplied by the average size of the nucleus (165 ␮m2 ) and the whole cell (1370 ␮m2 ), respectively. c The number of traversed particles can be paraphrased into the hit fraction (i.e., traversal of 0.04 particles/nucleus and 0.37 particles/cell means a single particle traversal of 4.48% of nuclei and 37.2% of cells, respectively). Therefore, the remaining 95.5% of nuclei (roughly 21 nuclei out of 22) and 62.8% of cells (roughly 5 cells out of 8) can be estimated to receive no particles.

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Fig. 2. (A) The clonogenic survival following broadfield irradiation. Confluent cultures were exposed to graded dose of ␥-rays (open triangles), carbon ions (open squares) or neon ions (open circles). (B) The clonogenic survival following microirradiation. A single site was targeted with 10 particles of carbon or neon ions (closed columns) or sham-irradiated (open columns). Within 1 h postirradiation, cultures were reseeded for colony formation. The data represent the means and standard errors of three or four independent experiments with quadruplicate measurements. *p < 0.05 compared with corresponding sham-irradiated controls.

specified. Statistical comparisons between groups were made by Student’s t-test, and a p value of 0.05 or less was considered to be significant.

indicated by a brown precipitate. The cells were counterstained with 0.5% methyl green, and viewed under an Olympus BX60 microscope after mounting. For each sample, 1000 or more cells were analyzed. Hoechst 33342 staining yielded apoptotic indices similar to those of TUNEL staining [37].

3. Results

2.6. Western blot analysis

3.1. Inactivation of clonogenic potential

Western blotting was done as previously described [38,39]. Briefly, cells were lysed in radioimmunoprecipitation assay buffer and cleared by centrifugation. The supernatants were used for western blotting after the protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL). Whole cell extracts (8 or 16 ␮g) were electrophoresed on SDS-polyacrylamide gels (BioRad, Hercules, CA) and electroblotted onto Immobilon-P (Millipore, Bedford, MA). The blots were reacted with polyclonal anti-serine-15-phosphorylated p53 (Cell Signaling Technology, Danvers, MA) or monoclonal anti-␣-tubulin antibodies (clone DM1A, Calbiochem, La Jolla, CA): the latter served as an internal standard to verify equal loading of the samples. After incubation with biotinylated secondary antibodies and streptavidin-alkaline phosphatase (GE Healthcare, Buckinghamshire, UK), the bands were visualized with the NBT/BCIP substrate (Roche, Indianapolis, MA) and analyzed by scanning densitometry using the ImageJ 1.37v freeware (W.S. Rasband, National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/).

To examine heavy ion-induced loss of clonogenicity in irradiated cells, confluent cultures exposed to two different broadbeams were reinoculated for colony formation (Fig. 2A). D10 of carbon and neon ions was 1.3 and 1.9 Gy, respectively (Table 2): for comparison, the relative biological effectiveness calculated relative to D10 of ␥-rays (5.1 Gy) was 3.9 and 2.7 for carbon and neon ions, respectively. Next, to assess the bystander effect for clonogenic survival, a single site was targeted with 10 particles of two different microbeams in situations whereby particle(s) traversed only 0.00026% of the cells. Fig. 2B illustrates that microirradiation led to almost 10% decreases in the survival irrespective of ion species. It should be stressed that these differences between microirradiated and sham-irradiated groups were small but statistically significant (p < 0.05).

2.7. Statistical analysis Data were calculated as the means and standard deviations of three or four repeated experiments except where otherwise

3.2. Apoptosis induction Cultures exposed to D10 of broadbeams were processed for the TUNEL assay to evaluate apoptosis in irradiated cells. Fig. 3A demonstrates that the apoptotic incidence increased with time at least up to 72 h

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postirradiation. The apoptotic index following carbon and neon irradiation rose 2.3- and 1.7-fold at 24 h postirradiation, and 3.5- and 3.0-fold at 72 h postirradiation, respectively over the control level. Subsequently, bystander-induced apoptosis was assessed. As is evident from Fig. 3B, exposure of a single site to 10 particles resulted in increased yields of apoptotic cells. Apoptotic frequency following carbon and neon microirradiation escalated 2.4- and 2.0-fold, respectively at 24 h postirradiation over the control level, but declined at 48 h postirradiation nearly to the control level. Thus, the temporal kinetics of apoptosis induction in bystander cells was distinct from that in irradiated cells. The apoptotic index in sham-irradiated controls remained unchanged during the time course (p > 0.2). 3.3. p53 phosphorylation Cultures exposed to D10 , 0.1 D10 or 0.01 D10 of broadbeams were subjected to western blotting probed with

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anti-serine 15-phosphorylated p53 antibody (Fig. 4A and B). Less cells should receive particle(s) at lower doses: in particular, this was the case for 0.01 D10 of neon ions, such that roughly 21 cell nuclei out of 22, or 5 cells out of 8, were estimated to receive no particles (see keynote c in Table 2). As shown in Fig. 4C and D, the levels of phosphorylated p53 increased even at 0.01 D10 and remained constant at 2 and 6 h postirradiation independently of dose and ion species. Next, bystander-induced p53 phosphorylation was examined in cultures whereby 10 particles were delivered to each of 1, 5 and 25 sites (Fig. 5A and B). As presented in Fig. 5C and D, phosphorylated p53 increased at 6 h postirradiation 1.9–2.0-fold and 1.6–1.7-fold over the control level responding to carbon and neon microirradiation, respectively. Such a rise at 6 h postirradiation arose regardless of the target numbers (1, 5 or 25 sites), namely, regardless of the fraction of targeted cells (0.00026, 0.0013 or 0.0066%): the difference between 1, 5 and 25 sites was statistically insignificant. The levels of phosphorylated p53 at 6 h

Fig. 3. (A) Apoptosis induction in broadbeam-irradiated cultures. Cultures were fixed at 12, 24, 36, 48 and 72 h after exposure to D10 (doses listed in Table 2). (B) Apoptosis induction in microirradiated cultures. Cultures were fixed at 12, 24 and 48 h after exposure of a single site to 10 particles. Afterward, fixed cells underwent the TUNEL assay. The data are shown as the means and standard deviations for three or four independent experiments, where 1000 or more cells were analyzed for each specimen. *0.01 ≤ p < 0.05; **p < 0.01 compared with sham-irradiated controls (open triangles) at each time point. ns, not significant.

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Fig. 4. p53 phosphorylation in broadbeam-irradiated cultures. Cultures exposed to carbon (A) or neon ions (B) were harvested at the specified times, followed by western blot analysis. The doses tested are listed in Table 2. Experiments were repeated three times with similar results, and representative blots are shown. ␣-Tubulin served as an internal control. Corresponding graphs in (C) and (D) show the alterations in the expression of serine 15-phosphorylated p53 in blots (A) and (B), respectively. Band intensity was densitometrically measured, and the relative expression of phosphorylated p53 was calculated as the intensity of each band divided by that of sham-irradiated control at 2 h postirradiation (leftmost lane in each blot) after normalization with ␣-tubulin. Open and filled columns denote the expression levels at 2 and 6 h postirradiation, respectively. The data are presented as the means and standard deviations of three independent experiments. *0.01 ≤ p < 0.05; **p < 0.01 compared to corresponding sham-irradiated controls. ns, nonsignificant between 2 and 6 h postirradiation.

postirradiation in microirradiated cultures were higher than those at 2 h postirradiation in microirradiated cultures and at 6 h postirradiation in controls (p < 0.05). In contrast, the difference versus controls at 2 h postirradiation did not reach statistical significance whilst there was a slight trend toward increased phosphorylation. As was the case for apoptosis induction (Fig. 3), the time course of p53 phosphorylation in bystander cells was different from that in irradiated cells. 4. Discussion Here we have investigated heavy ion-induced biological effects arising in irradiated and bystander fibroblasts, using broadbeams and precision microbeams. Exposure of only a very small fraction of cells reduced the survival (Fig. 2B), induced apoptosis (Fig. 3B) and increased serine 15-phosphorylated p53 proteins (Fig. 5). These measurable bystander responses may be attributable to a cascade of feed-forward signal amplification events,

such that signal(s) from irradiated cells are transduced into primary bystander cells, which in turn produce signals further transmissible to their secondary bystander cells one after another [21]. As regards the data in Fig. 5, we found delayed p53 phosphorylation in bystander cells. p53 phosphorylation on serine 15 plays pivotal roles in p53 stabilization and activation, and occurs most rapidly among multiple phosphorylation sites in response to DNA double-strand breaks (DSBs) and other stimuli [40]. Evidence is increasing for bystander-induced micronuclei [14–17] and chromosome-type breaks [5], both of which primarily arise from DSBs. Noteworthy is that bystander cells show delayed foci formation of phosphorylated histone H2AX (␥H2AX) [19] which forms discrete foci at the site of each nascent DSB [41,42]. These findings indicate that bystander-induced delayed p53 phosphorylation might be due to delayed DSB induction. Of further interest is the recent observation in regard to the temporal kinetics of foci formation of p53 binding

N. Hamada et al. / Mutation Research 639 (2008) 35–44

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Fig. 5. p53 phosphorylation in microirradiated cultures. Each of 1, 5 and 25 sites was targeted with 10 particles of carbon (A) or neon ions (B). Subsequently, cultures kept at 37 ◦ C for the stated period were processed for western blotting. Three or four independent analyses showed a consistent pattern of data, and representative blots are shown. ␣-Tubulin was used as an internal control. Corresponding graphs in (C) and (D) demonstrate the changes in the level of serine 15-phosphorylated p53 in blots (A) and (B), respectively. After normalization with ␣-tubulin, the relative expression of phosphorylated p53 was calculated as the intensity of each band divided by that of sham-irradiated control at 2 h postirradiation (leftmost lane in each blot). Open and solid columns represent the levels at 2 and 6 h postirradiation, respectively. The data shown are the means and standard deviations from three or four independent experiments. *0.01 ≤ p < 0.05; **p < 0.01 compared to corresponding sham-irradiated controls. # 0.01 ≤ p < 0.05; ## p < 0.01 compared between 2 and 6 h postirradiation. ns, nonsignificant.

protein 1 [18] which forms foci at DSB sites [43], in irradiated and bystander cells: whilst nuclear microirradiation increased the focal incidence comparably at 1 and 3 h postirradiation, cytoplasmic microirradiation did at 3 h postirradiation but not 1 h postirradiation. This is reminiscent of our data for the time course of p53 phosphorylation in bystander cells, such that its increase was subtle and insignificant at 2 h postirradiation, but more pronounced significantly at 6 h postirradiation (Fig. 5). It is hence conceivable that contribution of cytoplasmic irradiation was greater than that of nuclear irradiation in this study, where nuclei or cytoplasms were randomly exposed (see Section 2.3). On the other hand, little change in the levels of phosphorylated p53 was observed at 2 and 6 h after broadfield exposure (Fig. 4), even to 0.01 D10 of neon ions whereby about 63, 33 and 4% of nonirradiated, cytoplasm-irradiated, and both nucleusand cytoplasm-irradiated cells should coexist in the population, respectively. This raises the possibility that the influence of particle traversal through cell nuclei may

precede that through cytoplasms in such situations. However, it has also been reported that bystander-induced ␥H2AX foci formation occurs soon after irradiation [44,45]. Therefore, to test whether the different contribution of nuclear versus cytoplasmic damage is attributable to the different time course of p53 phosphorylation observed in irradiated and bystander cells, further experiments of ␥H2AX immunostaining in cytoplasm- and nucleus-irradiated cells, and their bystander cells by selectively targeting with more precise microbeams (e.g., the focused heavy ion microbeam system) are needed. It is known that normal human fibroblasts are susceptible to UV-induced apoptosis, but are resistant to ionizing radiation-induced apoptosis after irradiation with photons as well as heavy ions [46–48]. We also observed the low apoptotic indices in heavy-ion irradiated fibroblasts (Fig. 3A): for comparison, our previous work has shown the apoptotic index of 30% at 72 h after exposure of lymphocytes to D10 of carbon ions

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[49]. Nevertheless, we found bystander-induced apoptosis in human fibroblasts consistent with the earlier observation [50], and a difference in the timescale of apoptotic response between irradiated and bystander cells (Fig. 3B). It has been documented that whereas irradiated cells undergo both transient and permanent arrests in G1 phase in a p53 and p21Waf1 -dependent manner, transient but not permanent G1 arrest occurs in bystander cells [13], suggesting that most of bystander-induced DSBs may be reparable. This implies that such an event might be causative of temporally distinct response in irradiated and bystander cells. To scrutinize these molecular underpinnings, microarray gene expression analysis is ongoing. We also showed bystander-induced reductions in the survival (Fig. 2B). Cells were reseeded within 1 h postirradiation in these experiments, unlike the case for the data in Figs. 3–5 where cells were held in confluence throughout the time course. It is thence suggested that signals transmitted within 1 h postirradiation from irradiated to bystander cells initiated inactivation of clonogenic potential. Loss of clonogenicity should reflect the summed response of a plated parental cell (e.g., apoptosis and senescence-like growth arrest [51]) and its progeny (e.g., delayed reproductive death or lethal mutations [52]). In this light, mounting evidence now exists that the progeny of bystander cells express delayed phenotypes [10–12], and that the neighbors of the progeny of surviving cells show bystander responses [53,54]. These findings suggest that persistent spatiotemporal propagation of signals initially transmitted from irradiated to bystander cells may perpetuate the effects in their progeny over time. Accordingly, it seems very likely that bystander-induced reductions in the survival result from death of plated bystander cells (e.g., via apoptosis as presented in Fig. 3B) and delayed death of their descendants. Taken together, we recently reported clonal morphotypic heterogeneity and delayed loss of clonogenicity in colonies derived from carbon-irradiated cells [28,29]. Further experiments to test whether the same holds true for colonies arising from bystander cells are under way. Despite a series of studies on the bystander effect, very little is known concerning its LET dependency. Carbon and neon microirradiation similarly induced twofold increments in the apoptotic incidence (Fig. 3B), and around 10% reductions in the survival (Fig. 2B) so did for X-rays [6]. Such LET independence was in agreement with that for cell cycle-related bystander response [7] and bystander-induced chromosome aberrations [8]. However, a slight tendency toward less pronounced p53 phosphorylation by neon microirradiation than carbon

one (Fig. 5) is suggestive of potential LET dependence, as was for bystander-induced micronucleation [9]. Moreover, with respect to X-ray- and neon-induced bystander effect for chromosome aberrations, our recent work has shown the difference in the types of aberrations, but very little in the total aberration yields [8]. Taken together, bystander-induced reductions in the survival of normal human fibroblasts have been reported after exposure to helium ion broadbeam [55], but not after exposure to helium ion microbeam [56]. Altogether, these findings imply that LET dependency of bystander effect may vary between the endpoints, and thence needs to be more carefully examined under the comparable experimental conditions (e.g., consistent endpoint, dose, irradiation system, and cell types). In conclusion, we have demonstrated the bystander effect of heavy ions for inactivation of clonogenic potential, and the temporal difference in the response of irradiated and bystander cells to heavy ions in terms of apoptosis induction and p53 phosphorylation. These induced bystander responses might be a defensive mechanism that would avert or minimize further expansion of aberrant cells, thus maintaining the genome integrity and cellular homeostasis. A deeper understanding of the early- and late-arising effects of energetic heavy ions, to which normal tissues are exposed during manned space missions [24] or during cancer therapy [57], may reduce uncertainties in assessing risks to human health. Biological effectiveness of heavy ions has been shown to differ with ion species even at comparable LET [25,26]. An elucidation of LET and ion species dependence of heavy ion-induced biological effects and its underlying mechanisms encourages further investigation. Acknowledgements The authors are grateful to Drs. Takehiko Kakizaki, Yuichiro Yokota, Yoshihiro Hase, and the staff at the TIARA of JAEA for help with the heavy-ion irradiation. This work was supported by a Grant-in-Aid for the 21st Century COE Program for Biomedical Research Using Accelerator Technology from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by Nuclear Research of the MEXT based on the screening and counselling by the Atomic Energy Commission of Japan. References [1] H. Nagasawa, J.B. Little, Induction of sister chromatid exchanges by extremely low doses of ␣-particles, Cancer Res. 52 (1992) 6394–6396.

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