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Cell Cycle-Related Bystander Responses are not Increased with LET after Heavy-Ion IrradiationAuthor(s): C. Fournier, D. Becker, M. Winter, P. Barberet, M. Heiß, B. Fischer, J. Topsch, and G.Taucher-ScholzSource: Radiation Research, 167(2):194-206. 2007.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR0760.1URL: http://www.bioone.org/doi/full/10.1667/RR0760.1

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RADIATION RESEARCH 167, 194–206 (2007)0033-7587/07 $15.00� 2007 by Radiation Research Society.All rights of reproduction in any form reserved.

Cell Cycle-Related Bystander Responses are not Increased with LETafter Heavy-Ion Irradiation

C. Fournier,a,1 D. Becker,a M. Winter,a P. Barberet,b M. Heiß,b B. Fischer,b J. Topscha and G. Taucher-Scholza

a Department of Biophysics and b Department of Materials Research, Gesellschaft fur Schwerionenforschung, 64291 Darmstadt, Germany

Fournier, C., Becker, D., Winter, M., Barberet, P., Heiß,M., Fischer, B., Topsch, J. and Taucher-Scholz, G. Cell Cycle-Related Bystander Responses are not Increased with LET af-ter Heavy-Ion Irradiation. Radiat. Res. 167, 194–206 (2007).

Evidence has accumulated that irradiated cells affect theirunirradiated neighbors, so that they in turn display cellularresponses typically associated with direct radiation exposure.These responses are generally known as bystander effects. Inthis study, cell cycle-related bystander responses were inves-tigated in three strains of human fibroblasts after exposure todensely ionizing radiation. Varying the linear energy transfer(LET) from 11 to 15,000 keV �m�1 allowed a study of theimpact of the complexity of DNA damage in the inducing cellson the responses of bystander cells. Using both broad-beamand microbeam irradiation, transient bystander responseswere obtained for the induction of CDKN1A (p21). The latterwas also observed when the transmission of bystander signalswas limited to soluble factors. Targeted irradiation of singlecells in confluent cell monolayers revealed no correlation be-tween the amount of CDKN1A protein in the bystander cellsand the radial distance to the targeted cells. In line with theinduction of CDKN1A in bystander cells after irradiation withdifferent LETs, a transient delay in the first G1 phase afterirradiation of G0/G1 cells was observed. However, theCDKN1A induction revealed no significant effect on prema-ture terminal differentiation considered to underlie fibrosis inirradiated tissue. Thus the unchanged differentiation patternin bystander cells does not indicate pronounced, long-lastingeffects. � 2007 by Radiation Research Society

INTRODUCTION

Evidence has accumulated that unirradiated cells locatedadjacent to or in the same environment as irradiated cellscan exhibit cellular responses typically associated with di-rect radiation exposure. These so-called bystander respons-es have been under investigation for more than a decade.First, in vitro studies reported elevated frequencies of sisterchromatid exchanges (SCEs) in rodent cells exposed to lowfluences of � particles such that no more than 1% of thecell nuclei were expected to be hit (1). These observations

1 Address for correspondence: Planckstraße 1, D-64291 Darmstadt,Germany; e-mail: c.fournier@gsi.de.

have been confirmed in human cells (2) and challenged thefundamental paradigm of a direct nuclear exposure as theonly basis of radiation-induced cellular responses. The by-stander effects described include alterations in the cell cycleregulation, increases in SCEs and chromosomal aberrations,cell death, induction of gene mutation, and chromosomalinstability (3–5). Untargeted effects in vivo were impliedby the observation of ‘‘abscopal effects’’ in nonirradiatedsites after localized radiotherapy, showing that radiation-induced secreted factors can modulate effects outside theradiation field (5, 6).

The existence of extranuclear targets during the exposureof cells to radiation has been discussed mainly in terms ofthe potential impact on risk estimation (7, 8), but possibletherapeutic implications also need to be considered (9). Theincreasing application of heavy ions in radiotherapy pro-vides a strong motivation to elucidate the occurrence andthe mechanistic basis of bystander effects induced by ionswith higher linear energy transfer (LET), e.g. carbon ions.In contrast to the exponential decrease of the dose depositedby penetrating photons, the dose–depth profile of chargedparticles is inverted, resulting in maximum sparing of thehealthy tissue and the deposition of high doses within thetumor volume. In addition, the biological effectiveness isincreased in the tumor region owing to the very high ion-izing density of the stopping particles. In this context it isimportant to learn whether the exposure of tumor cells orsingle cells belonging to the normal tissue at the borderlineof a tumor could provoke persisting effects in the surround-ing tissue. It is noteworthy that the 5-year follow-up of thepatients with head and neck cancer who were treated at thecarbon-ion facility at GSI (Darmstadt, Germany) revealeda moderate acute and late toxicity with respect to the de-livered tumor dose (10, 11). In connection with plannedexpansions of the application of particle therapy, an under-standing of bystander effects on a molecular and cellularbasis will be of benefit, especially in understanding long-term effects. The study presented here is therefore focusedon the changes related to cell cycle progression of bystand-er cells and on their potential impact on the premature dif-ferentiation of fibroblasts. Radiation-induced premature dif-ferentiation is preceded by a persisting growth arrest underthe control of cell cycle-regulating proteins (12) and is be-

195CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

TABLE 1Energies and Respective LETs for the Ions Studied in Broad-Beam Experiments

IonEnergy on target(MeV nucleon�1)

LET(keV �m�1)

Fluence(p cm�2) Dose (Gy)

Mean number ofparticle traversals

per nucleus

Amount ofcells hit atleast once

Dose pertraversal (Gy)

Carbon 397 11 2.84 � 107–2.84 � 108 0.5–5 66.4–664 � 99% 0.00759.8 170 5 � 103–2 � 107 0.0014–5.44 0.01–39.2 1%–99% 0.13

Uranium 4.2 15,000 5 � 103–2 � 107 0.12–480 0.01–39.2 1%–98% 12

lieved to be one of the cellular events leading to fibrosis(13), one of the possible late effects in healthy tissue afterradiotherapy.

Generally, the bystander effects reported vary with thecell systems used and the biological end points studied (7).Multiple, still not fully elucidated signal transduction path-ways, including gap junction-mediated intercellular com-munication (GJIC), oxidative metabolism and secretion ofsoluble factors, are thought to be involved (5). Earlier stud-ies on the modulation of the expression of cell cycle-relatedproteins, e.g. TP53 and CDKN1A, in bystander cells afterexposure to low doses of � particles indicated changes inthe expression level of the proteins, but depending on theexperimental conditions, opposite effects have also beenobtained (14). Experiments allowing the transmission ofsignals via gap junctions, as in the case of broad-beam ex-posure, showed enhanced expression of cell cycle inhibitorsoccurring simultaneously with a transient cell cycle delay(15). In contrast, studies using a medium transfer techniqueto restrict the transmission of signals to soluble factors re-ported decreased expression of cell cycle inhibitors togetherwith enhanced proliferation activity in bystander lung fi-broblasts (16). However, since the transmission of signalsby soluble factors cannot be excluded using broad-beamexposure, the question about the underlying mechanismsremains open. More recently, for low-LET radiation (Xrays) and under conditions allowing signal transmissiononly via soluble factors, an enhanced level of the cell cycleinhibitor CDKN1A was observed in nonirradiated normalskin fibroblasts (17). Either the fact that different fibroblaststrains were used or differences in the radiation qualitycould account for this discrepancy. Up to now, no system-atic studies have been performed addressing the influenceof radiation quality on the possible mechanisms underlyingbystander effects. The newly developed heavy-ion micro-probe at GSI (18) allowed us to use this tool in parallelwith broad-beam exposure for selected experiments.

For broad-beam as well as microbeam exposure, we wereable to vary the particle’s LET and thus the ionizing den-sity. Increasing the ionizing density of the radiation leadsto increased amounts of unrepaired or misrepaired DNAdamage, which is generally accepted to be a consequenceof the enhanced complexity of the DNA lesions (19–21).The influence of impaired repair of DNA lesions on theinduction of bystander effects has been highlighted in me-dium transfer experiments in which cell strains with differ-

ent capacities to repair DNA damage were irradiated (22).The authors observed that repair-deficient human cells in-duced moderate to pronounced effects on cloning efficiencyin autologous or reporter bystander cells, whereas their nor-mal ‘‘repair-proficient’’ counterparts induced less severe orno bystander effects. The impact of the complexity of theDNA damage in the directly exposed cells on the bystanderresponses has been poorly investigated up to now. The firstideas on differential effects of the radiation quality werebased on experiments performed in human fibroblasts thatshowed a less pronounced effect for ultrasoft X rays thanfor helium ions (8). Here heavy ions were used as a toolto investigate the influence of an increasing LET on theinduction of responses in non-targeted bystander cells. Us-ing the single traversals of various particle species throughthe cells, we performed experiments on multiple biologicalend points over a range of LET values from 11 to 15,000keV �m�1. The main focus of our bystander study was toassess the changes in cell cycle-related proteins in fibro-blasts. The investigations of short-term changes in the cellcycle distribution were complemented by studies on pos-sible long-term consequences in bystander cells.

MATERIALS AND METHODS

Cell Cultures

All normal human fibroblasts were cultured under standard conditions(95% air/5% CO2, 37�C) in Eagle’s MEM (Bio-Whittaker) supplementedfor all cell strains with 10% or 20% fetal calf serum (Biochrom) forsubculturing and experiments, respectively (23). Cells of the foreskin fi-broblast cell strain AG1522C (Coriell Institute, Camden, NJ) at 20–25population doublings were used for all experiments. In addition, for theanalysis of protein expression, cells of two skin fibroblast cell strains,GM 5758 (Coriell Institute) and HSF-1 (kindly provided by H. P. Ro-demann, Universitat Tubingen, Germany), were used, both at a low pas-sage number. Cells were checked regularly and were found to be myco-plasma-free.

Irradiation

The cells were exposed to X rays (250 kV, 16 mA) or heavy ions.Carbon ions (397 MeV nucleon�1 on target) were produced at the SIS,and low-energy carbon and uranium ions (9.8 and 4.2 MeV nucleon�1 ontarget, respectively) were obtained at the UNILAC facility at GSI, Darm-stadt, Germany. The energies and respective LETs for the heavy ionsused in our broad-beam experiments are summarized in Table 1. Unlessstated otherwise, for experiments performed with carbon ions, the low-energy (9.8 MeV nucleon�1 on target) ions were used. For SIS and UN-ILAC, dosimetry relied on secondary electron transmission counting us-ing CR39 nuclear track detectors as reference.

196 FOURNIER ET AL.

For irradiation, confluent monolayers were used in which 85–90% ofthe cells were in the G0/G1 phase as determined by flow cytometry. Thisallowed us to control for cell cycle-dependent variations in the expressionlevels of TP53 and CDKN1A and for the transmission of bystander sig-nals by gap junctions. In all types of irradiation experiments, comparableconditions were maintained: The supernatant of the cells was replaced byfresh, serum-free medium before irradiation. This was necessary to avoida stimulus for cell cycle progression due to the special conditions at theUNILAC facility requiring submersion of the cells in a large amount ofmedium during the irradiation procedure [details described in ref. (23)].Special care was taken for the controls by using mock-irradiated samplesat each sampling time.

For broad-beam experiments with low-energy carbon and uraniumions, the fluences were chosen such that on average 1, 3, 10 or 98% ofthe cell nuclei would actually be traversed by a particle. The calculationof fluences was based on the stochastic dispersion of the number of par-ticle traversals per cell nucleus according to a Poisson distribution andthe mean size of the cell nuclei, which was determined to be about 234� 91 �m2 by quantitative analysis of 11,000 cell nuclei after nuclearstaining (Hoechst) as described below (quantification of immunofluores-cence intensities).

Coculture Experiments

To study whether secreted factors were responsible for effects in by-stander cells, confluent irradiated cells (referred to as donor cells) andconfluent nonirradiated cells (referred to as acceptor cells) were cocul-tured using a commercially available system (Falcon�, Becton DickinsonLabware, Franklin Lakes, NJ). The system comprises six-well cell cultureplates and transwell culture insert dishes with a permeable membrane(pore size 1 �m, density 1.6 � 106 pores cm�2) that restricts the inter-cellular communication between donor and acceptor cells to soluble fac-tors. Confluent cells in cell culture inserts were irradiated with X rays orcarbon ions, and immediately after irradiation the inserts were placed intothe wells of a six-well plate containing confluent cells. Both irradiatedand nonirradiated cells were cocultured 5 h after exposure under standardconditions.

Microbeam Irradiation

Targeted irradiation using carbon and argon ions (4.8 MeV nucleon�1

on target; LET 250 keV �m�1 and 1950 keV �m�1, respectively) wasperformed using the microbeam facility at GSI, Darmstadt, Germany[technical details are in ref. (18)]. The focused beam allows targetingwith an accuracy of 1.3 �m. For the targeted exposure, the area of theradiation chamber (d 8 mm) was divided within a virtual inner circle(d 5 mm) into 60 fields at a distance of 500 �m, each field containingabout 30 cells. The cell nuclei were stained with 200 nM Hoechst 33342for 1 h prior to irradiation and exposed to UV light to allow microscopicrecognition. Out of the cells in each field, one or six selected cells, cor-responding to approximately 0.5 or 3% of the cells of the confluent mono-layer, were mock-irradiated or irradiated. All the controls of the micro-beam experiments were subjected to exactly the same procedure as thecells belonging to the fields where single cells were irradiated. Generally,in one half of the fields, the nuclei of few cells were irradiated, deliveringfive ions per nucleus on a cross pattern (1.3 and 8 Gy per irradiatednucleus for carbon and argon ions, respectively). A typical complete ir-radiation procedure took about 10 min in total including the cell detectionby UV microscopy. Three hours after exposure and incubation, immu-nofluorescence staining was performed as described below. The irradiatedcells were revisited based on reference coordinates and unambiguouslyidentified due to the formation of -H2AX foci.

Dye Transfer Assay

The functionality of gap junctions in the fibroblast cell strains usedwas tested by dye transfer technique. Two fluorescent dyes [Vybrandt DiI

D-282 (DiI) and CalceinAM (CAM), Sigma], both of which can crossthe cell membrane irreversibly by diffusion, were used simultaneously.Only CAM dye is able to pass through gap junctions into adjacent cells,allowing the discrimination between staining and gap junction-mediateddye transfer. Confluent cells were loaded with DiI (50 mM) and CAM (5mM) for 30 min, washed with PBS, and exposed to radiation in serum-free cell culture medium. After irradiation, the cells were trypsinized andadded to a nearly confluent, unstained cell monolayer. After attachment,the (mock-) irradiated cells constituted approximately 3% of the totalnumber of cells in the monolayer. Since the assessment of the dye transferhas to be carried out in living cells, it was evaluated by visual inspectionof the cells.

Protein Quantification (CDKN1A) by Flow Cytometry

Confluent cell monolayers were harvested by trypsinization. After re-peated washing, the cells were fixed with 3.7% paraformaldehyde, per-meabilized with Triton X-100 (0.5% v/v in PBS), and blocked (0.5%w/v BSA in PBS). The cells were incubated with anti-CDKN1A (clone70, monoclonal mouse, Transduction Laboratories) and secondary anti-body (Alexa Fluor 488 conjugated anti-mouse). Counterstaining of thecell nuclei was performed with 1 �g ml�1 DAPI (in PBS). A PAS IIIflow cytometer (Partec, Germany) equipped with an argon laser operatingat 488 nm and a UV lamp was used for flow cytometry. Emission ofAlexa 488 fluorochrom was measured in the range of 500–560 nm, andemission of DAPI was detected at 420–480 nm. Only cells with a DNAcontent corresponding to G1, S and G2 amounts were included in theanalysis.

Flow Cytometry Analysis of Cell Cycle Distribution(Hoechst-BrdU Quenching)

Confluent cells were irradiated and reseeded 3 or 24 h after irradiationat low density (4,000 cells cm�2) in cell culture medium containing 3 �gBrdU ml�1. The cells were harvested from 18 h up to 96 h later, fixedand stained in 70% ethanol (in PBS). The cellular RNA was digestedwith 0.5 mg ml�1 ribonuclease (90 U mg�1, Roth, Germany) for 60 minat 37�C. Staining was performed in an ethanol/PBS solution (1:4) con-taining 16 �g ml�1 Hoechst 33258 and 1 mg ml�1 ethidium bromide. Forflow cytometry, a PAS III flow cytometer (Partec, Germany) was usedas described above. Hoechst and ethidium bromide fluorescence weremeasured at 420–480 nm and 620–750 nm, respectively, and the Hoechstand ethidium bromide fluorescence intensities of each measured cell wererecorded in two-dimensional histograms. The fluorescence of Hoechst isquenched by incorporation of BrdU and thus reflects the number of celldivisions since the beginning of the labeling period (24). The two-di-mensional histograms of the fluorescence intensities were analyzed witha software package (Multicycle, Phoenix Flow Systems Inc., San Diego,CA) discriminating the first, the second and the third cell cycle afterreseeding and cells with DNA content of G1, S and G2 phase.

Protein Quantification by Immunofluorescence

After targeted irradiation, information about the exact localization ofthe irradiated and the bystander cells had to be preserved. Therefore,protein quantification was performed by immunofluorescence staining ofthe adherent cells. Three or 24 h after exposure, the cells were fixed with4% paraformaldehyde, permeabilized with Triton X-100 (0.5% v/v inPBS), and blocked (0.5% w/v BSA in PBS). The cells were incubatedwith anti-CDKN1A (clone 70, monoclonal mouse, Transduction Labo-ratories), anti--H2AX (anti--H2AX Ser-139, rabbit polyclonal, UpstateUSA), anti-53BP1 (anti-rabbit-human 53BPI-IgG, Alexis), and secondaryantibody (Alexa Fluor 488 conjugated anti-mouse, Alexa Fluor 568 con-jugated anti-rabbit). Counterstaining of the cell nuclei was performedwith 0.2 �g ml�1 DAPI (in PBS) and stored in PBS at 4�C. Images fromat least 50 fields were acquired using a fluorescence microscope (DM,IRBE, Leica, Wetzlar, Germany) for each sample at identical exposure

197CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

and gain settings. The images were processed for quantification usingImageJ (NIH, Bethesda, MD). First, in DAPI images, a mask of the areasof individual nuclei was created by sequential steps of normalization,background subtraction, smoothing, edge detection, convolution andthresholding. This mask was used to define the fluorescence images forprotein analysis. The fluorescence intensities of the proteins were cor-rected for the background and inhomogeneous illumination of the fieldof view. The protein intensities were integrated for each nucleus.

Protein Quantification by Western Blotting and Immunodetection

Western blot analysis with subsequent immunodetection was performedto quantify the intracellular levels of TP53 and CDKN1A proteins. Irra-diated and unirradiated cells were harvested at different times after ex-posure. Protein lysates were prepared and SDS gel electrophoresis, West-ern blotting and immunodetection were performed as described elsewhere(23). For immunodetection, the membranes were probed with anti-TP53(Ab-6, monoclonal mouse, Oncogene), anti-CDKN1A (clone 70, mono-clonal mouse, Transduction Laboratories), and anti-�-tubulin (monoclonalmouse, Sigma) as a loading control. Secondary antibodies conjugatedwith horseradish peroxidase and the Enhanced Chemiluminescence De-tection System (ECL plus, Amersham Pharmacia Biotech) were used todetect the proteins. The protein levels were inferred by densitometricevaluation of film signals compensating for nonlinear film responses ac-cording to a procedure described elsewhere (25). The ratios of the valuesfor the CDKN1A or TP53 protein and �-tubulin for the same samplewere calculated. The ratios of irradiated samples were normalized to theratio of the corresponding control sample for each time.

Analysis of Differentiation Pattern by Morphological Features

Confluent cells were irradiated and reseeded 3 or 24 h after irradiationat low density (10 cells cm�2). After 14 days, the differentiation patternof the populations was analyzed based on morphological features as de-scribed elsewhere (26).

Statistical Analysis

Each sample of one Western blotting experiment was analyzed by re-peated loading (at least three times). The differentiation pattern was de-termined in a double-blind analysis three times independently. The num-bers of experiments and samples per data point are indicated in eachfigure. The data points of parallel experiments were averaged and thestandard error of the mean (SEM) was calculated. Significance tests (ttest) according to Baily (27) were performed for specific data points.

RESULTS

The aim of the present study was the assessment of cellcycle-related changes in bystander cells after exposure toheavy ions of different ionization densities. Broad-beam ir-radiation with low and high fluences was generally appliedto enable the analysis of a high number of cells. For themeasurement of the spatial distribution of intracellular lev-els of CDKN1A protein, targeted irradiation was performedusing the GSI microprobe. In addition, a coculture tech-nique was used to investigate the medium-mediated trans-mission of bystander signals.

Intercellular Communication in Bystander Cells isFunctional after Heavy-Ion Irradiation

The fibroblast cell strains used were first characterizedregarding a functional intercellular communication via gap

junctions after irradiation. Evidence for the latter was givenby fluorescent dye transfer after reseeding of irradiated,stained cells on a nearly confluent monolayer of unirradi-ated, unstained fibroblasts of the same or a different cellstrain. During visual inspection of the cells in 45-min in-tervals up to 5 h after exposure to X rays (0.5 and 5 Gy)and carbon ions (2 � 106 p cm�2), a rapid transfer of thedye (CAM) to the unstained cells was observed (notshown). A maximum transfer in terms of signal intensityin the adjacent cells and number of cells participating inthe expansion of the signal (roughly two-thirds of thestained cells) was observed 3 h after reseeding. It is note-worthy that the transfer was observed in all cell strains andoccurred independently of whether the stained cells wereirradiated or mock-irradiated.

Increasing Ionizing Density does not Result in IncreasedExpression of Cell Cycle-Related Proteins inBystander Cells

To assess a potential bystander effect with respect to theexpression of the cell cycle-regulating protein CDKN1Aand its transcription factor TP53, confluent monolayers ofthree fibroblast cell strains were exposed to a broad beamof carbon ions (170 keV �m�1). The amounts of cellularprotein were measured 3 to 24 h later using the Westernblot technique. A representative immunoblot is shown inFig. 1a. At low fluences when only 3 or 10% of the cellswere hit, the results revealed no significant changes in theamount of TP53 in AG1522 cells 3 h after exposure. Incontrast, an up to twofold increase in the level of CDKN1Aprotein was observed in samples in which only 1 to 10%of the cell nuclei were hit, thus indicating a response inbystander cells. The corresponding mean values for the ex-pression of CDKN1A protein are shown in Fig. 1b. Theamount of cellular protein after 10% of the cells were hitwas significantly different from controls (P 0.034) andwas higher than the amount expected for a linear dose re-sponse. The increased levels of CDKN1A protein in thenontargeted cells were still detectable up to 6 h later, but24 h after exposure they were always decreased to controllevels (not shown).

The induction of CDKN1A in bystander cells observedin AG1522 cells could be confirmed in cells of two otherfibroblast cell strains originating from human skin(GM5758 and HSF-1). In Fig. 1c the results for GM5758cells are shown. At very low fluences, when only 1 or 3%of the cells were hit, a twofold induction of CDKN1A pro-tein was detected, clearly exceeding an assumed linear doseresponse (P 0.02 and P 0.003, respectively). In con-trast to AG1522 cells, the maximum increase in CDKN1Aprotein was observed in these cells at 6 h rather than at 3h after exposure (not shown). Whether the lower level ofCDKN1A protein after 10% of the cell nuclei had been hitis significant cannot be deduced since only one experimentwas performed at this fluence.

198 FOURNIER ET AL.

←

FIG. 1. Expression of TP53 and CDKN1A after exposure to carbonions. Panel a: Representative Western blots showing the levels of TP53and CDKN1A protein in confluent monolayers of AG1522 cells 3 h afterexposure. �-Tubulin was used as a control for equal protein loading.Panels b and c: Data obtained from repeated Western blot analysis tostudy the expression of CDKN1A in AG1522 cells (panel b) and GMcells (panel c) 3 and 6h after exposure to carbon ions, respectively. Errorbars are �1 SEM with n 5 (AG1522 cells) and n 3 (GM 5758cells), except for the 10% hit cell data (GM 5758 cells). *Results aresignificantly different from control cells using Student’s t test (P � 0.05).The theoretical linear dose response is shown (dashed line) with the SEM(dashed-dotted lines). Panel d: Flow cytometry histograms depicting thedistribution of the amount of CDKN1A in AG1522 cells for the controls,the bystander cells, and cells exposed to low and high fluences of carbonions (one experiment). Cells were harvested 3 h after exposure. The re-spective lines for each population were drawn to guide the eyes and aresmoothed curves obtained by averaging 20 adjacent data points for eachof the 180 intensity values. The raw data points are shown for the controlsand the population where 3% of the cells were irradiated.

An induction of CDKN1A in bystander cells after car-bon-ion irradiation was also apparent when the amounts ofintracellular protein were measured by flow cytometry (Fig.1d). Although an increase in the mean expression level ofCDKN1A protein was observed when 3% of the cells wereactually traversed by a carbon-ion track, the intensity dis-tribution patterns of the protein were not significantlychanged in the bystander cells compared to the control pop-ulations. In addition, this method of analysis revealed abroad distribution of the intracellular protein levels in bothcontrol and irradiated AG1522 cell populations. Micro-scopic analysis showed that the heterogeneity of the cellularlevels of CDKN1A protein in GM 5758 cells was less pro-nounced than in AG1522 cells (not shown).

It has been shown that the repair capacity of donor cellstrains plays a role in the bystander response (22). In thepresent study, we asked whether the amount of unrepairableDNA lesions due to differences in damage complexity inthe donor cells has an impact on bystander responses. Anincreasing amount of nonrepairable DNA damage was in-duced by taking advantage of the increase in the ionizingdensity of heavy ions (28). To investigate the bystanderresponse, irradiation was performed such that only a fewcells of a monolayer (1, 3 and 10%) were hit by singleuranium ions depositing a 100-fold elevated dose comparedto carbon ions. The cells were assessed for the expressionof CDKN1A and TP53 as was done for carbon ions, butdifferent times (3 and 5 h after irradiation) were chosenaccording to the shift in time of the maximum response ofcells directly exposed to the heavier ions (23). The resultsfrom one representative experiment are shown in Fig. 2afor 3 h after exposure. Similarly elevated levels ofCDKN1A protein were also observed 5 h after exposure(not shown). For clarity, the corresponding mean values oftwo independent accelerator experiments for each timewere therefore pooled and are shown in Fig. 2b. Theamount of CDKN1A protein was enhanced compared to

199CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

FIG. 2. Expression of TP53 and CDKN1A after exposure to uraniumions. Panel a: Representative Western blot showing the levels of TP53and CDKN1A protein in AG1522 cells 3 h after exposure to uraniumions. �-Tubulin was used as a control for equal protein loading. Panel b:Data from repeated Western blot analyses to study the expression ofCDKN1A in AG1522 cells 3 and 5 h after exposure to uranium ions.The data obtained for both times are pooled. Error bars are �1 SEM withn 4 (two independent experiments). The theoretical linear dose re-sponse (dashed line) is shown; the dashed-dotted lines show SEM.

control cells but did not exceed a twofold increase. In thesame experiment, no significant changes were detected forthe intracellular amount of TP53 protein (Fig. 2a). As ex-pected from previous experiments (23), the levels of bothproteins were clearly increased when all cells were irradi-ated. Taken together, inducing DNA damage of substan-tially higher complexity in only a few cells leads to a pos-itive CDKN1A response in bystander cells, but the levelsdo not exceed those obtained after exposure to carbon ions.

To gain information about the spatial distribution of by-stander cells with increased levels of CDKN1A proteinwith respect to the irradiated cells, targeted irradiation ofAG1522 cells was carried out with carbon ions and argonions of higher ionization density using a microbeam probe(18). The targeted exposure comprised 60 fields at a dis-tance of 500 �m from each other, each field containingabout 30 cells. Out of the cells in each field, either one orsix cells were irradiated (corresponding to 0.5 or 3% of thecells) or mock-irradiated. The single targeted cells were tra-versed with five ions distributed on a cross pattern (fiveions per nucleus corresponding to 1.3 Gy and 8 Gy forcarbon and argon ions, respectively). Three hours after ex-

posure, the immunofluorescence staining was performed(Fig. 3a, shown for carbon ions). The irradiated cells wererevisited based on reference coordinates and unambiguous-ly identified due to the formation of -H2AX or 53BP1foci. For the radial distance analysis of the cellular levelsof CDKN1A protein, fields with single irradiated cells wereused. Five virtual concentric rings with an increasing radialdistance up to 200 �m were drawn around the irradiatedcells. The bystander cells were analyzed separately for eachvirtual ring. Up to a radial distance of 200 �m, the amountof CDKN1A protein per nucleus revealed no clusters ofcells bearing an increased CDKN1A protein level. This isshown in Fig. 3b for carbon ions. Similar results were ob-tained for argon ions (not shown). Moreover, this result isin accordance with the observation during visual inspec-tion. However, a very heterogeneous distribution of the in-tracellular levels of CDKN1A protein was observed, con-firming the results obtained by flow cytometry.

In addition, the overall induction of CDKN1A could beinferred from the comparison of the irradiated chamberscontaining bystander cells with control chambers. Analysisof the mean expression of the protein in chambers contain-ing a few irradiated cells revealed a 1.3-fold increase com-pared to mock-irradiated controls in separate chambers (twoexperiments, n 9, not shown). This value is in goodagreement with the results obtained after exposure of 1 to3% of the cells in the broad-beam experiments (see Fig.1b). A more detailed analysis of the enhancement of theCDKN1A expression in individual bystander cells was per-formed with the data obtained in specially constructed ir-radiation chambers, allowing a better control for identicalstaining conditions when comparing the overall protein lev-els of nontargeted bystander cells and nonirradiated con-trols. This special type of irradiation chamber (referred toas a divided chamber) has been described previously (29).Briefly it consists of two compartments that are completelyseparated during irradiation and incubation, while the phys-ical separation in the middle of the chamber can be re-moved during the staining procedure. First results obtainedwith carbon ions revealed a slight overall induction ofCDKN1A (29). The distribution of CDKN1A intensity percell nucleus for a representative divided chamber is shownin Fig. 4a and b for the control and the bystander cells.Intensity levels, which correspond to the light intensity col-lected by a CCD camera, have been grouped in bins toobtain a continuous distribution. Differences between con-trol and bystander cells are revealed over the whole rangeof intensities. Figure 4c shows the number of cells with ahigh level of CDKN1A expression. The mean values werecalculated for three independently exposed chambers. Tar-geted cells were excluded from evaluation. The number ofbystander cells expressing CDKN1A at a high level (binsabove the mean control value plus one standard deviation,shown for a typical experiment in Fig. 4a and b) is slightlyhigher than in the controls, although the significance levelis above 0.05 (t test, P 0.067). Figure 4d shows the

200 FOURNIER ET AL.

FIG. 3. Radial distribution of the expression of CDKN1A after targeted irradiation with carbon ions (microbeam): Panel a: Immunofluorescencestaining (merged) of fibroblasts for CDKN1A (green) and 53BP1 (red) after targeted irradiation of single AG1522 cells with carbon ions (arrow). Thecells were irradiated with five particles per nucleus in two crossed lines. Panel b: Immunofluorescence intensities of CDKN1A protein per cell nucleus,quantified using software packages. The distribution of the mean intensities according to the radial distance of up to 200 �m from the targeted cell isshown for a representative chamber (n 3; 300–500 cells per chamber; two independent experiments).

contribution of the highly expressing cells to the signal in-tensities of the entire populations, revealing a significantlyhigher contribution of cells with high-level intensities in thebystander populations compared to the control cells (t test,P 0.035).

Taken together, on the one hand, the results obtained byflow cytometry analysis after broad-beam exposure showthat the increase of the mean expression level of CDKN1Aprotein in bystander cells can be traced back to a generalshift from lower to higher levels. On the other hand, ac-cording to the results obtained after targeted irradiation, interms of CDKN1A-‘‘positive’’ cells, the increase can bepartially attributed to a higher number of cells with elevatedCDKN1A expression level. However, these highly inducingcells are not located adjacent to the irradiated cells.

CDKN1A Bystander Effects Mediated by Soluble Factorsafter Exposure to Low- and High-LET Radiation

Using coculture techniques, the signal transmission fromirradiated to bystander cells can be restricted to soluble fac-tors. We first used X rays to test our system of confluentfibroblasts (AG1522) regarding the induction of bystanderresponses under coculture conditions. After 3 h of cocul-turing after irradiation with 0.5 and 5 Gy X rays, a smallincrease in CDKN1A protein levels was observed in thebystander cells (not shown), in line with the published data(17), although different cell densities and Western blottinginstead of immunofluorescence were used for evaluation.Here the assessment of the induction of the cell cycle-re-lated proteins TP53 and CDKN1A using the coculture tech-nique was extended to a broad range of radiation qualitiesusing high- and low-energy carbon (397 and 9.8 MeV nu-cleon�1; LET 11 keV �m�1 and 170 keV �m�1, respec-tively) and uranium ions (4.2 MeV nucleon�1; LET 15,000keV �m�1) (Fig. 5). For a comparison of the results to theX-ray data, the doses applied ranging from 0.014 Gy to 5

Gy for carbon ions and 0.12 Gy for uranium ions are in-dicated in addition to the fluences. As depicted in Fig. 5for carbon ions, the levels of TP53 protein remained un-changed, whereas the levels of CDKN1A protein on theaverage increased up to twofold and 1.7-fold for 397 and9.8 MeV nucleon�1 carbon ions, respectively. However, forthe highest dose of low-energy/high-LET carbon ions (5Gy) and for uranium ions (0.12 Gy), the levels of CDKN1Aprotein were not elevated compared to control cells accord-ing to the densitometric evaluation. In both cases, the meandoses delivered per irradiated cell are high. After irradiationwith 5 Gy of 9.8 MeV nucleon�1 carbon ions, all cellsreceived a mean number of 39 traversals, whereas after ex-posure to uranium ions, only a few cells received one hit,corresponding to an average dose of 12 Gy. Taken together,for exposure to low- and high-LET radiation, a contributionof medium-mediated signaling to the observed bystandereffects could be shown. Increased protein levels in bystand-er cells were detected for the CDKN1A protein, but theirextent was related to both the dose and the LET of theradiation applied.

Transient Cell Cycle Delay after Exposure to LowFluences of Carbon Ions

The increase in CDKN1A in bystander populationsprompted us to explore the potential impact on cell cycleprogression. A detailed flow cytometry analysis of the cellcycle distribution with high time resolution was carried outafter broad-beam carbon-ion irradiation. Confluent mono-layers of fibroblast cells were exposed at a low fluence (3%of the cells actually hit) and reseeded several hours afterexposure, allowing for the transmission of signals from ir-radiated to bystander cells. The cell cycle distribution wasfollowed up to 96 h after reseeding. The use of BrdU-Hoechst staining enabled the discrimination between threesubsequent cell cycles and the respective classification of

201CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

FIG. 4. Intensity distribution of the expression of CDKN1A after targeted irradiation with carbon ions (microbeam). Representative data set of theCDKN1A intensity distributions 3 h after targeted exposure of AG1522 cells in one divided chamber, comprising 60 fields, each field containing eitherabout 30 control cells (panel a) or about 30 bystander plus irradiated cells (panel b). Approximately 3% of the total number of cells on one side wereirradiated and the same proportion of cells were mock-irradiated on the control side. The irradiated cells were excluded from the evaluation. The meanvalue for the control population plus one standard deviation are indicated by vertical dashed-dotted lines. Panel c: Percentage of cells with fluorescenceintensity above the mean plus one standard deviation, calculated from three different divided chambers as shown in panel a (see dashed-dotted lines).Panel d: Contribution to the global intensity of cells with fluorescence intensity above the mean plus one standard deviation (data set from panel c).

FIG. 5. Expression of TP53 and CDKN1A mediated by soluble factors after exposure to heavy ions. Representative Western blots from oneexperiment showing the protein levels of TP53 and CDKN1A in AG1522 bystander cells. Confluent monolayers of the cells were irradiated with either(panel a) carbon (11 keV �m�1/398 MeV nucleon�1 and 170 keV �m�1/9.8 MeV nucleon�1) or (panel b) uranium ions and cocultured with unirradiatedcells at 37�C. The unirradiated cells were harvested 5 h after exposure and the protein amounts were analyzed by Western blot analysis. �-Tubulinwas used as a control for equal protein loading.

202 FOURNIER ET AL.

FIG. 6. Cell cycle analysis after exposure to carbon ions. Flow cytometry analysis (Hoechst-BrdU quenching) of the fraction of AG1522 cellsretained in the first G0/G1 phase. Cells were harvested 24 h after exposure, reseeded sparsely and harvested again at various times. Panels a–c: Thehistograms show the DNA content for the cells of the first cycle at the time indicated after reseeding. Panel a: 18 h; in all populations, a large fractionof cells have not re-entered the cell cycle. Panel b: 44 h; for the populations in which 3 and 98% of the cells were hit, the number of cells delayedin the first G1 phase exceeds the number in the control population. Panel c: 96 h after reseeding; the delay in the first G1 phase persists only when98% of the cells are hit. Panel d: Pooled data from all experiments. In one out of three experiments the cells displayed a similar pattern of cell cycledistribution, but the cells re-entered the cell cycle earlier than 24 h, and these data points were considered only for the last time. Error bars are �1SEM with n 2–4.

G1, S and G2 phase in the individual cycles. The expressionof CDKN1A protein in the reseeded cells was assessed inparallel for selected times.

The relevant results for the cells reseeded 24 h after ir-radiation are shown for selected times in Fig. 6a–c. Asshown in Fig. 6a for control cells, at 18 h the cells of allpopulations have not entered the cell cycle. The summa-rized data are shown in Fig. 6d. The control cells re-enteredthe cell cycle between 18 and 24 h after reseeding, whereassubpopulations of the 3% and 98% irradiated cells weredelayed in the first G1 phase. When 3% of the cells wereirradiated, the fraction of cells additionally delayed com-pared to the control cells was larger than the fraction of3% irradiated cells (a representative measurement is shownfor 44 h in Fig. 6b). This delay in the bystander populationswas transient, because after 64 h the percentage of cellsretained in the first G1 phase was comparable to controlvalues. In contrast, a large fraction of cells in the irradiatedpopulation (98%) was still delayed at 96 h after reseeding(Fig. 6c). When only 3% of the cell nuclei were traversedby carbon ions, no retardation compared to the control cellswas observed for the cells entering the second and the thirdcell cycle after reseeding (not shown).

The analysis of protein expression in the proliferatingcultures performed in parallel to the cell cycle analysisshowed an enhanced expression of CDKN1A protein at 3h after reseeding of the cells. This induction of CDKN1Aprotein preceded the arrest of the cells in the first G1 phase.In agreement with the results obtained in confluent cells(see Fig. 1b), this increase was no longer detectable at 24h after reseeding (not shown).

Premature Differentiation is not Detected inBystander Cells

We set out to investigate whether the effect on cell cyclearrest in bystander cells could be linked to the induction ofpremature differentiation, since it has been reported previ-ously in irradiated cells (26, 30). Morphological featureswere used to determine the differentiation pattern after car-bon-ion exposure. The fractions of mitotically active (MF)and terminally differentiated, postmitotic (PMF) cell stageswere determined for the control cells and the populationsexposed to low fluences (1, 3 and 10% of the cells hit), inaddition to the cell samples where on average 98% of thecells were traversed by a carbon ion. The comparison is

203CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

FIG. 7. Differentiation pattern after exposure to carbon ions. Percent-age of mitotically active (MFII) and post-mitotic (PMF) AG1522 cells.(n 5–7, from one experiment; comparable results were obtained in arepeated experiment.) The cells were harvested 24 h after exposure, re-seeded sparsely and cultured for 2 weeks. Staining was performed (May-Gruenwald-Giemsa) and the differentiation pattern was assessed accord-ing to morphological features. The theoretical linear dose response(dashed line) is shown; the dashed-dotted lines correspond to the meanSEM.

shown in Fig. 7 for MFII, an important subgroup of MFcells, and PMF cells. The cells belonging to the mitoticallyactive stage MFII show a decrease in number upon irradi-ation, whereas the cells belonging to the postmitotic stagePMF increase in number compared to control cells. Thiswas clearly observed when 98% of the cell nuclei were hit.The results obtained when between 1, 3 and 10% of thecells were hit are in the range of the expected values basedon a hypothetical linear dose response. In summary, thetransient delay of bystander cells in their cell cycle pro-gression and the concomitant changes in the expression ofcell cycle-regulating proteins some hours after exposurehave no visible impact on the differentiation pattern. Theseresults are not indicative of a cell cycle-related, long-lastingbystander effect in fibroblasts after exposure to carbon ions.

DISCUSSION

Heavy ions of different LET allow investigation of theeffect of variations in the dose of single traversals. Increas-ing ionizing densities at constant fluences result at the sametime in a constant number of cells irradiated with an in-creasing dose. This has been accomplished in the studypresented here. A wide range of ionizing densities was cov-ered by using carbon ions of different energies and uraniumions. The impact of the quality of DNA damage induced inthe irradiated cells on the signal transmission to bystandercells has been assessed focusing on the evaluation of cellcycle-related effects.

Cell Cycle-Related Effects in Bystander Cells afterHeavy-Ion Exposure

The efficiency of densely ionizing radiation qualities ininducing lethal or unrepairable damage to cells is high com-

pared to sparsely ionizing radiation (31). The specificity ofthe response to high-LET radiation is attributed to the highcomplexity of the induced DNA damage. The quality andthe reparability of DNA damage varies depending on thelocal doses deposited within the particle tracks (28). Pre-viously, we reported that the expression of proteins regu-lating the cell cycle shows a pattern specific for high LET(23). Now we have addressed the question whether an in-crease in ionizing density, e.g. carbon ions, modifies thecapacity of directly irradiated cells to transmit bystandersignals compared to known data for � particles.

The results presented here indicate a clear induction ofCDKN1A in bystander cells when few cells have been ex-posed to carbon ions. The enhancement of the CDKN1Aprotein level is not very pronounced, but the significancecould be shown consistently in three different fibroblast cellstrains and using different methods (Western blot, flow cy-tometry, immunofluorescence). The observed induction ofCDKN1A is in line with changes after �-particle exposure(32, 33). The experimental conditions of the studies ex-ploring the effects on CDKN1A after �-particle and carbon-ion exposure are similar with respect to the cell type andthe ratios of irradiated and unirradiated cells, allowing acomparison of the results. The LET of carbon ions (170keV �m�1) is around 1.5-fold higher compared to the es-timated value for � particles, but the concomitantly higherionizing density of carbon ions definitely did not result ina more pronounced induction of CDKN1A in bystandercells compared to � particles. In addition, our results showa similarly increased expression of CDKN1A in bystandercells after exposure of a low fraction of cells to uraniumions. This indicates that the effect does not increase withionizing density, which has already been observed for theinduction of micronuclei in AG1522 cells comparing argonand neon ions (1260 and 380 keV �m�1, respectively) (34).We conclude that an induction of the cell cycle regulatorCDKN1A in bystander fibroblast cells is observed for allradiation qualities, but an increase in ionizing density (byvarying the LET about a factor of 100) neither prevents norincreases the effect in bystander cells.

Unlike reported elevated levels of TP53 protein after �-particle exposure (33), no significant accumulation of TP53protein after exposure to very low fluences of carbon anduranium ions was detected in the bystander cells. However,the result is not necessarily an indication of a TP53-inde-pendent induction of CDKN1A. It may indicate a weakresponse generally observed in this cell line (23). Sinceonly the overall protein level and not the post-translationalmodifications of TP53 in bystander cells have been inves-tigated, an activation of TP53 by post-translational modi-fications is still possible.

CDKN1A is one of the inhibitors of cyclin-CDK activ-ities and induces cell cycle delays in response to radiationexposure (35). The analysis of the time course of CDKN1Ainduction after carbon- and uranium-ion exposure showedthat the increase in protein levels was transient. This ob-

204 FOURNIER ET AL.

servation prompted us to investigate a possible effect onthe cell cycle distribution. Our results show a correlationbetween the transient induction of CDKN1A and the cellcycle progression of bystander cells. A larger fraction thanthe fraction of irradiated cells was retained transiently inthe first G1 phase for around 60 h after reseeding. Com-parable transient inhibition of cell proliferation was ob-served in S-phase labeling experiments after �-particle ex-posure using broad-beam (32) or microbeam irradiation(36). Taken together, the data from our carbon-ion studyand from the cited �-particle work are consistent with atransient cell cycle delay in bystander cells.

The fact that the elevation of CDKN1A in bystander cellsafter carbon-ion exposure is transient is suggestive of ashort-term rather than a long-term effect on the cell cycle.Further support for this hypothesis arises from the obser-vation that the analysis of the differentiation stages 2 weeksafter carbon-ion exposure did not reveal a premature dif-ferentiation in bystander compared to control cells.

Transmission of Bystander Signals after Exposure toHigh-LET Radiation

The bystander responses can be mediated by intercellularcommunication via gap junctions or by signal moleculesreleased to the surrounding medium. An involvement ofgap junctions in the transmission of bystander signals hasbeen reported after exposure to � particles or high-energycarbon ions by means of chemical inhibition (37) or theadditional use of CX43-deficient murine fibroblasts (33).The confluent monolayers of cells in our study are able toperform an efficient gap junction-mediated cell-to-cell com-munication as observed in our dye transfer experiments.However, the inhibition of GJIC using lindane to test for amodification of the effect did not provide clarification ofthe role of gap junctions after carbon-ion exposure becauseof the small magnitude of the CDKN1A response (notshown). The first experiments on the expression of one ofthe core proteins of gap junctions, connexin 43, did notreveal changes in the protein levels of bystander cells aftercarbon- and uranium-ion exposure (not shown).

In addition to the inhibitor experiments mentioned above,indirect evidence for the involvement of gap junctions inthe cell cycle-related responses of bystander cells after �-particle exposure arises from the observation of clusters ofcells with elevated levels of CDKN1A protein (33). Sincethese results were obtained in broad-beam experiments, theassignment of the cell clusters to the directly irradiated cellswas not possible. In our study, we intended to discriminatebetween heterogeneous protein expression at the level ofsingle cells and a radiation-induced bystander effect in thecells adjacent to the irradiated cells by performing targetedirradiation of single cells. The overall bystander responsefor CDKN1A expression as observed for broad-beam irra-diation was also confirmed for carbon ions under conditionsof microbeam irradiation. In the same samples we could

show that the bystander cells in proximity to the irradiatedcells did not have particularly elevated levels of CDKN1Aprotein and that the elevation of CDKN1A protein in by-stander cells is not correlated with the radial distance fromthe irradiated cells up to 200 �m. An influence of experi-mental conditions specific for the microbeam exposure canbe ruled out, because the results are in line with our pre-vious results obtained after broad-beam exposure to heavyions (LET 2100–4000 keV �m�1) and retrospective etching(38). It must also be considered that in control populationsthe analysis of the amount of CDKN1A by flow cytometryshowed a high heterogeneity. The distribution patterns weresimilar for the bystander populations, although the meanvalues were elevated compared to control populations. Sucha response would be indicative of medium-borne signals.

Therefore, the involvement of secreted factors after ex-posure to high-LET radiation was tested in this study inseparate experiments restricting the signal transmission onthe surrounding medium. Soluble factors have been shownto mediate signaling from irradiated to bystander cells inconnection with the observation that X-ray exposure in-duces an enhanced expression of CDKN1A protein in by-stander cells (17). The underlying experiments were per-formed using a coculture technique, and the CDKN1A-pos-itive cell nuclei were scored after immunofluorescencestaining. The results of our coculture experiments revealedenhanced overall levels of CDKN1A protein in bystandercells after exposure to X rays and carbon ions of low(‘‘X-ray like’’) and high LET. It is noteworthy that positivebystander effects were not observed when the doses perirradiated cell were high and the ionizing density of theradiation quality exceeded a critical value, as is the casewhen comparing low- and high-LET carbon ions. There-fore, we speculate that a homogeneous intracellular dosedistribution may be favorable for the transmission of by-stander signals by soluble factors. High doses in combi-nation with very high LET are delivered to the irradiatedcells with uranium ions, where one traversal corresponds toa high dose, even if the average dose is low because of thenumber of non-hit cells. Accordingly, no soluble factor-mediated bystander response was observed. However, a gapjunction-mediated transmission of the signals may still befunctioning as inferred from the results obtained for thebroad-beam uranium-ion irradiation. Therefore, the resultsindicate an involvement of medium-borne factors both afterlow- and high-LET irradiation, as long as the doses deliv-ered to the single cells and the ionizing density of the ra-diation quality are not too high. Interestingly, Sokolov etal. (39) also reported a lower efficiency of high comparedto low doses of X rays in inducing the formation of -H2AX foci in bystander cells. A general dose dependenceof non-cytotoxic bystander effects is currently under dis-cussion (40), but further investigation is needed to clarifythis assumption.

An indication for complementary roles of gap junction-and soluble factor-mediated signal transmission can be fur-

205CELL CYCLE-RELATED BYSTANDER RESPONSES AFTER HEAVY-ION IRRADIATION

ther inferred from the results obtained for uranium ions,where one traversal corresponds to a high dose and theintracellular dose distribution in the irradiated cells is lo-cally concentrated. An induction of CDKN1A after urani-um-ion exposure was observed only when the irradiatedcells were in direct contact with the bystander cells.

In summary, we showed in our study that the transmis-sion of bystander signals is not impaired by the very com-plex DNA damage induced in the directly irradiated cellsand that the resulting effects are of the same magnitudewhen comparing carbon and uranium ions. The observedchanges in the regulation and the progression of the cellcycle in bystander cells after carbon-ion irradiation are tran-sient. The fact that based on our experiments, no indicationwas found for the induction of cell cycle-related, persistingeffects in bystander cells is in line with the promising re-sults for carbon-ion therapy obtained over the last fewyears.

ACKNOWLEDGMENTS

This work was supported by the BMBF (02S8203) and EU Grant CEL-LION MRTN-CT-2003-503923. HSF cells were kindly provided by H. P.Rodemann, University of Tubingen, Germany. We would like to thankM. Scholz and E. Gudowska-Nowak for fruitful discussions, F. Knauf andC. Sieben for dedicated technical assistance, and K. Nixdorff for criticalreading of the manuscript. We also thank Wolfgang Becher, Gunther Lenzand the dosimetry team for excellent technical support during the exper-imental runs. Furthermore, we are grateful to Gerhard Kraft for his con-tinuous support.

Received: July 19, 2006; accepted: October 13, 2006

REFERENCES

1. H. Nagasawa and J. B. Little, Induction of sister chromatid exchangesby extremely low doses of alpha-particles. Cancer Res. 22, 6394–6396 (1992).

2. A. Deshpande, E. H. Goodwin, S. M. Bailey, B. L. Marrone andB. E. Lehnert, Alpha-particle-induced sister chromatid exchange innormal human lung fibroblasts: Evidence for an extranuclear target.Radiat. Res. 145, 260–267 (1996).

3. K. M. Prise, G. Schettino, M. Folkard and K. D. Held, New insightson cell death from radiation exposure. Lancet Oncol. 6, 520–528(2005).

4. P. J. Coates, S. A. Lorimore and E. G. Wright, Damaging and pro-tective cell signalling in the untargeted effects of ionizing radiation.Mutat. Res. 568, 5–20 (2004).

5. W. F. Morgan, Non-targeted and delayed effects of exposure to ion-izing radiation: I. Radiation-induced genomic instability and bystand-er effects in vitro. Radiat. Res. 159, 567–580 (2003).

6. S. A. Lorimore, J. M. Mc Ilrath, P. J. Coates and E. G. Wright,Chromosomal instability in unirradiated hemopoietic cells resultingfrom a delayed in vivo bystander effect of gamma radiation. CancerRes. 65, 5668–5673 (2005).

7. E. J. Hall, The bystander effect. Health Phys. 85, 31–35 (2003).8. K. M. Prise, M. Folkard and B. D. Michael, Bystander responses

induced by low LET radiation. Oncogene 22, 7043–7049 (2003).9. C. Mothersill, M. J. Moriarty and C. B. Seymour, Radiotherapy and

the potential exploitation of bystander effects. Int. J. Radiat. Oncol.Biol. Phys. 58, 575–579 (2004).

10. D. Schulz-Ertner, T. Haberer, M. Scholz, C. Thilmann, F. Wenz, O.Jakel, G. Kraft, M. Wannenmacher and J. Debus, Acute radiation-

induced toxicity of heavy ion radiotherapy delivered with intensitymodulated pencil beam scanning in patients with base of skull tu-mors. Radiother. Oncol. 64, 189–195 (2002).

11. D. Schulz-Ertner, A. Nikoghosyan, C. Thilmann, T. Haberer, O. Jakel,C. Karger, G. Kraft, M. Wannenmacher and J. Debus, Results ofcarbon ion radiotherapy in 152 patients. Int. J. Radiat. Oncol. Biol.Phys. 58, 631–640 (2004).

12. A. Di Leonardo, S. P. Linke, K. Clarkin and G. M. Wahl, DNAdamage triggers a prolonged p53-dependent G1 arrest and long terminduction of Cip 1 in normal human fibroblasts. Genes Dev. 8, 2540–2551 (1994).

13. K. H. von Wangenheim, H. P. Peterson and K. Schwenke, A majorcomponent of radiation action: Interference with intracellular controlof differentiation. Int. J. Radiat. Biol. 68, 369–388 (1995).

14. E. I. Azzam, S. M. de Toledo and J. B. Little, Oxidative metabolism,gap junctions and the ionizing radiation-induced bystander effect.Oncogene 22, 7050–7057 (2003).

15. E. I. Azzam, S. M. de Toledo, T. Gooding and J. B. Little, Intercel-lular communication is involved in the bystander regulation of geneexpression in human cells exposed to very low fluences of alphaparticles. Radiat. Res. 150, 497–504 (1998).

16. R. Iyer and B. E. Lehnert, Factors underlying the cell growth-relatedbystander responses to �-particles. Cancer Res. 60, 1290–1298(2000).

17. H. Yang, N. Asaad and K. D. Held, Medium-mediated intercellularcommunication is involved in bystander responses of X-ray-irradi-ated normal human fibroblasts. Oncogene 24, 2096–2103 (2005).

18. M. Heiß, B. E. Fischer, B. Jakob, C. Fournier, G. Becker and G.Taucher-Scholz, Targeted irradiation of mammalian cells using aheavy-ion microprobe. Radiat. Res. 165, 231–239 (2006).

19. B. Stenerlow, E. Hoglund, J. Carlsson and E. Blomquist, Rejoiningof DNA fragments produced by radiations of different linear energytransfer. Int. J. Radiat. Biol. 76, 549–557 (2000).

20. K. M. Prise, M. Pinto, H. C. Newman and B. D. Michael, A reviewof studies of ionizing radiation-induced double-strand break cluster-ing. Radiat. Res. 156, 572–576 (2001).

21. M. Lobrich, Induction and repair of DNA double-strand breaks inhuman fibroblasts after particle irradiation. Adv. Space Res. 22, 551–560 (1998).

22. C. Mothersill, R. J. Seymour and C. Seymour, Bystander effects inrepair-deficient cell lines. Radiat. Res. 161, 256–263 (2004).

23. C. Fournier, C. Wiese and G. Taucher-Scholz, Accumulation of thecell cycle regulators TP53 and CDKN1A (p21) in human fibroblastsafter exposure to low- and high-LET radiation. Radiat. Res. 161,675–684 (2004).

24. R. M. Bohmer and J. Ellwart, Cell cycle analysis by combining the5-bromodeoxyuridine/33258 Hoechst technique with DNA-specificethidium bromide staining. Cytometry 1, 31–34 (1981).

25. C. Fournier, S. Brons and G. Taucher-Scholz, Comparison of thequantification of chemiluminescent signals using photon-sensitivefilms or CCD-camera. Biotechniques 35, 284–292 (2003).

26. C. Fournier, M. Scholz, W-K. Weyrather, H. P. Rodemann and G.Kraft, Changes of fibrosis-related parameters after high- and low-LET irradiation of fibroblasts. Int. J. Radiat. Biol. 77, 713–722(2001).

27. N. T. J. Baily, Statistical Methods in Biology, 2nd ed. CambridgeUniversity Press, Cambridge, 1981.

28. G. Taucher-Scholz, J. Heilmann and G. Kraft, Induction of DNAstrand breaks by heavy ions. Nucl. Instrum. Methods Phys. Res. B107, 318–322 (1996).

29. P. Barberet, C. Fournier, F. Knauf, M. Heiß, B. E. Fischer and G.Taucher-Scholz, Studies on the induction of the cell cycle regulatorCDKN1A (p21) in bystander cells using the GSI heavy ion micro-beam. Radiat. Res. 166, 682–684 (2006). [extended abstract]

30. H. P. Rodemann, H. P. Peterson, K. Schwenke and K. H. von Wan-genheim, Terminal differentiation of human fibroblasts is induced byirradiation. Scan. Microsc. 5, 1135–1143 (1991).

31. W. K. Weyrather, S. Ritter, M. Scholz and G. Kraft, RBE for carbon

206 FOURNIER ET AL.

track-segment irradiation in cell lines of differing repair capacity. Int.J. Radiat. Biol. 75, 1357–1364 (1999).

32. E. I. Azzam, S. M. de Toledo, A. J. Waker and J. B. Little, High andlow fluences induce a G1 checkpoint in human diploid fibroblasts.Cancer Res. 60, 2623–2631 (2000).

33. E. I. Azzam, S. M. de Toledo and J. B. Little, Direct evidence forthe participation of gap junction-mediated intercellular communica-tion in the transmission of damage signals from �-particle irradiatedto non-irradiated cells. Proc. Natl. Acad. Sci. USA 98, 473–478(2001).

34. C. Shao, Y. Furusawa, Y. Kobayashi, T. Funayama and S. Wada,Bystander effect induced by counted high-LET particles in confluenthuman fibroblasts: A mechanistic study. FASEB J. 17, 1422–1407(2003).

35. J. Bartek and J. Lukas, Mammalian G1- and S-phase checkpoints inresponse to DNA damage. Curr. Opin. Cell Biol. 13, 738–747 (2001).

36. B. Ponnaiya, G. Jenkins-Baker, D. J. Brenner, E. J. Hall, G. Randers-

Pehrson and C. R. Geard, Biological responses in known bystandercells relative to known microbeam-irradiated cells. Radiat. Res. 162,426–432 (2004).

37. C. Shao, Y. Furusawa, M. Aoki and K. Ando, Role of gap junctionalintercellular communication in radiation-induced bystander effects inhuman fibroblasts. Radiat. Res. 160, 318–323 (2003).

38. B. Jakob, C. Fournier, M. Scholz and G. Taucher-Scholz, Cellularand subcellular response of CDKN1A (p21) after low-fluence heavy-ion irradiation. Radiat. Res. 161, 108–109 (2004).

39. M. V. Sokolov, L. B. Smilenov, E. J. Hall, I. G. Panyutin, W. M.Bonner and O. A. Sedelnikova, Ionizing radiation induces DNA dou-ble-strand breaks in primary human fibroblasts. Oncogene 24, 7257–7265 (2005).

40. C. Mothersill, F. Lyng, C. Seymour, P. Maguire, S. Lorimore and E.Wright, Genetic factors influencing bystander signaling in murinebladder epithelium after low-dose irradiation in vivo. Radiat. Res.163, 391–399 (2005).