INDUCTION AND REJOINING OF DNA DOUBLE STRAND BREAKS ASSESSED BY H2AX PHOSPHORYLATION IN MELANOMA CELLS IRRADIATED WITH PROTON AND LITHIUM BEAMS

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1 doi: /j.ijrobp Int. J. Radiation Oncology Biol. Phys., Vol. 74, No. 4, pp , 2009 Copyright Ó 2009 Elsevier Inc. Printed in the USA. All rights reserved /09/$ see front matter BIOLOGY CONTRIBUTION INDUCTION AND REJOINING OF DNA DOUBLE STRAND BREAKS ASSESSED BY H2AX PHOSPHORYLATION IN MELANOMA CELLS IRRADIATED WITH PROTON AND LITHIUM BEAMS IRENE L. IBAÑEZ, M.D., M.SC.,* y CANDELARIA BRACALENTE, M.SC.,* BEATRIZ L. MOLINARI, PH.D.,* y MÓNICA A. PALMIERI, M.SC., z LUCÍA POLICASTRO, PH.D.,* y ANDRÉS J. KREINER, PH.D.,* yx ALEJANDRO A. BURLÓN, PH.D.,* x ALEJANDRO VALDA, PH.D., x DANIELA NAVALESI, z JORGE DAVIDSON,PH.D., y MIGUEL DAVIDSON,PH.D., y MÓNICA VÁZQUEZ, B.S.,* MABEL OZAFRÁN,PH.D.,* AND HEBE DURÁN, PH.D.* yx *Comisión Nacional de Energía Atómica, San Martín, Argentina; y Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina; z Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; x Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, Argentina Purpose: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. Methods and Materials: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX (gh2ax) foci at 30 min and 6 h post-irradiation. Results: Survival curves showed the increasing effectiveness of radiation as a function of LET. gh2ax labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of gh2ax foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. Conclusions: Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of gh2ax foci. We conclude that gh2ax foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity. Ó 2009 Elsevier Inc. Ionizing radiation, LET, DNA DSB, Melanoma cells, gh2ax. INTRODUCTION The incidence of melanoma has substantially increased worldwide over the last 40 years. Although melanoma accounts for only 10% of skin cancer, it is responsible for at least 80% of skin cancer deaths. Most advanced melanomas respond poorly to radiotherapy and chemotherapy and no effective therapy exists to inhibit the metastatic spread of this cancer (1). Protons and lithium, and more generally charged hadrons, have a well-defined penetration depth, known as range, leaving toward the end of that range a maximum energy per traversed unit length. This generates a peak in the delivered dose, known as the Bragg peak, the dose being very small beyond that region. This behavior is completely different to that of photons (and neutrons), which involves an exponential attenuation in intensity from the surface towards the interior of tissue. Within this context, the utilization of hadron beams has important comparative advantages as far as dose localization is concerned. Thus, hadron therapy is becoming increasingly important for the treatment of tumors localized in the vicinity of vital and radiosensitive organs (2). In particular, for choroidal melanomas and certain tumors of the base of the skull, hadron therapy is recognized as the best alternative (3). Moreover, high linear energy transfer (LET) charged particles therapy is an interesting approach for the treatment of melanoma because of its increased potential to kill low- LET radioresistant cells. Reprint requests to: Hebe Durán, Ph.D., Comisión Nacional de Energía Atómica, Departamento de Radiobiología, Av. Gral. Paz 1499, (B1650KNA) San Martín, Provincia de Buenos Aires, Argentina. Tel: ; Fax: ; hduran@cnea.gov.ar 1226 Grants: CONICET (PIP-6134) and Universidad Nacional de San Martín (PROG07E/3). Conflict of interest: none. Received Nov 17, 2008, and in revised form Feb 23, Accepted for publication Feb 27, 2009.

2 gh2ax in melanoma cells irradiated with charged particle beams d I. L. IBAÑEZ et al Table 1. Irradiation energies, mean LET values on the cells midplane and the corresponding parameters of the survival curves of B16-F0 melanoma cells fitted to the linear quadratic model for gamma rays and plateau protons and to the exponential model for Bragg peak protons and lithium beams Survival curve parameters Mean energy [MeV] Mean LET [kev/mm] a (Gy -1 ) b (Gy -2 ) Gamma rays Protons Plateau Bragg peak Lithium ( 6 Li) Ionizing radiation produces a broad spectrum of molecular lesions to DNA, including single strand breaks (SSBs), double strand breaks (DSBs), and a great variety of base damages. DSBs are the most toxic form of DNA damage, because a single unrepaired DSB can lead to abnormal mitosis with losses of large fragments of DNA (4). It has been shown that the few DSBs that are produced by ionizing radiation correlate closely to the amount of induced cell death, whereas the 100-fold more abundant SSBs and base damage are of minor importance in relation to the cytotoxic effect. In the last decade, the phosphorylation of the histone protein H2AX (gh2ax) has been used as a measure of the formation and rejoining of DSB induced by different types of chemicals and radiations (5, 6). This approach is based on one of the early steps in the response of mammalian cells to DSBs. H2AX is a histone variant of the H2A family, which is phosphorylated at serine 139 within its conserved COOH-terminal region in response to the presence of DSBs. The induction of gh2ax by radiation is reported to be mediated mainly by ataxia telangiectasia-mutated protein and occurs at the sites of DSBs in the nuclear DNA (7). The number of gh2ax foci formed in this way has been shown to be directly proportional to the number of DSBs and their dephosphorylation has been correlated with repair of DSBs (8, 9). The local formation of gh2ax allows microscopic detection of distinct foci that most likely represent a single DSB (8). The potential to detect a single focus within the nucleus makes this the most sensitive method available for detecting DSBs (10). The detection of gh2ax offers the advantage over conventional methods of determining DNA damage and repair capacity in a dose range comparable with the doses per fraction used in radiotherapy (i.e., in the range of about 2 Gy). The aim of this study was to evaluate the response of a melanoma cell line to proton and lithium irradiations by determining DNA damage and survival. For this purpose, the formation and rejoining of DSBs was determined by the immunocytochemical detection of gh2ax foci at different times post-irradiation and survival curves were obtained by clonogenic assay. Considering that the immunofluorescence detection of gh2ax is unable to resolve DNA breaks that are close together in clusters induced by high LET radiations (11), both the number and size of gh2ax foci were considered contributory end points to evaluate more precisely the effects of the different quality radiations. METHODS AND MATERIALS Cell line and cell culture The mouse melanoma cell line B16-F0 (Molecular Oncology Laboratory, Quilmes National University, Argentina) was used. Cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 mg/ml streptomycin, at 37 C in a 5% CO 2 humidified atmosphere and were subcultured following standard procedures. Irradiation experiments The proton and lithium beams were produced by an electrostatic 20 MV vertical tandem accelerator (TANDAR accelerator, National Atomic Energy Commission, Argentina) as previously described (12). Dosimetry was performed using a dose-calibrated transmission ionization chamber, calibrated with a dose-calibrated Capintec PS-033 thin window parallel plate ionization chamber (13). This chamber was placed at exactly the same position as the cell culture plane and the Mylar cover of the dishes had the same thickness as the chamber window, ensuring that the incident energy on the cells matches that in the sensitive volume of the parallel plate ionization chamber. Table 1 shows the energy of the particles and mean LET values on the cells midplane, calculated as previously described (12). Typical fluences for a 1 Gy dose are shown in Table 2. A 137 Cs source (IBL-437C Irradiator; CIS Bio- International, CEBIRSA, Argentina) was used for gamma irradiations. Survival curves For clonogenic assays, cells from mid-log growing cultures were plated and incubated for 6 h. At the moment of irradiation, the medium was removed and the dish covers were replaced by 4-mm Mylar foils. Immediately after irradiation, complete fresh medium was added. Control cells were sham irradiated. Cells were incubated for 10 days at 37 C and 5% CO 2. Colonies were fixed and stained. The fraction of clonogenic cells was determined by scoring colonies containing at least 50 cells. Three independent experiments were performed in triplicates for each condition. Survival curves were fitted to the linear quadratic model (Eq. 1) for low LET radiation (gamma rays and plateau protons) or to the exponential model (Eq. 2) for intermediate (Bragg peak protons) and high-let radiation (lithium): S ¼ exp ad þ bd 2 (1) S ¼ exp ðadþ (2)

3 1228 I. J. Radiation Oncology d Biology d Physics Volume 74, Number 4, 2009 Table 2. Particle fluence for a dose of 1 Gy of plateau and Bragg peak protons and lithium beams, nuclear size of B16-F0 melanoma cells, calculated average number of hits per cell nucleus and the number of gh2ax foci per cell nucleus with a size larger than 1.45 mm 2 at 30 min post-irradiation Fluence (particles/ 100 mm 2 ) Nuclear size* (mm 2 ) Hits gh2ax Foci > 1.45 mm 2 * Protons Plateau Bragg peak Lithium * Mean SD. Immunofluorescence and quantification of gh2ax B16-F0 cells were irradiated at 70% confluence and handled as in clonogenic assays. After irradiation, cells were maintained at 37 C and5%co 2 until 4% paraformaldehyde fixation at either 30 min or 6 h post-irradiation. Cells were fixed for 15 min, washed with phosphate-buffered saline (PBS), permeabilized with 0.5% Triton X-100 in PBS during 15 min, washed and blocked with 5% fetal bovine serum in PBS during 30 min. After blocking, cells were incubated overnight at 4 C with a monoclonal anti-gh2ax antibody (Upstate, Lake Placid, NY), 1:500 in PBS, and washed and incubated with FITClabeled secondary antibody (Sigma) in the dark for 1 h at room temperature. Cells were then washed, counterstained, and mounted with 1 mg/ml 4 0,6-diamidino-2-phenylindole dihydrochloride dihydrate (DAPI) in an antifade solution, in the dark. Cells were examined in an Olympus BX51 epifluorescence microscope using immersion oil with a 100X (UPlanApo 100 X/1.35 oil) objective lens. For each treatment condition, FITC and DAPI images were serially captured by a CCD camera (Olympus DP70) and more than 50 fields containing approximately 20 cells each were stored. A code number was given to each image. Aleatory sampling methods were used to select the images and all the cells in each selected image were screened. An average of 150 cells was evaluated per each experimental condition. The gh2ax foci per nucleus were counted by eye by two scorers and results were crosschecked. The area of each gh2ax foci was quantified by using the National Institutes of Health Image J software. Two independent experiments were performed with triplicates for each condition. Statistical analysis The results are presented as mean SD. Significant changes were assessed using one-way analysis of variance and nonparametric Kruskal-Wallis test followed by Tukey s or Dunn s multiple comparison tests to determine significant differences between group means. p Values of less than 0.05 were considered significant for all tests. Regression analysis was performed when appropriate. RESULTS Survival curves were obtained to characterize the radiation response of B16-F0 cells (Fig. 1). The analysis of the fitted survival curves showed an increase of the a parameter value as a function of LET and the loss of the quadratic component of the survival curves (b = 0) at intermediate and high LET (Table 1). Nuclear gh2ax foci were determined to evaluate the formation and repair of DSBs. Figure 2 shows images of a representative experiment of cells irradiated with 1 Gy of different LET radiations. The quantification of the number of foci per nucleus (Fig. 3) demonstrated a significant increase (p < 0.01) of DSBs as a function of dose at 30 min post-irradiation for all the radiations evaluated. Regression analysis revealed a linear dose response at 30 min post-irradiation for plateau (r = 0.96, p < 0.01) and Bragg peak proton (r = 0.98, p < 0.01) and lithium irradiations (r = 0.95, p < 0.05). In the case of gamma irradiation linear fit results were inconclusive (r = 0.87, p = 0.06), probably because of the intrinsic variance of the data. Therefore, these results supported (for charged particles) and did not exclude (for gamma rays) a linear relationship between the number of foci at 30 min post-irradiation and the dose. A significant decrease in the number of foci was found at 6 h post-irradiation for 1 Gy (p < 0.01) and 2 Gy (p < 0.05) of low LET radiation vs. the number of foci at 30 min post-irradiation (Fig. 3a). The frequency histograms for low LET radiation show the increase of the number of cells with fewer foci at 6 h post-irradiation for 1 and 2 Gy (Fig. 3b). This reduction in the number of foci may be explained by the repair capacity of DSBs after low LET irradiation. Conversely, for intermediate or high LET radiation, the frequency histogram after 6 h is similar to that obtained 30 min after irradiation, revealing that the repair capacity of densely damaged DNA is significantly lower. These results are consistent with the survival curves obtained after Bragg peak proton and lithium irradiations, where the best fit for these data was to the first-order exponential function (Eq. 2). Considering that DSBs induced by high- LET radiations are densely concentrated in clusters, the size of gh2ax Fig. 1. Survival curves of B16-F0 melanoma cells after irradiation. (B) Gamma rays, ( ) plateau proton, ( ) Bragg peak proton, and (<) lithium beams. Data represent mean SD of a representative experiment out of three. Fitted curves are shown.

4 gh2ax in melanoma cells irradiated with charged particle beams d I. L. IBAÑEZ et al Fig. 2. Representative images of nuclear gh2ax foci. B16-F0 melanoma cells exposed to 1 Gy of different linear energy transfer (LET) radiations. An increase in the number of foci at 30 min post-irradiation for all types of radiation vs. unirradiated cells and a decrease in the number of foci at 6 h post-irradiation for low LET radiation vs. 30 min post-irradiation can be observed. DAPI: staining of nuclear DNA. gh2ax: FITC staining of gh2ax foci. foci was determined to evaluate the effects of the different quality radiations. Figure 4 shows representative images of cells irradiated with 0 4 Gy of different LET radiations, where variations in foci size can be visualized. A significant increase (p < 0.01) in foci size at 30 min post-irradiation in cells irradiated with lithium beams vs. cells irradiated with proton beams or gamma rays was demonstrated (Fig. 5), consistent with the highly concentrated DSBs in the clustered DNA damage induced by high LET radiation. At 6 h postirradiation, a significant increase (p < 0.01) in foci size was found not only for lithium irradiations, but also for Bragg peak protons and high doses (3 and 4 Gy) of plateau protons as compared to unirradiated cells, seen as a shift to the right in the frequency histograms (Fig. 5b). Thus, the increase in foci size after 6 h seems to be correlated with persistent DSBs. The relationship between the number of gh2ax foci induced by high LET radiation and the number of hits per nucleus was evaluated. The nuclear size and the particle fluence were considered to calculate the average number of hits per nucleus. A striking correspondence between the expected value of hits per nucleus and the number of foci larger than 1.45 mm 2 was found (Table 2, Fig. 6). Thus, these larger foci would be the direct result of the lithium particle track, seen as large clusters of DSBs. The smaller foci that increased the scored average number of foci could not be attributed to the primary particle track. To validate the use of gh2ax foci size as a marker of radiation response to different quality radiations, the correlation between surviving fraction and foci size at 6 h postirradiation was analyzed considering data obtained with gamma rays and proton and lithium beams (Fig. 7). Linear regression analysis revealed a significant correlation (r = 0.91, p < ) between both parameters. DISCUSSION In this study, we evaluated the response of B16-F0 melanoma cells to different quality radiations. We demonstrated:

5 1230 I. J. Radiation Oncology d Biology d Physics Volume 74, Number 4, 2009 Fig. 3. Quantification of nuclear gh2ax foci. B16-F0 melanoma cells were exposed to 1 4 Gy of different linear energy transfer (LET) radiations. Cells were fixed at (,) 30 min and (-) 6 h post-irradiation for gh2ax detection. (a) Average number of gh2ax foci per nucleus vs. dose. Data represent mean SD. *p < 0.01 vs. control; yp < 0.01 and yyp < 0.05 between 30 min and 6 h post-irradiation. (b) Frequency histograms of percentage of cells presenting 0 to more than 60 gh2ax foci per nucleus for the different doses and types of radiation utilized. Different scales were used to optimize visualization. (1) the increase in the number of gh2ax foci as a function of dose for all the radiations evaluated; (2) a marked decrease in the repair capacity as the LET of the radiation increased, revealed by the variations in the number of foci 6 h post-irradiation; (3) a strong increase in the size of the foci for high LET radiation; and (4) an increase in the foci size 6 h post-irradiation related to persistent DSBs for charged particle (proton and lithium) irradiations. Moreover, we confirmed in this cell line the greater effectiveness of intermediate or high-let radiation, revealed by the loss of the shoulder of the survival curve for Bragg peak proton and lithium irradiations with an increase in the a parameter and the disappearance of the b parameter of the fitted survival curves. Furthermore, we demonstrated a significant correlation between survival and gh2ax foci size at 6 h post-irradiation.

6 gh2ax in melanoma cells irradiated with charged particle beams d I. L. IBAÑEZ et al Fig. 4. Determination of gh2ax foci size. Representative images of nuclear gh2ax foci of B16-F0 melanoma cells exposed to 1 4 Gy of different linear energy transfer (LET) radiation, where variations in size can be observed: an increase in foci size 30 min and 6 h after high LET irradiation and an increase in foci size for Bragg peak protons and for high doses (3 and 4 Gy) of plateau protons only at 6 h post-irradiation. Cells were fixed at (a) 30 min post-irradiation and (b) 6 h postirradiation for gh2ax detection. DAPI: staining of nuclear DNA. gh2ax: FITC staining of gh2ax foci. Our results of gh2ax labeling in untreated melanoma cells showed a high number of foci per nucleus (mean range, 17 25). These values are consistent with previous reports that showed higher levels of gh2ax foci in human metastatic melanoma cells as compared with primary melanocytes (14) and the overexpression of gh2ax in human malignant melanocytic lesions (15). The chromosome rearrangements observed in melanoma cells likely require the induction of DSBs as intermediates (14). These authors demonstrated the colocalization of gh2ax foci with DNA repair complex proteins, suggesting that the gh2ax foci detected in melanoma cells are associated either with DNA DSBs or chromatin alterations recognized as DNA DSBs (14).

7 1232 I. J. Radiation Oncology d Biology d Physics Volume 74, Number 4, 2009 Fig. 5. Quantification of nuclear gh2ax foci size. (a) gh2ax foci size (top) and nuclear size (bottom) vs. dose for cells irradiated with (,) gamma rays, ( ) plateau protons, ( ) Bragg peak protons, and (-) lithium beams. Data represent mean SD. *p < 0.01 vs. control. (b) Frequency histograms: percentage of gh2ax foci of different sizes (0 >2.10 mm 2 ) for the different doses and types of radiation used. Cells were fixed at (,) 30 min and (-) 6 h post-irradiation for gh2ax detection. Different scales were used to optimize visualization.

8 gh2ax in melanoma cells irradiated with charged particle beams d I. L. IBAÑEZ et al Fig. 6. (a) Representative images of nuclear gh2ax foci larger than 1.45 mm 2 30 min post-irradiation in B16-F0 melanoma cells exposed to 1 Gy of lithium beams. Arrows indicate gh2ax foci larger than 1.45 mm 2. (b) Frequency histogram: number of foci of different sizes (0 >1.45 mm 2 ) in the arrowed cell. The average number of the large foci per nucleus corresponded to the expected average of hits per nucleus (Table 2). Neighboring smaller foci can also be visualized. It was reported that gh2ax is detectable within 1 3 min after irradiation and increases until a plateau is reached at min post-irradiation (16). The kinetics of the loss of gh2ax could be associated with the DSB repair process (17). In this study, we determined the induction of gh2ax foci at 30 min and the persistence of foci at 6 h post-irradiation for different quality radiations. We found a linear dosedependent increase in the number of gh2ax foci for charged particle irradiations. For gamma irradiation, the tendency was also linear with the regression value near significance. The number of foci decreased at 6 h post-irradiation for 1 and 2 Gy of low LET radiation. Particularly, we found a 50% decrease in the number of foci for 1 Gy, which is consistent with Olive et al. (17), who demonstrated that the gh2ax loss half-times are 2 7 h for cultured cells. The persistence of a higher number of foci for increasing dose could be showing nonrepairable or slowly rejoined DSBs because of greater DNA damage. Regarding different LET irradiations, no significant increase in the number of foci was found for increasing LET at 30 min post-irradiation. Conversely, at 6 h post-irradiation the number of foci for low LET radiation was significantly lower than for intermediate or high LET, revealing the differential repair capacity of the DSBs induced by the different quality radiations. These results reveal the greater complexity of DSB induced by high LET radiation, which potentially leads to increased mutagenicity and decreased repairability of the damaged site. Our results are consistent with previous reports (11, 18, 19), which showed that variations in the rate of dephosphorylation and the number of residual foci are dependent on radiation quality. Assuming that one DSB generates one focus, most of the reports have considered the number of foci per nucleus independently of the size of the foci. However, to evaluate gh2ax foci in cells exposed to charged particles, the size of the foci should be considered. Each high-let radiation track induces multiple DSBs along its core. Such clusters of DSBs cannot be resolved by optical microscopy and thus appear as a single focus (11, 20). Moreover, the number of H2AX molecules phosphorylated in response to a single DSB and indeed the positioning of the H2AX histone complement within the cell nucleus in comparison to the radiation tracks, may influence the size of the foci observed (18). These limitations to resolve DSB induced by charged particles could explain the lack of increase in the number of foci that we described herein for high-let vs. low-let radiations. Thus, we measured the size of the foci and we demonstrated that the foci size induced by lithium beams at 30 min post-irradiation triples that obtained in cells exposed to proton beams and gamma rays. Regarding the foci size measured for gamma irradiations, the values we obtained are in agreement with a previous study (21) that evaluated the size and distribution of gh2ax clusters by 4Pi and confocal microscopy. The increase in foci size induced by lithium beams at 30 min post-irradiation could be attributed to the densely damaged DNA. Within this context, the particle track structure of lithium ions was taken into account and Fig. 7. Correlation between survival and gh2ax foci size at 6 h post-irradiation. Data obtained for (o) gamma rays, ( ) plateau, and ( ) Bragg peak protons and (<) lithium beams were included. Data represent mean SD of surviving fraction and gh2ax foci size. A significant correlation between surviving fraction and the size of gh2ax foci resulted from the regression analysis (r = 0.91, p < ). The fitted regression line is shown.

9 1234 I. J. Radiation Oncology d Biology d Physics Volume 74, Number 4, 2009 a correspondence between the expected number of hits per nucleus and the number of gh2ax foci with a size larger than 1.45 mm 2 was found. Thus, after perpendicular exposure of the cells these larger foci would be the direct result of the lithium particle track and each larger focus would represent a cluster of DNA and chromatin damage (22). On the other hand, the smaller foci, that increased the value of the average number of foci per nucleus, would be the result of either the induction of DNA damage by d rays, secondary nuclear fragments, or possible bystander effects (11, 18). Other authors suggested that the smaller foci appear as a consequence of non specific binding of the antibodies or the presence of some cells in S-phase (22, 23). We demonstrated a marked increase in gh2ax foci size 6 h post-irradiation both for cells exposed to lithium and proton beams as compared with 30 min post-irradiation, related to the persistence of the foci. This result suggests that the increase in foci size would be a consequence of the highly complex DNA damage, difficult to repair or non-repairable. In relation with these results, Costes et al. (20) reported that the size and intensity of gh2ax foci in normal human fibroblasts increases twofold over a 2-h period after nitrogen beam irradiation, whereas no significant changes were found after X-ray exposure. The authors suggest that the progressively increasing size of gh2ax is due to further phosphorylation of H2AX, extending from the initial damaged area, instead of simple physical expansion of the initial focus. The formation of DSBs induces a local expansion of chromatin (9, 24) that has been proposed to activate the ataxia telangiectasiamutated kinase, triggering the cellular response to DNA damage (25). Thus, the increase in gh2ax foci size could be the result of the relaxation of chromatin at the site of damage, leading to more phosphorylation of H2AX. It has been reported that the positions of DSB-induced gh2ax can move to cluster together, supporting the notion that distant DSBs can be juxtaposed (26). This could be another explanation for the increase in foci size found herein. It has been previously demonstrated that mitotic cell death induced by ionizing radiation correlates with nonrepairable DNA damage (27). The analysis of the results presented herein reveals that nonrepairable or slowly rejoined DSBs would be an indicator of mitotic cell death, in agreement with previous reports that showed a correlation between the radiosensitivity of different human cell lines, tumors, and normal tissues and the residual level of gh2ax or the rate of loss of gh2ax (17, 28). In the present study, we found a strong correlation between survival and the size of persistent gh2ax foci. Considering that the size of foci could be directly linked with the extent of clusters of DNA damage along the particle track, this high concentration of DSBs would impair the efficient repair of DNA damage affecting cell survival. Thus, we suggest that gh2ax foci size is an accurate parameter to evaluate radiosensitivity to different LET radiations. 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