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Radiat Environ Biophys (2012) 51:23–32 DOI 10.1007/s00411-011-0398-1

ORIGINAL PAPER

Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams O. Zlobinskaya • G. Dollinger • D. Michalski • V. Hable • C. Greubel • G. Du • G. Multhoff • B. Ro¨per • M. Molls • T. E. Schmid

Received: 29 June 2011 / Accepted: 10 December 2011 / Published online: 7 January 2012 Ó Springer-Verlag 2012

Abstract In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose–effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of O. Zlobinskaya (&)  D. Michalski  G. Multhoff  B. Ro¨per  M. Molls  T. E. Schmid Klinikum rechts der Isar, Department of Radiation Oncology, Technische Universita¨t Muenchen, Ismaninger Strasse 22, 81675 Munich, Germany e-mail: [email protected] G. Dollinger  V. Hable  C. Greubel Institute for Applied Physics and Metrology, Universita¨t der Bundeswehr Mu¨nchen, 85577 Neubiberg, Germany G. Du Technische Universita¨t Mu¨nchen, Physik Department II, Garching, Germany

0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells. Keywords RBE  Protons  DNA DSB repair  Gamma-H2AX foci  Pulsed irradiation

Introduction Currently, the radiation treatment for tumors in human patients is in most cases performed with high-energy photon beams generated by clinical linear electron accelerators. Recently, high-energy proton or ion beams have been introduced as a promising alternative to classical radiotherapy treatment. Compared to the standard photon treatment, particle therapy can deliver improved dose distributions with less dose burden in healthy tissue (Schardt et al. 2010). Unfortunately, due to the fact that technology for particle acceleration and beam lines is rather complex and costly, this type of therapy is limited to a few centers worldwide only. Compact laser-driven accelerators (LDA) could circumvent this limitation and provide ion beam therapy to a broader range of patients (Ledingham et al. 2007). Laser technology is evolving very quickly, and specialized groups, mainly in the United States of America, Europe and Japan (Kraft et al. 2010; Yogo et al. 2009), are working on exploring the potential of high-intensity ultrashort laser pulses to accelerate ion beams, namely protons and carbon ions. It is proposed that hadron therapy will

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benefit from substantial reduction in building costs and space requirements usually connected with ion beam cancer treatment therapy (Kraft et al. 2010). Laser-driven accelerators are potentially capable of producing proton beams of high energy, for example, up to 60 MeV in single-shot mode (Bulanov et al. 2008) and up to 17 MeV in ultra-short pulses mode (Zeil et al. 2010). The laser intensities can only be achieved by an ultra-short laser pulse with a pulse length in the range of femtoseconds (10-15 s). Consequently, the ion beam produced by a laserdriven accelerator (LDA) is bound to be pulsed as well (Schell and Wilkens 2009). However, due to the energy spread of the produced particles, the dose is delivered in a pulse length of pico- or even nanosecond length. Consequently, this is the time scale at which a tissue ‘‘voxel’’ exposed to radiotherapy from an LDA proton or heavy ion beam receives a substantial part of the full dose, whereas with conventional proton or heavy ion irradiation from a synchrotron or cyclotron, the same dose is applied ‘‘continuously’’ over approximately 100 ms, which means a difference in dose rate of at least eight orders of magnitude (Schell and Wilkens 2009). Thus, it is mandatory to investigate the relative biological effectiveness (RBE) of the new beam quality before its use in a radiotherapeutic setting. Currently, it is imperative to determine (1)

(2)

whether or not a pulsed irradiation with high-energy particles or protons induces a different amount of damage in cells as compared to a continuous irradiation and whether repair or apoptosis pathways are also affected relative to the classical continuous irradiation.

The short-pulse effects are subject to our current investigations utilizing pulsed ion beams at the Munich tandem accelerator SNAKE (Superconducting Nanoprobe for Applied nuclear (Kern) physics Experiments) microprobe, where 105 high-energy protons can be packed into a single nanosecond pulse at a beam diameter of about 100 lm (Dollinger et al. 2009). These beam parameters are sufficient to irradiate cell cultures or tissue up to a dose of 5 Gy by a single pulse of protons. Using this system, in previous work, we reported that the induction of micronuclei in HeLa cells or in keratinocytes within 3D tissue was not significantly different between pulsed and conventional protons (Schmid et al. 2009, 2010b, 2011). This work was extended to the analysis of clonogenic survival of cells irradiated under both conditions (Auer et al. 2011), which is often considered to be of the most relevant when judging irradiation conditions. The RBE determined for both irradiation modes was compatible with the generally assumed RBE of 1.1, which is also applied in proton therapy (Paganetti 2003). In an analysis of chromosome aberrations, however, we found

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evidence that proton irradiation in the pulsed mode may be slightly less effective than in the continuous irradiation mode (Schmid et al. 2011). Among the many types of DNA damage that are induced by ionizing radiation in cells, double-strand breaks (DSBs) are generally recognized as the major initial lesions that can result in chromosome aberrations. However, a simple direct causal cannot be inferred, because the number of DNA DSBs produced by a dose of 1 Gy of low-LET ionizing radiation is much larger than the numbers of induced chromosome aberrations or cell reproductive death (Bedford 1991). Franken et al. found that the RBE value for induction of gamma-H2AX (phosphorylated histone H2AX) foci is much smaller than the values for induction of cell reproductive death or chromosome aberrations when irradiated with a-particles (Franken et al. 2011). Therefore, an important prerequisite for a better understanding of pulsed proton irradiation on the DNA is the mechanistic description of the processing of DNA DSBs. The induction and time-dependent loss of gamma-H2AX was quantitatively investigated in the present study as an indicator of DSB damage induced either by 20-MeV protons in pulsed or continuous mode or by X-rays from 70- to 200-kV electron beam tubes as the reference radiation. The cellular response to DSBs includes the very rapid phosphorylation of the histone H2AX (Rogakou et al. 1999). Phosphorylated H2AX forms microscopically visible foci, and the number of phosphorylated H2AX foci correlates well with the number of DSB for low linear energy transfer (LET) (Ismail and Hendzel 2008; Kinner et al. 2008). Since protons with energies of 20 MeV (LET = 2.65 keV/lm) is a low-LET irradiation, the number of gamma-H2AX foci can directly be compared with that of X-ray irradiation in order to determine the RBE values for DSB induction. It is proposed here to asses these irradiation modes by analyzing initial 1-h and late 24-h DNA DSB numbers in HeLa cells, in order to address foci numbers after different repair times. Schmid et al. showed that gamma-H2AX loss kinetics follows a bi-exponential decline with two distinct decay time constants independent of LET but with a larger fraction of damage belonging to the larger decay time constant for high-LET irradiation (Schmid et al. 2010a). In the current setting, these results were extended to assess the differences in temporal modes between pulsed and continuous irradiation (Auer et al. 2011).

Materials and methods Cell culture and irradiation conditions For the present experiments, human HeLa cells have been used. Since there are many isolates of HeLa cells grown in

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different laboratories all over the world, our HeLa subline has recently been characterized in detail and the appearance of the karyotype has been demonstrated (Schmid et al. 2009). Cell irradiation with pulsed and continuous proton beams

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Germany), an air gap of approximately 30 lm and the cell carrier foil, 6-lm Mylar. According to a SRIM (www.srim.org) simulation, the cumulative energy loss of 20-MeV protons until they reach the cells was 0.11 MeV. Thus, the LET increases by \0.5%. The delivered energy dose ED can be calculated from ED ¼

Since the layout and methods for irradiation of monolayer of cells have already been reported in detail, only a brief description is given here (Hauptner et al. 2004, 2006; Schmid et al. 2009). About 12 h before the irradiation experiments, cells were trypsinized, placed as monolayer on the carrier foil of the irradiation containers and allowed to adhere. Shortly before irradiation, about half of the culture medium was removed and the irradiation containers were tightly closed by clamping another Mylar foil and fixing a third metal plate. During irradiation at room temperature, the cell chamber was placed vertically in the focal plane of the ion microbeam; that is, the cells were not covered by medium, but residual medium in a reservoir ensures a saturated atmosphere. Typically, the total time required for sample preparation and irradiation, that is, the time outside of the CO2 incubator, is less than 15 min, including about 12 min in the upright position. Under these conditions, we did not observe enhanced numbers of gamma-H2AX foci in unirradiated cells compared to samples that remained in the incubator. In order to demonstrate reproducibility as well as the inter-test variability, two independent experiments I and II for exposure of HeLa cells to 20-MeV protons and as the reference to 70-kV X-rays in experiment I and 200-kV X-rays in experiment II were carried out during separated beam times. Three replicates were exposed to 1 Gy or 5 Gy at either continuous or pulsed proton irradiation mode (LET = 2.65 keV/lm) in each experiment. The pulsed proton beam with up to 105 protons per pulse focused into a spot of approximately 100 9 100 lm2 was prepared for cell irradiation experiments using the ion microprobe SNAKE at the Munich tandem accelerator. The pulse length was 1 ns in both experiments and characterized as described in (Dollinger et al. 2009). By putting single pulses in rectangular pattern, a dose distribution that is very close to a homogenous one was obtained. Roughly 60% of the total dose was delivered by one single proton pulse; the remaining dosage was delivered in smaller amounts by adjacent beam pulses. The homogeneity of the dose distribution was controlled by radiochromic films. During irradiation, the container with the cells attached to the carrier foil was mounted directly behind the beam exit nozzle, where protons leave vacuum through a thin foil. The protons transmit the vacuum exit window, 7.5-lm polyimide foil (Kapton, Goodfellow GmbH, Bad Nauheim,

N LET ; A q

ð1Þ

where LET is the linear energy transfer, q the target density (q = 1 g/cm3 assuming water as a tissue equivalent) and N/A the number N of particles that impinge on an area A. In pulsed mode, the area A is given by the product of the distances in X and Y directions between neighbored pulses and N by the number of particles per pulse. During beam scanning, the total irradiated area and thus the lateral distances were controlled with an accuracy of 1%. The number of protons N per pulse is given by N ¼ I=ðer Þ

ð2Þ

where I is the electric beam current induced by beam pulses repeating at a rate r and e is a unity charge. In our experiments, we used r = 5/128 MHz where 5 MHz is the base frequency of the pulses generated by the system and the pulse frequency was reduced by a factor of 27 = 128. For continuous irradiation, a quadratic homogeneously irradiated field of 2.0 9 2.0 mm2 was prepared with the installed slit system. The number of protons necessary to deliver a certain energy dose ED to this field of area A is given by Eq. 1. The number of protons was adjusted by the irradiation time, t, at a certain beam current, I, which could be measured: N ¼ ðI  tÞ=  e

ð3Þ

Applying a beam current of 70 pA for delivery of 1 Gy approximately 20 ms was necessary, and for 5 Gy approximately 100 ms. Such irradiation times are typically used to irradiate a certain ‘‘voxel’’ of a tumor in common treatment plans of raster scanned beams. The systematic error of the beam current measurement due to leakage currents and instrumental errors was estimated to be \5% (Schmid et al. 2009). For preparation of the pulsed and continuous beams for proton irradiation, the same setup was used. Thus, potential systematic errors were compensated by direct comparison of the results from the pulsed and the continuous beam experiments. X-ray irradiation for dose–response curve To obtain reference X-ray dose–response relationships of gamma-H2AX foci formation, HeLa cells were exposed to 70-kV X-rays during the first beam time (Philips RT100;

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Philips Medical Systems, Eindhoven, The Netherlands) in the dose range up to 2 Gy using a dose rate of approximately 1 Gy per min (10 mA, 2.0 mm Al, HVL = 1.9 Al) and a source-cell distance of 30 cm to a field of 20 cm 9 20 cm. Otherwise, cells were irradiated with 200-kV X-rays (RS225, Gulmay Medical, Surrey, United Kingdom) with a dose rate of 0.88 Gy per minute (15 mA, 0.8-mm Be and 0.5-mm Cu filter) and a source-cell distance of 50 cm using a field of 20 cm 9 20 cm during the second beam time and to obtain reference for the 24-h repair experiment. All X-ray irradiation experiments were performed using the same containers that were used for the proton irradiation at the same temperature (22°C).

minimum reduction in focus detection sensitivity, according to Bocker and Iliakis (2006). Nuclear boundaries and foci were automatically identified in images by a threshold algorithm. Minimum foci size of 5 9 5 (0.25 lm2) pixels and maximum foci size 200 pixel were selected. Cells with pan-nuclear staining or band-like staining of mitotic cells were not analyzed either. The latter cells accounted for 3 out of 15–20 cells per image, which agrees well with data on cell cycle analysis reported in Schmid et al. (2010a); more specifically, we found roughly 31% of cells in G2/S phase as determined using laser scanning cytometry (iCys, Compucyte, USA) with DAPI and CENP-F staining (Schmid et al. 2010a).

Analysis of gamma-H2AX

Statistical analysis

After irradiation, the remaining culture medium was replaced by fresh medium and cells were incubated either for 1 h or for 24 h at 37°C. A standard immunostaining protocol was applied to detect gamma-H2AX according to the method of Schmid et al. (2010a). The cells were permeabilized by three washing steps, and after blocking, the irradiated areas were incubated with 50 ll mouse antiH2AX antibody (Upstate, Charlottesville, Virginia, USA), diluted 1:200 in phosphate-buffered saline (PBS) at 4°C overnight in a humidified incubator. Unbound antibody was removed by several washing and goat-F(ab0 )2-antimouse antibody (1:500, Invitrogen, Karlsruhe, Germany) was applied as a secondary Alexa488-labeled antibody. For analysis, cells were counterstained with 40 ,6-diamidino-2phenylindole (DAPI) and a cover slip was mounted with a drop of Vectashield (Vector Laboratories, Burlingame, California, USA). At least 250 cells were analyzed per sample. Microscopic gamma-H2AX foci were immunolocalized, and images were acquired using epifluorescence sectioning microscopy (Zeiss Axiovert 200 M), a LCI Plan Neofluar 639 objective with 1.3 numerical aperture and a Zeiss AxioCam MRm resulting in a pixel size of 0.10 9 0.10 lm2. The Alexa488 and DAPI digital images were captured serially. Z-stacks of at least 15 different positions were taken from each sample with at least 15 slices per stack (20–25 cells per stack) at different positions. Data were analyzed on a ‘‘per nucleus basis’’ using NIH ImageJ software with a custom software macro FociCount designed to count particles. Results of the analysis are presented as a total foci number per image. The macro was partially adopted from Bhogal et al. (2009) and Cai et al. (2009). In short (see Fig. 1), it operates by evaluating the maximum intensity projection of a three-dimensional stack of images to produce a single two-dimensional intensity image, which is then processed. The images were normalized, and background noise was corrected with

Statistical analysis of the data was performed using SigmaStat software (Sigmaplot Software, ver. 11.0, Jandel Scientific Corp., Erkrath, Germany). To each data set (automated scoring vs. manual scoring), a linear fit was computed. The cosine of the angle between the identity line and the linear regression lines for both data sets was reported. To determine a statistical significance of the difference between samples irradiated with a pulsed proton beam and samples irradiated with a continuous proton beam, a paired t test was used. The results were considered statistically significant at a significance level of p B 0.05. Confidence limits for RBE values, shown in Table 2 as the empirical mean of RBE ± its estimated standard deviation (SD), were calculated using 5,000 independent Monte Carlo simulations. A weighted least-squares approximation was applied to fit the gamma-H2AX foci data with the linear-quadratic function y = y0 ? aD ? bD2 where y is the gamma-H2AX foci yield, D is the irradiation dose, y0 is the number of spontaneous gamma-H2AX foci, and a and b are coefficients (Markova et al. 2007).

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Results Foci scoring, validation of custom ImageJ macro For validation, individual nuclei were extracted from the original images with ImageJ software and analyzed separately each. There was a strong correlation between the number of gamma-H2AX foci per nucleus counted visually and scored using the ImageJ macro FociCount (coefficient of correlation, R2 ± 0.99 and R2 ± 0.99 for slide 1 and 2, respectively) (Fig. 2). The number of gamma-H2AX foci per nucleus was in the range from 10 to 60 foci per nucleus. No significant difference was found between manual and automatic foci counting (see Fig. 2). Based on the good correlation, visual counting can be effectively replaced

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Fig. 1 Main steps describing a custom ImageJ macro for counting gamma-H2AX foci: maximum projection of epifluorescent 3D images in Alexa 488 channel, gray scale images were inverted for better representation purposes (a) and mask of cell nuclei in DAPI channel (b). Foci mask with cell nuclei outline, cell nuclei are numbered (c) and digitally scored foci with cell nuclei mask generated by ImageJ and the customized macro (d)

with automated counting. There is also no obvious difference visible between continuous and pulsed proton irradiation. Reference data from 70- to 200-kV X-rays For experiments with 1-Gy proton irradiation and 1-h repair, the dose–response curve for reference was established in a dose range from 0 to 1.25 Gy with 70-kV X-ray in experiment I (Fig. 3a) and from 0 to 2 Gy with 200-kV in experiment II (Fig. 3b). A weighted least-squares approximation was applied to fit the gamma-H2AX foci data with the linear-quadratic function y = y0 ? aD ? bD2 where y is the gamma-H2AX foci yield, D is the irradiation dose, y0 is the number of spontaneous gamma-H2AX foci, and a and b are coefficients. Fit parameters for experiment I were y0 = 3.3 ± 0.82, a = (32.7 ± 3.1) Gy-1 and b = -(11.3 ± 2.3) Gy-2, and for experiment II y0 = 2.46 ± 1.24, a = (17.0 ± 2.9) Gy-1 and b = -(1.5 ± 1.3) Gy-2, respectively. In the unirradiated controls, that is, the shamtreated samples, the average number of spontaneous foci per cell was 3.80 ± 0.16 in proton experiment I and 2.97 ± 1.51 in proton experiment II. From these dose–response curves, a

Fig. 2 Comparison of manual gamma-H2AX foci scoring with automated scoring with a custom-made ImageJ macros. The automated gamma-H2AX yields are plotted versus the visually scored foci in 45 (sample 1) or 35 cells (sample 2) irradiated with 1-Gy pulsed or continuous protons and fixed 1 h after irradiation, respectively

yield of 24.8 ± 2.0 foci after 1-Gy irradiation in proton experiment I and 19.8 ± 3.0 in proton experiment II is calculated.

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In the other experiment with 5-Gy proton irradiation and 24-h repair time, the dose–response curve for the X-ray reference irradiation was established in a dose range from 0 to 12 Gy with 200-kV X-rays (Fig. 4). Here, the number of gamma-H2AX foci per cell followed a linear-quadratic relationship, as well (fit parameters were y0 = 0.02 ± 1.22, a = (1.22 ± 0.46) Gy-1 and b = (0.05 ± 0.03) Gy-2). In the unirradiated controls, that is, the sham-treated samples, the average number of spontaneous foci per cell was 0.91 ± 0.31. From the dose–response curve, a yield of 7.6 ± 2.0 foci residual after 5-Gy irradiation and 24-h repair was calculated. Data from proton irradiation One-hour repair time For the experiments with a proton dose of 1 Gy of 20 MeV at pulsed and continuous irradiation modes, mean numbers of gamma-H2AX foci per cell of 23.3 ± 2.0 and 26.5 ± 2.5 were obtained in proton experiment I and 19.5 ± 0.4 and 20.0 ± 0.5 in experiment II (Fig. 3a, b), respectively. The mean values (±SD) of gamma-H2AX foci data from two independent proton experiments, with three replicates (each at least 250 analyzed cells), are represented in Table 1. The mean value of 4.1 ± 0.2 for spontaneous induction of gammaH2AX foci in the sham-treated control group is consistent with the corresponding experiment using 70-kV X-ray. The RBE of 20-MeV protons is calculated as the ratio between the dose of the reference radiation (70- and 200-kV X-ray) and the dose of protons (20 MeV) which produced equal response. Accordingly, in experiment I, the corresponding RBE values at pulsed and continuous irradiation modes relative to the reference radiation of 70-kV X-ray are 0.97 ± 0.19 and 1.13 ± 0.21, while in experiment II, the corresponding RBE values relative to the reference radiation of 200-kV X-ray are 1.13 ± 0.098 and 1.16 ± 0.098, respectively. The difference in RBE values for pulsed and continuous proton irradiation is not significant (p = 0.21 and p = 0.16) (Table 2). Twenty-four-hour repair time For experiments with 20-MeV proton irradiation with a dose of 5 Gy at pulsed and continuous irradiation modes, mean numbers of gamma-H2AX foci per cell of 5.84 ± 1.23 and 5.37 ± 0.85 are obtained (Fig. 4). The corresponding RBE values relative to the reference radiation of 200-kV X-ray are 0.99 ± 0.29 for pulsed mode and 0.91 ± 0.23 for continuous irradiation mode. The difference is not significant (p = 0.22) (Table 2).

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Fig. 3 a Proton experiment I: 1 Gy of 20-MeV protons at pulsed or continuous irradiation mode after 1-h repair. Reference dose– response of 70-kV X-ray (open circle) and data for irradiation with 1-Gy continuous (filled square) and pulsed (filled triangle) proton beams. Data are represented as mean values of three independent replicates within an experiment ± SD. At least 250 cells were scored for each data point. Reference dose–response curve was fitted (R2 = 0.99) with a linear-quadratic equation y = y0 ? aD ? bD2, where y is the gamma-H2AX foci yield, D is the irradiation dose, y0 is the number of spontaneous gamma-H2AX foci, and a and b are coefficients. The SD for the control point has also been computed (mean = 3.8, SD = 0.16) and is hidden under the plotted point and can therefore not be seen on the figure. b Proton experiment II: 1 Gy of 20-MeV protons at pulsed or continuous irradiation modes after 1-h repair. Reference dose–response of 200-kV X-ray (open circle) and data for irradiation with 1-Gy continuous (filled square) and pulsed (filled triangle) proton beams. Data are represented as mean values of three independent replicates within an experiment ± SD. At least 250 cells were scored for each data point. Reference dose– response curve was fitted (R2 = 0.99) with a linear-quadratic equation y = y0 ? aD ? bD2, where y is the gamma-H2AX foci yield, D is the irradiation dose, y0 is the number of spontaneous gamma-H2AX foci, and a and b are coefficients

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Discussion When irradiating cell or tissue samples with high-energy protons generated by laser-driven accelerators, the dose is

Fig. 4 5 Gy of 20-MeV protons at pulsed or continuous irradiation modes after 24-h repair. Reference dose–response 200-kV X-ray curve (open circle) and data for irradiation with 5-Gy continuous (filled square) and pulsed (filled triangle) proton beams. Data are represented as mean values of three independent replicates within an experiment ± SD. At least 250 cells were scored for each data point. Reference dose–response curve was fitted (R2 = 0.98) with a linearquadratic equation y = y0 ? aD ? bD2, where y is the gammaH2AX foci yield, D is the irradiation dose, y0 is the number of spontaneous gamma-H2AX foci, and a and b are coefficients. The SD for the control point has also been computed (mean = 0.91, SD = 0.31), but is hidden under the plotted point and can therefore not be seen on the figure

applied in particle pulses of nanosecond duration, while in conventional proton sources, the dose is delivered within milliseconds or seconds. To date, however, the knowledge of the cytological or cytogenetic effects in cells following irradiation where a significant fraction of the full dose at one irradiation site is deposited by a few proton pulses of less than 1-ns duration is still very limited. Ionizing radiation induces a variety of DNA lesions, including single- and double-strand breaks, DNA–protein cross-links and various base lesions (Kobayashi et al. 2008). DNA DSBs are the most serious threats to cells because they can result in loss or rearrangement of genetic information that may lead to cell death and carcinogenesis (Wyman and Kanaar 2006). DSBs can be induced in the genome of eukaryotic cells by endogenous processes associated with oxidative metabolism, errors during DNA replication and various forms of site-specific DNA recombination, as well as by exogenous agents such as ionizing radiation (IR) and chemicals (Iliakis and Cheong 1999; Iliakis et al. 2004). Phosphorylation of H2AX is the most easily observed marker upon induction of DSB by radiation (Bouquet et al. 2006). The number of gamma-H2AX foci formed in this manner has been shown to be directly proportional to the number of DSB formed, and their dephosphorylation has been correlated with repair of DSB (Rothkamm et al. 2007; Rothkamm and Lo¨brich 2003). Additionally, it was shown that the rate of disappearance of radiation-induced gamma-H2AX foci correlates directly with the rate of DNA repair when fewer than 150 DSBs per genome are generated (Bouquet et al. 2006), which corresponds to approximately 6-Gy X-ray radiation. DSBs, if not repaired, may lead either to incorrect segregation during

Table 1 Gamma-H2AX foci per cell yield in three independent experiments after irradiation with pulsed or continuous proton beams (20 MeV) 1-Gy, 1-h Experiment I

1-Gy, 1-h Experiment II

5 Gy, 24 h

Mean, 3 samples

SD

Mean, 3 samples

SD

Mean, 3 samples

SD

Protons pulsed

23.3

±2.0

19.5

±0.4

5.8

±1.2

Protons continuous

26.5

±2.5

20.0

±0.5

5.4

±0.9

Shown are the mean values ± SD of gamma-H2AX foci data from two independent proton experiments, with three replicates (each at least 250 observations) of exposure of HeLa cells to 1 Gy of 20-MeV protons at pulsed or continuous irradiation modes after 1-h repair time. The data set in the third column represents mean values (±SD) of gamma-H2AX foci after 5 Gy of 20-MeV protons at pulsed or continuous irradiation modes after 24-h repair time

Table 2 RBE pulsed versus continuous proton beams (20 MeV) 1-Gy, 1-h Experiment I (SD)

1-Gy, 1-h Experiment II (SD)

5 Gy, 24 h (SD)

Protons pulsed

0.97 ± 0.19

1.13 ± 0.10

0.99 ± 0.29

Protons continuous

1.13 ± 0.21

1.16 ± 0.10

0.91 ± 0.23

Data represent the RBE after irradiation with continuous and pulsed proton beams calculated as ratio to X-ray reference irradiation. Confidence limits for RBE values are empirical mean of RBE ± its estimated SD

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mitosis or to chromosomal loss (Rogakou et al. 1998, 1999). It is known that the expression of gamma-H2AX protein in response to the induction of DNA DSBs is a kinetic event, which occurs within minutes and subsides due to its dephosphorylation (Chowdhury et al. 2005). In the present study, based on gamma-H2AX foci formation, no significant difference in the RBE between pulsed and continuous proton irradiation beams in HeLa cells has been detected at either 1- or 24-h repair time. We have presented dose responses for c-H2AX foci in HeLa cancer cells as measured at 1- and 24-h post-irradiation and have empirically determined that the dose response for the foci was better described by a linear-quadratic function than by a linear one, based on R2 statistics. It was previously shown that fitting depends on the cell-type irradiated and also on the time point after irradiation. Especially for HeLa cells, both linear fitting and linear-quadratic fitting were reported in previous studies (Markova et al. 2007). The negative beta value in the linear-quadratic function may reflect the limitation of the foci quantification at higher doses where the foci tend to overlap and, therefore, are difficult to resolve by the software used (Bladen et al. 2007). However, for the present RBE quantification, we are in the curve range where the dose relationship is still linear and the negative beta coefficient had no significant influence. The absolute numbers of foci formation per cell are well in line with previously published data (Kegel et al. 2007; Schweinfurth et al. 2004). The gamma-H2AX foci label the damage immediately after the induction of DNA DSBs and initiate the cellular repair machinery. For example, Schmid et al. (2010a) showed repair kinetics of DSBs with a fast and a slow exponentially decaying component of gammaH2AX foci intensity in HeLa cells after carbon ion or X-ray irradiation. Thus, the choice of a certain time point for enumerating gamma-H2AX foci decides on covering mainly the fast or slow component of the repair kinetics. However, in the present experiments, there was no evidence for differences in the repair between pulsed and continuous proton irradiation at either 1- or 24-h repair time. Our data support the idea that the amount of DNA damage inflicted is not decidedly affected by the ultrahigh dose rate of a pulsed delivery mode when compared to irradiation times in the millisecond range. Results of the present experiments with longer repair time showed no differences for pulsed or continuous protons after 24-h repair what is well line with our previous experiments (Schmid et al. 2010a) and with the experiments of Roig et al. (2009) in logarithmically growing cells and in 3D colon epithelial cell 24 h after irradiation with protons. Automated analysis as presently used for the quantification of gamma-H2AX foci has significant advantages over manual counting. The automated techniques can be performed in a consistent and reproducible manner and

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should not be compromised by investigator-introduced biases and artifacts (Bocker and Iliakis 2006). However, it should be kept in mind that this method seems to be partly dependent on the respective thresholds or gating values used. We cannot fully dismiss the possibility that the present gamma-H2AX data are affected by the automated quantification method. Especially, at high doses of 2 Gy a potential overlap of adjacent foci may not be accurately separated, resulting in an underestimation of foci counts. However, the influence on counted foci number should be the same when comparing different irradiation qualities. Thus, the relative number should not be affected by this systematic error. It is important to mention that visual inspection might worsen the reproducibility of the results, especially taking into account prolonged manual counting. Overexposed cells with pan-nuclear gamma-H2AX staining, which represent approximately 31% of total cell population, were excluded from the analysis. Such cells were also excluded during manual counting. According to the work of MacPhail et al. (2003a, b), some of these cells were in S phase and G2 phase of the cell cycle. We found that considerably more gamma-H2AX foci were observed when HeLa cells were grown in chamber slides during reference X-ray irradiations (unpublished data). Therefore, in our current experiment, all cells were grown on plastic foils for proton irradiation as well as X-ray reference experiments. This observation is well in line with the findings of Kegel et al. (2007) who determined that the number of gammaH2AX foci was doubled when growing on glass. Comparable RBE values for both pulsed and continuous 20-MeV proton-induced micronuclei data with respect to 70-kV X-rays were also obtained in our recent studies using HeLa cells with a modal number of 62 chromosomes (Schmid et al. 2009) or using keratinocytes of an EpiDermFTTM 3D reconstructed human skin tissue with 46 chromosomes (Schmid et al. 2010b). We extended these studies to the analysis of the clonogenic survival of cells irradiated under both conditions (Auer et al. 2011). This endpoint is often considered to be of utmost relevance when judging irradiation conditions. While the pulsed beam was slightly more efficient in cell killing, the difference was not significant. The RBE determined for both irradiation modes was compatible with the generally assumed RBE of 1.1, which is also applied in proton therapy (Paganetti 2003). Thus, these findings showed no evidence for a different RBE between pulsed and continuous irradiation mode, even when different biological endpoints or cell systems have been considered.

Conclusion No evidence for a different RBE for the induction and loss of gamma-H2AX in HeLa cells between pulsed or

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continuous irradiation modes was found. So far, from this and all our cell and tissue experiments (Schmid et al. 2009), there is no evidence for a substantially different radiobiology coming along with the ‘‘pulsed’’ proton irradiation mode. However, further experiments on animals are required before this irradiation mode can be considered safe for clinical applications of laser-induced protons with dose concepts obtained from our experience with ‘‘continuous’’ proton irradiation. Acknowledgments This work was supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics, by the EU-project EuroDyna, by the Maier Leibnitz Laboratory Munich, by the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0313909), by the Deutsche Forschungsgemeinschaft (DFG, SFB824), BMBF (MOBITUM, 01EZ0826; Kompetenzverbund Strahlenforschung, 03NUK007E) and by the European Union (EU-CARDIORISK, FP7-21103). Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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