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Purpose The scientific community shows great interest in the study of DNA damage induction, DNA damage repair, and the biological effects on cells and cellular systems after exposure to ionizing radiation. Several in silico methods have been proposed so far to study these mechanisms using Monte Carlo simulations. This study outlines a Geant4-DNA example application, named “molecularDNA”, publicly released in the 11.1 version of Geant4 (December 2022). Methods It was developed for novice Geant4 users and requires only a basic understanding of scripting languages to get started. The example includes two different DNA-scale geometries of biological targets, namely “cylinders” and “human cell”. This public version is based on a previous prototype and includes new features, such as: the adoption of a new approach for the modeling of the chemical stage, the use of the standard DNA damage format to describe radiation-induced DNA damage, and upgraded computational tools to estimate DNA damage response. Results Simulation data in terms of single-strand break and double-strand break yields were produced using each of the available geometries. The results were compared with the literature, to validate the example, producing less than 5% difference in all cases. Conclusion: “molecularDNA” is a prototype tool that can be applied in a wide variety of radiobiology studies, providing the scientific community with an open-access base for DNA damage quantification calculations. New DNA and cell geometries for the “molecularDNA” example will be included in future versions of Geant4-DNA.

Purpose: Track structure Monte Carlo (MC) codes have achieved successful outcomes in the quantitative investigation of radiation-induced initial DNA damage. The aim of the present study is to extend a Geant4-DNA radiobiological application by incorporating a feature allowing for the prediction of DNA rejoining kinetics and corresponding cell surviving fraction along time after irradiation, for a Chinese hamster V79 cell line, which is one of the most popular and widely investigated cell lines in radiobiology. Methods: We implemented the Two-Lesion Kinetics (TLK) model, originally proposed by Stewart, which allows for simulations to calculate residual DNA damage and surviving fraction along time via the number of initial DNA damage and its complexity as inputs. Results: By optimizing the model parameters of the TLK model in accordance to the experimental data on V79, we were able to predict both DNA rejoining kinetics at low linear energy transfers (LET) and cell surviving fraction. Conclusion: This is the first study to demonstrate the implementation of both the cell surviving fraction and the DNA rejoining kinetics with the estimated initial DNA damage, in a realistic cell geometrical model simulated by full track structure MC simulations at DNA level and for various LET. These simulation and model make the link between mechanistic physical/chemical damage processes and these two specific biological endpoints.

In the present hadrontherapy scenario, there is a growing interest in exploring the capabilities of different ion species other than protons and carbons. The possibility of using different ions paves the way for new radiotherapy approaches, such as the multi-ions treatment, where radiation could vary according to target volume, shape, depth and histologic characteristics of the tumor. For these reasons, in this paper, the study and understanding of biological-relevant quantities was extended for the case of 4He ion. Approach. Geant4 Monte Carlo based algorithms for dose- and track-averaged LET (Linear Energy Transfer) calculations, were validated for 4He ions and for the case of a mixed field characterised by the presence of secondary ions from both target and projectile fragmentation. The simulated dose and track averaged LETs were compared with the corresponding dose and frequency mean values of the lineal energy, and , derived from experimental microdosimetric spectra. Two microdosimetric experimental campaigns were carried out at the Italian eye proton therapy facility of the Laboratori Nazionali del Sud of Istituto Nazionale di Fisica Nucleare (INFN-LNS, Catania, I) using two different microdosimeters: the MicroPlus probe and the nano-TEPC (Tissue Equivalent Proportional Counter). Main results. A good agreement of and with and experimentally measured with both microdosimetric detectors MicroPlus and nano-TEPC in two configurations: full energy and modulated 4He ion beam, was found. Significance. The results of this study certify the use of a very effective tool for the precise calculation of LET, given by a Monte Carlo approach which has the advantage of allowing detailed simulation and tracking of nuclear interactions, even in complex clinical scenarios.

Accurately modeling the radiobiological mechanisms responsible for the induction of DNA damage remains a major scientific challenge, particularly for understanding the effects of low doses of ionizing radiation on living beings, such as the induction of carcinogenesis. A computational approach based on the Monte Carlo technique to simulate track structures in a biological medium is currently the most reliable method for calculating the early effects induced by ionizing radiation on DNA, the primary cellular target of such effects. The Geant4-DNA Monte Carlo toolkit can simulate not only the physical, but also the physico-chemical and chemical stages of water radiolysis. These stages can be combined with simplified geometric models of biological targets, such as DNA, to assess direct and indirect early DNA damage. In this study, DNA damage induced in a human fibroblast cell was evaluated using Geant4-DNA as a function of incident particle type (gammas, protons, and alphas) and energy. The resulting double-strand break yields as a function of linear energy transfer closely reproduced recent experimental data. Other quantities, such as fragment length distribution, scavengeable damage fraction, and time evolution of damage within an analytical repair model also supported the plausibility of predicting DNA damage using Geant4-DNA.The complete simulation chain application “molecularDNA”, an example for users of Geant4-DNA, will soon be distributed through Geant4.

CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) was the first Italian protontherapy facility dedicated to the treatment of ocular neoplastic pathologies. It is in operation at the LNS Laboratories of the Italian Institute for Nuclear Physics (INFN-LNS) and to date, 500 patients have been successfully treated. Even though proton therapy has demonstrated success in clinical settings, there is still a need for more accurate models because they are crucial for the estimation of clinically relevant RBE values. Since RBE can vary depending on several physical and biological parameters, there is a clear need for more experimental data to generate predictions. Establishing a database of cell survival experiments is therefore useful to accurately predict the effects of irradiations on both cancerous and normal tissue. The main aim of this work was to compare RBE values obtained from in-vitro experimental data with predictions made by the LEM II (Local Effect Model), Monte Carlo approaches, and semi-empirical models based on LET experimental measurements. For this purpose, the 92.1 uveal melanoma and ARPE-19 cells derived from normal retinal pigmented epithelium were selected and irradiated in the middle of clinical SOBP of the CATANA proton therapy facility. The remarkable results show the potentiality of using microdosimetric spectrum, Monte Carlo simulations and LEM model to predict not only the RBE but also the survival curves.

Even though protons show several benefits for the therapy of ocular tumors, the application of a single RBE value in eye treatment planning is still questionable and requires further investigation. In this work, the effects of protons were experimentally measured on 92.1, a specific uveal melanoma cell line. The chosen irradiation position was the middle of the 62 MeV therapeutic Spread-Out Bragg Peak. The normal, ARPE-19 retinal cells were also used to assess the behaviour of healthy tissue exposed to irradiation. Cells were irradiated with single doses ranging from 1 to 6 Gy, and the clonogenic assay was adopted to evaluate cell survival after irradiation. The radiobiological results suggest that proton irradiation can cause significant lethal damages in both normal and cancerous cells. The obtained experimental data from biological models will serve for comparisons with Monte Carlo simulations and LEM II calculations.

Ionising radiation induced DNA damage and subsequent biological responses to it depend on the radiation's track-structure and its energy loss distribution pattern. To investigate the underlying biological mechanisms involved in such complex system, there is need of predicting biological response by integrated Monte Carlo (MC) simulations across physics, chemistry and biology. Hence, in this work, we have developed an application using the open source Geant4-DNA toolkit to propose a realistic "fully integrated" MC simulation to calculate both early DNA damage and subsequent biological responses with time. We had previously developed an application allowing simulations of radiation induced early DNA damage on a naked cell nucleus model. In the new version presented in this work, we have developed three additional important features: (1) modeling of a realistic cell geometry, (2) inclusion of a biological repair model, (3) refinement of DNA damage parameters for direct damage and indirect damage scoring. The simulation results are validated with experimental data in terms of Single Strand Break (SSB) yields for plasmid and Double Strand Break (DSB) yields for plasmid/human cell. In addition, the yields of indirect DSBs are compatible with the experimental scavengeable damage fraction. The simulation application also demonstrates agreement with experimental data of gamma -H2AX yields for gamma ray irradiation. Using this application, it is now possible to predict biological response along time through track-structure MC simulations.

The specific advantages of ion beams for application in tumor therapy are attributed to their different macroscopic and microscopic energy deposition pattern as compared to conventional photon radiation. On the macroscopic scale, the inverted dose profile with a Bragg peak and small lateral scattering allow a better conformation of the dose to the tumor. On the microscopic scale, the localized energy deposition around the trajectory of the particles leads to an enhanced biological effectiveness, typically expressed in terms of the relative biological effectiveness (RBE). Experimental investigations reveal complex dependencies of RBE on many physical and biological parameters, as e.g., ion species, dose, position in the field and cell or tissue type. In order to complement the experimental work, different approaches are used for the characterization of the specific physical and biological properties of ion beams. In a set of two papers, which are linked by activities within a European HORIZON 2020 project about nuclear science and application (ENSAR2), we describe recent developments in two fields playing a key role in characterizing the increased biological effectiveness. These comprise the biophysical modeling of RBE and the microdosimetric measurements in complex radiation fields. This first paper gives a brief introduction into these fields and then focuses on aspects of biophysical modeling of RBE, specifically on semi-empirical approaches that are currently used in treatment planning for ion beam therapy. It summarizes the status and recent developments of the Local Effect Model (LEM) and its conceptual framework and shows examples of model validation using recent experimental data. The model is compared to other approaches, e.g., to the Microdosimetric-Kinetic Model (MKM), that builds the bridge to the experimental microdosimetric work. © Copyright © 2020 Scholz, Friedrich, Magrin, Colautti, Ristić-Fira and Petrović.

The aim of the present study was to investigate effects of pomegranate peel (PP) extract on different human cancer cell lines. MTT was performed to estimate cytotoxic effects of PP extract against HTB140, HTB177, MCF7, HCT116 human cancer cell lines and MRC-5 normal fibroblasts. Clonogenic assay was used to reveal cell survival after the treatment with PP extract. Cell cycle analysis was done using flow cytometry. Wound healing assay was applied to estimate inhibitory effects of PP extract on migration of cancer cells. The results showed that PP extract expressed selective cytotoxicity for cancer cells compared to normal cell line. Analyzed cancer cell lines displayed individual variations in sensitivity to PP extract reflected through changes in clonogenic survival, cell cycle distribution and migration, which may be due to the specific nature of each tested cell line. In conclusion, PP extract exhibits good inhibitory effects on tested cancer cell lines.

Purpose: Investigation of effects on DNA of γ-irradiated human cancer cells pretreated with free radical scavengers is aimed to create reference data which would enable assessment of the relative efficiency of high linear energy transfer (LET) radiations used in hadron therapy, i.e. protons and carbon ions. Materials and methods: MCF-7 breast and HTB177 lung cancer cells are irradiated with γ-rays. To minimize indirect effects of irradiation, dimethyl sulfoxide (DMSO) or glycerol are applied as free radical scavengers. Biological response to irradiation is evaluated through clonogenic cell survival, immunocytochemical and cell cycle analysis, as well as expression of proteins involved in DNA damage response. Results: Examined cell lines reveal similar level of radioresistance. Application of scavengers leads to the rise of cell survival and decreases the number of DNA double strand breaks in irradiated cells. Differences in cell cycle and protein expression between the two cell lines are probably caused by different DNA damage repair mechanisms that are activated. Conclusion: The obtained results show that DMSO and glycerol have good scavenging capacity, and may be used to minimize DNA damage induced by free radicals. Therefore, they will be used as the reference for comparison with high LET irradiations, as well as good experimental data suitable for validation of numerical simulations. © 2019, © 2019 Taylor & Francis Group, LLC.

The advancement of multidisciplinary research fields dealing with ionising radiation induced biological damage – radiobiology, radiation physics, radiation protection and, in particular, medical physics – requires a clear mechanistic understanding of how cellular damage is induced by ionising radiation. Monte Carlo (MC)simulations provide a promising approach for the mechanistic simulation of radiation transport and radiation chemistry, towards the in silico simulation of early biological damage. We have recently developed a fully integrated MC simulation that calculates early single strand breaks (SSBs)and double strand breaks (DSBs)in a fractal chromatin based human cell nucleus model. The results of this simulation are almost equivalent to past MC simulations when considering direct/indirect strand break fraction, DSB yields and fragment distribution. The simulation results agree with experimental data on DSB yields within 13.6% on average and fragment distributions agree within an average of 34.8%. © 2019 Associazione Italiana di Fisica Medica

The aim of this study was to investigate effects of irradiations with the therapeutic proton and carbon ion beams in two non-small cell lung cancers, CRL5876 adenocarcinoma and HTB177 large cell lung carcinoma. The DNA damage response dynamics, cell cycle regulation, and cell death pathway activation were followed. Viability of both cell lines was lower after carbon ions compared to the therapeutic proton irradiations. HTB177 cells showed higher recovery than CRL5876 cells seven days following the treatments, but the survival rates of both cell lines were lower after exposure to carbon ions with respect to therapeutic protons. When analyzing cell cycle distribution of both CRL5876 and HTB177 cells, it was noticed that therapeutic protons predominantly induced G1 arrest, while the cells after carbon ions were arrested in G2/M phase. The results illustrated that differences in the levels of phosphorylated H2AX, a double-strand break marker, exist after therapeutic proton and carbon ion irradiations. We also observed dose- and time-dependent increase in the p53 and p21 levels after applied irradiations. Carbon ions caused larger increase in the quantity of p53 and p21 compared to therapeutic protons. These results suggested that various repair mechanisms were induced in the treated cells. Considering the fact that we have not observed any distinct change in the Bax/Bcl-2 ratio following irradiations, it seemed that different types of cell death were involved in the response to the two types of irradiations that were applied.

Geant4 is a software toolkit for the simulation of the passage of particles through matter. It is used by a large number of experiments and projects in a variety of application domains, including high energy physics, astrophysics and space science, medical physics and radiation protection. Over the past several years, major changes have been made to the toolkit in order to accommodate the needs of these user communities, and to efficiently exploit the growth of computing power made available by advances in technology. The adaptation of Geant4 to multithreading, advances in physics, detector modeling and visualization, extensions to the toolkit, including biasing and reverse Monte Carlo, and tools for physics and release validation are discussed here.

Background and objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions (C-12) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon (C-12) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/mu m. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. Results: Cell viability experiments indicated strong anti-tumour effects of C-12 ions. The analysis of cell cycle showed that C-12 ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/mu m at the dose level of 16 Gy. Pro-apoptotic effects of C-12 ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NF kappa B). At the level of protein expression, the results indicated significant increases of p53, NF kappa B and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NF kappa B mRNA. Interpretation and conclusions: The present results indicated that anti-tumour effects of C-12 ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (gamma H2AX). Immunofluorescent staining visualizes formation of gamma H2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of gamma H2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to gamma-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of gamma H2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of gamma H2AX foci.

In most patients with lung cancer radiation treatment is used either as single agent or in combination with radiosensitizing drugs. However, the mechanisms underlying combined therapy and its impact on different modes of cell death have not yet been fully elucidated. We aimed to examine effects of single and combined treatments with gamma-rays and erlotinib on radioresistant CRL-5876 human lung adenocarcinoma cells with particular emphasis on cell death. CRL-5876 cells were treated with gamma-rays and/or erlotinib and changes in cell cycle, DNA repair dynamics, ultrastructure, nuclear morphology and protein expression were monitored at different time points. To reveal the relationship between types of cell death that arise after these treatments, autophagy was blocked with chloroquine. We found that higher dose of gamma-rays causes G2/M arrest while adding of erlotinib to this treatment decreases the number of cells in S phase. Impact of erlotinib on kinetics of disappearance of irradiation-induced DNA double strand breaks is reflected in the increase of residual gamma-H2AX foci after 24 h. gamma-rays provoke cytoprotective autophagy which precedes development of senescence. Erlotinib predominantly induces apoptosis and enlarges the number of apoptotic cells in the irradiated CRL-5876 cells. Chloroquine improved cytotoxicity induced by radiation and erlotinib, increased apoptosis and decreased senescence in the CRL-5876 cells. The results obtained on CRL-5876 cells indicate significant radiosensitizing effect of erlotinib and suggest that chloroquine in the combination with the above treatments may have an additional antitumor effect in lung adenocarcinoma.

Geant4 is a software toolkit for the simulation of the passage of particles through matter. It is used by a large number of experiments and projects in a variety of application domains, including high energy physics, astrophysics and space science, medical physics and radiation protection. Over the past several years, major changes have been made to the toolkit in order to accommodate the needs of these user communities, and to efficiently exploit the growth of computing power made available by advances in technology. The adaptation of Geant4 to multithreading, advances in physics, detector modeling and visualization, extensions to the toolkit, including biasing and reverse Monte Carlo, and tools for physics and release validation are discussed here.

Fluence, depth absorbed dose and linear energy transfer (LET) distributions of proton and carbon ion beams have been investigated using the Monte Carlo code Geant4 (GEometry ANd Tracking). An open source application was developed with the aim to simulate two typical transport beam lines, one used for ocular therapy and cell irradiations with protons and the other for cell irradiations with carbon ions. This tool allows evaluation of the primary and total dose averaged LET and predict their spatial distribution in voxelized or sliced geometries. In order to reproduce the LET distributions in a realistic way, and also the secondary particles contributions due to nuclear interactions were considered in the computations. Pristine and spread-out Bragg peaks were taken into account both for proton and carbon ion beams, with the maximum energy of 62 MeV/n. Depth dose distributions were compared with experimental data, showing good agreement. Primary and total LET distributions were analysed in order to study the influence of contributions of secondary particles in regions at different depths. A non-negligible influence of high-LET components was found in the entrance channel for proton beams, determining the total dose averaged LET by the factor 3 higher than the primary one. A completely different situation was obtained for carbon ions. In this case, secondary particles mainly contributed in the tail that is after the peak. The results showed how the weight of light and heavy secondary ions can considerably influence the computation of LET depth distributions. This has an important role in the interpretation of results coming from radiobiological experiments and, therefore, in hadron treatment planning procedures.

ELI-Beamlines is one of the pillars of the pan-European project ELI (Extreme Light Infrastructure). It will be an ultra high-intensity, high repetition-rate, femtosecond laser facility whose main goal is generation and applications of high-brightness X-ray sources and accelerated charged particles in different fields. Particular care will be devoted to the potential applicability of laser-driven ion beams for medical treatments of tumors. Indeed, such kind of beams show very interesting peculiarities and, moreover, laser-driven based accelerators can really represent a competitive alternative to conventional machines since they are expected to be more compact in size and less expensive. The ELIMED project was launched thanks to a collaboration established between FZU-ASCR (ELI-Beamlines) and INFN-LNS researchers. Several European institutes have already shown a great interest in the project aiming to explore the possibility to use laser-driven ion (mostly proton) beams for several applications with a particular regard for medical ones. To reach the project goal several tasks need to be fulfilled, starting from the optimization of laser-target interaction to dosimetric studies at the irradiation point at the end of a proper designed transport beam-line. Researchers from LNS have already developed and successfully tested a high-dispersive power Thomson Parabola Spectrometer, which is the first prototype of a more performing device to be used within the ELIMED project. Also a Magnetic Selection System able to produce a small pencil beam out of a wide energy distribution of ions produced in laser-target interaction has been realized and some preliminary work for its testing and characterization is in progress. In this contribution the status of the project will be reported together with a short description of the of the features of device recently developed.

Introduction: Proton radiation offers physical advantages over conventional radiation. Radiosensitivity of human 59M ovarian cancer and HTB140 melanoma cells was investigated after exposure to gamma-rays and protons. Material and methods: Irradiations were performed in the middle of a 62 MeV therapeutic proton spread out Bragg peak with doses ranging from 2 to 16 Gy. The mean energy of protons was 34.88+/-2.15 MeV, corresponding to the linear energy transfer of 4.7+/-0.2 keV/mu m. Irradiations with gamma-rays were performed using the same doses. Viability, proliferation and survival were assessed 7 days after both types of irradiation while analyses of cell cycle and apoptosis were performed 48 h after irradiation. Results: Results showed that gamma-rays and protons reduced the number of viable cells for both cell lines, with stronger inactivation achieved after irradiation with protons. Surviving fractions for 59M were 0.91+/-0.01 for gamma-rays and 0.81+/-0.01 for protons, while those for HTB140 cells were 0.93+/-0.01 for gamma-rays and 0.86+/-0.01 for protons. Relative biological effectiveness of protons, being 2.47+/-0.22 for 59M and 2.08+/-0.36 for HTB140, indicated that protons provoked better cell elimination than gamma-rays. After proton irradiation proliferation capacity of the two cell lines was slightly higher as compared to gamma-rays. Proliferation was higher for 59M than for HTB140 cells after both types of irradiation. Induction of apoptosis and G2 arrest detected after proton irradiation were more prominent in 59M cells. Conclusions: The obtained results suggest that protons exert better antitumour effects on ovarian carcinoma and melanoma cells than gamma-rays. The dissimilar response of these cells to radiation is related to their different features.

Molecular targeted cancer therapy is a promising treatment strategy. Considering the central role of the epidermal growth factor receptor in cell proliferation and survival, there are indications that targeted agents like tyrosine kinase inhibitors, i. e., erlotinib, may enhance the antitumor treatment by radiation. The aim of this study is to analyze the inactivation effects of gamma-rays and to test the radiosensitizing potential of erlotinib on human lung adenocarcinoma cells in vitro. Irradiations were performed with doses ranging from 1 Gy to 8 Gy. In order to increase the radiosensitivity of CRL-5876 lung adenocarcinoma cells, the cells were treated with a clinically relevant concentration of 2 mu M erlotinib. The effects of single and combined treatments were monitored using clonogenic survival, cell viability and proliferation assays at different time points. For the detection and visualization of the phosphorylated histone H2AX (gamma-H2AX), an important biological marker of DNA double-strand break formation, fluorescence inununocytochemistry, was performed. The response to the treatment was monitored at four time points: 30 min, 2, 6, and 24 h. Irradiations with gamma-rays resulted in significant cell inactivation regarding all analyzed biological endpoints. Combined treatments revealed consistent cell inactivation. Moreover, compared to gamma-rays alone, elevated levels of gamma-H2AX foci were observed after pretreatment with erlotinib, indicating radiosensitization through impaired DNA repair.

The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumour cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (gamma-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (gamma-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of gamma-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between gamma-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the database that might promote pulsed sources for medical treatments of malignant growths.