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The complex relationship between linear energy transfer (LET) and cellular response to radiation is not yet fully elucidated. To better characterize DNA damage after irradiations with therapeutic protons, we monitored formation and disappearance of DNA double-strand breaks (DNA DSB) as a function of LET and time. Comparisons with conventional γ-rays and high LET carbon ions were also performed. In the present work, we performed immunofluorescence-based assay to determine the amount of DNA DSB induced by different LET values along the 62 MeV therapeutic proton Spread out Bragg peak (SOBP) in three cancer cell lines, i.e. HTB140 melanoma, MCF-7 breast adenocarcinoma and HTB177 non-small lung cancer cells. Time dependence of foci formation was followed as well. To determine irradiation positions, corresponding to the desired LET values, numerical simulations were carried out using Geant4 toolkit. We compared γ-H2AX foci persistence after irradiations with protons to that of γ-rays and carbon ions. With the rise of LET values along the therapeutic proton SOBP, the increase of γ-H2AX foci number is detected in the three cell lines up to the distal end of the SOBP, while there is a decrease on its distal fall-off part. With the prolonged incubation time, the number of foci gradually drops tending to attain the residual level. For the maximum number of DNA DSB, irradiation with protons attain higher level than that of γ-rays. Carbon ions produce more DNA DSB than protons but not substantially. The number of residual foci produced by γ-rays is significantly lower than that of protons and particularly carbon ions. Carbon ions do not produce considerably higher number of foci than protons, as it could be expected due to their physical properties. In situ visualization of γ-H2AX foci reveal creation of more lesions in the three cell lines by clinically relevant proton SOBP than γ-rays. The lack of significant differences in the number of γ-H2AX foci between the proton and carbon ion-irradiated samples suggests an increased complexity of DNA lesions and slower repair kinetics after carbon ions compared to protons. For all three irradiation types, there is no major difference between the three cell lines shortly after irradiations, while later on, the formation of residual foci starts to express the inherent nature of tested cells, therefore increasing discrepancy between them. [ABSTRACT FROM AUTHOR]

Analysis of elimination of four human radioresistant malignant cell lines to mono-energetic and non mono-energetic incoming carbon ion beams, characterized by different linear energy transfer (LET) qualities is performed. Comparisons with protons from the middle of the therapeutic spread out Bragg peak (SOBP) and reference γ-rays are also included. HTB140 cells were irradiated at five positions, with different LET, along the 62 MeV carbon pristine Bragg peak. To provide reliable reproducibility of irradiations at INFN-LNS, as the carbon Bragg peak is very narrow, precise positioning of samples for desired LET value is complicated. The peak was slightly widened using two ripple filters. After defining irradiation position and LET at the peak itself where cell killing is almost the highest, irradiation position with the same LET value was found within somewhat broadened peak. HTB140, MCF-7, HTB177 and CRL5876 cells were irradiated at the two described positions. Additionally, irradiations in the middle of 62 MeV proton SOBP and reference γ-rays were performed. Doses ranged from 0.5 to 16 Gy. Cell survival and corresponding radiobiological parameters were assessed seven days after irradiations. When moving irradiation position along the carbon Bragg curve, LET rises from 85 to 747 keV/µm, while surviving fraction at 2 Gy (SF2) for HTB140 cells, falls from 0.72 to 0.57 further rising to 0.73 on the distal fall-off part of the curve. Improved cell radiosensitivity is seen for the doses below 4 Gy. Relative biological effectiveness (RBE) increases from 4.56 to 7.69 and drops to 4.23. Almost the highest cell killing LET, being ∼200 keV/µm, is used to irradiate HTB140, MCF-7, HTB177 and CRL5876 cells within the pristine and slightly broadened Bragg peak. After irradiations with protons of the mid SOBP, carbon ions of the pristine and slightly widened Bragg peak RBE ranges for HTB140 cells from 2.08, 4.81 to 7.06, for MCF-7 from 1.70, 3.28 to 4.17, for HTB177 from 1.98, 4.18 to 5.08 and for CRL5876 from 1.33, 2.57 to 3.51. Significant elimination of HTB140 cells is observed along the carbon Bragg curve. The highest one is achieved by LET that is at the level of already reported. For the same LET, mono-energetic carbon ions provide higher cell elimination than the non mono-energetic. For all cell lines, both carbon ion beams, more the monoenergetic one, express stronger killing rate than protons and especially γ-rays. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: To analyse changes of cell inactivation and proliferation under therapeutic irradiation conditions along the proton spread out Bragg peak (SOBP) with particular emphasis on its distal declining edge. Materials and methods: HTB140 cells were irradiated at four positions: plateau, middle, distal end and distal declining edge of the 62 MeV proton SOBP. Doses ranged from 2–16 Gy. They were normalised in the middle of SOBP and delivered following the axial physical dose profile. Survival, proliferation and cell cycle were assessed seven days after irradiation. Results: Moving from proximal to distal irradiation position surviving fractions at 2 Gy (SF2) decreased from 0.88–0.59. Increased radiosensitivity of the cells was noticed for the doses below 4 Gy, resulting in two gradients of cell inactivation, stronger for lower and weaker for higher doses. Relative biological effectiveness (RBE) increased from 1.68–2.84 at the distal end of SOBP. A further rise of RBE reaching 7.14 was at its distal declining edge. Following the axial physical dose profile of SOBP the strongest inactivation was attained at its distal end and was comparable to that at its declining edge. Conclusions: Survival data confirmed very high radioresistance of HTB140 cells. An effect similar to low-dose hyper radiosensitivity (HRS) was observed for order of magnitude larger doses. Better response of cells to protons than to γ-rays was illustrated by rather high RBE. Strong killing ability at the SOBP distal declining edge was the consequence of increasing proton linear energy transfer. [ABSTRACT FROM AUTHOR]

Purpose: To measure the ability of protons and γ-rays to effect cell viability and cell survival of human HTB140 melanoma cells. Materials and methods: Exponentially growing HTB140 cells were irradiated close to the Bragg peak maximum of the 62 MeV protons or with 60 Co γ-rays with single doses, ranging from 8 – 24 Gy. Cell viability using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay was evaluated at 6 h, 24 h, 48 h or 7 days after irradiation and clonogenic survival was assessed at 7 days after irradiation. Cell cycle phase redistribution and the level of apoptosis were evaluated at 6 h and 48 h after irradiation. Results: The study of cell viability as a function of time (cell survival progression) and cell survival, using a clonal assay, demonstrated the considerably stronger inactivation effect of protons compared to γ-rays with a relative biological effectiveness (RBE) of ∼⃒1.64. Cell cycle phase distribution and apoptosis levels with time enabled us to investigate the development and the character of the damage induced by irradiation. Due to the high radio-resistance of HTB140 cells, cell cycle phase redistribution exhibited only a modest cell accumulation in G2/M phase. Protons but not γ-rays induced apoptosis. Conclusions: It appears that protons reduce the number of HTB140 cells by apoptosis as well as by severe DNA damage, while γ-rays eliminate viable cells primarily by the production of irreparable DNA damage. Protons have an increased RBE relative to γ-rays. [ABSTRACT FROM AUTHOR]