Differences in gamma-H2AX foci formation after irradiation with continuous and pulsed proton beams

Introduction: Classical particle accelerators offer proton pulses of some milliseconds duration. In contrast, the new technology of the high-intensity laser acceleration will produce ultimately shorter particle packages (up to one nanosecond) with substantially lower pulse frequency and higher pulse-dose achievement. Very little is known about the relative biological effectiveness (RBE) of this new beam quality, which could be a possible future application in radiation oncology. In our present study we investigate possible differences based on quantitative analysis of γ-H2AX fluorescence - a known marker of DNA double strand breaks (DSBs). Methods: HeLa cells were irradiated with 1 Gy of 20 MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or at pulsed mode with a single pulse of 1 ns duration. A dose-effect-curve based on five doses of 75 kV x-rays served for reference. The total number of γ-H2AX foci per cell was determined using a self-developed macro (ImageJ, NIH, USA). Results: Quantitative analysis of γ-H2AX fluorescence revealed no significant difference (p=0.16) in yield of foci formation after irradiation with pulsed or continuous proton beams. γ-H2AX data for cell samples exposed to 1 Gy of 20 MeV protons at pulsed or continuous irradiation modes were 23.29 ± 2.04 and 26.54 ± 2.54 foci per cell, respectively. The corresponding RBE values for 20 MeV protons were 0.96 ± 0.18 and 1.13 ± 0.21 (p=0.21) for pulsed and continuous irradiation modes. However, the percentage of foci smaller than 5-10 pixels was slightly decreased and foci tended to cluster after irradiation with pulsed protons. Conclusions: Based on γ-H2AX foci formation no significant difference in the RBE between pulsed and continuous proton irradiation beams in HeLa cells has been detected so far. These results are well in line with our data on micronucleus induction in HeLa cells.