The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.

Summary

The biological effects of energetic heavy ions are attracting increasing interest for their applications in cancer therapy and protection against space radiation. The cascade of events leading to cell death or late effects starts from stochastic energy deposition on the nanometer scale and the corresponding lesions in biological molecules, primarily DNA. We have developed experimental techniques to visualize DNA nanolesions induced by heavy ions. Nanolesions appear in cells as “streaks” which can be visualized by using different DNA repair markers. We have studied the kinetics of repair of these “streaks” also with respect to the chromatin conformation. Initial steps in the modeling of the energy deposition patterns at the micrometer and nanometer scale were made with MCHIT and TRAX models, respectively.

Background

Treatment of moving target volumes with scanned particle beams benefits from treatment planning that includes the time domain (4D). Part of 4D treatment planning is calculation of the expected result. These calculation codes should be verified against suitable measurements. We performed simulations and measurements to validate calculation of the film response in the presence of target motion.

Methods

All calculations were performed with GSI's treatment planning system TRiP. Interplay patterns between scanned particle beams and moving film detectors are very sensitive to slight deviations of the assumed motion parameters and therefore ideally suited to validate 4D calculations. In total, 14 film motion parameter combinations with lateral motion amplitudes of 8, 15, and 20 mm and 4 combinations for lateral motion including range changes were used. Experimental and calculated film responses were compared by relative difference, mean deviation in two regions-of-interest, as well as line profiles.

Results

Irradiations of stationary films resulted in a mean relative difference of -1.52% ± 2.06% of measured and calculated responses. In comparison to this reference result, measurements with translational film motion resulted in a mean difference of -0.92% ± 1.30%. In case of irradiations incorporating range changes with a stack of 5 films as detector the deviations increased to -6.4 ± 2.6% (-10.3 ± 9.0% if film in distal fall-off is included) in comparison to -3.6% ± 2.5% (-13.5% ± 19.9% including the distal film) for the stationary irradiation. Furthermore, the comparison of line profiles of 4D calculations and experimental data showed only slight deviations at the borders of the irradiated area. The comparisons of pure lateral motion were used to determine the number of motion states that are required for 4D calculations depending on the motion amplitude. 6 motion states per 10 mm motion amplitude are sufficient to calculate the film response in the presence of motion.

Conclusions

By comparison to experimental data, the 4D extension of GSI's treatment planning system TRiP has been successfully validated for film response calculations in the presence of target motion within the accuracy limitation given by film-based dosimetry.

DNA double-strand breaks (DSBs) can induce chromosomal aberrations and carcinogenesis and their correct repair is crucial for genetic stability. The cellular response to DSBs depends on damage signaling including the phosphorylation of the histone H2AX (γH2AX). However, a lack of γH2AX formation in heterochromatin (HC) is generally observed after DNA damage induction. Here, we examine γH2AX and repair protein foci along linear ion tracks traversing heterochromatic regions in human or murine cells and find the DSBs and damage signal streaks bending around highly compacted DNA. Given the linear particle path, such bending indicates a relocation of damage from the initial induction site to the periphery of HC. Real-time imaging of the repair protein GFP-XRCC1 confirms fast recruitment to heterochromatic lesions inside murine chromocenters. Using single-ion microirradiation to induce localized DSBs directly within chromocenters, we demonstrate that H2AX is early phosphorylated within HC, but the damage site is subsequently expelled from the center to the periphery of chromocenters within ∼20 min. While this process can occur in the absence of ATM kinase, the repair of DSBs bordering HC requires the protein. Finally, we describe a local decondensation of HC at the sites of ion hits, potentially allowing for DSB movement via physical forces.

Background

Scanned ion beam therapy of intra-fractionally moving tumors requires motion mitigation. GSI proposed beam tracking and performed several experimental studies to analyse the dosimetric precision of the system for scanned carbon beams.

Methods

A beam tracking system has been developed and integrated in the scanned carbon ion beam therapy unit at GSI. The system adapts pencil beam positions and beam energy according to target motion.

Motion compensation performance of the beam tracking system was assessed by measurements with radiographic films, a range telescope, a 3D array of 24 ionization chambers, and cell samples for biological dosimetry. Measurements were performed for stationary detectors and moving detectors using the beam tracking system.

Results

All detector systems showed comparable data for a moving setup when using beam tracking and the corresponding stationary setup. Within the target volume the mean relative differences of ionization chamber measurements were 0.3% (1.5% standard deviation, 3.7% maximum). Film responses demonstrated preserved lateral dose gradients. Measurements with the range telescope showed agreement of Bragg peak depth under motion induced range variations. Cell survival experiments showed a mean relative difference of -5% (-3%) between measurements and calculations within the target volume for beam tracking (stationary) measurements.

Conclusions

The beam tracking system has been successfully integrated. Full functionality has been validated dosimetrically in experiments with several detector types including biological cell systems.