Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts

Abstract

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.

Stochastic Simulation of DNA Double-Strand Break Repair by Non-homologous End Joining Based on Track Structure Calculations

“…Several studies suggest that residual damage, represented by a subset of DSB that cannot be quickly repaired or rejoined and persist for a longer time, is an indicator of the cell killing probability (1)(2)(3)(4)(5). This is qualitatively consistent with observations after high-LET radiation, where typically the yield of initially induced DSB is similar to that of photon radiation (6), but the fraction of residual damage is increased as compared to low-LET radiation, in line with the higher cell killing efficiency of high-LET radiation (7).…”

“…Several hypotheses have been discussed concerning the biological interpretation of these two components, and some of these suggest the presence of DNA damages of different severity, which are processed with different kinetics. This hypothesis is also in line with the slower rejoining observed after exposure to high-LET radiations compared to photons (7,(9)(10)(11).…”

High content image cytometry in the context of subnuclear organization

“…At higher doses however (1 and 5Gy), a small dose-dependent number of residual persistent spots were detected. These observations are consistent with other reports on rejoining kinetics (34,35,45). It is plausible that the more persistent foci represent sites, where the repair machinery has difficulty repairing the breaks or where repair proceeds slower (46).…”

Unrepaired clustered DNA lesions induce chromosome breakage in human cells

“…Previously we reported that Si ion-induced DNA damage was repaired in a manner similar to repair of γ-ray-induced damage and that a larger fraction of Fe ion-induced DNA damage was irreparable (11). The difficulties associated with repair of DNA lesions induced by irradiation with Fe ions may be due to either the number of complex DNA lesions produced or the nature of clustered lesions induced.…”