Repair of HZE-Particle-induced DNA double-strand breaks in normal human fibroblasts

Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Radiation Research (Impact Factor: 2.45). 05/2008; 169(4):437-46. DOI: 10.1667/RR1165.1
Source: PubMed

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 γ 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 γ-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 γ 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 γ-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of γ-H2AX. Spatial co-localization analysis showed that unlike γ-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 γ 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.

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    • "Our results support previous studies which reported differential ATM kinase activation with varying radiation qualities [35]. It has been previously reported that DSB repair capacity of cells decrease with increasing LET [36]. The higher biological effectiveness of high LET radiation compared to ␥-rays is often explained theoretically by increased clustered ionizations within a small volume of the scale of the DNA helix and across the path of the particle increasing with LET [37]. "
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    ABSTRACT: Upon induction of DNA damage by ionizing radiation (IR), members of the phosphatidylinositol 3-kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM), DNA-PKcs, and ATM- and Rad3-related (ATR) maintain genomic integrity by mounting DNA damage response (DDR). Recent reports suggest that activation of ATM and ATR are oppositely regulated by the length of single stranded overhangs generated during end processing by nucleases at the break sites. These stretches of single stranded overhangs hold the clue for the transition from ATM to ATR signaling at broken DNA ends. We investigated whether differential processing of breaks induced by low and high LET radiation augments the phenomenon of switching from ATM to ATR kinase and hence a concomitant NHEJ to HR transition at the sites of DNA damage. 82-6 human fibroblasts were irradiated with 1 or 2Gy of γ-rays and particle radiation of increasing LET in order to increase the complexity and variability of DNA double strand breaks (DSB) structures. The activation kinetics of ATM and ATR kinases along with their downstream substrates were determined utilizing Western blotting and immunofluorescence techniques. Our data provide evidence of a potential switch from ATM to ATR kinase signaling in cells treated with γ-rays at approximately 2h post irradiation, with induction and completion of resection denoted by Rad51 foci resolution kinetics and observed with a significant decline of phosphorylated ATR kinase 8h after IR. On the other hand, irradiation with high LET 600MeV/u (56)Fe (180keV/μm) and 170MeV/u (28)Si (99keV/μm) particles show a similar Rad51 foci decay kinetics, however, exhibiting prolonged resection, evident by the persistent phosphorylated ATM and ATR kinase until 24h post irradiation. This residual effect, however, was significantly reduced for 250MeV/u (16)O particles of moderate LET (25keV/μm) and absent for γ-rays. Hence, our results support the hypothesis that the transition from ATM to ATR signaling at DNA break sites is extended for longer periods of time, indicated by sustained resection due to the complex type of damage induced, a hallmark of high LET radiation, which may contribute to its increased biological effectiveness.
    DNA repair 11/2013; 12(12). DOI:10.1016/j.dnarep.2013.10.004 · 3.36 Impact Factor
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    • "These proteins, which appear as foci when detected by immunofluorescence staining, are considered to be surrogate markers of DSBs. Using these techniques, experiments reveal important differences in the characteristics of the spatial distribution and repair kinetics of DSBs induced by low-and high-LET radiations (Prise et al 2001, Desai et al 2005, Asaithamby et al 2008). Previously, we estimated the number and distribution of DSBs within nuclei, using amorphous track calculations of the radial dose combined with chromosome models simulated by random walk (RW) (Cucinotta et al 1997, Ponomarev et al 2010, Costes et al 2007). "
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    ABSTRACT: The biological effects of high-linear energy transfer (LET) radiation are different from those caused by low-LET radiation due to the difference in the patterns of energy deposition in cells. In this work, we studied the role of the track structure in the spatial distribution of radiation-induced double-strand breaks (DSBs). In the first part, the irradiation of a cubic volume of 12 µm of side by 300 MeV protons (LET ∼0.3 keV µm(-1)) and by 1 GeV/amu iron ion particles (LET∼150 keV µm(-1)) was simulated with the Monte Carlo code RITRACKS (relativistic ion tracks) and the dose was calculated in voxels of different sizes. In the second part, dose calculations were combined with chromosomes simulated by a random walk (RW) model to assess the formation of DSBs. The number of DSBs was calculated as a function of the dose and particle fluence for 1 GeV protons, 293 MeV/u carbon, and 1 GeV/u iron particles. Finally, the DSB yield was obtained as a function of the LET for protons, helium, and carbon. In general, the number and distribution of calculated DSBs were similar to experimental DNA repair foci data. From this study, we concluded that a stochastic model combining nanoscopic dose calculations and chromosomes simulated by RWs is a useful approach to study radiation-induced DSBs.
    Physics in Medicine and Biology 09/2013; 58(18):6393-6405. DOI:10.1088/0031-9155/58/18/6393 · 2.92 Impact Factor
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    • "As expected, the voxels with the highest dose correspond to those with ion track cores and electron track ends. Additionally, the experiment and simulation results obtained are generally in good agreement with experimental data by Asaithamby et al. [41]. This indicated that more severe damage may be found in nuclei irradiated by high-LET radiation. "
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