The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions

Abstract

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/μm) and C ions (290 MeV; 50 keV/μm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.

FIG. 1

Colony survival assay. Clonogenic survival curves are shown for Chinese hamster cell lines, wild-type V79 and AA8 (panel A), HR-deficient irs1 and irs1SF (panel B). NHEJ-deficient XR1 and V3 (panel C) after irradiation with γ rays or 200 MeV protons and 290 MeV C ions with 6 cm SOBP. The curves were fitted by LQ model and the error bars represent SD.

FIG. 2

Relative biological effectiveness (RBE) and α values. RBE values at 10% (RBE_0.1) survival of protons and C ions and α values of all radiation types examined were calculated according to LQ model. Panel A: RBE_0.1 values of protons (●) and C ions (■) in wild-type V79 and AA8; HR-deficient irs1 and irs1SF; and NHEJ-deficient XR1 and V3 cell lines. Panel B: α values of γ rays (▼), protons (●) and C ions (■) in wild-type V79 and AA8 cells, HR-deficient irs1 and irs1SF and NHEJ-deficient XR1 and V3 cells. Student’s t test: ^##P < 0.01, ^#P <0.05 compared with wild-type and repair-deficient cells; **P < 0.01, *P < 0.05 compared with C ions. Error bars represent SD.

FIG. 3

Quantification of γ-H2AX foci. Time response of γ-H2AX foci after irradiation with 2 Gy of γ rays ( ), protons (●) and C ions (■). The percentage of foci per cell was plotted by normalizing the numbers at 30 min as 100% after subtracting the number of foci in 0 Gy irradiated cell. The γ-H2AX foci formation and dissolution in wild-type cell lines AA8 and V79 (panel A); HR-deficient cell lines irs1 and irs1SF (panel B); and NHEJ-deficient cell lines XR1 and V3 (panel C). Student’s t test: **P < 0.01, *P < 0.05 of C ions compared with protons; no significant differences were observed between C ions and γ rays. Error bars represent SD.

FIG. 4

Comparison of cell lines. Panel A: Survival curves of cell lines were plotted by each radiation type; γ rays, protons and C ions. The wild-type V79 (■) and AA8 (□) cells were most resistant, followed by HR-deficient irs1 ( ) and NHEJ-deficient XR1 (▼); HR-deficient irs1SF ( ) and NHEJ-deficient V3 (△) were found to be the most sensitive to all radiation types examined. Panel B: Formation and dissolution of γ-H2AX foci after irradiation with γ rays, protons and C ions. Student’s t test: **P < 0.01, *P < 0.05 of V3 compared with wild-type cell; ^##P < 0.01, ^#P < 0.05 of V3 compared with HR-deficient irs1SF cell. Panel C: Chromosome aberrations induced by 1 Gy γ ray, protons and C ions after 3 (filled column) and 16 h (gradient column) irradiations were plotted in each cell line examined after subtracting the number of 0 Gy irradiated cells. Student’s t test: **P < 0.01, *P < 0.05. Error bars represent SD.

FIG. 5

Chromosome and chromatid-type aberration at 16 h after irradiation. Panel A: The number of chromosome type aberrations and more frequently occurring chromosome type (break and ring with tail) aberrations per cell were plotted in each cell line after irradiation with γ rays (white column), protons (gray column) and C ions (black column). Panel B: The number of chromatid-type aberrations and more frequently occurring chromatid-type (break and triradial) aberrations per cell were plotted in each cell line after irradiation with γ rays (white column), protons (gray column) and C ions (black column). Student’s t test: **P < 0.01, *P < 0.05. The error bars represent SD.