DNA double-strand break induction in Ku80-deficient CHO cells following boron neutron capture reaction

Abstract

Background: Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He) particle and a recoiled lithium nucleus (7Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells.

Methods: Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity.

Results: Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity.

Conclusion: Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.

Figure 1

Cell survival curves following neutron irradiation. Survival curves are shown as a functional dose in Gy for CHO-K1(A) and xrs-5 (B) cells exposed to gamma-rays (open circle) and thermal neutrons. 25ppm of ¹⁰B BPA (black triangle) or boric acid (black square) was incubated on the cells for 1 hr before neutron irradiation. Each point and error bar represents the mean ± SE of three or more independent experiments.

Figure 2

Representative multi color images of nuclear γ-H2AX and 53BP1 foci in the CHO-K1 (A) and xrs-5 (B) cells at 30 and 60 min after the BNCR with 25ppm BPA. Nuclei were stained with DAPI (blue). γ-H2AX (green) and 53BP1 (red) foci are shown in the same photo. The bar represents 20 μm.

Figure 3

Induction and loss of induced nuclear γ-H2AX foci in CHO-K1 (A) and xrs-5 cells (B) determined up to 120 min after 1Gy of thermal neutron or gamma irradiation. In the BNCR study, 25 ppm of BPA (black diamond) and boric acid (black square) was used. The data represent the average number ± SE of γ-H2AX foci per cell in three or more independent experiments.

Figure 4

Representative images of nuclear γ-H2AX foci in the CHO-K1 (A) and xrs-5 (B) cells at 30 min after 2Gy of BNCR with 25ppm BPA compared with 1Gy gamma irradiation. Many large foci were observed in the cells exposed BNCR (open arrows). The bar represents 20 μm.

Figure 5

The histograms of γ-H2AX foci size at 30 min (white bar) and 120 min (black bar) after 1Gy in CHO-K1 (A) and xrs-5 cells (B). The upper graph shows the BNCR study and the lower graph shows the gamma-irradiation study. Each bar shows the average ± SE of three independent experiments.

Figure 6

Induction and loss of induced nuclear 53BP1 foci in CHO-K1 (A) and xrs-5 cells (B) determined up to 120 min after 1Gy of thermal neutron or gamma irradiation. In the BNCR study, 25 ppm of BPA (black diamond) and boric acid (black square) was used. The data represent the average number ± SE of 53BP1 foci per cell in three or more independent experiments.