DNA repair inhibitors sensitize cells differently to high and low LET radiation

Abstract

The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6-1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G₂/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was observed in the HEK293 by combining α-radiation and inhibitions. ATMi preferentially sensitized cancer cells and normal HEK293 cells to α-radiation. DNA-PKi and ATMi can sensitize cancer cells to X-ray, but the effectiveness was dependent on cancer cells itself. α-radiation reduced proliferation in primary fibroblast without G₂/M arrest.

Figure 1

Reduction of plating efficiency after DDR inhibitor treatment in various cancer cells.

Figure 2

DDR inhibitors sensitized cancer cells to α-radiation. Clonogenic survival for DNA-PKi (A) or ATMi (B). H460, A459, HCT116 and HEK293 cells were treated with 200 nM DNA-PKi and ATMi for 2 h prior to α-radiation, giving doses of 4, 8, 16 Gy, then incubated for 24 h and reseeded for colony formation. Statistical analysis: mean ± SEM, n ≥ 2, 2way ANOVA, Tukey's multiple comparisons test, (*) p < 0.05, (**) p < 0.01.

Figure 3

DDR inhibitors sensitized cancer cells to X-ray radiation. Clonogenic survival after combining DNA-PKi (200 nM) and radiation (A), ATMi (200 nM) and radiation (B). The cells were treated 2 h prior to radiation, then incubated for 24 h and reseeded for colony formation. Statistical analysis: 2way ANOVA, Tukey's multiple comparisons test; (*) p < 0.05, (**) p < 0.01, (****) p < 0.0001.

Figure 4

ATM inhibitor increased the formation of micronuclei after X-ray radiation in A549 (A) and in 22Rv1 (B). A549 and 22Rv1 cancer cell lines were treated with 200 nM DNA-PKi or ATMi for 2 h prior to X-ray exposure of 4 and 8 Gy, then were incubated an additional 24 h (48 h for 22Rv1) before analysis; statistical analysis: 2way ANOVA, Dunnett's multiple comparisons test: mean + SD, n = 2, (*) p < 0.05.

Figure 5

DDR inhibitors increased the formation of micronuclei after α-radiation in several cell lines. The micronucleus frequency for H460 cells (A), 22Rv1 (B), H1299 (C) and A549 (D) and HEK293 (E) treated with 200 nM DNA-PKi or ATMi for 2 h prior to α-radiation with activity of 1.3 kBq/cm² for 2, 4, 8 h, yielding doses of 4, 8 and 16 Gy incubated an additional 24 h (48 h for 22Rv1) before analysis; statistical analysis: 2way ANOVA, Dunnett's multiple comparisons test: mean + SD, n ≥ 2, (*) p < 0.05, (**) p < 0.01.

Figure 6

DDR inhibitors selectively affect cell cycle arrest in G₂/M after X-ray and alpha radiation in various cell lines. Cell cycle distributions for H460 (A), HEK293 (B) and MRC-5 (C) cells after treatment with 200 nM DNA-PKi M3814 and ATMi ADZ-1390 for 2 h prior to X-ray exposure or alpha radiation.