DNA-PKcs^−/− and WT MEF cells were kindly provided by Professor Gloria C. Li from Memorial Sloan-Kettering Cancer Center, USA (3,24,25). SUNE-1 cell line, derived from a patient with undifferentiated NPC (26,27), was a gift from Professor Tiebang Kang at Sun Yat-sen University Cancer Center. WT, DNA-PKcs^−/− MEF cells and human NPC SUNE-1 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin at 37°C in 5% CO₂.

The DNA-PK inhibitor NU7441 (Tocris Bioscience, Bristol, UK) was dissolved in dimethylsulfoxide (DMSO) as a 5 mmol/l stock solution and stored at −20°C. All drugs were added to cells with a final DMSO concentration of 0.5%. Cells were exposed to X-rays generated by a Rad Source RS2000 irradiator (Rad Source Technologies, Buford, GA, USA) operating at 25 mA with a 0.3 mm Al filter and effective photon energy of 160 kV. The dose rate at an irradiation distance of 48.6 cm was 1.31 Gy/min.

Clonogenic cell survival was determined by the colony formation assay as described previously (28). A total of 1.5×10⁵ exponentially growing WT, DNA-PKcs^−/− MEF cells and SUNE-1 cells were supplemented with control or NU7441 (1 µM)-containing medium for 1 h and then exposed to IR at the indicated dose. After IR, the cells were incubated with or without NU7441 for a further 24 h. Cells were harvested and plated in drug-free medium at 20–200 colonies per 100-mm dish in triplicate and left to develop colonies. Approximately 7–10 days later, the medium was discarded and the colonies were fixed and stained with crystal violet. Colonies consisting of >50 cells were counted. Surviving fractions were normalized by the plating efficiency of unirradiated controls.

Cells were exposed to IR of 5 Gy in the presence or absence of 2 µM NU7441 1 h pre-IR and collected at the indicated time. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 3% bovine serum albumin. The cover slips were incubated with anti-γH2AX antibody (1:200; Millipore, Billerica, MA, USA) overnight at 4°C, followed by incubation with conjugated secondary antibody for 1.5 h in the dark. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Roche, Basel, Switzerland). Images were captured using an LSM 710 confocal microscope (Zeiss, Oberkochen, Germany), with foci counted in 30 cells. Quantitative image analysis was performed by ImageJ (29).

To detect the percentage of cells with γH2AX, WT MEF cells were incubated with FBS-free medium to synchronize them into G1 phase. Twelve hours later, the FBS-free medium was removed and cells recovered in medium containing 10% FBS for another 24 h. The cells were irradiated with 5 Gy alone or in combination with 2 µM NU7441. Following treatment, the cells were trypsinized at the indicated time, fixed, treated with 0.2% Triton X-100, and blocked in 3% BSA. The cells were stained with anti-γH2AX antibody (1:100; Cell Signaling Technology, Inc., Danvers, MA, USA) and DAPI. A minimum of 10,000 labeled cells were acquired and analyzed by FlowJo 7.6.1 software (30).

For cell cycle analysis, SUNE-1 cells were exposed to irradiation of 5-Gy in the presence or absence of 2 µM NU7441. Cells were collected at indicated time post-IR and fixed with 70% ethanol. Cellular DNA was labeled with propidium iodide (PI). The cell cycle distribution was determined with 10,000 cells using FACS flow cytometry (Gallios; Beckman Coulter, Inc., Brea, CA, USA). The proportion of cells in different phases was gated and calculated using Flowjo 7.6.1 software.

Total protein and phosphorylated protein levels were analyzed by western blot analysis. Briefly, following treatment, cells were lysed and whole-cell lysates resolved by SDS-PAGE (6 or 10%) and immunoblotted with the indicated antibodies. The following primary antibodies were used for immunoblotting: anti-AKT, anti-phospho-Akt (S473), anti-CHK1, anti-phospho-CHK1 (S345), anti-CHK2, anti-ATR, anti-phospho-ATR (S428), anti-ATM, anti-rabbit/mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.), anti-Ku70, anti-phospho-CHK2 (T68) (Novus Biologicals LLC, Littleton, CO, USA), anti-phospho-ATM (S1981), anti-RAD51 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and anti-β-actin (Millipore).

The data are presented as mean ± SD of at least three independent experiments. The significance of G2/M phase arrest between IR and IR and NU7441 was determined by ANOVA and all other statistical tests were performed using Student's t-test in Sigma Plot 12.5. Significance was defined as p-values of <0.05.

To investigate the role of DNA-PK in response to IR, we utilized WT and DNA-PKcs^−/− MEF cells to determine differences in radiosensitivity in colony formation assay. Irradiation reduced cell survival in DNA-PKcs^−/− MEF cells. NU7441 (1 µmol/l) significantly enhanced radiosensitivity in WT MEF cells, whereas a modest effect was observed in DNA-PKcs^−/− MEF cells (Fig. 1A), supporting that potentiation was attributable to the inhibition of the DNA-PK complex involving in DNA-PKcs gene expression.

We assessed the capacity of DSB repair by counting cellular γH2AX foci, which is routinely performed to assess the amount of DSBs (31). As shown in Fig. 1B and C, IR led to a rapid peak of γH2AX foci formation (within 1 h). γH2AX foci in WT MEF cells gradually resolved to the baseline level at 6 h post-IR but declined with much slower kinetics in DNA-PKcs^−/− MEF cells. Notably, we observed considerable residual γH2AX foci in DNA-PKcs^−/− MEF cells compared to WT MEF cells at 1, 3, and 6 h after treatment (Fig. 1C), indicating that attenuation of the NHEJ pathway impedes DSB repair with highly significant radiosensitivity.

Based on our finding that DSBs are almost completely repaired in WT MEF cells but persist in DNA-PKcs^−/− MEF cells at 6 h post-IR, we evaluated the effect of NU7441 on DSBs at 6 h after irradiation. We detected a striking accumulation of γH2AX foci (5.3±0.3 foci per cell) in WT MEF cells treated with both IR and NU7441 compared to cells treated with IR alone (0.7±0.2 foci per cell; Fig. 2A and C). NU7441 moderately affected the level of radiation-induced γH2AX foci in DNA-PKcs^−/− MEF cells (Fig. 2B and C). In contrast, treatment with NU7441 alone had a negligible effect on γH2AX foci formation (Fig. 2). These results were mirrored by a corresponding decrease in viability (as measured by clonogenic survival assays).

In line with our previous data, deficiency of Ku heterodimer blocks cell cycle progression (9). NU7441 is designed to target the DNA-PK complex and may not only impair DSB repair, but also influence the cell cycle profile. In synchronized WT MEF cells, combined treatment with NU7441 and IR resulted in more γH2AX-positive cells than IR alone. The maximum level of γH2AX expression was detected at 3 h after combined treatment (Fig. 3A and B). Quantification of the percentage of cells with γH2AX in the G2/M phase showed a slight decrease 6 h after combined treatment (Fig. 3C).

Because IR-induced DSBs activate the DDR and amplify the cellular damage signal by phosphorylating cell cycle checkpoints (32), we assessed the role of NU7441 in the activation of DDR-related gene expression. Treatment with NU7441 was associated with a significant increase in phospho-CHK1 levels in response to radiation (Fig. 4A). Combined treatment with NU7441 and IR resulted in the inhibition of CHK2 phosphorylation compared to treatment with IR alone (Fig. 4B). IR-induced Akt activity was inhibited by NU7441 only in WT MEF cells (Fig. 4C). NU7441 potently inhibited the expression of RAD51, a marker of the HR pathway, suggesting that it may be an efficient inhibitor targeting both the NHEJ and HR repair pathways (Fig. 4C).

Clonogenic survival assays revealed that NU7441 markedly increased the cytotoxicity of IR (Fig. 5A). Treatment with IR and NU7441 resulted in more pronounced DSBs compared to treatment with IR alone (Fig. 5B and C) at 6 h or 12 h post-IR, whereas NU7441 alone did not affect γH2AX foci formation.

Our previous study showed that NU7441 alone did not induce cytotoxicity and DSB (25). Combination of IR and NU7441 significantly enhances radiosensitivity and induces more DSB than IR alone. It plays an important role in radiosensitizing cells but treatment of NU7441 alone might not influence the cell cycle progression. We aimed to compare the difference of cell cycle distribution between IR alone and IR + NU7441.

Irradiated cells undergo abnormal cell cycle progression in the absence of a proficient NHEJ factor (33). Cells are more sensitive to radiation in the G2/M phase of cell cycle than any other phases (34). We hypothesized that the addition of NU7441 would augment radiation-induced accumulation of SUNE-1 cells in G2 phase. As expected, the G2/M arrest was observed in SUNE-1 cells when exposed to IR with or without NU7441. The difference between the groups of IR alone and combination of IR and NU7441 was statistically significant at 24 and 48 h. The percentage of cells in the G2/M phase at 24 and 48 h for SUNE1 cells treated with IR and NU7441 was 76 and 65%, respectively; it was 53 and 27%, respectively, for the cells treated with IR alone (Fig. 6A and B).

IR induced cell cycle arrest at 16 h post-irradiation and then the cell cycle arrest gradually released in IR alone group, but the percentage of SUNE-1 cells in G2/M phase kept at a high level in IR and NU7441 group. Combined treatment of IR and NU7441 did not delay the peak of SUNE-1 cells at G2/M arrest but kept the percentage of cells in G2/M phase at a high level until 48 h post-irradiation. Comparison of the ratio of G2/M between IR and IR+NU at the same timing is aimed to examine the effect of NU7441 on IR-induced cell cycle arrest.

Next, we investigated the molecular basis in SUNE-1 cells. Levels of phosphorylated CHK1, ATR, CHK2 and ATM were significantly increased in cells treated with IR and NU7441 compared to cells treated with IR alone, indicating that the G2 cell cycle checkpoint is necessary to allow time to repair DNA damage. Treatment with NU7441 attenuated radiation-induced phosphorylation of Akt. The radiation-induced expression of RAD51 was decreased in the presence of NU7441 (Fig. 6C). Taken together, the results indicate that radiosensitive effect of NU7441 is mediated by cell cycle checkpoints by delaying DNA repair, blocking cell cycle progression, and promoting apoptosis. NPC cells are susceptible to combined treatment with NU7441 and irradiation due to a resulting deficiency in DSB repair and activation of the cell cycle checkpoint. The difference in DSB repair between normal and tumor cells in the suppression of DNA-PK activity offers promise for the development of new radiation-related therapeutic approaches.