The human cervical adenocarcinoma cell line HeLa was obtained from the China Center for Type Culture Collection (Wuhan, China). The HeLa/Ku80-siRNAcell line with Ku80 silenced by stable transfection with Ku80-targeted small interfering RNA, and it was confirmed that Ku80 protein expression was suppressed in the Ku80-siRNA stable cell line in our previous study (14). The cells were cultured in Dulbeccos modified Eagles medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin and 50 µg/ml streptomycin. All cells were maintained in a humidified 37°C incubator containing 5% CO₂, fed every 2-3 days with complete medium (containing 10% FBS).

Cells were plated in triplicate on 60-mm dishes at the required density to obtain between 50 and 100 colonies/dish and were allowed to attach for 24 h. HeLa and HeLa/Ku80-siRNA cells were exposed to 0, 2, 3, 4, 6 and 8 Gy X-ray radiation; the cells were cultured for between 10 and 14 days in 5% CO₂ to obtain viable colonies. Colonies were stained with 0.5 ml 0.01% crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) solution at room temperature for 1 h and enumerated using a light microscope (magnification, ×40). A viable colony was defined as having at least 50 cells after 10 days of growth. Colonies were counted from each triplicate sample and presented as the mean ± standard deviation (SD). The surviving fraction of treated cells was normalized to the plating efficiency of control (non-irradiated) cells. The cell survival ratio was obtained by means of clone formation. A one-hit multi-target model was fitted to the cell survival curve to calculate the dose quasithreshold (Dq), mean lethal dose (D0) and radiosensitivity parameter (N value). Cell survival was also plotted as a function of dose and fitted using the linear quadratic model SF=exp(−αD-βD²), where SF is the cell survival, D is the radiation dose, and α and β are constants. The surviving fraction of cells at 2 Gy (SF2) was calculated from the actual data when the cells received 2 Gy irradiation.

HeLa and HeLa/Ku80-siRNA cells at the exponential phase of growth were plated in 96-well plates at a density of 1×10⁴ cells/well and cultured in DMEM with 0, 0.5, 2, 5, 20 and 50 µg/ml cisplatin (Sigma-Aldrich; Merck KGaA) for 4 h. The medium was replaced with cisplatin-free medium. In total, ~10 µl (5 mg/ml) MTT (Sigma-Aldrich; Merck KGaA) was added to the wells when these cells were treated with cisplatin for 48 h and the plates were incubated in a 37°C incubator for 4 h. The blue formazan dye that had formed was dissolved in 150 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA). The absorbance (A) at 570 nm was recorded using an ELISA Multiskan reader (Thermo Fisher Scientific, Inc.). The inhibition rate was calculated as follows: Inhibition rate (%)=(1-A/A_0h) ×100%. In addition, the 50% inhibitory concentration (IC₅₀) was defined as the medicine concentration at which half of all cells were killed.

The two cell lines were plated in 6-well plates at 3×10⁵ cells/well, which corresponded to a density of between 70 and 80% at the time of treatment. Treatment was divided into three parts. In the first part, cells were exposed to 6 Gy 6 MV X-ray radiation (300c Gy/min). To determine the rate of apoptosis and cell cycle distribution, cells were harvested following irradiation for 1, 6, 12, 24, 48 and 72 h. In the second part, cells were first cultured in DMEM with 5 µg/ml cisplatin for 4 h, then the medium was replaced with cisplatin-free medium, and the cells were harvested for flow cytometric analysis following treatment with cisplatin for 6, 12 and 24 h. In the third part, the cells were exposed to X-ray radiation for 6 Gy, then treated with 5 µg/ml cisplatin (exposure for 4 h) for 23, 18 and 12 h following irradiation for 1, 6 and 12 h. All the cells were exposed to X-ray radiation and cisplatin for 24 h in total, prior to harvesting for flow cytometric analysis. The harvested cells were washed with PBS and fixed in 75% ice-cold ethanol overnight at −20°C. Following washing in PBS again, the cells were treated with 50 µg/ml propidium iodide and 1 mg/ml RNase A for 30 min at 37°C. Subsequent analyses of the rate of apoptosis (sub-G₁) and cell cycle distribution were performed using CellQuest software (Version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).

HeLa cells were grown on culture slides for two days until the density increased to 50% at the time of treatment. Following the aforementioned treatments, cells were washed in PBS, fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.5% Triton X-100/PBS for 15 min and blocked in blocking buffer [1% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)] in PBS) for 30 min. Immunostaining was performed using an anti-phosphorylated histone H2AX (γH2AX; Ser¹³⁹) antibody (mouse anti-human, monoclonal antibody, catalog no. 05-636, EMD Millipore, Billerica, MA, USA, at a dilution of 1:500) for 2 h at room temperature in a humidified chamber. Following three 10-min washes, the cells were incubated with the goat anti-mouse Alexa Fluor 568-conjugated secondary antibody (Invitrogen; Thermo Fisher Scientific, Inc.; catalog no. A11004, 1:1,000 dilution). The DNA was stained using a mounting medium with DAPI (Sigma-Aldrich; Merck KGaA). Immunofluorescence microscopic imaging was performed using a LSM 510 laser-scanning confocal microscope (Zeiss GmbH, Jena, Germany). All experiments were performed at least three independent times.

For western blotting analyses, the cell pellets were collected, and the total protein was isolated using M-PER™ mammalian protein extraction reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein concentrations were determined using a bicinchonic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturers protocol. An equal amount of total protein (30 µg) from each lysate was separated by SDS-PAGE (6–10% gel). Subsequently, the separated proteins were transferred onto nitrocellulose membranes, which were then blocked with 5% powdered non-fat milk in Tris-buffered saline containing Tween-20 (TBS-T) for 1 h at 37°C. Samples were incubated with mouse anti-human antibodies against Ku80 (cat no. KuAb-2; NeoMarkers, Inc., Portsmouth, NH, USA; at a dilution of 1:2,000), γH2AX or β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 1:1,000 dilution) at 4°C overnight. Following washing with TBS-T four times for 10 min each, the nitrocellulose membranes were incubated with a goat anti-mouse secondary antibody (cat no. ZB-2305; OriGene Technologies, Inc., Rockville, MD, USA; at a dilution of 1:500) for 1 h at room temperature and washed with TBS-T four times for 10 min each. Immunodetection was performed using the SuperSignal West Fem to Maximum Sensitivity substrate (Pierce; Thermo Fisher Scientific, Inc.).

Results are presented as the mean ± SD of at least three experiments, and a two-tailed unpaired Students t-test was used to compare the statistical significance of the differences in data from the two groups. A one-hit multi-target model SF=1-(1-exp(-D₀/D))^N was fitted to the cell survival curve to calculate Dq, D0 and N using SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA). The linear quadratic model SF=exp(−αD-βD²) was fitted to the cell survival curve and α, β and SF2 were calculated from the actual data using Sigma plot software (version 10.0; Systat Software Inc., Chicago, IL, USA), which was also used for all statistical procedures. P<0.05 was considered to indicate a statistically significant difference.

A dose of <4 Gy is insufficient to induce DSBs, and a dose of >10 Gy is considered to be a lethal dose, prone to induce cell death rather than DSBs; therefore, the majority of researchers select a dose of between 4 and 10 Gy to irradiate cells, and 6 Gy was considered to be a suitable dose for inducing DSBs (15). Following 6 Gy IR for 1, 6, 12 and 24 h, HeLa/Ku80-siRNA and HeLa cells exhibited similar rates of apoptosis (P>0.05), whereas at 48 and 72 h after irradiation, the rates of apoptosis of HeLa/Ku80-siRNA cells were increased compared with those of HeLa cells (48 h, t=6.293, P=0.003; 72 h, t=8.282, P=0.001; Fig. 1A).

Following 6 Gy IR, the proportion of cells in G₂/M phase gradually increased at 1, 6, 12 and 24 h, from ~15% following irradiation for 1 h to almost 70% following irradiation for 24 h. However, no significant differences between the two cell lines were identified at these time points (P>0.05; Fig. 1B and C). The proportion of the two cell lines in G₂/M phase were at their peak at 24 h, but decreased at 48 h and increased again at 72 h. The proportion of HeLa/Ku80-siRNA cells in G₂/M phase was decreased compared with that of HeLa cells at 48 and 72 h, but no significant difference between the two cell lines at these time points following irradiation was identified (P>0.05; Fig. 1B and C).

SPSS and Sigma plot software were used to calculate the radiobiological characteristics from the actual data (Table I). The SF2 and D₀ values of HeLa/Ku80-siRNA cells were decreased compared with that of HeLa cells, which suggests that HeLa/Ku80-siRNAcells exhibited more radiosensitivity than HeLa cells when Ku80 was silenced. The N value and Dq value of HeLa/Ku80-siRNA cells were decreased compared with those of the HeLa cells, indicating that when the shoulder area of HeLa/Ku80-siRNA cells becomes smaller, the ability to repair sub-lethal damage is weakened; in addition, the α value of HeLa/Ku80-siRNA cells also increased significantly, which indicated increased radiosensitivity of cells by inhibiting DNA DSB repair induced by a single ionizing particle energy deposition. However, the β value altered only minimally, which indicated that the inhibition of Ku80 had little influence on the repair of DSBs caused by double ionizing particle energy deposition.

According to the MTT assay results (Fig. 2A), the IC₅₀value of cisplatin for the HeLa cells was (5.83±1.06) µg/ml, whereas that for the HeLa/Ku80-siRNA cells was (5.17±0.99) µg/ml. No significant difference in the IC₅₀ value between the two cell lines was identified (t=0.788, P=0.475), so the concentration of cisplatin applied thereafter was 5 µg/ml, and the two cell lines were exposed to cisplatin for 4 h prior to replacement with fresh DMEM.

The rates of apoptosis of the two cell lines increased slowly following treatment with 5 µg/ml cisplatin for 6, 12 and 24 h (Fig. 2B), and no marked differences in cisplatin sensitivity between the two cell lines at the different time points were identified (P>0.05).

The proportions of cells in the G₂/M phase from the two cell lines treated with 5 µg/ml cisplatin for 0, 6, 12 and 24 h were all ~20%, and no significant differences were identified between the two cell lines at various time points (P>0.05; Fig. 2C).

Nevertheless, following irradiation (6 Gy) and treatment with 5 µg/ml cisplatin (4 h), the rates of apoptosis of the two cell lines at all time points were distinct (Fig. 3A). First, when the two cell lines received 6 Gy IR and cisplatin treatment in combination, they exhibited increased rates of apoptosis compared with the cell lines that only received 6 Gy IR or 5 µg/ml cisplatin alone (P<0.05). Secondly, compared with normal HeLa cells, HeLa/Ku80-siRNAcells exhibited increased rates of apoptosis, particularly following 6 Gy IR for 1 h and treatment with 5 µg/ml cisplatin for 23 h. The rate of apoptosis of HeLa/Ku80-siRNA cells was (11.92±1.02)%, which was markedly increased compared with that of normal HeLa cells (6.56±0.56)%, (t=7.637, P=0.002). The HeLa/Ku80-siRNA cells exposed to 6 Gy irradiation for 6 h and treated with 5 µg/ml cisplatin for 18 h exhibited increased apoptosis compared with normal HeLa cells that received the same treatment; their rates of apoptosis were (7.44±0.84)% and (5.43±0.42)%, respectively (t=3.707, P=0.021). Similar results were obtained when the two cell lines were exposed to 6 Gy irradiation for 12 h and treated with 5 µg/ml cisplatin for 12 h. The rates of apoptosis of HeLa/Ku80-siRNA and HeLa cells were (7.08±0.63) and (5.01±0.49)%, respectively (t=4.492, P=0.011). These results revealed that, following Ku80 inhibition and 6 Gy IR, the earlier the cisplatin was administered, the more apoptotic HeLa cells appeared.

Fig. 3B and C present the difference in the proportion of cells in the G₂/M phase in the two cell lines following receipt of different treatments for 24 h. The proportion of G₂/M phase cells was 70% following a single X-ray irradiation (6 Gy) for 24 h, whereas the proportion was only ~20% following the separate administration of 5 µg/ml cisplatin for 24 h, similar to the cells without any treatment, which suggested that cisplatin treatment for 24 h does not cause HeLa cells to undergo G₂/M cell cycle arrest. Following 6 Gy IR for 1 h plus cisplatin treatment for 23 h, the proportion of G₂/M phase cells remained close to 20%. Following 6 Gy IR for 6 h plus cisplatin treatment for 18 h, the proportion of cells in G₂/M phase increased to 30%, whereas following 6 Gy IR for 12 h plus cisplatin treatment for 12 h, the proportion of cells in G₂/M phase further increased to ~50%. However, the differences between the two groups were not significant (P>0.05).

At 24 h after treatment with 6 Gy IR and/or cisplatin, alterations in γH2AX phosphorylation were observed. There was no difference between the two cell lines following 6 Gy IR alone and cisplatin treatment alone; the two cell lines had few γH2AX foci remaining. However, when treated with 6 Gy IR and cisplatin, HeLa cells with Ku80 inhibition exhibited an increased number of remaining γH2AX foci, particularly following 6 Gy IR for 1 h plus cisplatin for 23 h, as presented in Fig. 4A and B. Western blotting also revealed the same results (Fig. 4C).