Dulbecco's modified Eagle's medium (DMEM) with high glucose and 10% fetal calf serum (FCS) was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) assay kits were obtained from Beijing Modern Gold Biotechnology Co., Ltd. (Beijing, China). The endonucleases and ligase enzymes used for molecular cloning technology were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Lipofectamine 2000, G418, CB1954 and MTT were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany).

The nfsB sequence was amplified by PCR from the Escherichia coli k12 genome using the following primers: 5′-CGGGATCCATGGATATCATTTCTGTCG-3′ and 5′-CGGAATTCTTACACTTCGGTTAAGGTG-3′. This sequence contained BamHI and EcoRI sites for insertion into the pcDNA3 plasmid. Following endonuclease digestion and DNA sequencing, the successful insertion of the nfsB gene was confirmed and was re-cloned into the pcDNA3 plasmid. The following the following thermocycling conditions: 94°C for 3 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 72°C for 5 min, 35 cycles. The resulting plasmid was termed pcDNA3-nfsB.

HeLa cells were maintained in DMEM, supplemented with 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin and were incubated at 37°C in a 5% CO₂ atmosphere. All of the cultures were demonstrated to be free of mycoplasma. The HeLa cells were transfected with the pcDNA3 plasmid and pcDNA3-nfsB with G418 selection at a concentration of 400 µg/ml. Following 1 month of selection, several independent clones were selected and the NTR mRNA and protein expression levels were determined. Subsequently, the concentration of G418 was reduced to 200 µg/ml.

Total RNA was extracted from the pcDAN3-nfsB-HeLa, pcDNA3-HeLa and non-transfected control HeLa cells using TRIzol reagent. RT was performed according to the manufacturer's protocol (Transgen Biotech Co., Ltd. (Beijing, China). The reaction mixture was placed in a 42°C water bath for 50 min, the temperature was increased to 70°C for 15 min to inactivate TransScript RT and synthesise cDNA. For nfsB, the following primers were used: Sense, 5′-ATGGACGATGTCTGGCTGAA-3′ and antisense 5′-AACGCTGTGATGACCTACCG-3′. Endogenously expressed human β-actin mRNA was used as an internal control with the following primers: Sense 5′-GGCATCCTCACCCTGAAGTA-3′ and antisense 5′-GGGGTGTTGAAGGTCTCAAA-3′. A DNA product of 208 base pairs (bp) was then amplified. A total of 500 ng cDNA were used. The following the following thermocycling conditions: 94°C for 3 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and 72°C for 5 min, 35 cycles. The PCR products of nfsB and β-actin were separated by electrophoresis on 1.5% agarose gel. The DNA bands were visualized and analyzed by staining with ethidium bromide. The proteins were collected from the three cell lines and SDS-PAGE was performed with a 10% acrylamide separating gel and 4% acrylamide, 10 µg protein were loaded per lane. The samples were prepared in a Tris-glycine buffer with 1% SDS at pH 8.8. Electrophoresis was conducted at a current of 10 mA for 5 h in electrophoretic Tris-glycine buffer with 0.1% SDS. Following electrophoresis, the gel sheets were stained for proteins with 0.25% coomassie brilliant blue-R250 and then were destained with 10% acetic acid and 20% methanol. Next, the protein bands were visualized and analyzed.

The pcDNA3-nfsB-HeLa, pcDNA3-HeLa and HeLa cells were incubated for 96 h and counted every 12 h. The number of cells was determined using trypan blue staining. Subsequently, a cell growth curve was generated and the differences between the three cell lines were compared. pcDNA3-nfsB-HeLa, pcDNA3-HeLa and HeLa cells of different concentrations were irradiated with different doses of γ-rays. The cells were subsequently incubated for 12 days and the survival fraction of the cells was detected by a colony-formation assay. The cell survival curve was generated using SPSS software, version 15.0 (SPSS, Inc., Chicago, IL, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA, USA).

Hoechst 33258/PI fluorescent vital stain was used to quantitatively determine the percentage of apoptotic cells. Briefly, the cells were washed with PBS, and Hoechst 33258 (15 µg/ml) was added. Following a 50 min incubation at ambient temperature, PI (10 µg/ml) was added, followed by another incubation for 10 min. The samples were analyzed using fluorescence microscopy. The nuclei of apoptotic cells appeared bright fluorescent blue, whereas the nuclei of necrotic cells were bright fluorescent red. The nuclei of normal cells appeared only weakly fluorescent blue (Fig. 1). This method was used to analyze the effects of the NTR/CB1954 suicide gene system in combination with γ-ray treatment on HeLa cells. Bradford assay (Leagene Biotech Co., Ltd., Beijing, China) was used for quantification.

The three cell lines were plated at a density of 1.0×10⁶ cells/well in a 6-well plate. Following incubation with CB1954 at various concentrations (0, 12.5, 25, 37.5 and 50 µmol/l) for 36 h, the cells were collected, washed twice with ice-cold PBS (pH 7.4), fixed with 70% alcohol for a minimum of 18 h and then stained with PI (50 µg/ml) in the presence of 20 µg/ml RNAse A for a minimum of 30 min prior to flow cytometric analysis. The data were analyzed with ModFit version 3.2 (Verity Software House, Inc., Topsham, ME, USA) and CellQuest version 7.5.3 (BD Biosciences, Franklin Lakes, NJ, USA).

pcDNA3-nfsB-HeLa cells in the exponential growth phase were incubated for 24 h at 37°C and CB1954 was added at concentrations of 0, 12.5 and 25 µmol/l. After 24 h the cells were irradiated at doses of 0, 2, 4 and 6 Gy with Co⁶⁰ γ-rays. Subsequently, the cells were incubated at 37°C with an atmosphere of 5% CO₂ for 48 h. Detection of apoptosis was performed using Hoechst/PI fluorescent vital staining.

Cells were cultured in 25 cm² flasks. Single suspension was obtained, the cells were diluted to 1×10⁴/ml and 1×10³/ml. Next, 0.2, 0.4, 0.6, 0.8 and 1 ml from the concentration of 1×10³/ml, and a cell suspension of 0.3, 0.8 and 1 ml from the concentration of 1×10⁴/ml, all of which were cultured in 50 cm² flasks, and the number of cells were 200, 400, 600, 800, 1,000, 5,000, 8,000 and 10,000 from low to high. Following the addition of CB1954 at concentrations of 0, 12.5 or 25 µmol/l, the cells were irradiated at doses of 0, 0.5, 1, 1.5, 2, 4, 6 and 8 Gy with Co⁶⁰ γ-rays, according to the number of cells from low to high. Next, the cells were incubated at 37°C with 5% CO₂ atmosphere for 2–15 days. Following this, the medium was removed, the cell culture plate was washed with PBS, and the cells were fixed in methanol for 15 min. The colonies were then stained with a solution of crystal violet for 20 min. Colonies that were visible to the naked eye were counted and the cell survival fraction (SF) was calculated using the following formulas: Plating efficiency (PE) = cell colonies formed / cells inoculated and SF = cell colonies formed / cells innoculated × PE.

Differences between treatment groups were determined using Student's t-test and one-way analysis of variance. Statistical analysis was performed using Statistics Analysis System version 8.0 (SAS Institute, Inc., Cary, NC, USA). Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

NTR mRNA expression level was determined using RT-PCR. In the pcDNA3-nfsB-HeLa cells, a 381 bp DNA fragment of nfsB was amplified, whereas no mRNA was detected in either the control pcDNA3-HeLa cells or in the non-transfected HeLa cells. A 208 bp human β-actin mRNA fragment, which was used as an internal control, was amplified in the three different cell lines (Fig. 2A). NTR protein expression level was determined using SDS-PAGE. In the pcDNA3-nfsB-HeLa cells, a 24 kDa protein was identified, which coincided with the size of NTR. However, in the control pcDNA3-HeLa cells and in the non-transfected HeLa cells, no corresponding protein band was detected (Fig. 2B). These observations indicated that NTR was stably and correctly expressed in the transfected HeLa cells.

The cell counting method was used following the transfection with the nfsB gene to observe the growth and proliferation of the three groups of cells. No significant differences were observed between the pcDNA3-nfsB-HeLa cells and the control cells (Fig. 3), which indicated that the nfsB gene did not affect the growth and proliferation of HeLa cells. Additionally, the apoptotic activity of the pcDNA3-nfsB-HeLa, pcDNA3-HeLa and non-transfected HeLa cells was examined following γ-ray irradiation (6 Gy) using Hoechst 33258/PI fluorescent vital staining. No significant differences were observed among the three different groups of cells in terms of apoptotic rate over time (0–72 h) (Fig. 4A). The cell survival fraction in the three groups of cells was also quantified. Following treatment with various doses of γ-ray irradiation, the cells were incubated for 12 days. No significant differences were identified among the three groups of cells in terms of the cell survival fraction (Fig. 4B). These observations demonstrated that transfection with the nfsB gene alone did not have an influence on the reaction of the cells to γ-rays.

The rate of apoptosis of pcDNA3-nfsB-HeLa, pcDNA3-HeLa and HeLa cells treated with CB1954 was determined using Hoechst 33258/PI fluorescent staining. Apoptosis was observed in the pcDNA3-nfsB-HeLa cells 12 h following treatment with CB1954 (Fig. 5A). No apoptosis was observed in the pcDNA3-HeLa cells or in the wild-type HeLa cells. The rate of apoptosis of the pcDNA3-nfsB-HeLa cells peaked at 36 h of treatment (Fig. 5A). Additionally, an MTT assay and flow cytometry were used to determine cell viability and apoptotic rates, with the observations consistent with the previous experiments (data not shown). The pcDNA3-nfsB-HeLa, pcDNA3-HeLa and non-transfected HeLa cells were treated with various concentrations of CB1954 (0, 12.5, 25 and 50 µmol/l) for 36 h. Flow cytometry was then used to analyze the number of cells in the different cell cycle phases, and the results demonstrated that the NTR/CB1954 suicide gene system primarily affected HeLa cells in S phase. When the concentration of CB1954 was 50 µmol/l, the percentage of cells in S phase was 70.51%, whereas no clear alterations were observed in the control cell groups, the pcDNA3-HeLa and HeLa cells (Fig. 5B).

pcDNA-nfsB-HeLa cells were treated with CB1954 at various concentrations (0, 12.5 and 25 µmol/l) for 16 h prior to γ-ray irradiation (0, 2, 4 and 6 Gy), and the rate of apoptosis was determined using Hoechst 33258/PI fluorescent vital staining. The cytotoxicity was proportional to the radiation dose in pcDNA-nfsB-HeLa cells and the CB1954 concentration used. Cytotoxicity increased with the combination of radiation and CB1954 at 12.5 and 25 µmol/l. For example, when the concentration of CB1954 was 12.5 µmol/l, the apoptotic rate of pcDNA3-nfsB-HeLa cells was 12.92% and at a dose of γ-ray irradiation of 6 Gy, the rate of apoptosis was 12.84%. However, with the combination of 12.5 µmol/l of CB1954 and 6 Gy of γ-ray irradiation, the rate of apoptosis rate increased to 39.9%, which was increased compared with irradiation or CB1954 alone for pcDNA3-nfsB-HeLa cells (Fig. 6A). These observations demonstrated that the cytotoxic effect on HeLa cells was due to the interaction between the suicide gene system NTR/CB1954 and radiation, and was not a simple additive effect. In addition, the present study determined the cell survival fraction with a colony-formation assay. The cells were treated with CB1954 at various concentrations (0, 12.5 and 25 µmol/l) for 16 h prior to the delivery of various doses of γ-rays (0, 0.5, 1, 1.5, 2, 4, 6 and 8 Gy). Next, the cell survival fraction was then determined using a colony-formation assay. Following treatment with 12.5 and 25 µmol/l of CB1954, reduced cell survival fraction was evident with the combination of radiation and CB1954. SER was obtained from the cell survival curve. At concentrations of 12.5 and 25 µmol/l CB1954 radiosensitivity ratios were 1.54 and 1.66, respectively (Fig. 6B). These results indicated that the NTR/CB1954 suicide gene system may significantly enhance the sensitivity of HeLa cells to radiation.