TE-11 cells, a cell line derived from a patient with human esophageal cancer, were purchased from the American Type Culture Collection and were maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. AZT was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and dissolved in PBS.

A total of 1×10⁴ cells/well were seeded in a 96-well plate. Cells were treated with 2, 20, 100 and 200 µM AZT for 24, 48, 72 and 96 h. The wells were subsequently replaced with 10 µl/well MTT solution (5 mg/ml) and incubated at 37°C for 4 h. The supernatant was removed and 100 µl/well dimethyl sulfoxide was added for 15 min. The spectrometric absorbance at a wavelength of 490 nm was measured on a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). As a control, TE-11 cells were additionally treated with DMEM alone.

TE-11 cells (2×10⁶) were cultured in 6-well plates and treated with 2, 20, 100 and 200 µM AZT for 48 h at 37°C. Lysates were prepared by treating cells for 30 min on ice with lysis buffer [10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 1 mM EGTA, 1% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, 10% glycerol, 5 mM β-mercaptoethanol and 0.1 mM phenylmethane sulfonyl fluoride]. Lysates were centrifuged at 15,000 × g for 20 min at 4°C. Supernatants were collected and the protein concentrations were determined using a Bicinchoninic Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Lysates were subsequently diluted to 10 mg/ml. Telomerase activity was measured by polymerase chain reaction (PCR) and the telomeric repeat amplification (TRAP) assay. The PCR reaction mixture was prepared using TRAP reaction buffer [20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20 and 1 mM EGTA]. The PCR reaction mixture was comprised of 312.5 µM dNTP, 0.625 µM telomerase substrate (TS) primer, the reverse primer for amplification (CX) and 1 U hot-start Taq DNA polymerase, in a total volume of 50 µl. In addition, each reaction mixture contained 0.625 µM internal control primer (NT) and 0.01 aM internal control template (TSNT) for amplification of a 36 bp internal standard. A total of 1 µl lysate was subsequently added to this mixture, which was placed in a thermal cycler. Primers were as follows: 5′-AATCCGTCGAGCAGAGTT-3′ for TS; 5′-CCCTTACCCTTACCCTTACCCTAA-3′ for CX; 5′-ATCGCTTCTCGGCCTTTT-3′ for NT and 5′-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′ for TSNT. Cycling conditions were as follows: An initial telomerase extension step at 30°C for 30 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for 30 sec and extension at 72°C for 1 min. For each sample, a total 10 µl PCR product was loaded onto 10% polyacrylamide gels and subjected to electrophoresis at 180–200 V for ~1 h in Tris/Borate/EDTA buffer. The gel was stained with GelRed™ (Biotium, Inc., Hayward, CA, USA) and visualized under an ultraviolet (UV) illuminator to determine telomerase activity. An internal control was included and was evident by a 36 bp PCR product. TE-11 cells cultured in DMEM medium alone were utilized as a control, and the band intensities were quantified with ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD, USA).

Following treatment with 2, 20, 100 and 200 µM AZT for 48 h, 2×10⁶ cells were washed twice with PBS. Following an overnight fixation in 70% ethanol at 4°C, cells were harvested by centrifugation at 200 × g for 10 min at room temperature and washed twice with PBS. Cells were subsequently stained with propidium iodide and analyzed on the BD FACScan™ system using BD Accuri C6 Software version 1.0.264.21 (BD Biosciences, Franklin Lakes, CA, USA).

TE-11 cells were cultured for 48 h in a 6 well plate and treated with 20, 100 and 200 µM AZT for 48 h. Additionally, cells treated with DMEM alone served as an untreated control, and cells treated with UV served as a positive control. The Comet assay was performed under alkaline conditions. Cells were resuspended in DMEM at a concentration of 1×10⁵/ml and were combined with molten LMAgarose (Trevigen, Gaithersburg, MD, USA) (at 37°C) at a ratio of 1:10, prior to pipetting 75 µl onto CometSlides™ (Trevigen). Slides were stored in the dark at 4°C for 30 min and immersed in alkaline solution (0.25 M NaOH containing 0.1 µM EDTA; pH 12.6) at 4°C for 2 h. Slides were gently removed from the lysis buffer and rinsed with distilled H2O. Slides were placed in freshly prepared alkaline solution at pH>13 for 30 min at room temperature. Gel electrophoresis was performed at 1 V/cm for 30 min. Subsequently, slides were washed in 70% ethanol, stained with GelRed^™ (Biotium, Inc.) and analyzed under a fluorescence microscope at ×400 magnification.

TE-11 cells were lysed with radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na₃VO₄) following treatment with 20, 100 and 200 µM AZT for 48 h. Protein concentrations were determined using the Bicinchoninic Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Heat-denatured protein samples (20 mg/lane) were loaded onto 10% gels and subjected to electrophoresis prior to transfer onto polyvinylidene fluoride membranes. The membranes were incubated with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. The membranes were then incubated at 4°C overnight with the following primary antibodies: Rabbit anti-phosphorylated-checkpoint kinase 2 [pChk2 (Thr68); cat. no. ab85743; 1:1,000; Abcam, Cambridge, MA, USA], mouse anti-γ-H2A histone family member X (γ-H2AX; cat. no. ab180651; 1:1,000; Abcam) and mouse anti-β-actin (cat. no. sc-8432; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, membranes were probed with goat anti-rabbit-horseradish peroxidase (−HRP; cat. no. sc2004; 1:5,000; Santa Cruz Biotechnology, Inc.) or goat-anti-mouse-HRP (cat. no. sc2005; 1:5,000; Santa Cruz Biotechnology, Inc.) secondary antibodies for 1 h at room temperature. Proteins were detected using the Enhanced Chemiluminescence Detection reagent (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol.

Data are expressed as the mean ± standard deviation of three experiments. Statistical differences were analyzed using one-way analysis of variance followed by Tukey's post-hoc test via GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) or Microsoft Excel 2007 software (Microsoft Corporation, Redmond, WA, USA).

TE-11 cell proliferation was inhibited following treatment with 20, 100 and 200 µM AZT for 0, 24, 48, 72 and 96 h, as determined by an MTT assay (Fig. 1). However, treatment with 2 µM of AZT did not have an inhibitory effect on TE-11 cells. Therefore, the inhibitory effect of AZT was time- and dose-dependent (Fig. 1; Table I).

The effect of AZT on telomerase activity in TE-11 cells was determined by a TRAP assay. Treatment with 20, 100 and 200 µM AZT for 48 h resulted in a dose-dependent decrease in telomerase activity (Fig. 2A and B) compared with the control. However, there was no significant difference in activity following treatment with 2 µM AZT.

The effect of AZT on cell cycle distribution was assessed by flow cytometric analysis. Representative cell cycle profiles of TE-11 cells treated with 2, 20, 100 and 200 µM AZT for 48 h are demonstrated in Fig. 3A-E. Treatment with AZT led to a marked dose-dependent decrease in the percentage of G1/G0 phase cells and a marked dose-dependent increase in S-phase cells, compared with the control (Fig. 3F).

Degradation of DNA is an irreversible event following apoptotic cascade events (17). To investigate whether treatment with AZT may induce DNA degradation, a comet assay was performed. As demonstrated in Fig. 4A, treatment of TE-11 cells with increasing concentrations of AZT for 48 h resulted in significant DNA damage compared with control cells; the comet tails of the treated cells demonstrate DNA migration out of the nucleus due to DNA breakage and loss of structure. Consistent with comet assay results, western blot analysis revealed that treatment with increasing concentrations of AZT resulted in enhanced expression levels of γ-H2AX and pChk2 (Fig. 4B), which are markers of the DNA damage response (DDR) pathway. In conclusion, AZT may induce DNA damage in TE-11 cells.