AZT was purchased from Sigma (St. Louis, MO, USA), telomerase ploymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) Kit from Boehringer Manheim (Germany), RPMI-1640 from Hyklong Company (USA), and trypsin from DIFCO Company (USA). Penicillin and streptomycin were the products of Pharmaceutical Factory of Haerbin, (China), fetal calf serum was obtained from Zhejiang Evergreen Company (China), 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) from Huamei Biological Company (China), and dimethyl sulfoxide from Jinshan Chemical Plant (Shanghai, China).

Ovarian cancer cell line HO-8910 was developed in the laboratory of the Department of Obstetrics and Gynecology, Xijing Hospital, China. HO-8910 cells were cultured in RPMI 1640 supplemented with 100mg/ml streptomycin, 100mg/ml penicillin and 10% fetal calf serum. The cells were incubated at 37°C in the presence of 5% CO₂. Logarithmically growing cells were used for subsequent experiments.

HO-8910 cells were randomly divided into experimental and the control groups. Cells in the experimental group were maintained in culture medium with supplemented with AZT at he concentrations of 0.5 mM, 0.8 mM, 1.0 mM and 1.5 mM for 24 h, 48 h and 72 h, respectively. The same culture medium without AZT was used for the cells in the control group. Cells were collected at different time points by centrifugation at 2000 g for 10 min at 4°C. The pelleted cells were resuspended in 200 μL lysis reagent and incubated on ice for 30 min followed by centrifugation at 12,000 g for 25 min at 4°C. In the control group, HO-8910 cells were treated for 10 min at 65°C. The telomerase activity of HO-8910 cells was measured using a telomerase PCR-ELISA Kit.

HO-8910 cells were cultured in RPMI 1640 containing 10% fetal calf serum, in which the cultured cells reached over 90% confluence in 96 well plates. Different concentrations of AZT (0.05 mM, 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1.0 mM, 1.2 mM, 1.25 mM, 1.6 mM, 2.0 mM) were added to the cells in the experimental group while equal volumes of culture medium were added to the cells in the control group. After 24, 48 and 72 h, 20 μL of MTT (5 mg/mL) was added to each well and the cells were incubated for another 4 h at 37°C. The supernatant was carefully removed, and 150 μL of dimethyl sulfoxide was added into each well and shaken for 10 min. Absorbance (A) of the samples was measured at 490 nm by an enzyme linked immunoassay meter. The inhibitory rate of tumor cells = (1 - A_AZT/A_control) ×100%.

HO-8910 cells were cultured in 100 mL flasks in RPMI 1640 containing 10% fetal calf serum. When the cells reached over 90% confluence, AZT was added to the cells in the experimental group at the final concentration of 0.8 mM or 1.2 mM, and equal volume of culture medium was added to the cells in the control group. Subsequently, cells in both groups were incubated for another 72 h, after which cells were transferred to a fresh tube and collected by centrifugation at 2000 g for 10 min. 2 mL of 20 g/L glutaraldehyde was then added and the cells were fixed for 2 h. After cells were embedded and ultrathin sections were cut, the ultrastructure of the cells were observed by transmission electron microscope.

HO-8910 cells (5 × 10⁵) in the experimental group were added to culture medium containing 0.8 mM or 1.2 mM AZT and the cells in the control group were added to culture medium without AZT. After incubation for 72 h, the cells in both groups were collected by centrifugation at 1000 g for 10 min and the pellet was carefully washed twice with 0.01 M phosphate buffered saline (pH7.3). Then 1 mL of 70% ethanol was added to the collected cells and the cells were kept at 4°C for further processing. After washed twice with 0.01 M phosphate buffered saline, the cells were stained with 300 μL of propidium iodide for 30 min. The cell cycle was detected by flow cytometer.

The SPSS 12.0 software was used to determine the statistical significance of the differences between the two groups. The result was examined by the Repeated Measure ANOVA. P value less than 0.05 was considered significant.

Telomerase activity of HO-8910 cells was inhibited by AZT at 0.5 mM for 24 h. The inhibitory effect was significantly increased with the elevation of drug concentration and elongation of exposure time. (P<0.05, Table 1).

The inhibition on HO-8910 cell growth of AZT at different concentrations for 24 h, 48 h and 72 h, respectively, was detected with MTT assay. The results showed that lower concentrations of AZT had a slight inhibitory effect on HO-8910 cells. With the increase of AZT concentration, the A value was decreased and the inhibitory rate was increased and the effect was dose-dependent. Similarly, with the elongation of acting time, the inhibitory rate was also increased in a time-dependent manner. The minimum inhibitory effect appeared with 0.8 mM at 24 h, with 0.5 mM AZT at 48 h, and with 0.1 mM AZT at 72 h (Fig. 1, Table 2).

The cells in the control group were observed to be polygon- or anomalous-shaped with various sizes by electron microscope. There were lots of long and thin microvilli on cellular surface (Fig. 2A). These cells had well-developed organelles, including mitochondria, rough endoplasmic reticulum, lysosomes and free ribosomes, etc. The nucleus was large with many karyokinesis, and abnormal nucleus mitosis was observed. After treated with AZT at 0.8 mM for 72 h, some HO-8910 cells became swollen with dilated endoplasmic reticulum and fewer mitochondria appeared when compared with the cells in the control group (Fig. 2B). After treated with AZT at 1.2 mM for 72 h, the microvilli of the cells were decreased in number and plenty of bubble-shaped convex appeared on cell surface. The chromatin was condensed and aggregated against the nuclear membrane, the structure of which was intact (Fig. 2C).

The cells treated with AZT at various concentrations had cell cycle profiles different from those of the cells in the control group. After exposure to 0.8 mM AZT for 72 h, the percentage of the cells in the phase G1 dropped from 57.6% (in control group) to 1.1%, the percentage of the phase G2/M cells rose from 15.5% to 57.6%, and the percentage of the phase S cells rose from 26.9% to 41.2%. When AZT was used at the concentration of 1.2 mM, the percentage of the phase G1cells rose to 67.7%, the phase G2 cells fell to 12.2%, and the phase S cells dropped to 20.1%. The apoptosis cusp was observed and the apoptotic rate was 14.2% in the group of 1.2 mM AZT (Fig. 3).