Long-term exposure to zidovudine delays cell cycle progression, induces apoptosis, and decreases telomerase activity in human hepatocytes

Abstract

Zidovudine (3'-azido-3'-deoxythymidine; AZT), which is currently used in the treatment of acquired immunodeficiency syndrome, has been shown to have anticancer properties. In the present study, we examined the mechanisms contributing to increased sensitivity of cancer cells to the growth-inhibitory effects of AZT. This was accomplished by incubating a hepatoma cell line (HepG2) and a normal liver cell line (THLE2) with AZT in continuous culture for up to 4 weeks and evaluating the number of viable and necrotic cells, the induction of apoptosis, cell cycle alterations, and telomerase activity. In HepG2 cells, AZT (2-100 microM) caused significant dose-dependent decreases in the number of viable cells at exposures > 24 h. During a 1-week recover period, there was only a slight increase in the number of viable cells treated with AZT. The decrease in viable cells was associated with an induction of apoptosis, a decrease in telomerase activity, and S and G2/M phase arrest of the cell cycle. During the recovery period, the extent of apoptosis and telomerase activity returned to control levels, whereas the disruption of cell cycle progression persisted. Western blot analysis indicated that AZT caused a decrease in checkpoint kinase 1 (Chk1) and kinase 2 (Chk2) and an increase in phosphorylated Chk1 (Ser345) and Chk2 (Thr68). Similar effects, to lesser extent, were observed in THLE2 cells given much higher concentrations of AZT (50-2500 microM). These data show that HepG2 cells are much more sensitive than THLE2 cells to AZT. They also indicate that a combination of a delay of cell cycle progression, an induction of apoptosis, and a decrease in telomerase activity is contributing to the decrease in the number of viable cells from AZT treatment, and that checkpoint enzymes Chk1 and Chk2 may play an important role in the delay of cell cycle progression.

FIG. 1.

Cell viability, as assessed by an MTT assay (A and B), and necrotic cell death, as assessed by an LDH release assay (C and D), in HepG2 cells (A and C), and THLE2 cells (B and D) following exposure to AZT. AZT was added at the beginning of the cultures. HepG2 cells were exposed to 0, 2, 20, or 100μM and THLE2 cells were exposed to 0, 50, 500, or 2500μM AZT for 24 h, 48 h, or 1 week (1 w). Cells were also exposed to AZT for 2, 3, and 4 weeks, with the cells being subcultured weekly. After being cultured for 4 weeks with AZT, the cells were switched to AZT-free medium for a 1-week recovery culture (Rec. w). MTT and LDH release assays were performed as described in “MATERIALS AND METHODS.” The data are normalized to the control value at each time point. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.

FIG. 2.

Determination of apoptotic cells by flow cytometry using an APO-BrdU assay in HepG2 cells (A) and THLE2 cells (B) treated with AZT. After treatment, floating and adherent cells were collected, fixed, and processed for APO-BrdU flow cytometry analysis as described in “MATERIALS AND METHODS.” The data are normalized to the control value at each time point. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.

FIG. 3.

Analysis of telomerase activity in HepG2 cells. Telomerase activity in HepG2 cells was measured using a real-time PCR-based TRAP assay in the presence of AZT as described in “MATERIALS AND METHODS.” The data are normalized to the control value at each time point. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.

FIG. 4.

Analysis of cell cycle distribution by flow cytometry. (A) Scatter plots depicting cell cycle distribution in HepG2 cells treated with different doses of AZT after 1 week of exposure. Bivariate analysis of the DNA content (FL3, PI staining) and the incorporation of BrdU (FL1, FITC-conjugated anti-BrdU staining) was performed. Cells were labeled with BrdU for 1 h after the treatment. Cells were then fixed, stained with FITC-coupled anti-BrdU antibody and PI, and analyzed by flow cytometry to determine the cell cycle distribution. As shown by the boxed regions, significant proportions of cells were found to occupy distinct cell cycle phases, including G1/G0, S, and G2/M. The number of cells for each gate was counted. Plots of percentage of cells in G1/G0 (B and E), S (C and F), and G2/M (D and G) in HepG2 cells and THLE2 cells treated with AZT. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.

FIG. 4.

Analysis of cell cycle distribution by flow cytometry. (A) Scatter plots depicting cell cycle distribution in HepG2 cells treated with different doses of AZT after 1 week of exposure. Bivariate analysis of the DNA content (FL3, PI staining) and the incorporation of BrdU (FL1, FITC-conjugated anti-BrdU staining) was performed. Cells were labeled with BrdU for 1 h after the treatment. Cells were then fixed, stained with FITC-coupled anti-BrdU antibody and PI, and analyzed by flow cytometry to determine the cell cycle distribution. As shown by the boxed regions, significant proportions of cells were found to occupy distinct cell cycle phases, including G1/G0, S, and G2/M. The number of cells for each gate was counted. Plots of percentage of cells in G1/G0 (B and E), S (C and F), and G2/M (D and G) in HepG2 cells and THLE2 cells treated with AZT. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.

FIG. 5.

Western blotting of Chk1, Chk2, pChk1 (Ser345), pChk2 (Thr68), and β-actin using whole cell lysates from HepG2 cells and THLE2 cells treated with different doses of AZT. Equal amounts (50 μg) of the whole cell lysate protein were loaded in each lane. Immunoblotting for each protein was done in triplicate, using lysates prepared independently. All primary antibodies were incubated with the same membrane after consecutive stripping. The intensity of each band was quantified by densitometry, and the relative protein levels were calculated using β-actin as the internal reference. (A) Immunoblotting for Chk1, Chk2, pChk1 (Ser345), pChk2 (Thr68), and β-actin, using whole cell lysates from HepG2 cells treated with different doses of AZT after a 1-week exposure. Relative levels of Chk1 (B and F), Chk2 (C and G), pChk1 (Ser345) (D and H), and pChk2 (Thr68) (E and I) in HepG2 cells and THLE2 cells treated with AZT. The data are normalized to the control value at each time point. Columns and bars are means and SD for three separate experiments. Asterisks denote significant difference (p < 0.05) compared with the control cultures at the same time point.