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. 2008 Oct 20;24(2):133–141. doi: 10.1093/mutage/gen059

Interference of cell cycle progression by zidovudine and lamivudine in NIH 3T3 cells

Jia-Long Fang 1,*, Lynda J McGarrity 1, Frederick A Beland 1
PMCID: PMC2720688  PMID: 18936108

Abstract

Zidovudine (3′-azido-3′-deoxythymidine; AZT) and lamivudine [(−)2′,3′-dideoxy-3′-thiacytidine; 3TC] are nucleoside reverse transcriptase inhibitors used to treat and prevent human immunodeficiency virus-1 infections. In short-term incubations (<48 h), AZT, but not 3TC, has been shown to interfere with cell cycle progression. In the present study, we examined if these alterations persist during long-term incubations in which cells were exposed to AZT (0–1000 μM) or 3TC (0–500 μM) in continuous culture for up to 5 weeks. In addition, we investigated the reversibility of these effects upon removal of the drugs. Both drugs caused concentration- and time-dependent decreases in the number of viable cells, with the effect being more pronounced with AZT. There was only a slight increase in the number of viable cells treated with AZT for 5 weeks and then allowed a 1-week recovery period; cell viability in cells treated with 3TC returned to control levels during the recovery period. The decrease in viable cells was not due to apoptotic or necrotic cell death, but rather was associated with S and G2/M phase cell cycle arrest. Western blot analysis indicated that AZT treatment caused a decrease in checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) at all time points. Cyclin-dependent kinase 1 was decreased at later time points, while cyclin A was increased at early times. These data indicate that AZT and, to a lesser extent, 3TC interfere with cell growth by slowing cell cycle progression and that checkpoint proteins Chk1 and Chk2 may play an important role in this delay.

Introduction

The nucleoside reverse transcriptase inhibitors zidovudine (3′-azido-3′-deoxythymidine; AZT) and lamivudine [(−)2′,3′-dideoxy-3′-thiacytidine; 3TC] are used extensively in the treatment of acquired immunodeficiency syndrome (AIDS) and in the prevention of the mother-to-child transmission of human immunodeficiency virus-1 (HIV-1) (1–3). Although AZT and 3TC are the most common components of the highly active antiretroviral therapy and increase the survival of AIDS patients and asymptomatic HIV-1-positive patients, toxic side effects, including myopathy, cardiomyopathy, lipodystrophy, haematological toxicities and hepatotoxicity associated with mitochondrial DNA depletion (4–8), have been observed in patients treated with therapeutic doses. These side effects remain a limiting factor in the clinical management of AIDS.

AZT is a derivative of 2′-deoxythymidine, in which the 3′-hydroxy group is substituted with an azido function, while 3TC is the (−)enantiomer of a dideoxy analogue of cytidine (Figure 1). Both drugs act as DNA chain terminators. Intracellularly, AZT and 3TC are phosphorylated to active triphosphate derivatives. The triphosphate derivatives efficiently inhibit HIV-1 reverse transcriptase by acting as competitive inhibitors of normal nucleotides (9–11) and causing proviral DNA termination, thus inhibiting retroviral replication. In addition to inhibiting viral reverse transcriptase, the triphosphates of AZT and 3TC also are weak inhibitors of mammalian DNA polymerases α, β and γ, which catalyse DNA replication in vivo (12–15). There is evidence that AZT and 3TC can become incorporated into cellular DNA (13,16–19). This may result in the occurrence of irreversible DNA damage, which can lead to impaired cellular function and contribute to the genotoxic effects of the drugs. Several studies have demonstrated that AZT is clastogenic and capable of inducing genetic mutations in mammalian cells (20–25). Moreover, AZT has been reported to be a transplacental carcinogen in mice and rats (23,26–29). The International Agency for Research on Cancer has classified AZT as a group 2B carcinogen, designated as possibly carcinogenic to humans (25).

Fig. 1.

Fig. 1

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Chemical structures of AZT and 3TC.

In short-term in vitro incubations (<48 h), AZT has been demonstrated to have anti-proliferation effects in mammalian cells, including human cells (30–32). This has been attributed to interference with cell cycle progression, with a decrease in the percentage of cells in the G0/G1 phase and an increase in the percentage of cells in S phase (30–32), and has been suggested to be a consequence of AZT being incorporated into cellular DNA (32). Based upon analysis of gene expression, the S-phase blockade induced by AZT was correlated with an up-regulation of cyclin D1 and a down-regulation of cyclin A2 and the cyclin D1-associated inhibitors P18 and P57 (32). In contrast to AZT, 3TC did not inhibit cell cycle progression in these short-term in vitro incubations (32).

In the present study, we examined if these cell cycle perturbations persist during long-term incubations in which cells were exposed to AZT (0–1000 μM) or 3TC (0–500 μM) in continuous culture for up to 5 weeks. In addition, we investigated the reversibility of the effects upon removal of the drugs. Our data indicate clearly that AZT and, to a lesser extent, 3TC interfere with cell growth by slowing cell cycle progression and that the checkpoint kinases checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) may play an important role in this delay.

Materials and methods

Chemicals

AZT and 3TC were obtained from Cipla Ltd (Mumbai, India). The purities, as assessed by high-performance liquid chromatography (HPLC), were 99.7% for AZT and 99.6% for 3TC. [3H]AZT (12.7 Ci/mmol, radiochemical purity >99.8% by HPLC) was obtained from Moravek Biochemicals Inc. (Brea, CA). Tetrachlorohydroquinone, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), 5-bromo-2′-deoxyuridine (BrdU), bovine serum albumin (BSA) and RNase A were obtained from Sigma (St Louis, MO). Dulbecco's modified Eagles medium (DMEM), penicillin–streptomycin and 2.5% trypsin were purchased from Fisher Scientific (Pittsburgh, PA). Dulbecco's phosphate-buffered saline (PBS, minus calcium chloride and magnesium chloride) and calf serum were obtained from Invitrogen (Carlsbad, CA). The protein assay kit was purchased from Bio-Rad (Hercules, CA). All other chemicals and biochemicals were of analytical grade and used without further purification.

Antibodies

Monoclonal antibodies to Chk2, β-actin, anti-mouse IgG peroxidase conjugate and anti-rabbit IgG peroxidase conjugate were purchased from Sigma. Monoclonal antibodies to Chk1 and Wee1 and rabbit antisera against cyclin-dependent kinase 1 (Cdk1) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-BrdU antibody (clone B44) was purchased from BD Biosciences (San Jose, CA).

Cell culture and treatments

These experiments were conducted in NIH 3T3 cells because these cells have been shown to incorporate AZT into their nuclear DNA (19). NIH 3T3 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% calf serum and antibiotics at 37°C in a humidified atmosphere containing 95% air and 5% CO2. AZT and 3TC were dissolved in PBS. The drug concentrations were determined spectrophotometrically at 266 nm for AZT and 272 nm for 3TC. Cells were plated at cell densities of 4 × 104, 2 × 104 and 1 × 103 cells/cm2 culture surface and cultured in the presence of 20, 100 or 1000 μM AZT or 10, 50 or 500 μM 3TC for 24 h, 48 h, 1 week or 5 weeks. The ratio of the drugs was selected based upon the fact that in clinical settings, AZT is typically used at twice the level of 3TC. Drugs were added at the beginning of the cultures and, when appropriate, fresh medium and AZT or 3TC were added every other day. For the 5-week incubation, the cells were subcultured weekly. Following the 5 weeks of culturing in the presence of AZT or 3TC, the cells were then allowed to recover in drug-free complete medium for one week. Control cells were fed with complete culture medium free of AZT and 3TC. Each of the incubations, with the exception of the one with [3H]AZT, was performed three separate times, and all the measurements described below were conducted independently for each of the experiments.

Determination of AZT incorporation into DNA

AZT incorporation into DNA was measured in NIH 3T3 cells exposed to 20, 100 or 1000 μM [3H]AZT (diluted to a specific activity of 20 mCi/mmol with unlabelled AZT) in media for 48 h. After treatment, the cells were harvested and DNA was isolated using a conventional phenol–chloroform extraction method. The radioactivity incorporated into the DNA was determined by liquid scintillation counting.

MTT assay

An MTT reduction assay was used to measure the cell viability. Briefly, cells were cultured and exposed to AZT or 3TC in triplicate wells of a six-well plate. After the specified treatment period, the cells were washed and incubated for 4 h with MTT (500 μg/ml) dissolved in the complete culture medium. An equal volume of MTT solubilization solution (anhydrous isopropanol containing 10% Triton X-100 and 0.1 N HCl) was then added to dissolve the formazan salts. After complete solubilization of formazan salts, absorbance of converted dye was measured spectrophotometrically at 570 nm, with background subtraction at 690 nm.

Lactate dehydrogenase release assay

The cell culture medium was collected and cell debris in the medium was removed by centrifugation at 300× g for 5 min. Lactate dehydrogenase (LDH) released into the medium was measured by determining the absorbance at 490 nm, using an LDH assay kit (Sigma) according to the manufacturer's instructions.

Determination of apoptotic cells

After treatment, cells were trypsinized and washed twice in PBS. Cells (2 × 106) were then fixed in 4 ml of freshly prepared paraformaldehyde (1% w/v) in PBS. After a 1-h fixation period on ice, the cells were washed twice in PBS, centrifuged and the pellet was re-suspended in 100 μl of ice-cold PBS followed by the addition of 4.0 ml of ice-cold 70% (v/v) ethanol. The fixed cell suspensions were stored at −20°C until analysed.

Apoptotic cells were analysed using an APO-BrdU kit (BD Biosciences) according to the manufacturer's instructions. Briefly, the fixed cells (2 × 106) were washed twice with PBS and re-suspended in 51 μl of DNA-labelling solution that included BrdU–triphosphate and terminal deoxynucleotidyl transferase. After incubation for 1 h at 37°C, the cells were rinsed twice with PBS, re-suspended in 100 μl of antibody solution that included FITC-labelled anti-BrdU monoclonal antibody and incubated for 30 min at room temperature. Cell suspensions were mixed with 500 μl PBS containing 2 μg/ml PI and 50 μg/ml RNase A and then analysed by flow cytometry.

Individual samples were analysed on a Becton Dickinson FACScan flow cytometer (Franklin Lakes, NJ), with an argon ion laser tuned to 488 nm. Data were acquired using CellQuest Software. The forward-scatter (FSC) signal and the side-scatter (SSC) signal were measured in the linear mode, with an amplification of 1.5 for FSC and 1.0 for SSC. FL1 (green, FITC) fluorescence was detected on a logarithmic scale and FL3 (red, PI) fluorescence with linear amplification. A total of 25 000 cells was analysed for each sample and doublets were eliminated by gating on the DNA area signal versus the DNA width signal.

Cell cycle analysis

At the end of treatment, cells were trypsinized and washed once in complete medium. Cells (2 × 106) were re-suspended in 10 ml of complete medium containing 10 μM BrdU and incubated for 1 h at 37°C. Cells were then washed twice in PBS and fixed with 70% ethanol. The fixed cells were washed once in PBS, re-suspended in 4 ml of 2 N HCl and 0.5%Triton X-100 and incubated for 30 min at room temperature to denature the DNA. Cells were pelleted by centrifugation and neutralized with 4 ml of 0.1 M Na2B4O7 at pH 8.5 for 10 min at room temperature. After washing with 4 ml of PBS that contained 1% BSA, the cells were centrifuged, and the pellets were re-suspended in 50 μl of PBS containing 1% BSA and 0.5% Tween-20. Twenty microlitres of FITC-conjugated monoclonal anti-BrdU antibody (clone B44) was added to the cell suspensions, and, after a 1-h incubation at room temperature in the dark, the cells were washed once in PBS and re-suspended in 300 μl of PBS containing 5 μg/ml PI. Cells were then analysed on a FACScan flow cytometer as described above.

Western blot analysis

After treatment, cells were trypsinized and washed twice in PBS. Approximately 107 cells were lysed in buffer containing 20 mM MOPS (pH 7.2), 0.5% Nonidet P40, 2 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 5 mM ethylenediaminetetraacetic acid, 50 μg of phenylmethylsulphonyl fluoride/ml and a complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) for 30 min on ice. After sonicating three times for 10 sec each time, the cell lysate was centrifuged at 105 000× g for 1 h at 4°C in a 50 Ti rotor (Beckman, Fullerton, CA). The resulting supernatant fraction was collected and stored at −80°C in 50 μl aliquots. The protein concentration was determined with a Bio-Rad protein assay kit. Fifty micrograms of cell lysate protein was subjected to electrophoresis in 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The resolved proteins were electrophoretically transferred onto an Immun-Blot PVDF™ membrane (Bio-Rad). Both electrophoresis and blotting were performed with a Mini-PROTEAN® 3 electrophoresis system (Bio-Rad). Blots were blocked with 5% milk and probed with anti-Chk1 (1:300 dilution), anti-Chk2 (1:1000 dilution), anti-cyclin A (1:500 dilution), anti-Cdk1 (1:2500 dilution), anti-Wee1 (1:500 dilution) or anti-β-actin (1:2500 dilution), followed by a secondary antibody to IgG conjugated to horseradish peroxidase. The proteins were then detected by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). The intensity of each band was quantified by densitometry (Kodak Digital Science Image Station 440CF, using Kodak 1D Image Analysis Software, Rochester, NY), and the relative protein levels were calculated using β-actin as the internal reference. All primary antibodies were incubated with the same membrane after consecutive stripping using Restore Western Blot Stripping Buffer (Pierce). Densitometric results were always consistent irrespective of the exposure time.

Data analysis

Data are expressed as mean ± standard deviation. Comparisons among doses were conducted by two-way analysis of variance, with pair-wise comparisons being performed by Student–Newman–Keuls method. When necessary, the data were log transformed to maintain an equal variance or normal distribution. The results were considered significant at P <0.05.

Results

Incorportation of AZT into DNA of NIH 3T3 cells

In order to insure that the experimental conditions would permit the incorporation of AZT into DNA, an experiment was conducted in which NIH 3T3 cells were incubated with 20, 100 and 1000 μM [3H]AZT for 48 h. Following isolation of the DNA, the extent of radioactivity was assessed by liquid scintillation counting, which indicated values of 26, 170 and 330 AZT molecules/106 nucleotides.

Anti-proliferative effects of AZT and 3TC in NIH 3T3 cells

Treatment of NIH 3T3 cells with AZT caused significant concentration- and time-dependent decreases in the number of viable cells, with the effect being greatest at the 1- and 5-week time points (Figure 2A). Compared to the control cultures, growth rates at 20, 100 and 1000 μM AZT were 66.6, 39.2 and 16.4%, respectively, after a 1-week exposure, and 51.4, 36.8 and 23.7%, respectively, after 5 weeks of exposure. In cells allowed a 1-week recovery after being treated for 5 weeks with AZT, there was only a slight increase in the number of viable cells compared to the 5-week time point.

Fig. 2.

Fig. 2

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Cell viability, as assessed by an MTT assay (A and B), and cell death, as assessed by an LDH release assay (C and D), in NIH 3T3 cells following exposure to either AZT (A and C) or 3TC (B and D). The drugs were added at the beginning of the cultures. Cultures of NIH 3T3 cells were exposed to 0, 20, 100 or 1000 μM AZT or 0, 10, 50 or 500 μM 3TC for 24 h, 48 h or 1 week. Cells were also exposed to AZT and 3TC for 5 weeks, with the cells being subcultured weekly. After being cultured for 5 weeks with AZT and 3TC, the cells were switched to AZT- and 3TC-free medium for a 1-week recovery culture. 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 standard deviations for three separate experiments. Asterisks denote significant difference (P < 0.05) compared to the control.

3TC also caused concentration- and time-dependent decreases in the number of viable cells, with the effect being greatest at the 1- and 5-week time points (Figure 2B). However, compared to cells treated with AZT, the effect was much less pronounced and upon a 1-week recovery period, the cell viability returned to control levels.

The decrease in the number of viable cells could be due to an increase in necrotic cell death, an induction of apoptosis and/or an impairment of the cells transiting through the cell cycle. Each of these pathways was considered separately.

Effect of AZT and 3TC on necrotic cell death in NIH 3T3 cells

To establish whether the decrease in the number of viable cells was due to necrosis, an LDH release assay was conducted. LDH release assays have been used extensively as a marker for cell death both in vitro and in vivo (33). The release of LDH into the culture medium accurately reflects necrotic cell death in vitro. In NIH 3T3 cells treated with AZT, there was a dose- and time-dependent decrease in LDH release (Figure 2C), with the maximum decrease being observed after 1 week of treatment. In cells treated with 3TC, a decreased release of LDH also occurred, but only at the highest concentration and after 1 and 5 weeks of incubation (Figure 2D). These findings of a decreased release of LDH indicated that the doses used in the study did not cause cytotoxicity and that necrotic cell death did not contribute to the inhibitory effect on cell growth resulting from the AZT and 3TC treatments.

Effect of AZT and 3TC on apoptotic cell death in NIH 3T3 cells

To evaluate whether or not AZT and 3TC induced apoptosis, an APO-BrdU assay, a staining method to detect apoptotic cells by flow cytometry, was performed. One of the biochemical features of apoptosis is the cleavage of the chromatin structure of DNA into nucleosomal size fragments, which can be detected by end labelling with BrdUTP. The amount of incorporated BrdU is then estimated following the binding of FITC-labelled anti-BrdU mAb.

Figure 3A shows a dot plot of NIH 3T3 cells after exposure to 100 μM tetrachlorohydroquinone for 17 h. In this sample, which served as a positive control, 34.5% of the cells were apoptotic, a clear indication that NIH 3T3 cells retain apoptotic machinery. Neither AZT (Figure 3B) nor 3TC (Figure 3C) had a consistent effect upon the induction of apoptosis in NIH 3T3 cells. This finding was further confirmed by FITC–Annexin V assay using flow cytometric analysis (data not shown). These data indicate clearly that apoptosis does not contribute to the decrease in the number of viable cells resulting from AZT and 3TC treatments.

Fig. 3.

Fig. 3

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Detection of apoptotic cells by flow cytometry using an APO-BrdU assay. (A) Scatter plot of apoptotic and non-apoptotic cells in NIH 3T3 cells exposed to 100 μM tetrachlorohydroquinone for 17 h. The DNA content of 25 000 cells is plotted on the x-axis (FL3, PI staining) and BrdU content is plotted on the y-axis (FL1, FITC anti-BrdU staining). The number of apoptotic and non-apoptotic cells in each sample was determined from the regions indicated. The percentage of apoptotic cells in TCHQ-treated cells is 34.5%. (B and C) Data obtained from NIH 3T3 cells treated with AZT and 3TC, respectively. The data are normalized to the control value at each time point. The percentage of apoptotic cells across all treatments and times was 0.08 ± 0.01%. Columns and bars are means and standard deviations for three separate experiments. Asterisks denote significant difference (P < 0.05) compared to the control.

Cell cycle analysis of NIH 3T3 cells treated with AZT and 3TC

Based on the above findings, we hypothesized that AZT and 3TC may induce cell cycle arrest in NIH 3TC cells. To estimate the effect of AZT and 3TC on cell cycle progression, we utilized a flow cytometric technique with BrdU. Representative flow scatter plots depicting the cell cycle distribution in NIH 3T3 cells following a 1-week exposure to AZT (0, 20, 100 and 1000 μM) are shown in Figure 4A. As shown by the regional gates applied to the PI versus FITC–BrdU dot plot, flow cytometric analysis of cells stained with PI and FITC–BrdU allowed discrimination of cell subsets that resided in G0/G1, S and G2/M phases of the cell cycle.

Fig. 4.

Fig. 4

Fig. 4

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Analysis of cell cycle distribution by flow cytometry. (A) Scatter plots depicting cell cycle distribution in NIH 3T3 cells treated with different doses of AZT after a 1-week exposure. Bivariate analysis of the DNA content (FL3, PI staining) and the incorporation of BrdU (FL1, FITC anti-BrdU staining) was performed. Cells were labelled with BrdU for 1 h after the treatment. Cells were then fixed, stained with FITC-coupled anti-BrdU antibody and PI and analysed 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 G0/G1, S and G2/M. The number of cells for each gate was counted. Percentage of cells in G1/G0 (B and E), S (C and F), and G2/M (D and G) phases of the cell cycle. Columns and bars are means and standard deviations for three separate experiments. Asterisks denote significant difference (P < 0.05) compared to the control.

In NIH 3T3 cells treated with AZT, there was a significant dose- and time-dependent decrease in the percentage of G0/G1 cells (Figure 4B). The effect was most pronounced after 24 h of incubation and decreased with the length of treatment. Upon removal of the AZT, the percentage of G0/G1 cells returned closer to the control value, although there was still a significant decrease in the cells that had been treated with 100 and 1000 μM AZT. Concomitant with decrease in G0/G1 cells, AZT treatment caused a marked dose- and time-dependent increase in the percentage of cells in the S phase (Figure 4C). There were also changes in the percentage of cells in the G2/M phase (Figure 4D), with the effect being most pronounced after 1 and 5 weeks of treatment. These data indicate clearly that AZT-mediated growth inhibition of NIH 3T3 cells is correlated with S phase and G2/M phase cell cycle arrest.

The effect of 3TC upon the cell cycle distribution of NIH 3T3 cells treated with 3TC is shown in Figures 4E–G. As was observed with AZT, 3TC caused a decrease in the percentage of cells in the G0/G1 phase (Figure 4E) and an increase in cells in the S phase (Figure 4F); however, the effect was clearly attenuated compared with AZT, with the change only being significant at the highest dose of 500 μM and beyond 1 week of treatment.

Western blotting of cell cycle-related proteins

The passage of a cell through the cell cycle is controlled by proteins such as cyclin and cyclin-dependent kinase complexes, cell cycle division (Cdc) control proteins and cell cycle checkpoint kinases. Because AZT had a very pronounced effect upon the cell cycle in NIH 3T3 cells, the effects of AZT on the cell cycle-related proteins, including Wee1, Chk2, Chk1, Cyclin A, Cdk1, and β-actin, were analysed by western blotting. As shown in Figure 5A, antibodies to these cell cycle proteins were specific and gave a distinct band for each protein.

Fig. 5.

Fig. 5

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Western blotting of Chk1, Chk2, Cdk1, Wee1, cyclin A and β-actin using whole-cell lysates from NIH 3T3 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, Cdk1, Wee1, cyclin A and β-actin using whole-cell lysates from NIH 3T3 cells treated with different doses of AZT after a 1-week exposure. Relative levels of proteins of Chk1 (B), Chk2 (C), Cdk1 (D), Wee1 (E), and Cyclin A (F). The data are normalized to the control value at each time point. Columns and bars are means and standard deviations for three separate experiments. Asterisks denote significant difference (P < 0.05) compared to the control.

In NIH 3T3 cells treated with AZT, there was a time-dependent decrease in the relative levels of Chk1 (Figure 5B). The maximum effect was observed after 1 week of treatment, with all doses of AZT giving a similar reduction in the levels of Chk1. AZT treatment also decreased the relative levels of Chk2 (Figure 5C); this occurred at all time points, with the maximum response being observed at doses of 100 μM AZT and above. In an additional experiment, 30 cell cycle-related proteins were analysed in control and 1000 μM AZT samples from the 48-h and 5-week treatments using a western multiblot-based Kinetworks™ KCCP-1.0 Cell Cycle Protein Screen (Kinexus Bioinformatics Corporation, Vancouver, Canada). In accord with western blotting results, the AZT-treated samples exhibited an ∼50% decrease in the levels of Chk1 and Chk2 (data not shown).

AZT treatment significantly decreased the relative levels of Cdk1 in a time-dependent manner, with the maximum effect being observed after 5 weeks of treatment (Figure 5D). After a 1-week recovery period, the relative level of Cdk1 in AZT-treated cells approached that of the control value. The level of Wee1 was similar to the control value at each of the doses and sampling times (Figure 5E). By comparison, cyclin A was significantly elevated in AZT-treated cells at early treatment times (24 and 48 h) but was not changed after prolonged exposures (Figure 5F).

Discussion

In this study, we demonstrated that AZT and, to a lesser extent, 3TC decreased the number of viable NIH 3T3 cells. The decrease was not due to necrotic or apoptotic cell death, but rather was associated with S phase and G2/M phase cell cycle arrest. S phase cell cycle arrest has been observed previously in cells exposed to AZT for short periods (<48 h) (30–32). In previous work, cell cycle arrest was not observed with 3TC (32), and in our study, 3TC only caused changes in cell cycle progression at the highest dose of 500 μM and after 48 h of exposure.

A depletion of cells in the G0/G1 phase, when accompanied by the S and G2/M phase cell cycle arrest, typically indicates retardation of mitosis. The impairment of the cell cycle progression that we observed may be explained by the ability of AZT and 3TC to be phosphorylated intracellularly to the active triphosphate derivatives. The triphosphate derivatives can be incorporated into replicating DNA, which leads to DNA chain termination. The incorporation of AZT into NIH 3T3 cell DNA has been demonstrated previously (19), and when we incubated NIH 3T3 cells with 20–1000 μM [3H]AZT for 48 h, DNA incorporation levels (26–330 AZT molecules/106 nucleotides) similar to those observed in the previous study were obtained.

Specific checkpoints exist to restrict the progression of damaged cells through the cell cycle. Chk1 and Chk2 are the components of the DNA damage checkpoint pathway that prevent transmission of altered genetic information to progeny; as such, they are involved in regulating cell cycle arrest. The activation of Chk1 and Chk2, in response to DNA damage, involves phosphorylation of Ser345 and Thr68, respectively. Activated Chk2 phosphorylates Ser123 of Cdc25A, targeting Cdc25A for degradation. This, in turn, prevents the activation of Cdk2, which results in cells arresting in the S phase. Activated Chk1 and Chk2 also inactivate Cdc25C via phosphorylation at Ser216, blocking the activating dephosphorylation of Cdk1 and the transition of G2 phase cells into M phase. Several phospho-Chk1 (Ser345) and phospho-Chk2 (Thr68) antibodies, obtained from Cell Signaling Technology Inc. (Danvers, MA) and Santa Cruz Biotechnology Inc., were screened for cross-reactivity against phosphorylated Chk1 (ser345) and Chk2 (Thr68) in NIH 3T3 cells; however, no cross-reactivity was detected. Although phosphorylated Chk1 and Chk2 could not be analysed, the decrease in Chk1 and Chk2 protein levels associated with the AZT-mediated S phase and G2/M phase cell cycle arrest may be due to phosphorylation of these proteins. Since levels of Wee1, a tyrosine-specific kinase that mediates the inhibitory phosphorylation of Cdk1, were not altered, a reduction in the activated form of Cdk1 was probably the result of the activation of the checkpoint pathway involving Chk1 and Chk2. The change in the amount of the active dephosphorylated Cdk1 further supported the role of Chk1 and Chk2 in the AZT-induced S and G2/M cell cycle arrest.

Based upon these data, we hypothesize that when a cell starts replicating chromosomes in S phase, the damaged DNA induced by AZT is sensed by repair mechanisms with a consequent delay in cell cycle progression until the damage is repaired. Two facts from our study suggest that removal of AZT from DNA occurred. The first is the activation of a checkpoint pathway that arrests the cell cycle to permit DNA repair and the transcription of genes that facilitate DNA repair (34,35), and the second is that the cell cycle progression was partially restored during the recovery period. A study conducted by Slameňová et al. (36) has indicated that AZT-induced DNA damage is repairable; however, to date, only a few studies have been performed that address the mechanisms underlying the removal of AZT from DNA. In Escherichia coli, it has been shown that AZT does not cause DNA lesions that are repairable by excision repair and/or error-free post-replication repair processes, but rather an SOS response appears to be induced by DNA chain termination leading to the inhibition of DNA replication (37). Furthermore, an enzyme with exonucleolytic activity has been reported to remove AZT from chain-terminated DNA in eukaryotic cell line K562 (13). Additional studies to understand the mechanisms of AZT–DNA repair are clearly warranted.

In conclusion, we have demonstrated that exposure of NIH 3T3 cells to AZT and, to a much lesser extent, 3TC affects cell growth due to alterations in the transit through the cell cycle and that checkpoint enzymes Chk1 and Chk2 may play an important role in this delay. Further studies that aim to understand the mechanisms of AZT–DNA repair are under way in our laboratory.

Funding

Interagency Agreement 224-07-0007 between National Center for Toxicological Research, U.S. Food and Drug Administration and the National Institute for Environmental Health Sciences/National Toxicology Program.

Acknowledgments

This article is not an official guidance or policy statement of U.S. Food and Drug Administration. No official support or endorsement by the U.S. Food and Drug Administration is intended or should be inferred.

Conflict of interest statement: None declared.

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