Mutagenesis Advance Access originally published online on March 22, 2005
Mutagenesis 2005 20(2):139-146; doi:10.1093/mutage/gei019
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Published by Oxford University Press on behalf of the UK Environmental Mutagen Society 2005
Zidovudine induces S-phase arrest and cell cycle gene expression changes in human cells
Carcinogen–DNA Interactions Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA and 1Laboratory of Molecular Oncology, Quilmes National University, Bernal, Argentina
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Abstract |
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Antiretroviral therapy for the human immunodeficiency virus-1 (HIV-1) typically includes two nucleoside reverse transcriptase inhibitors (NRTIs). 3'-Azido-3'-deoxythymidine (AZT, Zidovudine) plus 2'-deoxy-3'-thiacytidine (3TC, Lamivudine) is a combination that is used frequently. The NRTIs are mutagenic nucleoside analogs that become incorporated into DNA and terminate replication. We therefore hypothesized that exposure to this class of drug may alter cell cycle parameters. We used flow cytometry to examine the cell cycle in human epithelioid carcinoma (HeLa) cells exposed to AZT and 3TC alone, as well as a series of AZT/3TC dose combinations: (A) 125.0 µM AZT/12.5 µM 3TC; (B) 250.0 µM AZT/25.0 µM 3TC; and (C) 500 µM AZT/50 µM 3TC. At 24 h, at all doses, there was a good cell viability (
68%), and incorporation of AZT into nuclear DNA. Using flow cytometry, a dose-related increase in the percentage of cells in S phase, from 9.5% with no drug, to 36.0% with dose C, was observed in cells exposed for 24 h (P = 0.001, ANOVA). A concomitant decrease in the percentage of cells in G1 phase, from 82.6% with no drug to 58.5% with dose C, was observed in cells exposed for 24 h (P = 0.017, ANOVA). A similar S phase arrest was seen in cells exposed to 125, 250 and 500 µM AZT alone, but there was no S phase alteration with 50 µM 3TC alone, suggesting that AZT is responsible for the accumulation of cells in S phase. To elucidate the accumulation of cells in S phase and explore the cell cycle gene expression changes induced by AZT and 3TC, we used c-DNA microarray, Cell Cycle Super Array and real-time PCR. There was a strong upregulation of the DNA damage-inducible transcript 3 (DDIT3 or GADD153) in NRTI-exposed cells. In addition, AZT induced an upregulation of cyclin D1 accompanied by a downregulation of the cyclin D1-associated inhibitors P18 and P57, and the G1-S check point gene P21, the net effect of which would be to foster a cell progression into S phase. Cyclin A2 was down-regulated in cells exposed to AZT, suggesting a block in S–G2–M progression that would also be consistent with the accumulation of cells in S phase. Overall, the study demonstrates that AZT, but not 3TC, causes an arrest of cells in S phase with a consistent alteration in the expression of several cell cycle genes. |
Introduction |
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Highly active antiretroviral therapy (HAART), the most effective treatment for human immunodeficiency virus-1 (HIV-1), typically consists of drug combinations that include two NRTIs, a non-nucleoside reverse transcriptase inhibitor, and a protease inhibitor. These drugs are dideoxy-nucleosides that become incorporated into DNA, function as DNA elongation chain terminators, and produce genotoxic manifestations that include mutagenesis, chromosomal aberrations and telomere shortening (1
). Common NRTI components of HAART therapy include 3'-azido-3'-deoxythymidine (AZT) and 2'-deoxy-3'-thiacytidine (3TC). AZT-DNA incorporation has been reported in nuclear and mitochondrial DNA from mice, monkeys and humans (2–4), as well as in DNA from cultured cells (2,5). Chromosomal aberrations have been found in lymphocytes from patients receiving AZT therapy (6) and in bone marrow of AZT-exposed mice (7). A positive correlation has been found between the incorporation of AZT into DNA and induced mutant frequency in TK6 cells (8). Furthermore, Meng et al. (9) reported a multiplicative synergistic enhancement of AZT-DNA incorporation and mutant frequency in response to the exposure of human lymphoblastoid cells to the combination of AZT plus Didanosine (ddI).
In this study, we hypothesized that AZT and other NRTIs might alter cell cycle parameters, and we therefore used flow cytometry to examine the cell cycle profiles of human cervical epithelioid carcinoma (HeLa) cells exposed for 24 h to either AZT or 3TC alone, or the combinations AZT plus 3TC. Under the chosen experimental conditions, the cells showed good survival (68% at 24 h) and values for AZT-DNA incorporation were similar to those documented previously (2). Upon finding that AZT, but not 3TC, induces an accumulation of cells in S phase, we used microarrays and real-time PCR to examine the gene expression of cell cycle-related genes in order to elucidate the underlying molecular events.
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Materials and methods |
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Cell culture conditions and exposure
HeLa cells were cultured in minimum essential medium Eagle (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (ATCC). AZT (Sigma, St Louis, MO) and 3TC (Moravek Biochemicals, Mountain View, CA) were dissolved in phosphate-buffered saline and the final concentration was determined by spectrophotometry at 266 nm for AZT and 272 nm for 3TC. Single drug treatments were: 125, 250 and 500 µM AZT and 1, 10 and 50 µM 3TC. For the purpose of simplicity, the combination doses will be defined as: A = 125 µM AZT/12.5 µM 3TC; B = 250 µM AZT/25 µM 3TC; and C = 500 µM AZT/50 µM 3TC.
Cell viability assays
A commercial proliferation assay, CellTiter 96TM (Promega, Madison, WI), was used to assess cell viability. Cells were exposed to the above mentioned doses in triplicate wells of a 12-well plate. After 24 and 48 h, aliquots were transferred to 96-well plates and incubated for 3 h with a tetrazolium salt. The reaction was terminated, and transformation of tetrazolium salt into a blue colored formazan product was captured by a visible light plate reader (Spectra MAX 190, Molecular Devices, Sunnyvale, CA) at 570 nm with background correction at 730 nm. Survival was calculated by comparing the emissions from exposed cells and unexposed cells. A negative control, consisting of cells killed by freezing/thawing cycles, was assayed in three separate wells, and the values of these wells were averaged and subtracted from values for wells containing unknown experimental samples.
Cell cycle analysis
Analysis of the cell cycle was carried out by flow cytometry. In two independent experiments, HeLa cells were plated in 6-well plates, and for each plate three wells were unexposed, and three wells were exposed. After exposure to the above mentioned doses at 24 h, 
106 cells were harvested, pelleted and washed with culture medium without serum. Cells were fixed in 1 ml of ice-cold 70% ethanol dropped while vortexing. Following an overnight fixation at 4°C, the cells were pelleted by centrifugation and incubated with 50 µl (100 U) of RNAse (Qiagen, Valencia, CA) at room temperature for 20 min. Propidium iodide (500 µl of 10 µg/ml) (Sigma) was added to each cell suspension and the cells were kept in the dark at 4°C overnight. Cells were passed through a fluorescence activated cell sorter (FACSCalibur, BD Biosciences, San Jose, CA) using the doublet discrimination module, and data were acquired using CellQuest (BD Biosciences) software. The cell cycle was modeled using ModFit software (Venty Software, Topsham, ME). Percentages of cells in S and G1 phases were calculated directly by the software.
Radioimmunoassay (RIA) of AZT and 3TC incorporated into DNA
High molecular weight HeLa DNA was prepared with a non-organic kit from Intergen Company (Purchase, NY). DNA samples, quantified by spectrophotometry at 260 nm, were diluted to a final concentration of 25–30 µg DNA/ml in Tris–EDTA buffer (10 mM Tris and 1 mM EDTA). DNA samples were sonicated for 45 s, boiled for 10 min, placed immediately on ice and assayed by competitive RIA. Standard curves consisted of serial dilutions of the drug to which a consistent quantity of boiled and sonicated carrier DNA, matching the quantity of sample DNA, had been added.
The AZT-RIA has been described previously (2). Briefly, a rabbit polyclonal anti-AZT antibody (Sigma), which also recognizes AZT in DNA, reconstituted and diluted to 1:7500, was incubated with HeLa DNA for 90 min at 37°C. Next, 100 µl [3H]AZT (16 Ci/mmol, Moravek Biochemicals) containing 20 000 c.p.m., was added along with 100 µl goat anti-rabbit immunoglobulin G (Sigma), and the mixture was incubated for 25 min at 4°C. After centrifugation (2000 g for 15 min at 4°C), the resulting supernatant was decanted, the pellets were dissolved in 0.1 M NaOH and counted in a liquid scintillation counter. The concentration of standard AZT, added to 3 µg of carrier calf thymus DNA, required to inhibit antibody binding by 50% was 4.67 ± 1.22 pmol AZT (mean ± SE, n = 3). The lower limit of detection was 18.8 molecules of AZT/106 nt. Each sample was assayed in three separate RIAs.
The 3TC-RIA protocol was adapted from Robbins et al. (10) with some modifications. Briefly, 2.5 µg of sample DNA was incubated with an anti-3TC antibody (Cayman Chemical, Ann Arbor, MI) diluted to 1:700 with sample buffer (25 mM potassium phosphate, pH 7.4), and a radiolabeled tracer [3H]-3TC (Cayman Chemical), for 2 h at room temperature with gentle agitation. A reconstituted goat anti-rabbit immunoglobin G (Sigma) was added and the mixture was incubated for an additional 30 min at room temperature. Tubes were centrifuged (room temperature for 30 min at 2000 g), the supernatant decanted and the pellet was dissolved in 0.1 N HCl before counting in a scintillation counter. The concentration of standard 3TC, added to 2.5 µg of carrier calf thymus DNA, required to inhibit antibody binding by 50% was 5.3 ± 1.1 pmol 3TC (mean ± SE, n = 3). The lower limit of detection was 50 molecules of 3TC/106 nt. Each sample was assayed in 3 separate RIAs.
cDNA microarray analysis
HeLa cells were exposed on two separate occasions to the drug combination C (500 µM AZT/50 µM 3TC) for 24 h, and total RNA from the unexposed cells or drug-exposed cells was extracted on each occasion using Trizol (Invitrogen, Carlsbad, CA). Using 20 µg of total RNA, c-DNA probes were generated by indirect labeling using the green color Cy3 dye (Amersham, Buckinghamshire, UK) for unexposed cells and the red color Cy5 dye (Amersham) for drug-exposed cells. Labeled probes were denatured and hybridized to cDNA microarrays containing 9600 human immobilized cDNA elements (Microarray Facility, Advanced Technology Center, NCI). Following hybridization, slides were washed with sodium saline citrate (SSC), spun, dried and scanned using a Gene Pix 4000 A scanner. Spots with yellow color indicated no changes in gene expression. Increases in Cy5 (red color) indicated an upregulation in drug-exposed cells, and increases in Cy3 (green color) indicated a downregulation in drug-exposed cells. Analysis of gene expression changes was performed by the National Cancer Institute Microarray Database System. Comparative analysis of gene expression was performed on replicate RNA samples from each of the two different exposures. Hierarchical clustering was performed using the National Cancer Institute Microarray Database System, which allowed the visualization of groups of genes organized into a mock phylogenetic tree based on similar expression levels.
Cell cycle super array
Specific pathway gene expression was interrogated for 23 genes involved in the G1–S transition phase of the cell cycle using printed membranes (SuperArray, Bioscience Corporation, Frederick, MD). Genes of interest were printed in duplicate along with non-specific genes (plasmid c-DNA) and housekeeping genes were used for normalization purposes. Reverse transcription of cellular RNA was carried out with the RT-Labeling Kit (SuperArray, Bioscience Corporation) according to the manufacturer's instructions. The biotinylated probes from NRTI-exposed and unexposed cells were hybridized overnight to separate membranes at 60°C, washed with SSC/SDS solutions, incubated with the avidin–alkaline phosphatase conjugate and exposed to a chemiluminescent substrate. Images of the hybridized spots were captured with a LumiImager (Roche, Indianapolis, IN). Analysis of the images and quantitation of the spots was achieved by the ScanAlyze 2.5 software, and normalization of the values and comparison of the intensities of the spots in both membranes was achieved using the GE ArrayAnalyzer 1.3 (SuperArray Bioscience Corporation) software.
Real-time PCR
Total RNA (1 µg) from exposed and unexposed cells was heat denatured for 3 min at 85°C in the presence of 5 µM Random Decamers and reverse transcribed using 200 U of mouse mammary leukemia virus–reverse transcriptase, 10 U of RNase inhibitor and 0.5 mM dNTP mix in 1x RT buffer at 42°C for 1 h, according to the manufacturer's (RETROscript, Ambion, Austin, TX) instructions. The RT in the reaction mix was inactivated by heat denaturation at 92°C for 10 min and the vials were stored at –20°C until real-time PCR evaluation as follows. Highly purified salt-free primer for: target gene 1 (CCNE2, cyclin E2: forward primer, TTG GCT ATG CTG GAG GAA GT; reverse primer, CCT GGT GGT TTT TCA GTG CT), target gene 2 (CCND1, cyclin D1: forward primer, CGT GGC CTC TAA GAT GAA GG; reverse primer, CCA CTT GAG CTT GTT CAC CA), target gene 3 (CDKN1A, p21, Cip 1: forward primer, GGA AGA CCA TGT GGA CCT GT; reverse primer, AAT CTG TCA TGC TGG TCT GC), target gene 4 (DDIT3, DNA-damage inducible transcript 3: forward primer, GCG CAT GAA GGA GAA AGA AC; reverse primer, TCA CCA TTC GGT CAA TCA GA), target gene 5 (CCNA2, cyclin A2: forward primer, CCT GCA AAC TGC AAA GTT GA; reverse primer, AAA GGC AGC TCC AGC AAT AA), target gene 6 (CCND2, cyclin D2: forward primer, TAC CTT CCG CAG TGC TCC TA; reverse primer, TCA CAG ACC TCC AGC ATC CA) and reference gene (18S RNA,18S subunit ribosomal protein mRNA: forward primer, GGA CAC GGA CAG GAT TGA CA; reverse primer, AGA CAA ATC GCT CCA CCA AC) (Invitrogen) were optimized to an equal annealing temperature of 64°C. PCR reaction conditions were optimized in a gradient cycler (Smartcycler, Cepheid, Sunnyvale, CA) using 5.0 µl of mastermix (Premix Taq: Ex Taq R-PCR Version, TaKaRa, Shiga, Japan), 2.5 µl of SYBR Green I (Invitrogen, 25 000 dilution), 0.5 µl of forward primer (0.4 µM), 0.5 µl of reverse primer (0.4 µM), 2.0 µl of cDNA (equivalent to 5.0 ng reverse transcribed total RNA) and 14.5 µl of H2O to make a 25 µl reaction mix. The reaction mix was transferred to the SmartCycler tube, topped with mineral oil, centrifuged and placed into the instrument. The SmartCycler experimental run protocol was as follows: denaturation cycle (95°C for 3 min), 40 cycles of amplification (95°C for 3 s, 64°C for 6/s, 72°C for 10 s with fluorescence measurement during annealing), and melting curve analysis (60–95°C, increasing temperature at the rate of 0.2°C/s with continuous fluorescence measurement). Each sample was assayed in triplicate and the cycle threshold (CT) values were normalized to the housekeeping gene (18S RNA), while the fold change was calculated using the 2–AACT method. Specificity of real-time PCR products was determined by melting curve analysis. Gene specific amplicons gave single melting temperatures and no primer-dimers were generated.
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Results |
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Cytotoxicity and NRTI-DNA incorporation in HeLa cells
Cell viability was determined in HeLa cells either unexposed or exposed for 24 (Table I) and 48 h to the AZT/3TC combination doses A, B and C. When untreated cells were considered 100% viable, cell viability at 24 h was 67, 77 and 87% for the A, B and C doses, respectively. All the doses showed
50% viability at 48 h (data not shown).
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Incorporation of AZT and 3TC into cellular DNA, intended to validate the genotoxicity of these exposures, was measured by drug-specific RIAs. Using DNA extracted from cells exposed for 24 h to AZT/3TC combinations A, B and C, AZT-DNA was measurable at all three doses but 3TC-DNA was measurable only with dose B (Table I).
Cell cycle changes in HeLa cells, measured by flow cytometry
Flow cytometry was used to examine the changes in cell cycle induced in HeLa cells that were either unexposed, or exposed for 24 h to: 125, 250 or 500 µM AZT alone; 1, 10 or 50 µM 3TC alone; or doses A, B and C of the combination AZT/3TC (Figure 1, Table I). Cells were exposed on two separate occasions, and for each experiment the distribution of cells at different phases of the cell cycle was determined either in duplicate for drug-exposed cells, or triplicate for unexposed cells. Separation of cells in G0/G1, S phase and G2/M was based upon linear fluorescence intensity after staining with propidium iodide. Representative profiles are shown in Figure 1. The large initial peak (left) represents cells in G0/G1, the intervening area represents cells in S phase and the final tail/small peak (right) represents cells in G2/M.
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The percentage of cells in S phase increased from 9.5 to 32 (Figure 1a and d) in HeLa cells exposed to 0, 125, 250 or 500 µM AZT for 24 h. In HeLa cells exposed to the AZT/3TC combination at doses A, B and C (Figure 1b), a similar dose-related increase in the percentage of cells in S phase was observed with the highest dose having 36% of cells in S phase (Table I). For 3TC doses of 0, 1.0, 10.0 and 50 µM, the cell cycle changes were minimal with 12% of cells in S phase at the highest dose (Figure 1c). The data indicate that AZT, and not 3TC, is responsible for the S phase accumulation seen in NRTI-exposed cells.
A decrease in the percentage of cells in G0/G1 phase accompanied the AZT-induced accumulation of cells in S phase. Unexposed HeLa cells had 82.6% of cells in G1, while cells exposed to the A, B and C combination doses had 63.4, 63.0 and 58.5% of cells in G1, respectively (Table I). Furthermore, decreases in G1 were observed with AZT alone (Figure 1a) but not with 3TC alone (Figure 1c). The data indicate that AZT, and not 3TC, is responsible for the decrease in the percentage of cells in G0/G1 phase.
Gene expression changes in HeLa cells determined by cDNA microarray
Cells were exposed to combination dose C (500 µM AZT/50 µM 3TC) for 24 h on two separate occasions and RNA was extracted from exposed and unexposed cells. Among the 9600 elements hybridized, 5 were downregulated 2-fold or more, and 38 were upregulated 2-fold or more. An ontogenic analysis revealed that 23 elements were involved in metabolic pathways, 11 elements were related to cell growth or maintenance, 8 elements were involved in cell communication, and of the 16 elements that were related to external stimulus response (including stress), 8 had involvement in the death process.
For hierarchical clustering, elements were grouped based on the similarity of their expression profiles, and a pseudocolor image representing the degree of induction (red) or repression (green) occurring as a result of the combined drug exposure was produced. Figure 2 shows a hierarchical clustering of all elements changed by 2-fold in cells exposed to dose C; each lengthwise row of the panel shows a separate microarray. Remarkable consistency was observed for the four microarrays, where each RNA was subjected to two microarrays with reciprocal Cy3 and Cy5 labeling.
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cDNA microarray and Super Array changes in cell cycle genes
Having observed the AZT-induced accumulation of cells in S phase, we hypothesized that AZT exposure might also change the expression of cell cycle genes. To address this question we first queried the cDNA microarray data base (above), obtained from HeLa cells exposed to either 0 or combination dose C, for cell cycle gene expression changes. Finally, we examined the same RNA samples using the Cell Cycle Super Array. With minor exceptions, the two array platforms are fundamentally different and do not query the same genetic elements.
Using the cDNA microarray data base, cell cycle genes altered 1.7-fold were examined (Table II). The most highly upregulated gene was ‘DNA damage inducible transcript 3’ (DDIT3), otherwise known as ‘growth arrest and DNA damage 153’ (GADD153). This stress-response gene, known to inhibit protein synthesis leading to growth arrest and apoptosis (11,12), was induced 4-fold. A second highly upregulated gene was cyclin D1, which initiates the cell cycle and downstream events, being responsible for progression through the G0–G1/S phase transition (13). Furthermore, the cyclin-dependent kinase inhibitors P18 and P57, which inhibit cyclin D, were downregulated, presumably allowing for an enhanced cyclin D activity. Cyclin A2, which regulates S/G2 progression (13), was downregulated, potentially slowing progression of cells out of S phase. Furthermore, the observed downregulation of dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), necessary for the synthesis of nucleic acids, could slow the progression of cells through S phase.
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To supplement the cDNA microarray data, the Cell Cycle Super Array was used to query changes in gene expression
1.7 fold, and the data are also shown in Table II. No upregulation was found using this array, but the observed downregulation of several cell cycle check points, including p21 (CDKN1A), P107 (RBL1), P130 (RBL2) and P27 (Kip 1, CDKN1B), would favor cell cycle progression in the presence of DNA damage. One gene, P57 (Kip2, CDKN1C), was present in both array platforms and showed similar downregulation (Table II).
Real-time PCR
To confirm the cDNA microarray and Super Array data, real-time PCR was performed using primers for p21 (CDKN1A) and the DNA damage inducible gene (DDIT3), as well as cyclins D1 (CCND1), D2 (CCND2), A2 (CCNA2) and E2 (CCNA2), and the values for fold change are shown in Table III. A pictorial comparison of the cDNA microarray and real-time PCR data is shown (Figure 3) for 2-fold change in gene expression in cells exposed to 500 µM AZT, 50 µM 3TC and 500 µM AZT/50 µM 3TC. Real-time PCR confirmed a strong upregulation of the DNA damage inducible (DDIT3) gene, initially observed by cDNA microarray in cells exposed to dose C, and showed additionally that this gene is upregulated by AZT and 3TC alone. Upregulation of cyclin D1 was found in cells exposed to dose C which also confirmed the cDNA microarray. Furthermore, this gene was upregulated by AZT alone, but not by 3TC alone. The real-time PCR showed a strong downregulation of cyclin E2 and D2 in all three exposure groups, while cyclin A2 and p21 were either downregulated or showed no change.
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Discussion |
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Cultured HeLa cells exposed to AZT, 3TC or combinations of AZT and 3TC, were subjected to flow cytometric analysis, which revealed an accumulation of cells in S phase only for cells exposed to AZT or AZT/3TC. Since this phenomenon was not observed in unexposed cells or cells exposed to 3TC alone, it was concluded that the accumulation of cells in S phase was largely attributable to the presence of AZT. In order to investigate changes in gene expression associated with the S phase accumulation, RNA samples were subjected to two different microarray platforms, cDNA Microarray and Cell Cycle Super Array, which showed changes in DNA damage response and cell cycle genes. Several of the most strongly altered genes were selected for confirmation by real-time PCR. Some, but not all, of the alterations in gene expression were consistent with the accumulation of cells in S phase observed by flow cytometry.
The antiretroviral nucleoside analog drugs, AZT and 3TC, become phosphorylated and incorporated into both viral and host nuclear and mitochondrial DNA (1,14). While the phosphorylated drugs incorporate into nascent DNA chains, they lack the necessary ribose moieties required for DNA chain extension and therefore act as DNA chain terminators. Various genotoxic manifestations (clastogenicity, mutagenicity) resulting from such events, have been documented (1). Incorporation of AZT into DNA has been reported in different mammalian cell lines (2), in mouse and monkey tissues (3,7,15), and in adult and infant human lymphocyte DNA (4). Previous studies, using thymidine-starved human HL60 cells exposed to 800 µM AZT for 4 h, reported the AZT-DNA incorporation of 107 molecules AZT/106 nt (2). In these experiments, the cells were not thymidine-starved and the dose was lower, but the 68 molecules AZT/106 nt observed here for the 500 µM AZT/50 µM 3TC combination was comparable.
The dose-related S phase cell cycle accumulation described in these experiments appears to be primarily the result of AZT exposure. A similar cell cycle pattern has been documented for AZT in studies performed in H9 cells (16), WiDr human colon cancer cells (17) and MCF-7 and K562 cells (18). A synergistic S phase accumulation was reported in human peripheral blood leukocytes exposed to AZT and ddC (19). A delay in progression through S phase is consistent with the known NRTI chain termination activity, as the truncated DNA chains would require some time for repair before S phase could be completed. In the presence of continued drug exposure the open nascent chains might also be vulnerable to continuing chain termination.
In this study, we have demonstrated AZT-induced expression changes in DNA damage-response, cell cycle check point/progression and nucleic acid synthesis genes, many of which were consistent with the observed AZT-induced S phase arrest. The most strongly upregulated gene was DDIT3, otherwise known as GADD153. This gene acts in response to the stress of chemical exposure, radiation and other events, blocking proliferation at G1 and G2 check points. The DDIT3 gene product binds to various transcription factors, resulting in the inhibition of protein synthesis and induction of apoptosis (11,12). A second upregulated gene, cyclin D1, initiates the cell cycle, being responsible for phosphorylation of Rb, induction of the transcription factor E2F and many downstream cell cycle activities (13,20,21). In this study, cyclin D1 was upregulated 2-fold while the cyclin-dependent kinase inhibitors P18 and P57, which inhibit cyclin D, were downregulated. Taken together, these changes would potentially allow for an enhanced cyclin D activity and cell cycle stimulation. The downregulation of p21, may have allowed the progression from G1 to S phase in the presence of DNA damage, as this protein is responsible for cell cycle arrest as a consequence of DNA damage (22). The downregulation of cyclin A2 is also consistent with the observed cell cycle arrest as the cyclin A family regulates S/G2 progression. Furthermore, as DHFR and TYMS are key components in the synthesis of nucleic acids, the downregulation of these enzymes may have slowed the progress of DNA replication required for the progression of cells through S phase (23). The downregulation of cyclin E2 (CCNE2) is consistent with studies showing that this gene plays a role in initiating resting cells into the cell cycle, controlling progression from G0 to G1 (24–27). Since the HeLa cells used here were already progressing through the cycle, there was no need for cyclin E to be expressed. Overall, the gene expression data presented here reveal several cell cycle alterations that are consistent with the observed accumulation of cells in S phase induced by AZT.
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Acknowledgments |
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We greatly appreciate the editorial assistance of Mrs Bettie Sugar and Ms Mary Velthuis.
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* To whom correspondence should be addressed at: Building 37, Room 4032B, NIH, 37 Convent Drive, MSC-4255, Bethesda, MD 20892-4255, USA. Tel: +1 301 435 7843; Fax: +1 301 402 8230; Email: oliveroo{at}exchange.nih.gov
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Received on January 20, 2005; revised on February 16, 2005; accepted on February 21, 2005.
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