Relationships between DNA Incorporation, Mutant Frequency, and Loss of Heterozygosity at the TK Locus in Human Lymphoblastoid Cells Exposed to 3'-Azido-3'-deoxythymidine

Quanxin Meng*, Ting Su*,{dagger}, Ofelia A. Olivero{ddagger}, Miriam C. Poirier{ddagger}, Xiaochu Shi*, Xinxin Ding*,{dagger} and Vernon E. Walker*,{dagger},1

* Wadsworth Center, New York State Department of Health, Albany, New York 12201–0509; {dagger} School of Public Health, State University of New York at Albany, Albany, New York 12203; and {ddagger} Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received September 13, 1999; accepted November 23, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes tumors in mice exposed as adults or in utero. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of AZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant frequencies (Mfs), and determining the percentage of loss of heterozygosity (LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid cell line, TK6. Cells were exposed to 300 µM AZT for 0, 1, 3, or 6 days, or to 0, 33, 100, 300, or 900 µM AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK Mfs were assessed by a cell-cloning assay. AZT was incorporated into DNA in a dose-related manner at concentrations up to 300 µM, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0.903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10–6/7.4 x 10 –6) of AZT-induced mutants were attributable to LOH, consistent with the known mechanism of AZT as a DNA chain terminator. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further study.

Key Words: 3'-azido-3'-deoxythymidine; human lymphoblastoid cells; DNA incorporation; thymidine kinase; mutant frequency; loss of heterozygosity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the treatment of AIDS patients and for prevention of the onset of AIDS in HIV-seropositive individuals, causes vaginal epithelial cell tumors in adult mice and rats, and lung, liver, and female reproductive system tumors in mice exposed in utero (Ayers et al., 1996Go; Diwan et al., 1999Go; Olivero et al., 1997Go). While the long-term effects of AZT exposure in humans are unknown, in AIDS patients AZT treatment causes dose-limiting bone marrow toxicity, including anemia, neutropenia, and thrombocytopenia (Fischl et al., 1987Go; Pizzo, et al., 1988Go; Richman et al., 1987Go). The mechanisms of bone marrow toxicity and potential long-term effects of AZT exposure in humans have been extensively studied, and two major mechanisms have been proposed. The first hypothesis is that the imbalance of deoxyribonucleotide pools resulting from the inhibition of thymidine kinase by AZT-monophosphate causes the bone marrow toxicity of AZT (Furman et al., 1986Go; Hao et al., 1988Go). However, experimental approaches have produced conflicting results, and the association between the perturbation of deoxyribonucleotide pools and AZT-related bone marrow toxicity is still under debate (Darnowski and Goulette, 1994Go; Frick et al., 1988Go; Fridland et al., 1990Go; Furman et al.,1986Go; Sommadossi et al., 1989Go; Vazquez-Padua et al., 1990Go; Hao et al., 1988Go). In contrast, substantial evidence is accumulating to support a second hypothesis that genetic damage results from the incorporation of AZT-triphosphate into cellular DNA (Vazquez-Padua et al., 1990Go). Incorporation of AZT into cellular DNA has been demonstrated in several cell-culture systems and in transplacentally exposed mice and monkeys (Darnowski and Goulette, 1994Go; Olivero et al., 1994Go, 1997Go; Vazquez-Padua et al., 1990Go). Recently, Olivero et al. (1999) found that the majority of leukocyte samples from peripheral blood of AZT-exposed adults or cord blood of infants had detectable AZT-DNA levels. A study conducted by Sommadossi et al. (1989) indicated a highly significant relationship between the extent of AZT incorporation into cellular DNA and the clonogenic survival fraction of human bone marrow progenitor cells. However, the relationships between AZT therapy, DNA incorporation of AZT into host cells, and the mutagenic potential of AZT have not been adequately addressed in terms of the potential for long-term side effects in humans.

Our laboratory has launched a series of studies aimed at evaluating the effects of duration and concentration of AZT exposure on the AZT-DNA incorporation, mutation induction at multiple reporter gene loci, and molecular nature of mutations in the human lymphoblastoid cell line, TK6. Previously, AZT was found to accumulate in DNA of TK6 cells with increase in exposure time, leading to a significant increase in mutant frequency (Mf) over background at the X-linked hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus (Sussman et al., 1999Go). The current study assessed (a) the effects of exposure concentration on incorporation of AZT into DNA, (b) the effects of duration and concentration of AZT exposure on Mfs at an autosomal gene locus, thymidine kinase (TK), and (c) the role of loss of heterozygosity (LOH) in the induction of mutation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and exposures.
TK6 B-lymphoblastoid cells were grown in suspension in 75 cm2 tissue culture flasks containing RPMI 1640 medium supplemented with 10% FBS, 12.5 mM HEPES buffer, 4 mM L-glutamine, 100 µM non-essential amino acids, and 100U/ml pencillin/streptomycin. Cells were subcultured daily at 4 x 105 cells/ml. Prior to experimental exposure, cells were grown for 2 days in medium containing CHAT (cytidine, hypoxanthine aminopterin, and thymidine) and 1 day in medium containing THC (thymidine, hypoxanthine, and cytidine) to reduce the background TK Mf (Liber and Thilly, 1982Go). All cultures (40 ml, 5 x 105 cells/ml) were exponentially growing at the time of treatment. AZT (Sigma, St. Louis, MO) was dissolved in culture medium for exposures.

To evaluate the effects of exposure duration on TK Mf, cultures (n = 5 flasks/group) were exposed to 300 µM AZT for 0, 1, 3, or 6 day(s). Immediately after exposure, cells were washed and plated at a density of 2 or 4 viable cells/well in 96-well U-bottom microtiter plates in the presence of 4 x 104 lethally irradiated feeder cells to determine relative survival of treated cells versus unexposed cells by measuring cloning efficiency. The remaining cells were subcultured daily for 3 days to allow expression of the mutant phenotype, and seeded in 96-well plates as described below, to measure TK Mfs.

To determine the effects of exposure concentration on TK Mfs, cultures (n = 5 flasks/group) were exposed to 0, 33, 100, 300, or 900 µM AZT for 3 days. Aliquots of cells were plated as described above for a determination of relative survival. In addition, aliquots of cells from each flask (10 x 106 cells/flask) were washed and the pellets frozen for DNA isolation and AZT-DNA incorporation studies. The remaining cells were subcultured and plated to measure TK Mfs. To generate more independent mutant colonies for molecular analysis, 25 additional cultures (15 control, 10 exposed to 300 µM AZT for either 3 or 6 days, 10 ml/flask) were plated.

Study of AZT incorporation into cellular DNA.
Genomic DNA was isolated from TK6 cells using an Oncor non-organic extraction procedure (Oncor, Gaithersburg, MD), followed by RNase treatment of the extracted DNA. A competitive anti-AZT radioimmunoassay (AZT-RIA) was used to measure incorporation of AZT into genomic DNA as previously described (Olivero et al., 1994Go). Briefly, a rabbit polyclonal anti-AZT antibody (diluted 1:5000) was incubated with either standard AZT plus 3 µg of calf thymus carrier DNA, or 3 µg sample DNA from untreated or AZT-treated cells, for 90 min at 37°C. [3H]AZT tracer (20Ci/mmol) and the secondary antibody, goat anti-rabbit immunoglobulin G, were then added and incubated for 25 min at 4°C. The mixture was subjected to centrifugation at 2000 x g for 15 min at 4°C. The resulting supernatant was decanted and the pellets were dissolved in 100 mM NaOH and counted in a liquid scintillation counter.

The amount of AZT in 3 µg of biological sample DNA was obtained by comparing DNA from untreated cells with DNA from treated cells, calculating the percent inhibition of antibody binding, and reading the amount of AZT from the plotted standard curve. Typically, the control sample has no inhibition or minimum inhibition, and it is similar to the value of maximum binding between the tracer and the antibody for the generation of a standard curve in every assay. Each sample was assayed in 2 separate radioimmunoassays and calculated against the control sample (with the control value set at "zero"). Levels of incorporation were expressed as molecules AZT/106 nucleotides (Olivero et al., 1997Go).

Cell-cloning assay for TK mutant frequencies.
After exposure, cells were washed and subcultured in non-selective medium for 3 days to allow phenotypic expression of TK mutations. To determine the TK Mfs, a cell-cloning assay was employed as previously described by Liber and Thilly (1982). Briefly, two 96-well U-bottom microtiter plates per sample were seeded with 2 viable cells/well in the presence of 4 x 104 lethally irradiated feeder cells/well to determine cloning efficiencies. To evaluate the TK Mf for each sample, ten 96-well plates were seeded at 4 x 104 cells/well in the presence of 1 µg/ml trifluorothymidine (TFT). The TK-negative colonies were scored at 10, 21, 28, or 35 days after plating to define the time course of colony formation. Mf was calculated as the ratio of mean cloning efficiency in selective medium to that in non-selective medium (Sussman et al., 1999Go). One to 3 TK mutants from each independent culture were transferred to 12-well plates, expanded in fresh medium and collected after 1 week for molecular analysis.

Southern-blot analysis for determining loss of heterozygosity.
DNA samples were extracted from the propagated mutant colonies using an automated DNA extraction procedure (Walker et al., 1992Go), and used for Southern-blotting analysis as previously described (Yandell et al., 1986Go). Briefly, a full length TK cDNA purified as a BamH1-Sma1 fragment from plasmid ptk11 was used as a hybridization probe. Twenty µg of Sac1 digested genomic DNA was electrophoresed in a 0.8% agarose-TAE buffer gel and transferred onto Gene Screen Plus membrane by capillary blotting. Hybridizations were carried out overnight at 65°C in 1% SDS, 1 M NaCl, 10% dextran sulfate, and 0.5 mg/ml herring sperm DNA. Twenty-five ng of [{alpha}-32P]dATP (New England Nuclear) random primer-labeled hybridization probe was used. Stringent washes were conducted to remove non-specific probe binding from Gene Screen Plus membranes. Films were allowed 1–2 days for exposure.

Statistical analyses.
Statistical significance of the differences in Mf values between control and AZT-treated groups were determined using the Mann-Whitney U-statistic. The correlation between AZT incorporation and AZT-induced Mfs was determined by the Pearson Product Moment Correlation test. The Chi-square test was used to measure the significance of the differences of LOH spectra between control and AZT-treated cells. A P-value <= 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of AZT Exposure on TK6 Cell Survival
Relative cell survival was evaluated by comparing the cloning efficiency in AZT-exposed cells versus that in unexposed cells immediately after exposure (with the control value set at 100%). Cell survival declined with the extension of exposure duration to 300 µM AZT up to 6 days (Fig. 1Go), or with the increase of exposure concentration up to 900 µM for 3 days (Fig. 2Go). The levels of cell survival indicated that there were sufficient viable cells after AZT-exposure to allow meaningful measurements of TK Mf.



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FIG. 1. Effects of the duration of 3'-azido-3'-deoxythymidine (AZT) exposure on the relative survival of TK6 cells. Cultures (n = 5/group) were exposed to 300 µM AZT for 0, 1, 3, or 6 days. Relative cell survivals were ratios of cloning efficiency in treated versus control samples determined immediately after exposure. Points, averages; bars, SE.

 


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FIG. 2. Effects of the concentration of 3'-azido-3'-deoxythymidine (AZT) exposure on the relative survival of TK6 cells. Cultures (n = 5/group) were exposed to 0, 33, 100, 300, or 900 µM AZT for 3 days. Relative cell survivals were ratios of cloning efficiency in treated vs. control samples determined immediately after exposure. Points, averages; bars, SE.

 
Effect of AZT-Exposure Concentration on the Incorporation of AZT into DNA
The relationship between exposure concentration and incorporation of AZT into the genomic DNA of the TK6 cell was assessed after exposure to 0, 33, 100, 300, or 900 µM for 3 days. DNA samples from each exposure concentration were coded and analyzed in a blind fashion. The DNA samples yielding the minimal inhibition (representing zero value) proved to be from unexposed control cells, and the resulting inhibition values for these samples were subtracted from those found for the remaining samples. AZT incorporation increased in a near-linear fashion with exposure concentrations up to 100 µM, and then increased more slowly to reach a plateau at 300 µM AZT (Fig. 3Go).



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FIG. 3. The relationship between concentration of 3'-azido-3'-deoxythymidine (AZT) exposure and AZT incorporation into cellular DNA. Cultures (n = 5/group) were exposed to 0, 33, 100, 300, or 900 µM for 3 days. After exposure, genomic DNA was extracted and assayed by AZT-RIA for AZT incorporation into DNA. Points, averages; bars, SE.

 
Effects of AZT Exposure Duration and Concentration on TK Mutant Frequency
To determine the effects of AZT exposure duration on TK Mfs, lymphoblastoid cells were exposed to 300 µM AZT for 0, 1, 3, or 6 days. The cloning efficiencies ranged from 52.1% to 78%, and were indistinguishable between control and treated cells. AZT produced significant increases in TK Mfs over the control value of 6.8 ± 0.2 x 10–6 (average ± SE), with the observed TK Mfs being 9.8 ± 0.7 x 10–6 (P = 0.004), 13.3 ± 1.0 x 10–6 (P = 0.004), and 15.1 ± 1.8 x 10–6 (P = 0.004) for exposure days 1, 3, and 6, respectively (Fig. 4Go).



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FIG. 4. The relationship between duration of 3'-azido-3'-deoxythymidine (AZT) exposure and AZT-induced TK mutant frequency in TK6 cells. Cultures (n = 5/group) were exposed to 300 µM of AZT for 0, 1, 3, or 6 days. A cell-cloning assay was used to measure the frequency of TK mutants. Mutant frequencies were significantly increased over background after 1 or more days of exposure (P values = 0.004). AZT-induced mutant frequencies were obtained by subtracting the background mutant frequencies from the frequency in AZT-exposed samples. Points, averages; bars, SE.

 
Liber et al. (1989) classified TK mutants as normal growth mutants (mutant colonies observed at day 10 after plating), and slow growth mutants (mutant colonies observed between days 10 and 21 after plating). To explore further the time course of TK mutant-colony formation, trifluorothymidine (TFT) selection plates for cells unexposed or exposed to 300 µM AZT for 1, 3, or 6 days were scored at 10, 21, 28, or 35 days after plating. TK-mutant colonies continued to appear up to 35 days after plating (Fig. 5Go). A few samples were also scored at 42 days after exposure, but the additional net increase of Mfs was less than 0.1 x 10–6 (data not shown). If TK Mfs at 35 days after plating were designated 100%, the Mfs obtained at 10, 21, or 28 days after plating would be, respectively, 37, 72, and 94% of the 35-day value. Considering the percentage of mutant colonies scored and the time factor, 10 and 28 days after plating seemed to be optimal sampling times for scoring normal- and slow-growth TK mutants, respectively.



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FIG. 5. The time course of the TK mutant colony formation following exposure of TK6 cells to 3'-azido-3'-deoxythymidine (AZT). Cultures (n = 5/group) were exposed to 0 or 300 µM AZT for 0, 1, 3, or 6 days. Cells were allowed 3 days for phenotypic expression of TK mutations and then plated in 96-well U-bottom plates. Plates were scored at 10, 21, 28, and 35 days after plating and TK mutant frequencies were determined.

 
To define the dose-response relationship between AZT exposure and TK Mfs, TK6 cells were exposed to 0, 33, 100, 300, or 900 µM AZT for 3 days. The cloning efficiencies ranged between 52.1% and 92.7%, and there were no significant differences between control and AZT-treated samples. Based on the time course of TK mutant-colony formation, as mentioned above, plates were scored at 10 and 28 days after plating, for normal- and slow-growth mutants, respectively. In AZT-exposed cultures, TK Mfs were significantly increased over the control value of 6.5 ± 0.2 x 10–6 (average ± SE) at all the exposure concentrations (P values ranging from 0.004 to 0.008) (Fig. 6Go); the normal-growth mutants constituted about 46%, while the slow-growth mutants made up about 54% of the total AZT-induced Mfs. In addition, there was a significant positive correlation (coefficient = 0.903, P = 0.036, Pearson Product Moment Correlation test) between AZT incorporation into nuclear DNA and AZT-induced TK Mfs in cells exposed to 0, 33, or 100, 300, or 900 µM AZT. If AZT incorporation at 900 µM was excluded, because AZT incorporation into DNA reached a plateau after 300 µM, the correlation was even more significant (coefficient = 0.996, P = 0.004). This high degree of correlation is illustrated by the near plot of the relationship between DNA incorporation of AZT at exposures between 33 and 300 µM and AZT-induced TK Mfs (Fig. 7Go).



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FIG. 6. The relationship between concentration of 3'-azido-3'-deoxythymidine (AZT) exposure and AZT-induced TK mutant frequency in TK6 cells. Cultures (n = 5/group) were exposed to 0, 33, 100, 300, or 900 µM of AZT for 3 days. A cell-cloning assay was used to measure the frequency of TK mutants. Mutant frequencies were significantly increased at all exposure concentrations (P values ranging from 0.004 to 0.008). AZT-induced mutant frequencies were obtained by subtracting the background mutant frequencies from that in AZT-exposed samples. {circ}, normal growth mutants; {blacksquare}, slow growth mutants; •, total. Points, averages; bars, SE.

 


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FIG. 7. The relationship between DNA incorporation of AZT and AZT-induced TK mutant frequencies in TK6 cells. Cultures (n = 5/group) were exposed to 0, 33, 100, or 300 µM of AZT for 3 days. An AZT-RIA and a cell-cloning assay were used to measure AZT incorporation into cellular DNA and TK mutant frequencies, respectively. AZT-induced mutant frequencies were obtained by subtracting the background mutant frequencies from that in AZT-exposed samples.

 
Southern Blot Analysis of TK Mutant Colonies
The TK-6 cell line is heterozygous for a SacI restriction fragment length polymorphism (RFLP) at the TK locus, which allows the functional and nonfunctional TK alleles to be differentiated by Southern-blot analysis (Yandell et al., 1986Go). Figure 8Go shows a Southern blot analysis of SacI-digested genomic DNA from a set of TK mutants. The bands at 14.8- and 8.4-kb are a polymorphic pair resulting from a SacI RFLP at the TK locus. The LOH mutants contain only the 8.4-kb fragment corresponding to the nonfunctional allele.



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FIG. 8. Loss of heterozygosity (LOH) in TK mutants analyzed by Southern blot. Genomic DNA from each of the mutants was digested with SacI. A human thymidine kinase cDNA was used as a hybridization probe. Bands appearing at 14.8 and 8.4 kb correspond to the functional and non-functional alleles, respectively. Samples missing bands at 14.8 kb indicate LOH.

 
For each independent culture, 1–3 mutant colonies that scored on day 10 after plating (normal-growth mutants), and 1–3 mutant colonies that appeared between 28 and 35 days after plating (a portion of slow-growth mutants) were propagated and analyzed for LOH by Southern blotting, with subsequent hybridization using a full length TK cDNA probe. Mutational specificity other than LOH was not characterized. The percentages of LOH in control and AZT-induced mutants are presented in Table 1Go. In spontaneous mutants, the occurrence of LOH in slow-growth mutants (85.4%) was much higher than that in normal-growth mutants (44.9%), which is consistent with the previous findings (Yandell et al., 1986Go, 1990Go). The Chi-square test of the homogeneity of control and AZT-treated groups demonstrated that changes in LOH due to AZT-treatment were significant (P = 0.034) in slow growth mutants but not in normal growth mutants (Table 1Go). The mutant fraction [i.e., (average observed Mf) x (average percent of total mutants for a class of mutation)] can be used to estimate the induced Mf of each class of mutation (i.e., AZT-treated mutant fraction – spontaneous mutant fraction) and provides further evidence that the increase in Mf over background can be mostly accounted for by LOH at the TK locus. The AZT-induced mutant fraction values shown in Table 1Go indicate that LOH accounted for 44% (1.2 x 10–6/2.7 x 10–6) of total AZT-induced Mfs in normal growth mutants versus 100% (0.52 x 10–6/0.52 x 10–6) in slow growth mutants. If mutants analyzed here were representative of all the AZT-induced mutants (normal + slow growth rate), 84% (6.2 x 10–6/7.4 x 10–6) of AZT-induced mutants would be attributable to LOH.


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TABLE 1 Loss of Heterozygosity in TK Mutant from Control and 3'-Azido-3'-Deoxythymidine-treated TK6 Cells
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The genotoxicity of AZT has been variably demonstrated in human cell lines, mice, and HIV-infected patients using multiple end-points, including micronuclei, sister-chromatid exchange, chromosomal aberrations, and gene mutations (Agarwal and Olivero, 1997Go; Ayers et al., 1996Go; Dertinger et al., 1996Go; Gonzales-Cid and Larripa, 1994Go; Grdina et al., 1992Go; Oleson and Getamn, 1990Go; Phillips et al., 1991Go; Shafik et al., 1991Go). AZT incorporation into DNA, a potential mechanism underlying AZT genotoxicity, has also been detected in cell culture from multiple species, animal models, and most importantly, in blood cells from AZT-exposed adults and newborn infants (Darnowski et al., 1994; Olivero et al., 1997Go, 1994Go, 1999Go; Sommadossi et al., 1989Go; Vazquez-Padua et al., 1990Go). However, these AZT genotoxicity studies and the AZT-DNA incorporation measurements were conducted in different samples, which precluded a direct evaluation of the correlation between AZT-DNA incorporation and AZT-induced genotoxic effects. In the current report, AZT-DNA incorporation and AZT-induced TK Mfs were measured using aliquots of the same AZT-exposed TK6 human lymphoblastoid cells, and the effects of duration and concentration of AZT exposure on DNA incorporation and TK mutation induction were compared. The studies demonstrated a direct correlation between DNA incorporation of AZT and AZT-induced mutagenicity.

An apparent linear relationship between the extracellular concentration of [3H]AZT and the amount of AZT incorporated into DNA was previously observed in human colon tumor cells exposed to up to 100 µM AZT for 5 days in the presence of 0.1 µM or 50 µM deoxythymidine (Darnowski et al., 1994). In human bone marrow cells, more sensitive to AZT cytotoxicity, a linear relationship between the initial AZT exposure concentration and AZT incorporation into genomic DNA was seen at exposure concentrations of 1, 5, 10, 15, or 25 µM (Sommadossi et al., 1989Go). In the current study, there appeared to be a linear relationship between exposure and DNA incorporation at AZT concentrations up to 100 µM in TK6 cells. However, there were insufficient exposure concentrations in this dose range to conduct a test for linearity. The plateau of DNA incorporation reached at 300 µM AZT in TK6 cells may have been related to AZT-induced cytotoxicity and/or saturation of metabolic phosphorylation pathways. Cells with higher levels of AZT incorporation died, and they were not represented in cells used for DNA extraction and AZT-radioimmunoassay.

TK6 cells are heterozygous at the TK locus and hemizygous at the HPRT locus. Many mutagenesis mechanisms that involve homologous interaction, such as gene conversion and mitotic recombination, cannot occur at the X-linked HPRT locus. In addition, multi-locus deletions are likely to be lethal in the HPRT gene, because these gross deletions may span the adjacent genes essential for cell survival. Therefore, if the primary mechanism of mutation induction by a chemical implicates homologous interaction or large deletion, the mutagenicity of that agent will be significantly underestimated by the HPRT mutation assay. Previously, whole gene deletions were found to account for 64% of AZT-induced HPRT Mfs in exposed TK6 cells (Sussman et al., 1999Go), and it was reasonably hypothesized that a greater mutagenic response might be seen at the TK locus. Indeed, the average AZT-induced TK Mf (6.5 x 10–6) was 2.2-fold greater than AZT-induced HPRT Mf following simultaneous Mf measurements in aliquots of TK6 cells exposed to 300 µM for 3 days.

The study conducted by Sommadossi et al. (1989) indicated a highly significant relationship (P = 0.001) between the AZT incorporation into cellular DNA of human bone marrow progenitor cells and the inhibition of clonal growth. The present study showed that there was a positive correlation between the extent of AZT incorporation into genomic DNA and TK mutation induction. At 900 µM AZT, more than half of the cells apparently died from AZT cell toxicity; however, the raw number of mutant colonies observed was corrected by the cloning efficiency, which measures cell viability without selection pressure. Therefore, the TK Mfs continued to increase, even after AZT incorporation into cellular DNA reached a plateau at 300 µM AZT. Based on our experience, Mf can be accurately measured with the adjustment of the simultaneously determined cloning efficiency value if more than 20% of exposed cells remain viable.

Liber et al. (1989) first described two distinct phenotypic classes of TK mutants: normal-growth and slow-growth mutants. While the normal-growth TK mutants had a doubling time similar to parental TK6 cells (14–18 h), the doubling time for slow-growth mutants was longer (21–44 h). The presence of slow-growth mutants was hypothesized to be related to a growth gene, which was linked to the TK locus on chromosome 17. A gene mutation that affected both this growth gene and the TK gene would result in a slow-growth-rate mutant colony. The scoring time for normal- and slow-growth mutants was set at 10 and 21 days after plating, respectively, based on the findings that, from 6 plates of cells exposed to X-rays, the first group of mutants (normal growth rate) was microscopically visible at around 10 days after plating, and the appearance of more mutant colonies reached a plateau at about 20 days after plating (Liber et al., 1989Go). However, in the current study, formation of TK mutant colonies was found to be a continuous process, and there was not a clear cut-off time point between normal growth and slow-growth mutants. TK mutants continued to appear through 35 days after plating, with nearly 30% more of the total mutants scored on day 28 (94% of total) compared with day 21 after plating (73% of total). The discrepancies between our observations and the findings of Liber et al. (1989) were probably related either to differences in the treatments cells received (X-rays versus AZT) and/or the sample size (6 plates versus 200 plates).

The rate of LOH was determined for two reasons. First, LOH is an important mechanism for the loss of gene function in recessive genes such as "tumor suppressor" genes. It has been shown that LOH is a common event in numerous types of cancer, including colorectal tumor, osteosarcoma, etc. (Hansen et al., 1985Go; Fearon and Vogelstein, 1990Go). Second, AZT incorporation into DNA terminates chain elongation, which is a major mechanism for the induction of LOH. As discussed above, the formation of TK mutant colonies was a continuous process. Therefore, to determine whether there were any differences in the percent of LOH between mutants scored earlier and those scored later, the first group of mutants (scored at 10 days after plating, so-called normal-growth mutants) and the latter group (which appeared between 28 and 35 days after plating, a portion of slow-growth mutants) were analyzed separately. While less than half of normal-growth mutants exhibited LOH, the majority of the last group of mutants had LOH. It is conceivable that LOH in mutants that appeared between 10 and 28 days after plating would fall within this range. These findings were consistent with the "growth gene" theory, i.e., TK mutant cells with LOH (which have lost both the TK gene and the growth gene) tend to grow more slowly than cells with an intact growth gene (Liber et al., 1989Go).

There were no differences in LOH in TK-normal growth mutants collected from control cells versus those collected from AZT-treated cells. However, considering the 2-fold increase in Mfs in normal growth mutants, 44% (1.2 x 10–6/2.7 x 10–6) of AZT-induced normal-growth mutants were apparently due to LOH. In contrast, there was a significant difference in mutational spectra between control and AZT-treated slow growth mutants (P = 0.034, Chi-square test), and all the AZT-induced slow-growth mutants were attributable to LOH. As noted above, total gene deletions accounted for 64% of AZT-induced HPRT Mfs in TK6 cells exposed 300 µM for 3 days (Sussman et al., 1999Go), while 84% of the total AZT-induced TK Mfs from similarly exposed cells was attributable to LOH. The detection of lower AZT-induced Mfs at HPRT compared with TK was probably related to the occurrences of multi-locus deletions that were lethal for HPRT mutant cells (and were thus not recoverable in the HPRT mutation assay). Nevertheless, molecular analyses of both HPRT and TK mutants indicate a dominant role for LOH in AZT-induced mutagenesis. Considering the importance of LOH in human carcinogenesis, AZT-induced LOH warrants further investigation.


    ACKNOWLEDGMENTS
 
We thank Dr. Howard L. Liber (Massachusetts General Hospital) for providing the TK cDNA probe. We gratefully acknowledge the use of the services of the Wadsworth Center Media and Glassware Support Service Group for preparing media components. This work was supported, in part, by NIH grant HD33648 (to V.E.W.) from the National Institute of Child Health and Human Development, National Cancer Institute, and Office of AIDS Research, and by NIH grant ES07462 (to X.D.).


    NOTES
 
The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

1 To whom correspondence should be addressed. Fax: (518) 486-1505. E-mail: walker{at}wadsworth.org. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Agarwal, R. P., Olivero, O. A. (1997). Genotoxicity and mitochondrial damage in human lymphocytic cells chronically exposed to 3'-Azido-2',3'-dideoxythymidine. Mutat. Res, 390, 223–231.[ISI][Medline]

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