* Wadsworth Center, New York State Department of Health, Albany, New York 122010509;
School of Public Health, State University of New York at Albany, Albany, New York 12203; and
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
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ABSTRACT |
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Key Words: 3'-azido-3'-deoxythymidine; human lymphoblastoid cells; DNA incorporation; thymidine kinase; mutant frequency; loss of heterozygosity.
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INTRODUCTION |
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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., 1999). 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.
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MATERIALS AND METHODS |
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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., 1994). 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., 1997).
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., 1999). 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., 1992), and used for Southern-blotting analysis as previously described (Yandell et al., 1986
). 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 [
-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 12 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.
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RESULTS |
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DISCUSSION |
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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., 1989). 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., 1999), 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 106) 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 (1418 h), the doubling time for slow-growth mutants was longer (2144 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., 1989). 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., 1985; Fearon and Vogelstein, 1990
). 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., 1989
).
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 106/2.7 x 106) 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., 1999), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (518) 486-1505. E-mail: walker{at}wadsworth.org.
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