Suppression of spontaneous and hydrogen peroxide-induced mutagenesis by the antioxidant ascorbate in mismatch repair-deficient human colon cancer cells

Warren E. Glaab,1, Rosina B. Hill and Thomas R. Skopek

Department of Genetic and Cellular Toxicology, Merck Research Laboratories, WP45-320, West Point, PA 19486, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Genomic instability associated with deficiencies in mismatch repair (MMR) plays a critical role in tumorigenesis. Here we have investigated the contribution of oxidative damage to this instability in MMR-defective cells. Treatment with H2O2 produced less cytotoxicity in MMR-deficient cells than in those proficient in MMR, supporting a role for MMR in the recognition and/or processing of oxidative damage. Additionally, growth of MMR-defective cells in the presence of the antioxidant ascorbate (500 µM) reduced the spontaneous mutation rate at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus by up to 50% and reduced microsatellite instability by 30%. Induction of HPRT mutants by exogenously added H2O2 was also significantly suppressed by ascorbate. Collectively, these results suggest that (i) oxidative damage contributes significantly to the spontaneous mutator phenotype in MMR-defective cells, (ii) this damage may select for MMR-deficient cells due to their increased resistance to cell killing and (iii) dietary antioxidants may help to suppress the mutator phenotype and resulting carcinogenesis in individuals with compromised MMR.

Abbreviations: DMSO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution; HPRT, hypoxanthine-guanine phosphoribosyl transferase locus; MMR, mismatch repair; 8-oxo-G, 8-oxoguanine; 6-TG, 6-thioguanine.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Maintaining genomic integrity is fundamental in protecting cells from neoplastic transformation. Mismatch repair (MMR) has been shown to play a crucial role in maintaining genomic stability (13). Lack of MMR results in the failure to correct spontaneous polymerase misincorporation errors or strand slippage events during replication and consequently results in a hypermutable state. This is evidenced by increased rates of mutation at both microsatellite sequences and endogenous loci in cells deficient in MMR (4,5). Loss of MMR and the associated genomic instability have been implicated in the formation and progression of a variety of sporadic and hereditary cancers (13).

In addition to elevated spontaneous mutation rates, cells defective in MMR display greater resistance to the toxic effects of certain DNA-damaging agents (model alkylating and chemotherapeutic agents) as well as greater susceptibility to their mutagenic effects (68). This alkylation resistance/hypermutable phenotype may be due to lack of recognition of the damaged base prior to replication and/or the lack of repair of damage-induced mismatches. Although this resistance/ hypermutable phenotype has been demonstrated with a variety of exogenous chemical mutagens, endogenous DNA-damaging agents may also behave in a similar manner and thus play a critical role in `spontaneous' mutation and tumorigenesis in MMR-deficient cells. Reactive oxygen species produced in the cell represent an important class of endogenous DNA-damaging agents. Recent studies have shown that oxidative damage can induce microsatellite instability and increase point mutations in MMR-defective cells (912). Furthermore, it is thought that the majority of spontaneous mutations that occur even in MMR-proficient cells are due to oxidative damage to DNA (13). Thus, oxidative stress may be responsible for a significant percentage of the elevated level of spontaneous mutations occurring in MMR-deficient cells.

Here we investigate the cellular response of MMR-deficient cells to oxidative damage. Data are presented that show that MMR status influences the response of cells to H2O2-induced damage and that the oxygen radical scavenger ascorbate has a protective effect against genomic mutations both spontaneously and induced by H2O2.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cell lines
The HCT116 colon cancer cell line and the HCT116 chromosome 3 transfer cell line clone 6 (designated HCT116+ch3) were obtained from the laboratories of T.A. Kunkel and J.C. Barrett (NIEHS, Research Triangle Park, NC). The HCT116 cell line is defective in the MMR genes hMLH1 (14) and hMSH3 (15). Chromosome 3 complements the hMLH1 defect in the parental line and restores all MMR activities (16). Cell lines were grown in DMEM/F-12 (1:1) + 10% dialyzed fetal bovine serum (HyClone) and chromosome transfer cell lines maintained with G418 (400 µg active/ml; Gibco BRL).

H2 O2 cytotoxicity
The cytotoxic response to H2O2 exposure was determined for both cell lines by colony-forming ability. For each dose level three 10 cm dishes were seeded with ~500 cells/dish. Cells were exposed to various concentrations of H2O2 <24 h after plating. For serum-free treatment conditions, cells were exposed to H2O2 in Hanks' balanced salt solution (HBSS) for 30 min at 37°C. Following treatment, H2O2-containing HBSS was removed and replaced with complete medium. For treatment in the presence of 10% serum, H2O2-containing normal medium was added to the cells and the plates returned to 37°C. Cells were allowed to grow for 12–14 days and colonies visualized by staining with 0.5% crystal violet (in 50% methanol) (Sigma). Colonies with >=50 cells were counted and survival expressed as a percentage of untreated control plates. Each cytotoxicity determination was performed three times and the average of all independent experiments are reported.

Mutation rate at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT)
Mutation rate determinations at HPRT were determined as previously described (17). Cell populations were first cleansed of pre-existing HPRT mutants by culturing in HAT medium (100 µM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine; Sigma) for 15 days. While cells were being cleansed of HPRT mutants by HAT, either ascorbate or dimethyl sulfoxide (DMSO) was also added to the cultures so that new `spontaneous' mutation rates could be established. Following HAT removal, the initial HPRT mutant frequency was determined by plating 106 cells in 40 µM 6-thioguanine (6-TG) (Sigma) at a density of 5x104/10 cm dish, while 2–3x106 cells were subcultured. Mutant frequency plates were incubated for 12–14 days and 6-TG-resistant colonies were visualized by staining. Five to six additional mutant frequencies were then obtained while maintaining the cells in logarithmic growth. Population doublings were calculated between mutant frequency determinations. Mutation rate was then obtained by plotting the observed mutant frequency as a function of population doubling and calculating the slope by linear regression. The slope of the curve yields the mutation rate (mutations/cell/generation). Mutation rate determinations were performed in duplicate.

Induced mutant frequency
The induced mutagenic response at HPRT was determined as described previously (18). Cell populations were cleansed of pre-existing HPRT mutants by culturing in HAT medium as indicated above. Twenty-four hours prior to removing HAT medium, either 250 or 500 µM ascorbate was added to the medium to allow for intracellular equilibration. HAT-containing medium was then removed from the cultures and the cells plated in separate 175 cm2 flasks at a density of 1.5x106 cells/flask in medium containing either 250 or 500 µM ascorbate. Twenty-four hours after plating cultures were treated with various concentrations of H2O2 (2 flasks/dose) in HBSS alone for 30 min. The cells were maintained in logarithmic growth during a 10 day expression period, after which the frequency of 6-TG-resistant cells (40 µM) was measured.

Microsatellite instability analysis
Cell cultures of HCT116 grown in medium containing either ascorbate or DMSO were maintained in logarithmic growth for 30 days prior to assessing microsatellite stability. Cells were passed every 3–5 days with large dilutions (1:20, ~5x105/passage). Single cell clones from each culture were then isolated by limiting dilution. Cells were plated in 96-well plates at 0.5 cells/well. Clones were expanded and transferred to 24-well plates and treated with proteinase K to isolate DNA as described (19). Approximately 40 clones were isolated per culture condition. Microsatellite instability was assessed at multiple loci: BAT-25, BAT-26, D2S123, D5S346, D10S89, D13S175, D17S250 and RB. Primer sequences for all loci were as previously reported (20). Primers were labeled with specific fluorescent markers and alterations in tract length determined by GeneScan (Applied Biosystems).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Although the genomic instability imposed by MMR defects alone appears to be sufficient for progression to neoplasia, additional factors are likely to contribute to tumor progression. Factors such as endogenous DNA damage may select for MMR-defective cell populations while enhancing the mutagenic burden. It is clear that certain methylating and chemotherapeutic agents select for MMR-deficient cells while increasing mutagenesis within these populations (68). Therefore, it is plausible that endogenous DNA-damaging agents such as reactive oxygen species may play a more integral role in MMR-mediated tumorigenesis. Here we investigate the role that oxidative damage plays in the generation of genomic instability in MMR-defective cells.

We first determined the cytotoxic effects of increasing doses of hydroxyl radical-producing H2O2 in the MMR-defective cell line HCT116 and the MMR-proficient chromosome transfer cell line HCT116+ch3. Assuming that serum in the medium may act to scavenge hydroxyl radicals produced by H2O2 exposure, we carried out colony-forming assays with and without serum present in the medium during treatment. In both cases, survival of the chromosome transfer cell line, HCT116+ch3, following H2O2 treatment was significantly lower than that observed in the parental line, HCT116 (Figure 1Go). That is, the MMR-proficient cells were more sensitive to H2O2-induced cytotoxicity than cells deficient in MMR. The differential cytotoxicity between cell lines was clear under both assay conditions (Figure 1A and BGo). Survival was significantly different between HCT116 and HCT116+ch3 at all doses above 20 µM (P < 0.05, t-test), regardless of the culture conditions during treatment.



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Fig. 1. H2O2 cytotoxicity in HCT116 and HCT116+ch3 cell lines. Graphs represent the average percent survival (colony-forming ability) from at least three independent determinations. (A) H2O2 treatment in normal medium containing serum; (B) H2O2 treatment in HBSS.

 
The finding of differential cytotoxicity between MMR-deficient and MMR-proficient cells following treatment with H2O2 implies a role of MMR in the processing of H2O2-induced damage. Perhaps this processing involves recognition of the H2O2-induced DNA lesion 8-oxoguanine (8-oxo-G). Two other studies have demonstrated similar differences in cytotoxicity between MMR-deficient and MMR-proficient cells following ionizing radiation (21,22). 8-oxo-G is a common DNA lesion resulting from ionizing radiation, suggesting a role for this lesion in the observed cellular response. Recently it was shown that MMR proteins can recognize and bind to 8-oxo-G lesions that are base paired normally with cytosine or in a mismatched configuration with adenine (10). This suggests that MMR proteins may act as sensors for 8-oxo-G damage in DNA, thereby promoting cell cycle signaling and/or apoptosis or the recruitment of other repair pathways, such as base excision repair (10,23,24). Hardman et al. recently illustrated similar differences in H2O2-induced cytotoxicity between MMR-proficient and MMR-deficient cell lines and implicated a dysregulation of apoptosis as the causal mechanism (25). Clearly, a lack of functional MMR reduces the ability of the cell to recognize and repair oxidative damage such as 8-oxo-G lesions. Under conditions of oxidative stress the differential cytotoxicity data presented here and by others suggest that MMR-deficient cells may escape the pathways that inhibit cell growth and allow for selection of cell populations which have a growth advantage over MMR-proficient cells.

One could argue that the dose of H2O2 and the overall level of toxicity needed to observe a difference in survival between MMR-proficient and MMR-deficient lines is too great to be of biological relevance. However, one should remember that external application of a short-lived radical-forming agent could have very different effects than reactive oxygen species generated internally in the cell. For example, the level of external H2O2 needed to damage DNA in the cell may produce significant toxicity due to collateral damage to other structures, such as the cell membrane. Nonetheless, a significant difference in the cytotoxic effects of H2O2 was seen between MMR-proficient and MMR-deficient cell lines, implicating differences in the response to induced DNA damage, which may be even more pronounced when oxygen radicals are produced internally.

Next, the role that oxidative damage plays in the spontaneous mutagenic response at HPRT was determined. Specifically, the HPRT mutation rate was obtained in the HCT116 cell line cultured in normal medium and in medium containing oxygen radical scavengers. Conditions tested included 250 µM ascorbate, 500 µM ascorbate and 1% DMSO (two independent cultures each). For HCT116 cultures containing ascorbate there was a dose-dependent decrease in HPRT mutation rate relative to normal medium (Table IGo). This decrease was statistically significant at both doses of ascorbate (P < 0.05, ANOVA). In the 500 µM ascorbate cultures a 50% decrease in mutation rate was observed. This implies that at least half of all spontaneous mutations occurring at HPRT result from spontaneous oxidative damage. For MMR-proficient cells it has been shown that the majority of spontaneous mutations are due to oxidative stress (13). The significant contribution of oxidative damage to the spontaneous HPRT mutation rate seen in MMR-deficient cell lines suggests that there may be an exaggerated effect of oxidative stress on the observed mutator phenotype in cells defective in MMR. Recently, a suppression of HPRT mutation rate in HCT116 cells was also shown for several oxygen radical scavengers, including ascorbate, supporting the findings presented here (26).


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Table I. Mutation rates at the HPRT locus in HCT116 cells under various culture conditions
 
Surprisingly, the cultures containing 1% DMSO as an oxygen radical scavenger resulted in HPRT mutation rates that were elevated relative to the normal medium control (Table IGo). This corresponded to a 1.8-fold increase in mutation rate, which was statistically significant (P < 0.05, ANOVA). Upon observing this increase, rather than the hypothesized decrease, we investigated potential mechanisms by which DMSO could increase mutations. It appears that by scavenging a hydroxyl radical, DMSO produces a methyl radical from the reaction (27). Although the hydroxyl radical is more reactive, perhaps the effects seen here are due to the generation of DNA damage induced by this methyl radical. Methyl radicals can methylate DNA bases (28), which may account for the mutation rates observed here, since methylation tolerance and associated hypermutability are well characterized in MMR-defective cells (7,29). There have been several reports demonstrating that DMSO is mutagenic in microbial mutation assays, consistent with the data presented here (30,31). DMSO can also act as a transcriptional activator and has been used to induce cells to differentiate in culture (27). Here, 1% DMSO was present in the cultures for 15 days and, therefore, the effect on mutation rate may be a result of induced transcripts that act to promote genomic instability. For either situation, the net result is that DMSO was mutagenic in MMR-deficient cells.

After observing the significant decrease in mutation rate at the HPRT locus by the antioxidant ascorbate, we then evaluated its effect on microsatellite instability. Recently, Jackson et al. demonstrated that oxidative damage can induce microsatellite instability (9). Since HCT116 cells exhibit elevated microsatellite instability spontaneously (3), we wished to determine the contribution of endogenous oxidative damage. HCT116 cells were cultured for 30 days in 250 or 500 µM ascorbate or 1% DMSO and single cell clones isolated. Microsatellite instability was assessed at a variety of loci and the number of clones having alterations in tract length was determined and compared with that arising in normal medium. As observed at HPRT, there was a dose-dependent decrease in the level of instability observed in cultures containing ascorbate (Table IIGo). Summing all unstable loci and determining the percentage of `instability' revealed that an average of 28.4% of the clones showed instability at these particular markers in normal medium, while 20.4% showed instability in medium containing 500 µM ascorbate. This corresponded to an ~30% reduction in microsatellite instability (P < 0.05, one-sided Tukey's studentized range test, HSD, a procedure for multiple comparison of population means). Although a decrease was observed at both mono- and dinucleotide loci, mononucleotide instability was decreased to a greater extent (Table IIGo). This implies that a significant fraction of microsatellite instability occurring spontaneously in these cells involves oxidative damage.


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Table II. Microsatellite instability in single cell clones of HCT116 under various culture conditions and in HCT116+ch3
 
Cultures grown in 1% DMSO displayed 36.3% instability at microsatellite sequences compared with 28.4% in normal medium (Table IIGo). Although this increase was borderline statistically different, this result nevertheless is consistent with the data concerning the effect of DMSO on HPRT mutation frequency (presented above) and again appears contrary to what one would have predicted given the ability of DMSO to scavenge hydroxyl radicals. For the possible reasons outlined previously, DMSO appears to promote genomic instability in MMR-defective cells rather than reduce it.

Finally, we investigated the mutagenic response at HPRT in HCT116 induced by exogenous H2O2 and tested whether this response could also be ablated by ascorbate administration. The data obtained from these experiments are presented in Table IIIGo. Under normal medium conditions we observed a significant induction of mutant frequency at both 25 and 50 µM exposures to H2O2 relative to the control, illustrating a significant mutable response induced by H2O2 in MMR-deficient cells. This induced response correlated with 110 and 190 mutants/106 cells for 25 and 50 µM H2O2, respectively. By pretreating the cells with 250 µM ascorbate prior to exposure to H2O2 we found that the induced mutagenic response to the low dose of H2O2 was entirely suppressed and the induced mutant frequency at the high dose of 50 µM H2O2 was decreased to 130/106 cells relative to controls. For the 500 µM ascorbate cultures we observed complete suppression of the H2O2-induced mutagenic response at both doses of H2O2. The difference in the induced mutagenic response due to culture conditions (normal versus 250 µM ascorbate and normal versus 500 µM ascorbate) was statistically significant at both doses of H2O2 (P < 0.05, t-test).


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Table III. H2O2-induced mutant frequencies at HPRT in HCT116 cells under various growth conditionsa
 
The finding of a significant mutable response to H2O2 in MMR-deficient cells supports the role of the MMR pathway in recognizing and/or repairing oxidative stress-induced mutagenic lesions, such as 8-oxo-G (10,21,22). This hypermutability induced by H2O2 in MMR-defective cells has been seen by others, however, an even more mutagenic response was observed when MMR deficiencies were coupled with defects in p53 (12). Taken together with the results of Hardman et al. (25), who found abnormalities in the apoptotic response following exposure to H2O2 in MMR-defective cells, it can be suggested that the H2O2-induced mutable and cytotoxic response observed in MMR-deficient cells may result from an inability to recognize induced damage and signal apoptosis. Since this H2O2-induced mutagenic response can be completely suppressed by the presence of the antioxidant ascorbate, the hydroxyl radical and the resulting induced 8-oxo-G lesions are implicated as responsible.

To summarize, the suppression of spontaneous mutation at the endogenous HPRT and at several microsatellite loci by the antioxidant ascorbate supports the theory that endogenous oxidative damage contributes significantly to the mutagenic burden in MMR-defective cells. Supporting evidence was provided by the observation that ascorbate could also ablate the increase in mutation frequency induced by low levels of exogenous H2O2 added to the cultures.

Although in vitro culture conditions have significantly elevated oxygen concentrations relative to those found in vivo, these results nevertheless suggest that continuous cell proliferation in the presence of even low levels of oxidative damage may contribute significantly to the level of genetic damage. Since MMR-deficient cells are more resistant to the cytotoxic effects of reactive oxygen and are more susceptible to its mutagenic effects, in vivo conditions may simultaneously select for the MMR-deficient phenotype and also promote genomic instability within them.

The data presented here have obvious health implications for individuals with MMR deficiencies. An increase in dietary radical scavengers may decrease the probability of tumor formation in these individuals by removing the selective pressure for the MMR phenotype and the accompanying increases in mutation rate due to reactive oxygen species. This theory may be tested in an appropriate transgenic model for MMR deficiencies.


    Notes
 
1 To whom correspondence should be addressedEmail: warren_glaab{at}merck.com Back


    Acknowledgments
 
We thank Dr Sheila Galloway for helpful discussions and for critical evaluation of this manuscript. We thank Xiaoli (Shirley) Hou for her help with the statistical analyses performed on this data set. We also thank Drs John Deluca and Diane Umbenhauer for critical evaluation of this manuscript.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received May 3, 2001; revised June 14, 2001; accepted June 19, 2001.