Oxidative stress inactivates the human DNA mismatch repair system

Christina L. Chang1,*, Giancarlo Marra2,*, Dharam P. Chauhan1, Hannah T. Ha1, Dong K. Chang1, Luigi Ricciardiello1, Ann Randolph1, John M. Carethers1, and C. Richard Boland1

1 Department of Medicine and Cancer Center; University of California at San Diego, La Jolla, California 92093 - 0688; and 2 Institute of Medical Radiobiology, University of Zürich, 8008 Zürich, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the human DNA mismatch repair (MMR) system, hMSH2 forms the hMutSalpha and hMutSbeta complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer. These complexes, together with other components in the MMR system, correct single-base mismatches and small insertion/deletion loops that occur during DNA replication. Microsatellite instability (MSI) occurs when the loops in DNA microsatellites are not corrected because of a malfunctioning MMR system. Low-frequency MSI (MSI-L) is seen in some chronically inflamed tissues in the absence of genetic inactivation of the MMR system. We hypothesize that oxidative stress associated with chronic inflammation might damage protein components of the MMR system, leading to its functional inactivation. In this study, we demonstrate that noncytotoxic levels of H2O2 inactivate both single-base mismatch and loop repair activities of the MMR system in a dose-dependent fashion. On the basis of in vitro complementation assays using recombinant MMR proteins, we show that this inactivation is most likely due to oxidative damage to hMutSalpha , hMutSbeta , and hMutLalpha protein complexes. We speculate that inactivation of the MMR function in response to oxidative stress may be responsible for the MSI-L seen in nonneoplastic and cancer tissues associated with chronic inflammation.

hMutSalpha ; hMutSbeta ; hMutLalpha ; inflammation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXIDATIVE STRESS is a state in which the production of reactive oxygen species (ROS) exceeds the capacity of the antioxidant defense system in cells and tissues (21). ROS are free radicals that can damage nearby macromolecules. For example, ROS can attack proteins, which leads to denaturation and loss of function. ROS can also attack nucleic acids, resulting in a variety of alterations, including base modifications, double-base lesions, and strand breaks (21). Among ROS, hydroxyl radical is the most potent and can be generated from H2O2 by the Fenton reaction (16). H2O2 is a common intermediate generated by multiple oxidative pathways. Failure to remove and/or repair ROS-initiated damage can be either mutagenic or lethal to cells (19).

Inflammatory diseases create significant oxidative stress to cells and tissues. Increased incidences of cancer are detected in patients with chronic gastritis, chronic pancreatitis, and inflammatory bowel disease. Tissues from patients with these diseases display insertion and/or deletion in the microsatellite regions (5, 6, 29, 37), which has been termed microsatellite instability (MSI) (5, 37). Microsatellites are simple, tandemly repeated DNA sequences that are composed of one to six nucleotides and are widely dispersed throughout the human genome. MSI is associated with a defective DNA mismatch repair (MMR) system (23).

The MMR system maintains genomic integrity by correcting replicative errors (27). During DNA replication, a noncomplementary base can be erroneously introduced into the newly synthesized strand (i.e., single-base mismatch), or a loop containing a few extrahelical bases (i.e., insertion/deletion loop or IDL) may form in one of the two DNA strands. IDLs typically occur in microsatellites. If not repaired, the former lesion results in a point mutation, whereas the latter leads to an insertion or deletion in 50% of the progeny DNA. In the human DNA MMR system, hMSH2 forms hMutSalpha and hMutSbeta protein complexes with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the hMutLalpha heterodimer (2, 31). Together, the hMutSalpha and hMutLalpha complexes correct single-base mismatches and IDLs, whereas the hMutSbeta and hMutLalpha complexes correct mainly the loops (10, 11, 18, 26, 28). Several accessory proteins, including proliferating cell nuclear antigen and DNA polymerase delta , are also required in the mismatch repair process (24, 39).

MSI can be divided into "high frequency" (i.e., MSI-H) and "low frequency" (i.e., MSI-L) categories, in which either >= 40 or <= 20% of assayed microsatellites have been mutated, respectively (4). MSI-H occurs in the colon and endometrial tumors of patients with hereditary nonpolyposis colorectal cancer (1, 33) and is strongly associated with germline mutations in hMSH2 or hMLH1 (3, 30). MSI-H also occurs in about 10% of sporadic colorectal cancers, where it is associated with epigenetic silencing of the hMLH1 expression due to hypermethylation of its promoter (3, 17). However, 15-20% of sporadic colorectal cancers with MSI-L show no evidence of mutational inactivation of any known component of the MMR system and are only rarely associated with hypermethylation of the hMLH1 promoter (12, 13). Moreover, MSI-L also has been found in gastric cancer (13, 32), adenocarcinoma of the esophagus (15, 20), and colon cancers associated with ulcerative colitis (37). Perhaps more surprisingly, MSI-L can also be found in chronically inflamed nonneoplastic ulcerative colitis tissues (6), as well as in pancreatic secretions obtained from patients with chronic pancreatitis (5). Thus MSI-L, in which mutational inactivation of the MMR system is not evident, can be observed in several settings that are associated with inflammation.

We hypothesize that oxidative stress created in the inflammatory settings reduces DNA MMR function. In this study, we utilized H2O2 as an exogenous source of oxidative stress, because it readily crosses the cell membrane and causes damage to macromolecules after being converted to a hydroxyl radical (16). Previously, we characterized the MMR system in the MMR-proficient human erythroleukemia (HEL) cell line (25) and demonstrated the regulation of the hMSH2 gene throughout the cell cycle. Here, we further utilized the HEL cell line as a model system to examine the effects of oxidative stress on MMR function. We found that noncytotoxic levels of H2O2 inactivated MMR function, most likely via oxidative damage to MMR protein components. We speculate that alteration in DNA MMR activity may be a link between oxidative damage and the occurrence of MSI-L in nonneoplastic and cancer tissues associated with chronic inflammation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture, H2O2 treatment, cell viability, and metabolic rate. HEL cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 4 mM glutamine, and 2 mM pyruvate (all from GIBCO BRL) in 7% CO2. Exponentially growing HEL cells were washed and resuspended in PBS at a density of 5 × 105 cells/ml before being treated with various concentrations of H2O2 (Sigma) for 1 h. At the end of treatment, cells were washed with PBS, resuspended in growth medium at a density of 4 × 105 cells/ml, and allowed to recover from oxidative stress. During recovery, cell samples were collected at various time points for analysis. Cell viability was determined by trypan blue exclusion.

The metabolic rates of HEL cells treated with or without H2O2 were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HEL cells (in 100 µl) treated with H2O2 were placed into each well of a 96-well plate. MTT (Sigma) dissolved in PBS was added to each well at a final concentration of 1 mg/ml. After a 2-h incubation at 37°C, 100 µl of solubilizing solution (10% SDS, 0.01 N HCl) was added to each well to lyse the cells. Colored formazan converted from MTT by viable cells was measured at 570 nm by a microplate reader.

Expression and purification of recombinant hMutSalpha , hMutSbeta , and hMutLalpha . Baculovirus vectors carrying cDNA inserts encoding the hMSH6, hMSH3, hMSH2, hMLH1, and hPMS2 proteins were constructed to express DNA MMR proteins (26). To maintain protein stability, hMutSalpha , hMutSbeta , and hMutLalpha recombinant protein complexes were purified from Sf9 cells after coinfection with hMSH2 and hMSH6, hMSH2 and hMSH3, or hMLH1 and hPMS2 expression constructs, respectively (26).

In vitro DNA mismatch repair assay. Twenty-four hours after HEL cells had been exposed to specified concentrations of H2O2 for 1 h, cytoplasmic extracts were prepared as described previously (25) from each sample containing 5 × 108 HEL cells. The extracts, each containing 50 µg of proteins, were used to repair 1 fmol of the M13mp2 heteroduplex containing a G/T mismatch or a loop with two extrahelical nucleotides (38). In the complementation studies, the repair assays were carried out as described above, except that the extracts (50 µg) were supplemented with 0.1 µg of recombinant hMutSalpha , hMutSbeta , and/or hMutL. The repaired M13mp2 DNA was subsequently purified and electroporated into the NR9162 strain of Escherichia coli (mutS) and plated on soft agar containing CSH50 E. coli strain, isopropyl beta -D-thiogalactoside (Sigma), and 5-bromo-4-chloro-3-indolyl beta -D-galactoside (Sigma). Under these plating conditions, if no DNA repair occurs, a high percentage of mixed plaques containing both blue and colorless progeny will be observed. A reduced percentage of mixed plaques and a concomitant increase in blue plaques are indicative of DNA repair. The DNA mismatch repair activity was calculated from the following formula: 100% × [1 - (percentage of mixed colored plaques developed from the reaction containing the extract/percentage of mixed colored plaques developed from the reaction containing no extract)].

Western blot analysis. Of total proteins extracted from each cell sample, 100 µg were resolved by 8% SDS-PAGE before transfer onto a polyvinylidene difluoride membrane (PVDF) (Millipore, Bedford, MA), as previously described (8). Anti-hMSH2, anti-hMLH1, anti-hPMS2 (all from Calbiochem), anti-hMSH6 (Santa Cruz Biotechnology), or anti-hMSH3 antibodies were used separately for immunodetection with an enhanced chemiluminescence system (Amersham) following the manufacturer's recommendations.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Noncytotoxic levels of H2O2 reduce the metabolic rate of HEL cells. Exponentially growing HEL cells, at a density of 5 × 105 cells/ml, were treated with various concentrations of H2O2 in PBS for 1 h, and their metabolic rates were measured by the MTT assay, which reflects the activity of succinate dehydrogenase in mitochondria. At low concentrations of H2O2 such as 1 and 10 µM, there was no significant change in the metabolic rate of HEL cells (Fig. 1). However, the metabolic rate of HEL cells was reduced by 10, 33, and 54% at 0.1, 1, and 10 mM H2O2, respectively (Fig. 1). The results indicate that a dose-dependent reduction in the metabolic rate of HEL cells occurred from 0.1 to 10 mM H2O2.


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Fig. 1.   Effect of H2O2 on the metabolic rate of human erythroleukemia (HEL) cells. HEL cells, at a density of 5 × 105 cells/ml, were treated with the indicated concentrations of H2O2 in PBS for 1 h. Metabolic rate was subsequently determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and expressed as an optical density (OD) at 570 nm. Each data point is the average of triplicate samples per experiment. Results were confirmed in 3 independent experiments.

To avoid cytotoxic levels of H2O2 for HEL cells, we determined the metabolic rate and cell viability at periods up to 24 h after H2O2 removal. At the end of a 1-h exposure to specified H2O2 levels ranging from 0.1 to 10 mM, HEL cells were washed, seeded at a density of 4 × 105 cells/ml, and returned to normal growth conditions for 0, 1, 3, 6, and 24 h. Up to 6 h posttreatment, both untreated and treated HEL cells had no significant recovery in their metabolic rates (Fig. 2). At 24 h posttreatment, untreated cells increased their metabolic rates approximately twofold, whereas 0.1 mM H2O2 reduced recovery of the metabolic rate of HEL cells by ~30% compared with untreated cells (Fig. 2). When HEL cells were exposed to 1 mM H2O2 for 1 h, the metabolic rate decreased immediately by ~50%, and there was no recovery during posttreatment up to 24 h (Fig. 2). Exposure to 10 mM H2O2 was cytotoxic to HEL cells, because the cells died during posttreatment periods (data not shown).


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Fig. 2.   Recovery of the metabolic rate of HEL cells after 1-h treatment with H2O2. HEL cells were treated for 1 h in PBS with 0, 0.1, or 1 mM H2O2, as indicated. Subsequently, HEL cells were returned to the normal growth conditions for various time intervals after H2O2 removal. Metabolic rate was determined by MTT assay. Each data point is the average of triplicate samples per experiment, and results were confirmed in 3 independent experiments.

To determine whether the reduced metabolic rate was due to diminished cell viability, we compared the metabolic rate with the number of viable HEL cells 24 h after H2O2 removal. In parallel with the changes observed in the metabolic rate, untreated HEL cells doubled their numbers within 24 h posttreatment. Relative to untreated cells, a reduction of ~20 and 40% in cell numbers was detected 24 h after removal of 0.1 and 1 mM of H2O2, respectively (Fig. 3). A 1-h exposure to 0.1 or 1 mM H2O2 reduced the metabolic rate of HEL cells by ~35 and 65%, respectively, at 24 h posttreatment (Figs. 2 and 3). The observation that H2O2 had a greater effect in reducing the metabolic rate than the reduction in numbers of HEL cells suggests that the amount of oxidative stress created by 0.1 and 1 mM H2O2 may overwhelm the antioxidant defense system.


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Fig. 3.   Comparisons of viability and the metabolic rate of HEL cells 24 h after H2O2 removal. Both the metabolic rate and viable cell numbers of untreated HEL cells doubled 24 h posttreatment, relative to time zero, and their values were set at 100 for subsequent comparisons. Metabolic rate was determined by MTT assay, whereas the number of viable cells was determined by the dye exclusion method. Triplicate samples of each H2O2 concentration were used per experiment, and error rates were <12%. Results were confirmed in 2 independent experiments.

H2O2 reduces single-base MMR activity by damaging hMutSalpha and hMutLalpha . We next examined whether the single-base MMR function would be damaged by H2O2 at 24 h posttreatment, a time at which cell doubling had occurred in untreated HEL cells. In the in vitro MMR function assay, a cell extract containing 50 µg of proteins from each sample was used to repair 1 fmol of the M13mp2 heteroduplex containing a G/T single-base mismatch (38). Relative to untreated cells, 0.1 and 1 mM H2O2 reduced the MMR activity in HEL cells by ~60 and 86%, respectively (bars 1, 3, and 7 in Fig. 4).


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Fig. 4.   H2O2 reduces the single-base mismatch repair activity in HEL cells by damaging hMutSalpha and hMutLalpha . Cell extracts were prepared from HEL cells 24 h posttreatment with specified H2O2 concentrations. The G/T single-base mismatch repair activity was reduced by H2O2 in a dose-dependent fashion (bars 1, 3, and 7). Complementation of H2O2-treated cell extract with 0.1 µg of recombinant hMutSalpha and/or 0.1 µg of recombinant hMutLalpha (bars 2, 4-6, and 8-10) restored the activity that was reduced by H2O2. Results are averages of 2 independent experiments, and error bars are shown.

To determine whether hMutSalpha and hMutLalpha complexes were damaged by H2O2, resulting in a reduced single-base MMR activity, we complemented H2O2-treated HEL extract with recombinant hMutSalpha and hMutLalpha protein complexes, individually and in combination. To the extract prepared from HEL cells 24 h after the removal of 0.1 mM H2O2, addition of 0.1 µg of recombinant hMutSalpha complex restored the single-base mismatch repair activity from 40 to 77% of the activity present in untreated cells (bar 3 vs. 4 in Fig. 4). The addition of 0.1 µg of recombinant hMutLalpha to this extract restored the activity from 40 to 91% of the control (bar 3 vs. 5 in Fig. 4). To the extract prepared from HEL cells 24 h after the removal of 1 mM H2O2, the addition of 0.1 µg of recombinant hMutSalpha or hMutLalpha restored the activity from 14 to 58 and 43% of the control, respectively (bars 7, 8, and 9 in Fig. 4). When both 0.1 µg of hMutSalpha and 0.1 µg of hMutLalpha complexes were added to the extract prepared from HEL cells at 24 h posttreatment with 0.1 or 1 mM H2O2, the activity was restored to ~99 and 92% of the activity present in untreated cells, respectively (bars 6 and 10 in Fig. 4). As expected, the addition of 0.1 µg of hMutSalpha and 0.1 µg of hMutLalpha to the MMR-proficient extract prepared from untreated HEL cells did not increase the existing single-base MMR activity significantly (bar 1 vs. 2 in Fig. 4).

H2O2 reduces IDL repair activity by damaging hMutSalpha , hMutSbeta , and hMutLalpha . The effect of H2O2 on IDL repair activity was determined by the ability of cell extract containing 50 µg of proteins to repair 1 fmol of the M13mp2 heteroduplex containing a loop with two extrahelical bases (38). H2O2 reduced the IDL repair function in a dose-dependent manner, resulting in a repair efficiency of only 26 and 4% of the untreated HEL cells at 0.1 and 1 mM H2O2 levels, respectively (bars 1, 4, and 10 in Fig. 5).


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Fig. 5.   H2O2 reduces the insertion/deletion loop repair activity of HEL cells by damaging hMutSalpha , hMutSbeta , and hMutLalpha . Cells extracts were prepared from HEL cells 24 h posttreatment with specified H2O2 concentrations. Repair of a loop with two extrahelical bases was reduced by H2O2 in a dose-dependent fashion (bars 1, 4, and 10). Complementation of H2O2-treated cell extract with 0.1 µg of recombinant hMutSalpha , 0.1 µg of recombinant hMutSbeta , and/or 0.1 µg of recombinant hMutLalpha (bars 2, 3, 5-9, and 11-15) restored the loop repair activity that was reduced by H2O2. Results are averages of 2 independent experiments, and error bars are indicated.

Because IDLs are repaired by both hMutSalpha and hMutSbeta in conjunction with hMutLalpha , we further determined whether reduced IDL repair was a result of oxidative damage to any of these heterodimers. To the extract prepared from HEL cells 24 h after the removal of 0.1 mM H2O2, the addition of 0.1 µg of recombinant hMutSalpha , hMutLalpha , or hMutSbeta complex restored the repair activity from 26 to 38, 44, and 67% of the activity present in untreated cells, respectively (bars 4, 5, 6, and 7 in Fig. 5). To the extract prepared from HEL cells 24 h after the removal of 1 mM H2O2, the addition of 0.1 µg of recombinant hMutSalpha , hMutLalpha , or hMutSbeta restored the loop repair activity from 4 to 25, 9, and 60% of the control, respectively (bars 10, 11, 12, and 13 in Fig. 5).

When both 0.1 µg of hMutSalpha and 0.1 µg of hMutLalpha complexes were added to the extract prepared from HEL cells 24 h posttreatment with 0.1 or 1 mM H2O2, the loop repair activity was restored to 72 and 67% of the activity present in untreated cells, respectively (bars 8 and 14 in Fig. 5). When both 0.1 µg of hMutSbeta and 0.1 µg of hMutLalpha complexes were added to the extract prepared from HEL cells at 24 h posttreatment with 0.1 or 1 mM H2O2, the activity was restored to 93 and 76% of the activity present in untreated cells, respectively (bars 9 and 15 in Fig. 5). In combination, 0.1 µg of hMutSalpha and 0.1 µg of hMutLalpha or 0.1 µg of hMutSbeta and 0.1 µg of hMutLalpha did not significantly affect the loop repair activity of the extract prepared from untreated HEL cells (bars 2 and 3 in Fig. 5).

H2O2 degrades hMSH6 and hPMS2 proteins. To determine whether reduced MMR activity by H2O2 is due to oxidative damage to the MMR proteins components, we compared the steady-state levels of hMSH2, hMSH3, hMSH6, hMLH1, and hPMS2 proteins at 24 h posttreatment. Equal amounts of total protein extracted from HEL cells exposed to 0, 0.1, or 1 mM H2O2 were resolved by 8% SDS-PAGE and subjected to Western blot analysis. No significant change in the protein levels of hMSH2 and hMLH1 was detected in HEL cells after normalization with beta -actin (Fig. 6). The steady-state protein levels of hPMS2 were substantially decreased by 1 mM H2O2 treatment, and hPMS2 degradation was suggested because of faster electrophoretic mobility (Fig. 6). We also detected a dramatic reduction in the level of hMSH6 protein in HEL cells exposed to 0.1 and 1 mM H2O2. However, we could not determine the effect of H2O2 on the hMSH3 protein because it was below the detection level in HEL cells compared with that in HCT116 + chr3 colorectal cancer cell line examined on the same PVDF membrane (data not shown).


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Fig. 6.   Steady-state protein levels of mismatch repair components at 24 h posttreatment with H2O2. HEL cells were treated with 0, 0.1, or 1 mM H2O2 for 1 h and then returned to normal growth conditions for 24 h. An equal amount of total protein extracted from each sample was resolved by 8% SDS-PAGE, transferred to the polyvinylidene difluoride (PVDF) membrane, and subjected to Western blot analysis. Antibodies specific for hMSH6, hMSH2, hMLH1, hPMS2, and beta -actin were used for immunodetection with an enhanced chemiluminescence system. Results were confirmed in 2 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MSI-L has been found not only in cancer tissues associated with inflammation (13, 15, 20, 32, 37) but also in chronically inflamed nonneoplastic tissues (6). The lack of mutational inactivation of the MMR genes in several inflammatory settings points to a potential role of oxidative stress in the inactivation of MMR function. In this study, we demonstrated that noncytotoxic levels of H2O2 dramatically reduced the activities of the MMR system in repairing both single-base and IDL mismatches in a dose-dependent manner.

For repairing single-base mismatches, both hMutSalpha and hMutLalpha complexes are required. Results from the single-base MMR assay showed that individual recombinant hMutSalpha and hMutLalpha protein complexes could partially restore the MMR function that was inactivated by oxidative stress. In combination, both recombinant hMutSalpha and hMutLalpha protein complexes were able to restore almost completely the single-base MMR activity present in untreated HEL cells. These findings suggest that H2O2-inactivated single-base repair activity is a result of oxidative damage to both complexes. Both hMutSalpha and hMutSbeta participated in correcting IDL mispairings in the presence of a functional hMutLalpha complex (11). Results from the loop repair assay indicate that H2O2 also damaged hMutSbeta in addition to hMutSalpha and hMutLalpha (26), and this finding could explain why we (6) and others (5, 12, 13, 15, 20, 32, 37) have observed MSI-L in nonneoplastic and tumor tissues associated with chronic inflammation. Indeed, we have observed that 1 mM H2O2 increased in vivo frame-shift mutations fourfold in MMR-proficient HCT116 + chr3 cells (14).

ROS produced during oxidative stress damage macromolecules in close proximity. On the basis of a quantitative RT-PCR, H2O2 did not significantly alter the steady-state mRNA levels of hMSH2, hMSH3, hMSH6, hMLH1, and hPMS2 24 h after exposure to either 0.1 or 1 mM H2O2 (data not shown). However, Western blot analysis indicates that oxidative stress greatly reduced hMSH6 and hPMS2 protein steady-state levels. The observed reduction in hMSH6 and hPMS2 levels may be due to protein degradation; in fact, hPMS2 degradation was indicated by its increased electrophoretic mobility. The derivatives of ROS are known to modify amino acid residues, especially aromatic and sulfur-containing residues, which mark proteins for degradation (35, 36).

Although there were no significant changes in the steady-state protein levels of hMSH2, hMLH1, and to a lesser degree, hPMS2, our complementation assays strongly indicate that these proteins were inactivated by H2O2 via denaturation. For example, the protein levels of both hPMS2 and hMLH1 remained unchanged after exposure to 0.1 mM H2O2; however, recombinant hMutLalpha comprised of these two proteins was able to increase the single-base MMR activity in H2O2-treated cells from 40 to 91% of the activity of untreated cells (bars 3 and 5 in Fig. 4). Moreover, recombinant hMutLalpha also restored the single-base MMR activity to 43 from 16% in cells exposed to 1 mM H2O2 (bars 7 and 9 in Fig. 4). Because hMSH6 protein was undetectable in cells that had been treated with either 0.1 or 1 mM H2O2 (Fig. 6), we expect that the addition of recombinant hMutLalpha should restore to almost 100%, and not to only 91 and 43%, respectively, of the single-base MMR activity seen in untreated cells, unless detectable hMSH2 protein was denatured by H2O2 in a dose-dependent fashion. Oxidative denaturation of hMSH2 could affect not only its function but also its interaction with hMSH6 and hMSH3 to form hMutSalpha and MutSbeta heterodimers, respectively, resulting in reduced activity for repairing single-base mismatches and IDLs in cells. Although our antibody was unable to detect hMSH3 protein in HEL cells, the inability of recombinant hMutSbeta and hMutLalpha to enhance loop repair efficiency in the cell extract (bar 1 vs. 3 in Fig. 5) suggests the presence of hMSH3 protein. This is further supported by a detectable level of hMSH3 mRNA in HEL cells, based on our RT-PCR analysis (not shown).

It is currently unclear why hMSH2 and hMLH1 proteins are more stable than hMSH6 and hPMS2, considering that these four proteins have similar percentages of aromatic and sulfur-containing residues. Differential susceptibility to oxidative modification has also been reported in plasma and mitochondrial proteins (7, 34). On the basis of in vitro and in vivo observations (9, 26, 31), individual components such as hMSH6, hMSH3, and hPMS2, which are present in the hMutSalpha , hMutSbeta , and hMutLalpha heterodimers, are unstable without their partners. In addition, we (9) and others (11) have shown that hMSH2 and hMLH1 proteins are stoichiometrically more abundant than hMSH6 and hPMS2. It is possible that hMSH2 and hMLH1 are modified by H2O2 in such a way that their heterodimerization abilities are affected, facilitating the degradation of hMSH6 and hPMS2. Alternatively, hMSH2 and hMLH1 may be shielded by hMSH6 and hPMS2 from direct contact with ROS. In any case, it is difficult to test these possibilities in vitro because individual components of the MMR system cannot be stably expressed and purified (9).

It has been shown that 250-400 µM H2O2 permanently arrests the growth of Chinese hamster ovary fibroblasts, embryonic mouse fibroblasts, Chinese hamster lung fibroblasts, and rat liver epithelial cells, whereas the cells die when the concentration of H2O2 is 1 mM or higher (40). In HEL cells, however, l mM H2O2 results in ~40% cell death, and the surviving cells are able to proliferate within a few days of recovery. Colorectal cancer cell lines, such as HCT116 + chr3 (22), also tolerate similar doses of H2O2, as do HEL cells (14). The ability of cells to proliferate when the MMR activity is inactivated by oxidative stress would presumably facilitate the introduction of additional mutations, which may explain why MSI-L has been detected in nonneoplastic ulcerative colitis tissues (6) and in pancreatic secretions obtained from patients with chronic pancreatitis (5).

Inactivation of DNA MMR function in response to oxidative stress has important implications for carcinogenesis. Reduced MMR activity may cause somatic mutations in hMSH3, hMSH6, and hPMS2 genes, which contain (A)8, (C)8, and (A)8 tracts in their coding regions, respectively. Mutations in these genes could further impair MMR activity, which would subsequently augment microsatellite instability in other target genes that also contain microsatellites in their coding regions. It is known that adenomatous polyposis coli (APC) harbors exonic (AG)5 and (A)6 sequences, whereas transforming growth factor-beta receptor type II (TGF-beta RII) contains an (A)10 tract in its coding region, and both APC and TGF-beta RII play important roles in cancer development.

In summary, we have described that noncytotoxic H2O2 levels inactivate the MMR function in a dose-dependent fashion in HEL cells. The reduced MMR activity was likely due to oxidative damage to hMutSalpha , hMutSbeta , and hMutLalpha complexes. On the basis of these findings, we propose that the MMR function reduced by oxidative stress may play a role in the low frequency of MSI detected in inflamed tissues, which might eventually lead to tumorigenesis.


    ACKNOWLEDGEMENTS

We express our gratitude to Drs. Markus Räschle and Patrick Dufner (Institute of Medical Radiobiology, University of Zürich) for providing recombinant MMR heterodimers.


    FOOTNOTES

*  C. L. Chang and G. Marra contributed equally to this work.

This work was supported by the Research Service of the Department of Veterans Affairs and by National Cancer InstituteGrant RO1-CA-72851 to C. R. Boland.

Address for reprint requests and other correspondence: C. R. Boland, 4028 Basic Science Bldg., 9500 Gilman Drive, La Jolla, CA 92093-0688 (E-mail: crboland{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00422.2001

Received 4 September 2001; accepted in final form 15 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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