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
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ABSTRACT |
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In the human DNA mismatch repair
(MMR) system, hMSH2 forms the hMutS and hMutS
complexes with
hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2 form the
hMutL
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 hMutS
, hMutS
, and hMutL
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.
hMutS; hMutS
; hMutL
; inflammation
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INTRODUCTION |
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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 hMutS and hMutS
protein complexes
with hMSH6 and hMSH3, respectively, whereas hMLH1 and hPMS2
form the hMutL
heterodimer (2, 31). Together, the
hMutS
and hMutL
complexes correct single-base mismatches
and IDLs, whereas the hMutS
and hMutL
complexes correct mainly
the loops (10, 11, 18, 26, 28). Several accessory proteins, including proliferating cell nuclear antigen and DNA polymerase
, 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.
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MATERIALS AND METHODS |
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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 hMutS, hMutS
,
and hMutL
.
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, hMutS
,
hMutS
, and hMutL
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 hMutS, hMutS
, 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
-D-thiogalactoside (Sigma), and
5-bromo-4-chloro-3-indolyl
-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.
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RESULTS |
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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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H2O2 reduces single-base MMR activity by
damaging hMutS and hMutL
.
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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H2O2 reduces IDL repair activity by
damaging hMutS, hMutS
, and hMutL
.
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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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 -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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DISCUSSION |
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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 hMutS and hMutL
complexes are required. Results from the single-base MMR assay showed
that individual recombinant hMutS
and hMutL
protein complexes could partially restore the MMR function that was inactivated by
oxidative stress. In combination, both recombinant hMutS
and hMutL
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
hMutS
and hMutS
participated in correcting IDL mispairings in the
presence of a functional hMutL
complex (11). Results
from the loop repair assay indicate that H2O2
also damaged hMutS
in addition to hMutS
and
hMutL
(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 hMutL 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 hMutL
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 hMutL
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 hMutS
and MutS
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 hMutS
and hMutL
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 hMutS, hMutS
, and hMutL
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-
receptor type II (TGF-
RII) contains an (A)10 tract in
its coding region, and both APC and TGF-
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 hMutS, hMutS
, and hMutL
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.
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ACKNOWLEDGEMENTS |
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We express our gratitude to Drs. Markus Räschle and Patrick Dufner (Institute of Medical Radiobiology, University of Zürich) for providing recombinant MMR heterodimers.
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FOOTNOTES |
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* 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.
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