Microsatellite Instability and Suppressed
DNA Repair Enzyme Expression in
Rheumatoid Arthritis
This information is current as
of June 18, 2017.
Sang-Heon Lee, Dong Kyung Chang, Ajay Goel, C. Richard
Boland, William Bugbee, David L. Boyle and Gary S.
Firestein
J Immunol 2003; 170:2214-2220; ;
doi: 10.4049/jimmunol.170.4.2214
http://www.jimmunol.org/content/170/4/2214
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References
The Journal of Immunology
Microsatellite Instability and Suppressed DNA Repair Enzyme
Expression in Rheumatoid Arthritis1
Sang-Heon Lee,2 Dong Kyung Chang, Ajay Goel, C. Richard Boland, William Bugbee,
David L. Boyle, and Gary S. Firestein3
R
heumatoid arthritis (RA)4 is a chronic inflammatory disease characterized by invasion of synovial tissue into cartilage and bone. Fibroblast-like synoviocytes (FLS) contribute to this process and exhibit some features of transformed
cells, including anchorage-independent growth in vitro, loss of
contact inhibition, and expression of several oncogenes in situ (1–
3). The most compelling evidence of invasive behavior by RA
synoviocytes was demonstrated in a SCID mouse model using FLS
that were coimplanted with cartilage (4). RA FLS invade the cartilage explants, whereas osteoarthritis (OA) and normal FLS do
not. The mechanism of autonomous behavior may involve permanent genetic changes in RA synovial cells, including somatic
mutations in the p53 tumor suppressor gene (5– 8). Mutations
Division of Rheumatology, Allergy, and Immunology, Division of Gastroenterology,
and Department of Orthopedics, University of California at San Diego School of
Medicine, La Jolla, CA 92093
Received for publication October 9, 2002. Accepted for publication December
10, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant AR45347 and a
Veterans Affairs Hospitals Merit Review award. S.-H.L. was supported, in part, by
postdoctoral fellowship programs of the Catholic University of Korea and Korea
Science and Engineering Foundation.
2
Current address: Department of Internal Medicine, the Catholic University of Korea,
College of Medicine, Seoul, Korea.
3
Address correspondence and reprint requests to Dr. Gary S. Firestein, Division of
Rheumatology, Allergy, and Immunology, University of California at San Diego
School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656. E-mail address:
[email protected]
4
Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like synoviocyte; HNPCC, hereditary nonpolyposis colon cancer; MMR, mismatch repair;
MSI, microsatellite instability; MSI-H, MSI high; MSI-L, MSI low; MSS, microsatellite stable; OA, osteoarthritis; RNS, reactive nitrogen species; ROS, reactive oxygen
species; SNAP, S-nitroso-N-acetylpenicillamine.
Copyright © 2003 by The American Association of Immunologists, Inc.
have also been observed in other genes in RA, including hprt
and H-ras (9, 10).
The presence of somatic mutations in RA synovium might result
from local production of genotoxic intermediates such as reactive
oxygen and nitrogen species. Inflammation-induced genetic damage is not unique to RA, and might contribute to mutations in
ulcerative colitis that ultimately lead to neoplasia (11–13). However, the human genome is also monitored by several homeostatic
systems that repair mutations. Highly efficient repair mechanisms
present in the nucleus, including the mismatch repair (MMR) system, protect DNA from sequence alterations (14, 15). In eukaryotes, the heterodimeric complexes MutS␣ and MutS␤ play
essential roles in this process. Both contain hMSH2, which can
pair with either hMSH6 (MutS␣) or hMSH3 (MutS␤). The former
primarily repairs single base mismatches, while the latter repairs
larger insertion/deletion loops. Abnormalities of DNA repair produce a hypermutable state, which can account for mutations found
in some cancers (16, 17).
Progress in understanding DNA damage and repair in cancer
can potentially be applied to inflammatory diseases. In colorectal
cancer, for instance, the presence of microsatellite instability
(MSI) is evidence of ongoing mutagenesis (18, 19) that causes
insertion or deletion mutations in mononucleotide (e.g., An) and
dinucleotide repeat (e.g., [CA]n) sequences (20, 21). MSI often
results from defective human DNA MMR function (22–25), and
well-defined criteria for quantifying MSI phenotype have been developed. Based on the presence of somatic mutations in RA synovial cells, we sought evidence of DNA damage in synovium by
assessing MSI. Our studies demonstrated marked MSI in RA compared with noninflamed synovium. To explore potential mechanisms of DNA damage, the expression and regulation of MMR
enzymes were then evaluated. Because reactive oxygen can suppress DNA repair activity in some tumor cell lines (26), we determined the effect of NO, a common reactive intermediate produced in the joint, on MMR expression. Most notably, the key
0022-1767/03/$02.00
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Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in
key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during
DNA replication. Key members of the MMR system include MutS␣ (hMSH2 and hMSH6) and MutS␤ (hMSH2 and hMSH3). To
provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was
examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer
sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis
tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA
synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from
synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine,
the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax
the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base
mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially
mutagenic, but also suppresses the mechanisms that limit the DNA damage. The Journal of Immunology, 2003, 170: 2214 –2220.
The Journal of Immunology
MMR enzyme MSH6 was decreased in RA synovium. The expression of this enzyme was also suppressed in cultured FLS after
exposure to reactive nitrogen species (RNS). We propose that oxidative stress not only creates DNA adducts that are mutagenic, but
also relaxes the mechanisms that ordinarily repair DNA damage.
Materials and Methods
Synovial tissue and FLS
Microsatellite analysis
MSI status was determined following DNA extraction from synovial tissue
and paired autologous PBMC collected during joint replacement surgery
(seven RA and five OA; see characteristics of patients above) using the
genomic DNA separation kit (Qiagen, Valencia, CA). A panel of five microsatellite markers was used comprising three dinucleotide repeats
(D2S123, D5S346, D17S250) and two mononucleotide repeats (BAT25
and BAT26), which serve as a National Cancer Institute MSI reference
panel for colorectal cancer (18). Changes in the electrophoretic mobility of
amplified PCR products are used to assess the microsatellite status. PCR
amplification was performed with 100 ng of purified genomic DNA isolated from snap-frozen synovial tissue or the PBMCs in a final reaction
volume of 20 ␮l using an MJ Research Thermocycler (PTC100; MJ Research, Watertown, MA). The primers for PCR were end labeled with
[␥-32P]ATP. Products were denatured, electrophoresed on 8% denaturing
polyacrylamide gels, and visualized by autoradiography. MSI was readily
determined by the presence of clearly visualized altered allelic shifts in the
PCR products in the synovial tissue specimens when compared with the
paired control DNA derived from autologous PBMC. MSI status was independently evaluated by two blinded observers. These criteria were defined and validated by a National Cancer Institute workshop chaired by one
of us (C. R. Boland) (18). Synovial tissues exhibiting allelic shifts in ⱖ2
of the 5 markers were considered as microsatellite high (MSI-H), as previously defined in colorectal cancer. A low level of MSI (MSI-L) was
defined by a shift in only 1 of the 5 markers. Synovial tissues that did not
exhibit any allelic shifts compared with the control DNA were defined as
microsatellite stable (MSS).
clonal anti-hMSH2 (Calbiochem-Novabiochem, La Jolla, CA), goat polyclonal anti-hMSH6 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit
polyclonal anti-hMSH3, mouse monoclonal anti-hMLH1 (CalbiochemNovabiochem), or mouse monoclonal anti-hPMS2 (BD PharMingen, San
Diego, CA). Blots were washed with PBS-T and incubated for 2 h at room
temperature with HRP-conjugated secondary anti-mouse IgG Ab for
hMSH2, hMLH1, and hPMS2; anti-goat IgG Ab for hMSH6; or anti-rabbit
IgG Ab (Santa Cruz Biotechnology) for hMSH3. The proteins were visualized using hydrogen peroxide and luminal as a substrate (Cell Signaling
Technology, Beverly, MA), with Kodak X-OMAT AR film (Eastman
Kodak, Rochester, NY). To quantify the amount of MMR proteins, each
band density was normalized to actin protein and digitized using National
Institutes of Health image analyzer software (Bethesda, MD).
Induction of oxidative stress in vitro
To induce oxidative stress, FLS were washed with serum-free DMEM and
treated with 0.1 mM of H2O2 (Sigma-Aldrich, St. Louis, MO) for 1 h or 1
mM of S-nitroso-N-acetylpenicillamine (SNAP; Sigma-Aldrich) for 3 h.
Cells were then washed with DMEM and allowed to recover in DMEM
supplemented with 10% FCS. During recovery, cell lysates were collected
at various time points (0, 6, 12, 24, and 48 h after the initial stress) for
analysis and stored at ⫺70°C.
Statistics
Data are expressed as mean ⫾ SEM. Statistical analysis was performed by
Fisher’s exact test for MSI analysis. Student’s t test was used to compare
relative MMR expression in synovial tissues. Repeated measure ANOVA
was used to perform the time point analysis for MMR regulation by RNS
or reactive oxygen species (ROS). Values of p ⬍ 0.05 were considered
statistically significant.
Results
Presence of MSI in RA synovial tissue
To determine genomic stability in RA synovial tissue, we evaluated the five microsatellite markers used in colorectal cancer associated with increased reactive intermediates and MMR defects
(18). The techniques and criteria for defining mutations and MSI
using gel electrophoresis were in agreement with previously defined and validated methods. Because microsatellite sequences are
highly polymorphic among the population, DNA was extracted
from autologous PBMC served as controls. Fig. 1A shows a representative MSI analysis of two loci, D5S346 and D17S250, for
two RA and two OA patients (note that MSI was only present in
the RA samples). MSI was observed in all seven RA synovial
tissues tested, with five meeting criteria for MSI-H (MSI in two or
more loci) and two patients meeting criteria for MSI-L (MSI in 1
locus) (see Fig. 1B). In contrast, no MSI was found in the OA
samples ( p ⫽ 0.0025 for RA vs OA).
MMR protein expression in synovial tissue
Based on the known relationship between MSI and MMR defects
in colorectal cancer, we evaluated the expression of MMR enzymes in RA and OA synovial tissues. As shown in Fig. 2, hMSH6
expression was significantly lower in RA synovial tissues compared with OA ( p ⫽ 0.02), while hMSH3 levels were significantly
higher in RA ( p ⫽ 0.03). There was also a trend toward increased
hMSH2 levels in RA that did not reach statistical significance.
Western blot analysis of MMR proteins
MMR protein expression in FLS
Tissue homogenates (100 mg) or cells (⬃106 cells) were suspended in cold
PBS/0.1% sodium deoxycholate, frozen for 2 h at ⫺20°C, and thawed for
30 min on ice. After a second freeze-thaw cycle, the membranes were
gently broken by mechanical pipetting (5). Protein concentrations were
measured with the BSA protein assay kit (Bio-Rad Laboratories, Hercules,
CA). Protein samples from FLS lysates (30 ␮g) or synovial tissue lysates
(100 ␮g) were separated by electrophoresis using a SDS-8% polyacrylamide gel, and transferred onto a nitrocellulose membrane at 140 mA in 25
mM Tris Cl (pH 8.3), 200 mM glycine, and 20% methanol. Membranes
were blocked with 5% nonfat dry milk in PBS-0.1% Tween 20 (PBS-T) for
1 h. This was followed by overnight incubation at 4°C with mouse mono-
Variations in cellular composition could contribute to differences
in the expression of MMR proteins between OA and RA. High
expression of the MMR enzymes in the intimal lining compared
with the rest of the synovium (29) led us to examine MMR expression in synoviocytes using Western blot analysis. Fig. 3 shows
that the levels of several MMR proteins in cultured FLS, including
hMSH2, hMSH3, and hMSH6 as well as hMLH1 and hPMS2, are
similar in RA, OA, and normal cultured FLS ( p ⬎ 0.10). Therefore, the differences between OA and RA synovium are most likely
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RA and OA synovial tissues were obtained at joint replacement surgery
from patients with destructive arthritis. The diagnoses conformed to the
1987 revised ACR criteria (27). None of the patients had been treated with
alkylating agents. For the MSI studies, the average ages were 75 (52–94)
years and 55 (32– 87) years for OA (n ⫽ 5) and RA (n ⫽ 7), respectively.
The patients were female, and the synovial tissues were derived from the
knees or hips, except for one RA patient who had shoulder surgery. Of the
RA samples, five were rheumatoid factor positive. Four patients received
methotrexate, three received leflunomide, three received low dose prednisone, and one received etanercept. OA patients were treated with analgesics and nonsteroidal anti-inflammatory drugs. Informed consent was
obtained, and the protocol was approved by the University of California at
San Diego Human Subjects Research Protection Committee. For microsatellite analysis, synovial tissue was snap frozen and aliquoted at the time
of surgery. FLS were prepared as previously described (28). Briefly, the
tissues were minced and incubated with 1 mg/ml collagenase in serum-free
DMEM (Life Technologies, Grand Island, NY) for 2 h at 37°C, filtered
through a nylon mesh, extensively washed, and cultured in DMEM supplemented with 10% FCS (Life Technologies; endotoxin content ⬍0.006
ng/ml), penicillin, streptomycin, gentamicin, and L-glutamine in a humidified 5% CO2 atmosphere. After overnight culture, nonadherent cells were
removed and adherent cells were cultivated in DMEM plus 10% FCS. At
confluence, cells were trypsinized, split at a 1:3 ratio, and recultured in
medium. Synoviocytes were used from passages 3 through 9, in which they
comprised a homogeneous population of FLS (⬍1% CD11b, ⬍1% phagocytic, and ⬍1% Fc␥RII receptor positive).
2215
2216
DNA REPAIR IN RA
due to local environmental influences or variations in cellular composition rather than generalized suppression of MMR expression
in RA.
Effect of RNS and ROS on MutS MMR proteins in FLS
Because no differences in basal MMR expression were observed
between OA- and RA-cultured FLS, we studied the effect of various mediators on the components of the MMR enzyme complexes
in vitro. No changes in hMSH2, 3, or 6 were observed in FLS that
had been stimulated with IL-1 (1 ng/ml) or TNF-␣ (100 ng/ml) for
up to 24 h (data not shown). We then used an in vitro model of
oxidative stress to determine whether MMR expression in cultured
FIGURE 2. Expression of DNA MMR proteins in synovial tissues. Synovial tissue extracts
from RA and OA were evaluated by Western blot
analysis. hMSH6 protein expression was significantly less in RA than in OA, while hMSH3 expression was higher in RA in comparison with
OA (ⴱ, p ⬍ 0.05). RA synovium from lanes 1– 4
were MSI-H, and OA synovium from lanes 2– 4
were MSS. Although there was a trend for higher
hMSH2 expression in RA, the differences did not
reach statistical significance.
RA FLS is regulated by RNS or ROS. FLS were cultured in the
presence of the NO donor SNAP, and expression of MMR enzymes was assessed by Western blot analysis (n ⫽ 5 separate
lines). As shown in Fig. 4, pretreatment of FLS with SNAP for 3 h
significantly decreased hMSH6 expression 6 –12 h after exposure
(62% decrease at 6 h, p ⫽ 0.002). hMSH3 levels increased during
the same period of time (201% increase at 6 h, p ⫽ 0.047), indicating that the effect on hMSH6 was not due to nonspecific toxicity
(Fig. 4A). hMSH2 expression was not significantly changed, although there was a trend toward a modest increase after RNS exposure. Alterations in MMR protein expression were transient, and
expression returned to baseline within 24 – 48 h. No differences
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FIGURE 1. MSI in RA. A, MSI analysis using primers for two microsatellite loci, D5S346 and D17S250. Note that patterns for autologous PBMC differ
from synovial tissue (ST) from the same patient in RA. In contrast, band patterns in OA synovium matched autologous PBMC. B, Frequencies of MSI in
RA (n ⫽ 7) and OA (n ⫽ 5) for five standard microsatellite markers. MSI-H (MSI in two or more loci) is observed in RA synovium, while OA are MSS
( p ⫽ 0.0025).
The Journal of Immunology
2217
FIGURE 3. Expression of DNA MMR enzymes in cultured FLS. RA (2 lines), OA (2
lines), and normal (1 line) FLS constitutively express hMSH2, hMSH6, hMSH3, hMLH1, and
hPMS2.
Effect of dose and duration of NO exposure on hMSH6
expression in FLS
Kinetics and dose-response experiments were then performed to
characterize the effects of RNS on MMR expression. To evaluate
the concentration of NO required to suppress hMSH6, RA FLS cell
lines (n ⫽ 3) were exposed to various concentrations of SNAP
(0.01, 0.1, 1.0 mM) for 3 h. Minimal or no effect was observed for
the lower concentrations, while 1 mM clearly suppressed hMSH6
( p ⬍ 0.05) (Fig. 5). We then treated RA FLS cell lines with 1 mM
of SNAP for 0.5–3 h, and analyzed hMSH6 protein levels. As
illustrated in Fig. 6, suppression of hMSH6 correlated with the
duration of exposure to SNAP ( p ⬍ 0.05).
Discussion
Genotoxic adducts, especially ROS and RNS, are released as a
result of cell activation and can damage genomic DNA. One such
product, NO, is especially prevalent in chronic inflammatory diseases such as ulcerative colitis (30) and RA (31–33). In the former,
local NO production induces somatic mutations that can ultimately
lead to neoplasia (34). DNA damage and mutation have also been
documented in the synovial intimal lining in rheumatoid synovium, albeit without resultant tumor formation (35). To determine
the mechanisms of DNA damage in inflamed tissues, we evaluated
rheumatoid synovium for evidence of mutagenesis by performing
FIGURE 4. Effect of RNS and ROS on MutS MMR proteins. SNAP
was used as an NO donor. Hydrogen peroxide was used as ROS stress.
Cells were incubated with SNAP (1 mM for 3 h) or hydrogen peroxide (0.1
mM for 1 h), and then allowed to recover up to 48 h. A, SNAP treatment
significantly decreased hMSH6 expression at 6 –12 h, while hMSH3 was
increased 6 h after NO stress. B, Hydrogen peroxide also decreased
hMSH6 at 6, 12, and 48 h, while hMSH2 was not significantly changed (ⴱ,
p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 compared with baseline).
FIGURE 5. Regulation of hMSH6 by NO in FLS. RA synoviocytes
were treated with 0.01, 0.1, and 1.0 mM of SNAP for 3 h and then allowed
to recover for various time intervals, as described in Materials and
Methods.
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were observed between RA and OA FLS with regard to their response to SNAP (data not shown). Hydrogen peroxide treatment
also modestly decreased hMSH6 (Fig. 4B, p ⬍ 0.05), although the
effect was more prolonged. hMSH2 was not significantly changed
by ROS exposure. These data indicate that the MMR pattern observed in RA synovial tissue, i.e., low hMSH6 and high hMSH3,
was reproduced in cultured FLS after oxidative stress. This amount
of MMR enzyme decrease is known to suppress DNA repair activity in cells, supporting the functional relevance of this phenomenon (26).
2218
FIGURE 6. Kinetics of hMSH6 regulation by NO. RA FLS were treated
with 1 mM SNAP for 0.5 to 3 h, and then hMSH6 was analyzed after 6 h.
hMSH6 expression was determined by Western blot analysis and demonstrates that prolonged exposure was required for maximum MMR
suppression.
hMSH2 is a subunit of both dimers and is the most common defective gene in HNPCC (22). It can pair with hMSH6 to form
MutS␣, while hMSH3 and hMSH2 form MutS␤. MutS␣ mainly
repairs single base mispairs, while MutS␤ repairs larger insertion/
deletion mispairs (39). Because MSH2 is the common component,
balance between repair of single base mismatches or longer insertion/deletion loops depends on the ratio of MSH6 and MSH3. In
cells that overexpress MSH3, the available MSH2 protein is sequestered into MutS␤. This leads to degradation of the partnerless
MSH6 and defective repair of single base mismatches (40). Therefore, MMR deficiency can arise not only through mutation or transcriptional silencing of MMR genes, but also from an imbalance in
the relative amounts of the MSH3 and MSH6 proteins.
Based on our observation that RA synovium is MSI-H, we assessed the expression of MMR proteins. hMSH2 and another
MMR enzyme, hMLH1, are expressed by intimal lining cells in
RA, as determined by immunohistochemistry (38). However, there
is no information on the other key enzymes or the relative amounts
compared with OA. Decreased hMSH6 was observed in the rheumatoid synovium compared with OA, although hMSH3 expression
was significantly higher in RA. Variations in the cellular content of
synovium in the two diseases might contribute, but the high expression of MMR proteins in the intimal lining suggests this region, comprising macrophage and FLS in RA and OA, is the major
site. The observed pattern of MMR enzyme expression provides a
potential explanation for the type of mutation observed in the intimal lining of RA, where single base pair transition substitutions
account for the vast majority of abnormalities (5, 29). Also, transition mutations are characteristic of MMR defects and represent
almost all of the RA mutations identified to date.
The MMR differences between OA and RA synovium led us to
explore the expression and regulation of these enzymes in FLS.
Basal of MMR protein expression was similar in RA and OA FLS,
indicating that the abnormalities observed in the tissue were not
simply due to a generalized suppression as observed in HNPCC.
The pattern of MMR expression in FLS changed when they were
stressed by RNS, with induction of hMSH3 and suppression of
hMSH6. The decrease in hMSH6 levels was dose dependent and
varied with the duration of NO exposure. The fact that other MMR
proteins or housekeeping genes such as actin were unaffected indicates that hMSH6 suppression was not caused by nonspecific
toxicity. NO donors such as SNAP serve as physiologic sources of
NO and permit accurate assessment of reactive nitrogen effects.
The results with SNAP were largely confirmed using hydrogen
peroxide, which is a standard reagent that exposes cells to ROS.
Although the mechanism of MMR regulation is currently unknown, the MMR protein levels in other cells correlate with
mRNA expression and suggest that transcriptional regulation most
likely participates (41). ROS was recently noted to decrease expression of hMSH6 in erythroleukemia cells to an extent similar to
FLS (26). More important, hydrogen peroxide suppressed DNA repair
activity, as determined with functional assays. This defect was reversed by the addition of rMMR enzymes to the cell extracts of
stressed cells, indicating the functional relevance of the observation.
The cultured FLS studies showed that the pattern of MMR expression in RA synovium (high hMSH3/low hMSH6) was reproduced in synoviocytes that were exposed to genotoxic stimuli. This
tissue culture model obviously does not completely mimic the situation in chronically inflamed synovium, and other cell types can
contribute to MSI or MMR protein expression in RA synovium.
Nevertheless, the levels of RNS and ROS exposure that alter MSH
expression are relevant to the in vivo situation (33, 42). Suppression of hMSH6 by RNS appears paradoxical at first. However, it
might represent a mechanism that protects the cell from major
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standard microsatellite analysis. Microsatellites are tandem repeated sequences in the DNA that are primarily (but not exclusively) located in noncoding regions. Instability is defined as a
microsatellite insertion or deletion mutation in genomic DNA isolated from the target tissue or tumor compared with an autologous
control (usually peripheral blood) (18). Many studies demonstrate
an association among MSI, DNA damage, and development of
neoplasia (19, 36, 37).
Criteria for MSI-H and MSI-L phenotypes, respectively, and
MSS phenotype have been carefully defined for colon tumor loci
utilizing the same techniques used in our study (18). Five specific
microsatellites can be evaluated for evidence of mutations and the
number of mutations quantified. The phenotypes are: 1) MSI-H,
with mutations in ⱖ40% of loci; 2) MSI-L with mutations in
ⱕ20% of loci; and 3) MSS, with no microsatellite mutations. Our
initial studies were performed on RA and OA synovial tissues and
used the loci validated for colon inflammation because the mechanisms of mutagenesis are most likely similar. These experiments
demonstrated clear evidence of the MSI-H genotype in RA using
the same methodology and criteria used to define MSI in neoplastic disease. In contrast, OA samples were MSS. The presence of
microsatellite mutations in RA is consistent with previous studies
demonstrating somatic mutations and increased DNA strandbreaks
in rheumatoid synovium (5, 35). It extends our hypothesis that
DNA damage and mutations can occur randomly throughout the
genome in RA; when nonconserved base changes occur in key
genes such as p53 that provide a selective advantage, local expansion of individual clones can occur. Our recent microdissection
studies on RA synovium demonstrating islands of p53 mutant cells
are consistent with this observation (29).
This area, like other studies examining mutations and DNA
damage in RA, is not without controversy. A previous report by
Muller-Ladner and colleagues (38) suggested that microsatellites
are stable in long-term cultured RA synoviocytes. Our MSI analysis focused on fresh tissue rather than cultured cells, which we
believe provides a more accurate indication of the situation in situ.
Their patient population also did not have evidence of p53 mutations (7), in contrast to patients from other groups (5, 6, 8). Although we do not know why our data differ, it could be related to
greater severity of disease in our patients because we primarily
examined destructive arthritis requiring total joint replacement.
This is clearly an area that requires additional investigation, and
we are currently evaluating the relationship between duration of
disease, severity, and DNA damage.
The mechanisms of MSI have been evaluated in other settings,
especially in hereditary nonpolyposis colon cancer (HNPCC).
These patients have many mutations in the colon and frequently
develop colon cancer in early adulthood. The etiology of the mutagenic propensity in HNPCC is defective DNA MMR enzymes
due to inactivating germline mutations in MMR genes (22, 25, 36).
This complex repair system involves many enzymes, including
two key heterodimeric complexes known as MutS␣ and MutS␤.
DNA REPAIR IN RA
The Journal of Immunology
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Acknowledgments
We thank Zuoning Han for expert assistance.
26.
References
1. Cifone, M. A., and I. J. Fidler. 1980. Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma. Proc.
Natl. Acad. Sci. USA 77:1039.
2. Lafyatis, R., E. F. Remmers, A. B. Roberts, D. E. Yocum, M. B. Sporn, and
R. L. Wilder. 1989. Anchorage-independent growth of synoviocytes from arthritic and normal joints: stimulation by exogenous platelet-derived growth factor
and inhibition by transforming growth factor-␤ and retinoids. J. Clin. Invest.
83:1267.
3. Muller-Ladner, U., J. Kriegsmann, R. E. Gay, and S. Gay. 1995. Oncogenes in
rheumatoid arthritis. Rheum. Dis. Clin. N. Am. 21:675.
4. Muller-Ladner, U., J. Kriegsmann, B. N. Franklin, S. Matsumoto, T. Geiler,
R. E. Gay, and S. Gay. 1996. Synovial fibroblasts of patients with rheumatoid
27.
28.
29.
arthritis attach to and invade normal human cartilage when engrafted into SCID
mice. Am. J. Pathol. 149:1607.
Firestein, G. S., F. Echeverri, M. Yeo, N. J. Zvaifler, and D. R. Green. 1997.
Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium. Proc. Natl. Acad. Sci. USA 94:10895.
Inazuka, M., T. Tahira, T. Horiuchi, S. Harashima, T. Sawabe, M. Kondo,
H. Miyahara, and K. Hayashi. 2000. Analysis of p53 tumor suppressor gene
somatic mutations in rheumatoid arthritis synovium. Rheumatology 39:262.
Kullmann, F., M. Judex, I. Neudecker, S. Lechner, H. P. Justen, D. R. Green,
D. Wessinghage, G. S. Firestein, S. Gay, J. Scholmerich, et al. 1999. Analysis of
the p53 tumor suppressor gene in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 42:1594.
Reme, T., A. Travaglio, E. Gueydon, L. Adla, C. Jorgensen, and J. Sany. 1998.
Mutations of the p53 tumor suppressor gene in erosive rheumatoid synovial tissue. Clin. Exp. Immunol. 111:353.
Cannons, J. L., J. Karsh, H. C. Birnboi, and R. Goldstein. 1998. HPRT-mutant T
cells in the peripheral blood and synovial tissue of patients with rheumatoid
arthritis. Arthritis Rheum. 41:1772.
Roivainen, A., J. Jalava, L. Pirila, T. Yli-Jama, H. Tiusanen, and P. Toivanen.
1997. H-ras oncogene point mutations in arthritic synovium. Arthritis Rheum.
40:1636.
Suzuki, H., N. Harpaz, L. Tarmi, J. Yin, H. Y. Jiang, J. D. Bell, M. Hontanosas,
G. M. Groisman, J. M. Abraham, and S. J. Meltzer. 1994. Microsatellite instability in ulcerative colitis-associated colorectal dysplasias and cancers. Cancer
Res. 54:4841.
Kern, S. E., M. Redston, A. B. Seymour, C. Caldas, S. M. Powell, S. Kornacki,
and K. W. Kinzler. 1994. Molecular genetic profiles of colitis-associated neoplasms. Gastroenterology 107:420.
Brentnall, T. A., D. A. Crispin, M. P. Bronner, S. P. Cherian, M. Hueffed,
P. S. Rabinovitch, C. E. Rubin, R. C. Haggitt, and C. R. Boland. 1996. Microsatellite instability in nonneoplastic mucosa from patients with chronic ulcerative
colitis. Cancer Res. 56:1237.
Boland, C. R. 1996. Roles of the DNA mismatch repair genes in colorectal tumorigenesis. Int. J. Cancer 69:47.
Boland, C. R., and L. Ricciardiello. 1999. How many mutations does it take to
make a tumor? Proc. Natl. Acad. Sci. USA 96:14675.
Loeb, L. A., C. F. Springgate, and N. Battula. 1974. Errors in DNA replication
as a basis of malignant changes. Cancer Res. 34:2311.
Loeb, L. A. 1991. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res. 51:3075.
Boland, C. R., S. N. Thibodeau, S. R. Hamilton, D. Sidransky, J. R. Eshleman,
R. W. Burt, S. J. Meltzer, M. A. Rodriguez-Bigas, R. Fodde, G. N. Ranzani, et
al. 1998. A National Cancer Institute Workshop on Microsatellite Instability for
cancer detection and familial predisposition: development of international criteria
for the determination of microsatellite instability in colorectal cancer. Cancer
Res. 58:5248.
Boland, C. R., J. Sato, K. Saito, J. M. Carethers, G. Marra, L. Laghi, and
D. P. Chauhan. 1998. Genetic instability and chromosomal aberrations in colorectal cancer: a review of the current models. Cancer Detect. Prev. 22:377.
Thibodeau, S. N., G. Bren, and D. Schaid. 1993. Microsatellite instability in
cancer of the proximal colon. Science 260:816.
Ionov, Y., M. A. Peinado, S. Malkhosyan, D. Shibata, and M. Perucho. 1993.
Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558.
Fishel, R., M. K. Lescoe, M. R. Rao, N. G. Copeland, N. A. Jenkins, J. Garber,
M. Kane, and R. Kolodner. 1993. The human mutator gene homolog MSH2 and
its association with hereditary nonpolyposis colon cancer. Cell 75:1027.
Leach, F. S., N. C. Nicolaides, N. Papadopoulos, B. Liu, J. Jen, R. Parsons,
P. Peltomaki, P. Sistonen, L. A. Aaltonen, M. Nystrom-Lahti, et al. 1993. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell
75:1215.
Papadopoulos, N., N. C. Nicolaides, Y. F. Wei, S. M. Ruben, K. C. Carter,
C. A. Rosen, W. A. Haseltine, R. D. Fleischmann, C. M. Fraser, M. D. Adams,
et al. 1994. Mutation of a mutL homolog in hereditary colon cancer. Science
263:1625.
Nicolaides, N. C., N. Papadopoulos, B. Liu, Y. F. Wei, K. C. Carter, S. M. Ruben,
C. A. Rosen, W. A. Haseltine, R. D. Fleischmann, C. M. Fraser, et al. 1994.
Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.
Nature 371:75.
Chang, C. L., G. Marra, D. P. Chauhan, H. T. Ha, D. K. Chang, L. Ricciardiello,
A. Randolph, J. M. Carethers, and C. R. Boland. 2002. Oxidative stress inactivates the human DNA mismatch repair system. Am. J. Physiol. Cell. Physiol.
283:C148.
Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries,
N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al. 1988.
The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31:315.
Alvaro-Gracia, J. M., N. J. Zvaifler, and G. S. Firestein. 1990. Cytokines in
chronic inflammatory arthritis. V. Mutual antagonism between interferon-␥ and
tumor necrosis factor-␣ on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by
rheumatoid arthritis synoviocytes. J. Clin. Invest. 86:1790.
Yamanishi, Y., D. L. Boyle, S. Rosengren, D. R. Green, N. J. Zvaifler, and
G. S. Firestein. 2002. Regional analysis of p53 mutations in rheumatoid arthritis
synovium. Proc. Natl. Acad. Sci. USA 99:10025.
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DNA damage while permitting single base pair changes. This process could provide short-term survival benefits to an organism by
shifting the balance from MutS␣ to MutS␤. It also might serve as
a mechanism for establishing a mutator phenotype, which can have
additional survival benefits (43).
Exposure to reactive species, especially RNS, can be directly
mutagenic and can increase the likelihood of single base mismatches in RA through oxidative deamination (44). In addition,
suppression of MMR mechanisms could contribute to a hypermutable state and increase the likelihood for the accumulation of somatic mutations (17). In RA, ROS production is increased by the
hypoxic environment mediated by activated leukocytes, ischemiareperfusion injury, an increased metabolic rate, and reduced capillary density (45, 46). Expression of iNOS is also markedly increased in RA, especially in the synovial intimal lining (32). Local
NO production results in nitrosylation of synovial proteins and can
produce mutagenic nucleotide adducts (44). Synovial expression
of proinflammatory cytokine genes probably contributes to cell
activation, reactive oxygen production, and iNOS induction. The
cytokine networks that perpetuate disease in RA thereby provide a
link between synovial inflammation, production of reactive intermediates that have mutagenic potential, and relaxation of the DNA repair
system. However, a direct influence of cytokines such as IL-1 and
TNF-␣ on MMR expression in cultured FLS was not observed.
We have not formally examined in vivo correlations between
synovial cytokine production or clinical disease activity and the
extent of MSI. Because the proinflammatory milieu is directly related to both processes, they probably move in tandem over time.
The corollary is that suppression of cytokine networks and control
of inflammation with TNF-␣ inhibitors might also have long-term
benefits by protecting DNA integrity in the synovium. Because all
of the patients evaluated in the current study had longstanding
disease requiring total joint replacement, it is difficult to correlate
disease activity with MSI. The latter integrates accumulated damage in resident cells over many years, while the former represents
a more immediate assessment of disease activity. Future studies on
early RA samples are planned to evaluate this relationship.
Relaxation of MMR in stressed synovium has parallels in other
diseases. For instance, MSI has been described in ulcerative colitis
and may be associated with functional MMR suppression (47). RA
is the first chronic inflammatory disease associated with MSI, somatic mutations of key genes, and suppression of DNA repair
mechanisms, but that does not lead to cancer. Instead, this process
might alter the natural history of RA and cause local invasion and
an aggressive FLS phenotype. It also provides potential therapeutic
targets to modify the long-term destructive potential of RA synovium. By limiting local RNS or ROS production, mutagenesis can
potentially be minimized. Alternatively, replacing or augmenting
MMR expression could mitigate the genotoxicity in the synovium
or in other chronically inflamed sites.
2219
2220
39.
40.
41.
42.
43.
44.
45.
46.
47.
Microsatellite analysis in rheumatoid arthritis synovial fibroblasts. Ann. Rheum.
Dis. 59:386.
Kolodner, R. D., and G. T. Marsischky. 1999. Eukaryotic DNA mismatch repair.
Curr. Opin. Genet. Dev. 9:89.
Marra, G., I. Iaccarino, T. Lettieri, G. Roscilli, P. Delmastro, and J. Jiricny. 1998.
Mismatch repair deficiency associated with overexpression of the MSH3 gene.
Proc. Natl. Acad. Sci. USA 95:8568.
Chang, D. K., L. Ricciardiello, A. Goel, C. L. Chang, and C. R. Boland. 2000.
Steady-state regulation of the human DNA mismatch repair system. J. Biol.
Chem. 275:18424.
Borderie, D., H. Le Marechal, O. G. Ekindjian, and A. Hernvann. 2000. Nitric
oxide modifies glycolytic pathways in cultured human synoviocytes. Cell Biol.
Int. 24:285.
Chang, D. K., D. Metzgar, C. Wills, and C. R. Boland. 2001. Microsatellites in
the eukaryotic DNA mismatch repair genes as modulators of evolutionary mutation rate. Genome Res. 11:1145.
Wiseman, H., and B. Halliwell. 1996. Damage to DNA by reactive oxygen and
nitrogen species: role in inflammatory disease and progression to cancer. Biochem. J. 313:17.
Mapp, P. I., M. C. Grootveld, and D. R. Blake. 1995. Hypoxia, oxidative stress
and rheumatoid arthritis. Br. Med. Bull. 51:419.
Tak, P. P., N. J. Zvaifler, D. R. Green, and G. S. Firestein. 2000. Rheumatoid
arthritis and p53: how oxidative stress might alter the course of inflammatory
diseases. Immunol. Today 21:78.
Jackson, A. L., R. Chen, and L. A. Loeb. 1998. Induction of microsatellite instability by oxidative DNA damage. Proc. Natl. Acad. Sci. USA 95:12468.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
30. Singer, I. I., D. W. Kawka, S. Scott, J. R. Weidner, R. A. Mumford, T. E. Riehl,
and W. F. Stenson. 1996. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111:871.
31. Sakurai, H., H. Kohsaka, M. F. Liu, H. Higashiyama, Y. Hirata, K. Kanno,
I. Saito, and N. Miyasaka. 1995. Nitric oxide production and inducible nitric
oxide synthase expression in inflammatory arthritides. J. Clin. Invest. 96:2357.
32. Grabowski, P. S., P. K. Wright, R. J. Van’t Hof, M. H. Helfrich, H. Ohshima,
and S. H. Ralston. 1997. Immunolocalization of inducible nitric oxide synthase in synovium and cartilage in rheumatoid arthritis and osteoarthritis.
Br. J. Rheumatol. 36:651.
33. Hilliquin, P., D. Borderie, A. Hernvann, C. J. Menkes, and O. G. Ekindjian. 1997.
Nitric oxide as S-nitrosoproteins in rheumatoid arthritis. Arthritis Rheum. 40:
1512.
34. Hussain, S. P., P. Amstad, K. Raja, S. Ambs, M. Nagashima, W. P. Bennett,
P. G. Shields, A. J. Ham, J. A. Swenberg, A. J. Marrogi, et al. 2000. Increased
p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancerprone chronic inflammatory disease. Cancer Res. 60:3333.
35. Firestein, G. S., M. Yeo, and N. J. Zvaifler. 1995. Apoptosis in rheumatoid arthritis synovium. J. Clin. Invest. 96:1631.
36. Dietmaier, W., S. Wallinger, T. Bocker, F. Kullmann, R. Fishel, and J. Ruschoff.
1997. Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression. Cancer Res. 57:4749.
37. Wong, N. A., and D. J. Harrison. 2001. Colorectal neoplasia in ulcerative colitis:
recent advances. Histopathology 39:221.
38. Kullmann, F., T. Widmann, A. Kirner, H. P. Justen, D. Wessinghage,
W. Dietmaier, J. Ruschoff, S. Gay, J. Scholmerich, and U. Muller-Ladner. 2000.
DNA REPAIR IN RA
The Journal of Immunology
CORRECTIONS
Boyoun Park, Sungwook Lee, Euijae Kim, and Kwangseog Ahn. A Single Polymorphic Residue Within the PeptideBinding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence. The Journal of Immunology
2003;170:961–968.
In the original article, both the concepts and language in the last paragraph of the Discussion were taken without
attribution from the previous publications (References 3 and 9). We deeply regret and apologize for this lack of appropriate attribution.
Sang-Heon Lee, Dong Kyung Chang, Ajay Goel, C. Richard Boland, William Bugbee, David L. Boyle, and Gary S.
Firestein. Microsatallite Instability and Suppressed DNA Repair Enzyme Expression in Rheumatoid Arthritis. The Journal of Immunology 2003;170:2214 –2220.
In Figure 2B, the mismatch repair enzyme expression for osteoarthritis (OA) and rheumatiod arthritis (RA) was
compared in a bar graph. The designation for RA and OA were reversed in the figure. The corrected figure is shown below.
Copyright © 2003 by The American Association of Immunologists, Inc.
0022-1767/03/$02.00