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Carcinogenesis |
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan 812-8582 [N. N., Y. N.]; and Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatobiology, Mayo Clinic/Foundation/Medical School, Rochester, Minnesota 55905 [M. J., N. F. L., G. J. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have been interested in how NO functions as a mutagen using biliary
epithelial cells (cholangiocytes) and cholangiocarcinoma cell lines as
model systems (11
, 12)
. Our studies have demonstrated that
iNOS is not expressed in normal cholangiocytes but is expressed in
cholangiocytes of patients with chronic biliary tract inflammation such
as primary sclerosing cholangitis, a disease known to predispose
individuals to the development of cholangiocarcinoma (11)
.
The expression of iNOS in cholangiocytes is also associated with
accumulation of 8-oxodG lesions; 8-oxodG lesion is a dominant oxidative
DNA lesion that is highly mutagenic because it predisposes to the
development of GC
TA transversions (13
, 14)
. Because
8-oxodG is efficiently repaired in humans by the BER pathway (15
, 16)
, the accumulation of this oxidative DNA lesion in
iNOS-expressing cholangiocytes suggests an inhibition of DNA repair.
Indeed, we have shown inhibition of both global and base excision DNA
repair in cholangiocytes and cholangiocarcinoma cell lines by a
NO-dependent mechanism (12)
. The inhibition of DNA repair
by NO could be blocked by NO scavengers but not by inhibitors of the
NO-mediated, cGMP-dependent signal transduction pathway
(12)
. These observations suggest that NO may directly
inhibit DNA repair enzymes including those responsible for BER. Given
the importance of the BER pathway in preventing mutagenesis after
oxidative DNA damage, more information is needed with regard to how NO
disrupts this pivotal antimutagenesis safeguard.
Oxidative DNA damage is predominantly repaired by BER enzymes. The predominant oxidative mutagenic lesion 8-oxodG in humans is removed by a BER glycosylase termed hOgg1 that is ubiquitously expressed and maps to chromosome 3p26.2 (15 , 16) . Studies from mice deficient in the gene for murine Ogg1 demonstrate that this enzyme is responsible for >95% of BER activity in mammalian cells (17) . Two isoforms of hOgg1 are expressed due to alternative splicing of a single mRNA gene product. The hOgg1-1a protein is localized to the nucleus, and the hOgg1-2a is localized to the mitochondria (18) . hOgg1 contains critical thiol moieties that are necessary for catalytic activity (19) . The hOgg1 protein has two distinct DNA-binding motifs, a helix-hairpin-helix motif and a C2H2 zinc finger motif (15) . Like other zinc finger proteins (20) , hOgg1 is potentially susceptible to nitrosylation of its thiol moieties (21 , 22) . Thiol nitrosylation can disrupt the zinc finger motif, resulting in loss of the zinc ion and irreversible disruption of catalytic activity (23) .
Thus, the objective of the current study was to determine whether hOgg1-1a is a direct target for nitrosylation with resultant loss of catalytic activity. We used a cholangiocarcinoma cell line for these studies as a model system because the biliary epithelium is a target of chronic inflammation in a variety of cholangiopathies that predispose individuals to the development of cancer. We demonstrate that cellular hOgg1-1a-mediated BER activity is inhibited during exposure to NO. This inhibition was associated with protein nitrosylation and ejection of zinc. These data suggest that NO generated during human inflammatory diseases not only causes oxidative DNA damage but also prevents DNA repair by directly inhibiting key DNA repair enzymes. The inability to repair oxidative DNA lesions would be predicted to be mutagenic and increase the risk of carcinogenesis.
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MATERIALS AND METHODS |
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Transfection of KMBC Cells with hOGG1-1a.
The corresponding cDNA for human Ogg1 protein was engineered into
mammalian expression plasmid pcDEB
-Ogg1a (25)
. The
plasmid was constructed by inserting the corresponding cDNA fragment,
to which a sequence for the HA epitope was introduced
(25)
. Transfection of the KMBC cells was carried out using
a 4:1 (w/w) ratio of LipofectAMINE Plus (Life Technologies,
Inc.):plasmid DNA. The lipid-DNA complexes were overlaid on 70%
confluent KMBC cells and incubated at 37°C with 5%
CO2 and 95% humidity in serum-free media for
24 h. After transfection, the cells were incubated for an
additional 24 h with complete growth media. The transfection
efficiency was determined by transfecting cells with pEGFP-CI
(Clontech, Palo Alto, CA) and assessing the percentage of
GFP-expressing cells by fluorescence microscopy; the transfection
efficiency was approximately 60%. The transfected cells were incubated
in the presence of a mixture of inflammatory cytokines (0.5 ng/ml
rhIL-1ß, 500 units/ml rhTNF-
, and 100 units/ml rhIFN-
) known to
induce iNOS expression in KMBC cells (11)
, in the presence
or absence of 10 mM DTT, a NO scavenger, or 50
µM 1400W
[N-(3-(aminomethyl)-benzyl)acetamide], a highly selective
iNOS inhibitor. The transfected cells were also treated with 300
µM SNAP, a pharmacological NO donor, as a
control for the complex signaling events occurring during incubation of
the cells with the cytokine mixture. WCEs were prepared as described
previously (26
, 27)
.
hOgg1-1a Immunofluorescence in Single Cells.
KMBC cells were transfected with HA-hOGG1-1a as described previously.
The cells were washed with PBS and fixed in 0.1 M PIPES (pH
6.5), 1 mM EGTA, 3 mM
MgSO4, 0.01% gluteraldehyde, and 2%
paraformaldehyde at 37°C. The cells were permeabilized in 0.2%
Triton X-100 and incubated with a 1:100 dilution of rabbit anti-HA
(Santa Cruz Biotechnology, Santa Cruz, CA) primary antibody to bind
specifically to the HA-tagged protein and subsequently incubated with a
goat antirabbit Texas Red-tagged secondary antibody (1:400 dilution).
The cells were mounted and visualized on a fluorescence microscope.
Western Blot Analysis.
Cells were harvested by trypsinization and lysed in ice-cold lysis
buffer containing 100 mM Tris-HCl (pH 7.5), 0.5
mM EDTA, 0.5 mM EGTA, 2 mM DTT
protease inhibitors (5 mg/ml leupeptin, pepstatin, and chymostatin and
87 mg/ml phenylmethylsulfonyl fluoride), and 1% Triton X-100 for 20
min. Whole cell lysates were boiled in Laemmli buffer [200
mM Tris-HCl (pH 8.0), 8% SDS, 1% bromphenol blue and 400
mM DTT]. Protein samples (40 µg/lane) were loaded on a
7.5% SDS-polyacrylamide gel and separated electrophoretically. The
proteins were transferred to a nitrocellulose membrane (Schleicher and
Schuell, Keene, NH) overnight at 90 mA in a Hoefer Scientific TransBlot
cell (Hoefer Scientific Instruments, San Francisco, CA). The membrane
was blocked with 5% nonfat dried milk in TTBS for 1 h. The
primary antibody for HA (Santa Cruz Biotechnology) was applied at a
1:5000 dilution for 2 h. The membrane was washed three times in
TTBS for 10 min each before applying the secondary goat antirabbit
antibody (Transduction Laboratories, Kensington, KY) at a 1:5000
dilution for 1 h. The blot was washed in TTBS four times for 10
min each. It was then incubated in commercial enhanced
chemiluminescence reagent (Amersham, Buckinghamshire, United Kingdom)
and exposed to Kodak X-OMAT AR photographic film (Eastman Kodak
Company, Rochester, NY).
IP of hOgg1-1a Protein.
Cells were harvested by trypsinization and lysed in ice-cold lysis
buffer containing 100 mM Tris-HCl (pH 7.5), 0.5
mM EDTA, 0.5 mM EGTA, 2 mM DTT
protease inhibitors (5 mg/ml leupeptin, pepstatin, and chymostatin and
87 mg/ml phenylmethylsulfonyl fluoride), and 1% Triton X-100 for 20
min on ice. After the protein concentration was measured, the HA
antibody was added in a ratio of 2:1 by weight, respectively, and cells
were incubated with a gentle rolling action overnight at 4°C.
The lysate was centrifuged at 13,000 x g for
10 min, and the supernatant was transferred to a
dolphin-nosed IP tube (PEG Scientific, Frederick, MD). Sepharose A (0.1
part by volume) and Sepharose G (0.05 part by volume) were added to the
supernatant, which was then incubated at 4°C with gentle
rolling action for 3 h. The beads and protein mixture were washed
with radioimmunoprecipitation assay buffer [50
mM Tris-HCl (pH 7.5), 150
mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate, and 0.1% SDS], agitated with rolling action at 4°C,
and centrifuged at 600 x g. This step was
repeated five times. The HA-tagged protein was recovered by elution
with Laemmli buffer and incubation at 85°C for 5 min, followed by
centrifugation at 13,000 x g for 1 min at
room temperature. The supernatant was then loaded on a 12%
SDS-polyacrylamide gel and separated electrophoretically. The proteins
were transferred to a nitrocellulose membrane (Schleicher and Schuell)
overnight at 90 mA in a Hoefer Scientific TransBlot cell (Hoefer
Scientific Instruments). The membrane was first exposed to 0.1% FAST
Green FCF (Sigma Chemical Co., St. Louis, MO) in 20% methanol
and 5% acetic acid to determine the presence of the single band
corresponding to the immunoprecipitated protein. The membrane was
destained with 1x TTBS and blocked with 5% nonfat dried milk in TTBS
for 1 h. The membrane was probed with primary antibody for HA
(Santa Cruz Biotechnology) as described above.
Treatment of hOgg1-1a Immunoprecipitated Protein with NO and
Peroxynitrite.
Gaseous NO and peroxynitrite were applied to the protein solution using
ultrafiltration cups (Ultrafree-MC; Millipore, Bedford, MA). The
protein was placed in the filter cup, and the pharmacological chemical
solution producing NO was placed in the filtrate collection tube. SNAP
and SNOC were used as NO donors, and peroxynitrite was generated using
SIN-1 (Cayman Chemicals, Ann Arbor, MI). To maintain an optimum
distance of 2 mm (head space gas volume) between target protein on the
cellulose membrane and gaseous producing chemicals in the filtrate, 400
µl of 300 µM SNAP, 100 mM SNOC, or 20
µM SIN-1 solution was used. The cup was closed and
incubated for 2 h at 4°C. The NO scavenger, 100 µM
C-PTIO, the peroxynitrite scavenger, 40 µM MnTBAP,
and the iNOS inhibitor, 50 µM 1400W, were added to the
enzyme buffer.
Specific DNA Repair Assay.
hOgg1-1a activity was assayed by determining the ability of WCEs and
immunoprecipitated hOgg1-1a protein to recognize and excise 8-oxodG. A
24-base DNA oligonucleotide,
5'-GAACTAGTG8oxoGATCCCCCGGGCTGC-3'
(Trevigen, Gaithersburg, MD), containing an 8-oxodG oxidative lesion at
position 10 from the 5' end and its complement were used as substrates.
The calculated ratio of enzyme:substrate was 
2.5:1. A total of 50
pmol of the 24-bp oligonucleotide substrate with the 8-oxodG lesion was
radiolabeled with 4 µM [-32P]ATP (6000 mCi/mol; New England Nuclear Life Sciences
Products, Boston, MA) using polynucleotide kinase and incubated for 45
min at 37°C. The specific repair activity was assayed by incubating
0.5 pmol of radiolabeled oligonucleotide with 1.5 pmol of its
complement in 10 mM Tris (pH 7.5), 1 mM EDTA,
and 50 mM NaCl at 37°C for 2 h. Positive control
experiments with bacterial 8-oxodG repair enzyme, formamidopyrimidine
glycosylase, were run simultaneously. The reaction was loaded
onto a 15% acrylamide/7 M urea/1x Tris-borate EDTA gel.
The gel was exposed to a storage screen (PhosphorImager; Molecular
Dynamics) for 4 min. Autoradiograms were analyzed using ImageQuant
software (Molecular Dynamics).
Assay for Protein S-Nitrosylation.
S-Nitrosylation of hOgg1 was assessed using both an
immunochemical assay and a biochemical assay. An immunochemical assay
(28)
was used to detect the formation of
S-nitrosylated hOgg1-1a protein. HA-tagged hOgg1-1a protein
was immunoprecipitated from control cells and cells treated with SNAP,
cytokine mixture, and cytokine mixture plus 1400W. The purified protein
was first treated with 20 mM methyl
methanethiosulfonate (Pierce, Rockford, IL) for 1 min at room
temperature. Next, 25% SDS was added to block free thiols, and the
mixture was incubated at 50°C for 20 min. The methyl
methanethiosulfonate was then removed by desalting three times with the
microBioSpin6 (Clontech) column. The nitrosothiol bonds in the hOgg1
protein were selectively reduced with 1 mM sodium
ascorbate to thiols. The thiols were reacted at 25°C for 1 h
with 4 mM Biotin-HPDP (Pierce), a
sulfydryl-specific biotinylating reagent (29)
. Because the
cysteine biotinylation in this assay is reversible, SDS-PAGE sample
buffer was prepared without reducing agent and loaded for
electrophoresis on SDS-PAGE gel at room temperature, and the
above-mentioned steps were carried out with minimum exposure to light.
The biotinylated cysteines were immunoblotted with 3 µg/ml
anti-biotin (Pierce) following the Western blot procedure described
above.
The Saville method (30)
was also used to biochemically
detect the formation of S-nitrosothiol adducts on cysteine
residues in the hOgg1-1a protein. The Saville method works on the
principle that protein S-nitrosylation adducts replaced with
mercuric ions will react with sulfanilamide under acidic conditions to
produce a diazonium salt. The formation of this salt can be monitored
spectrophotometrically after its reaction with the aromatic amine
(N-(1-naphthyl)-ethylenediamine) using an absorbance maxima
of 540 nm (
= 50,000 M-1
cm-1). Purified hOgg1-1a protein was treated
with NO generated from 100 mM SNOC (30 min) and
300 µM SNAP (2 h) and peroxynitrite generated
from 20 µM SIN-1 (1 h), using ultrafiltration
cups as described above. As demonstrated previously (11
, 12
, 31)
, approximately 60–80 µM
nitrite/nitrate is generated by this treatment. This method prevents
contamination of the target protein solution with decomposition
byproducts of SNAP, SNOC, and SIN-1. To 1 mg/ml purified hOgg1-1a
protein in 100 mM Tris-Cl (pH 6.8) buffer, an
equivalent volume of 1% sulfanilamide dissolved in 0.4
M HCl or an equivalent volume of 0.2%
HgCl2 in 1% sulfanilamide dissolved in 0.4
M HCl was added. The protein solution was
incubated at room temperature for 10 min and mixed with an equal volume
of a 0.02% solution of N-(1-naphthyl)-ethylenediamine
dihydrochloride dissolved in 0.4 M HCl (Greiss
reagent). The sample absorbance was then read spectrophotometrically
between 400 and 700 nm. The S-nitrosylation of the protein
was quantified as the difference in absorbance between solution with
HgCl2 subtracted from one without
HgCl2. The experimental results were standardized
with glutathione ( - Glu-Cys-Gly) as described
previously (32)
.
Quantification of Zinc Release from Purified hOgg1-1a.
Approximately 500 µg of hOgg1-1a protein was immunoprecipitated from
hOgg1-1a-transfected KMBC cells in 100 mM Tris-Cl (pH 7.0).
Gaseous NO was applied to the protein solution through ultrafiltration
cups with a Mr 10,000 nominal
molecular weight off (Ultrafree-MC; Millipore) as described above.
SNAP, freshly prepared SNOC, and SIN-1 were used. SNOC (100
mM) was prepared fresh from an equimolar solution
of 100 mM sodium nitrite
(NaNO2) and 100 mM cysteine
hydrochloride as described previously (31)
. To maintain
optimum distance of 2 mm (head space gas volume) between target protein
on the cellulose membrane and NO-producing chemicals in the filtrate,
400 µl of 300 µM SNAP or 100
mM SNOC or 20 µM SIN-1
solution was used. The cup was closed and incubated for 30 min (SNOC),
2 h (SNAP), and 1 h (SIN-1), respectively, at 4°C. After
incubation, the protein solution was filtered by centrifugation (30 min
at 5000 x g) to remove protein. The zinc
concentrations in the filtrate were determined by monitoring the
complexation of Zn2+ by PAR at
A500 nm using
ZnSO4 as standards as described previously
(31)
.
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RESULTS |
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; thus, the HA-tagged hOgg1-1a protein was expressed and
appropriately localized in the nucleus of transfected cells. We had
previously established that these cells express iNOS and generate NO
after stimulation with a cytokine mixture (11
, 12) . To
ensure that cytokines did not influence the expression of hOgg1-1a, we
transfected cells in the presence of these compounds. Cytokine mixture
and 1400W, a specific iNOS inhibitor, had no effect on hOgg1-1a protein
expression after transfection as assayed by immunoblot analysis (Fig. 1B)
. Purified protein was obtained by IP using HA antibody.
The isolated protein was analyzed by Western blot to determine the
purity of the IP procedures. Only a single
Mr 36,000 protein band corresponding
to HA-tagged hOgg1-1a was identified on the blot with preliminary FAST
Green FCF staining (data not shown) and confirmed with HA
immunoblotting (Fig. 1C)
. Thus, hOgg1-1a can be efficiently
expressed and immunoprecipitated from this cholangiocyte-derived cell
line.
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, and 100 units/ml rhIFN-) to induce iNOS or
treated with SNAP, a pharmacological NO donor, BER activity was
inhibited (Fig. 2A)
. Coincubation with DTT, a scavenger for NO, or 1400W, an
iNOS inhibitor, prevented loss of BER activity. (Fig. 2A)
.
To more directly examine the effects of NO on hOgg1 activity, HA-tagged
hOgg1-1a protein was immunoprecipitated from cells transfected with its
expression vector. Immunoprecipitated hOgg1-1a protein isolated from
cells incubated with cytokine mixture was unable to excise 8-oxodG
lesions (Fig. 2B)
. In contrast, immunoprecipitated hOgg1-1a
from cells incubated with cytokine mixture in the presence of 1400W
manifested normal BER activity (Fig. 2B)
. These data
indicate that NO generated from iNOS inhibited hOgg1-1a activity.
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. MnTBAP, a superoxide mimetic, was also
effective in preventing inhibition of enzyme activity during treatment
with SIN-1 (Fig. 3B)
. The results suggest that either NO or
peroxynitrite is capable of inhibiting the BER activity of hOgg1.
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Immunoprecipitated hOgg1-1a was treated with SNOC, SNAP, and SIN-1, and
the number of S-nitrosothiol adducts was determined using
the Saville reaction. Approximately 0.24, 0.30, and 0.17
µM S-nitrosothiol adducts form per
milligram of purified hOgg1-1a protein after treatment with SNOC, SNAP,
and SIN-1, respectively (Fig. 4B)
. Control experiments with untreated protein demonstrated
that essentially no S-nitrosothiol adducts were formed.
Thus, NO is capable of directly nitrosylating hOgg1.
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= 66,000 M-1 cm-1).
Using this experimental approach, we estimated the release of 0.58,
0.61, and 0.38 µg of Zn2+ from 500 µg of
HA-tagged hOgg1 treated with SNOC, SNAP, and SIN-1, respectively
(Fig. 5)
. Minimal release of zinc was observed in control experiments. These
data suggest that nitrosylation of hOgg1 results in expulsion of zinc
from its zinc finger motif configuration.
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DISCUSSION |
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We have previously shown that proinflammatory cytokines inhibit BER activity in cholangiocytes and cholangiocarcinoma cells by a NO-dependent process. The current study significantly extends these observations by identifying hOgg1 as a molecular target for NO inhibition of BER activity. Furthermore, the data provide mechanistic insight into NO-mediated inhibition of hOgg1 activity. Studies by others have shown that NO can reversibly or irreversibly inhibit their catalytic activity (21 , 33 , 34) . NO inhibition of glycosylases has been attributed to S-nitrosothiol and 3-nitrotyrosine adduct formation (22 , 35) . The present data demonstrate the formation of S-nitrosothiol adducts in hOgg1-1a protein after treatment with NO donors. In contrast, 3-nitrotyrosine residues could not be detected by immunoblot analysis in the experimental paradigms used (data not shown). The formation of these S-nitrosothiol adducts was also associated with the loss of zinc ions from the protein. The release of zinc ions measured in response to S-nitrosothiol formation strongly suggests that loss of the active site cysteine 4-type zinc finger motif causes irreversible loss of hOgg1 tertiary structural integrity and hence loss of enzyme function.
The present findings suggest that either NO or peroxynitrite may inhibit hOgg1. For example, SNAP, a pharmacological NO donor, and SIN-1, a peroxynitrite donor, both inhibited hOgg1 activity. These results were bolstered by showing that C-PTIO, a NO scavenger, and MnTBAP, a peroxynitrite scavenger, protected the enzyme from loss of activity by SNAP and SIN-1, respectively. These results indicate that NO generation during inflammation may be sufficient to inhibit repair activity, but peroxynitrite generated from NO and superoxide in inflamed tissues may also contribute to inhibition of this enzyme. Scavengers for both reactive nitrogen species are therefore expected to help preserve base excision DNA repair activity in chronic inflammatory diseases.
We acknowledge that our results are based largely on in vitro biochemical assays. Optimally, a specific assay for hOgg1 activity in living cells would be ideal to confirm these observations. However, a robust assay specific for specific hOgg1 activity in living cells has not been developed and is difficult to conceive, given current technological limitations. Therefore, extrapolation of our data to tissues in vivo must take these considerations into account.
The hOGG1 gene maps to chromosome 3p26.2. This region
shows loss of heterozygosity in a variety of human cancers accompanied
by frequent GCTA transversions and may therefore function as a tumor
repressor (15
, 18
, 36)
. Mutations in the hOGG1
gene have also been identified in gastric cancer (37)
and
in lung and kidney tumors (38)
, further suggesting that
loss of hOgg1 function contributes to the carcinogenesis process. Our
previous studies, along with the current findings, suggest that in
addition to mutations in the hOGG1 gene, posttranslational
modification of hOgg1 may also contribute to the initiation, promotion,
and progression of human cancers. The latter mechanisms may be
especially important in chronic inflammatory diseases in which iNOS is
induced and relatively high concentrations of NO are generated. This
concept suggests that iNOS inhibition would have merit as a
chemopreventive strategy to decrease cancer development in chronic
inflammatory diseases of the biliary tract and perhaps in other organs
as well. Chemopreventive approaches may include inhibition of iNOS
expression with minocycline (39)
, inhibition of iNOS
activity with selective nontoxic inhibitors (40
, 41)
, or
administration of NO scavengers (42)
. Animal models of
carcinogenesis and cancer progression in wild-type and iNOS knockout
animals coupled with studies of BER activity of hOgg1 will be useful to
further test these concepts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants DK59427 (to G. J. G.)
and DK24031 (to N. F. L.) and by the Mayo Comprehensive Cancer
Center, Rochester, MN. 
2 To whom requests for reprints should be
addressed, at Mayo Medical School, Clinic, and Foundation, 200 First
Street SW, Rochester, MN 55905. Phone: (507) 284-0686; Fax:
(507) 284-0762; E-mail: gores.Gregory{at}mayo.edu
3 The abbreviations used are: iNOS, inducible
nitric oxide synthase; 8-oxodG, 8-oxoguanine; BER, base excision
repair; HA, hemagglutinin; IP, immunoprecipitation; MnTBAP,
Mn(III)tetrakis(4-benzoic acid) porphyrin; SNAP,
S-nitroso-N-acetyl-D-L-pencillamine;
SIN-1, 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride; SNOC,
S-nitrosocysteine; WCE, whole cell extract; hOgg1, human
8-oxoguanine glycosylase; rhIL-1ß, recombinant human interleukin
1ß; rhTNF-, recombinant human tumor necrosis factor ;
rhIFN-, recombinant human IFN-; TTBS, 20 mM Tris,
0.05% Tween, and 0.5 M NaCl (pH 7.0); Biotin-HPDP,
N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propioamide-biotin;
PAR, 4-(2-pyridyalzo) resorcinol; PTIO,
2-phenyl-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide.
Received 3/26/01. Accepted 7/ 3/01.
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R. De Bont and N. van Larebeke Endogenous DNA damage in humans: a review of quantitative data Mutagenesis, May 1, 2004; 19(3): 169 - 185. [Abstract] [Full Text] [PDF]
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 Y. NAKABEPPU, D. TSUCHIMOTO, A. ICHINOE, M. OHNO, Y. IDE, S. HIRANO, D. YOSHIMURA, Y. TOMINAGA, M. FURUICHI, and K. SAKUMI Biological Significance of the Defense Mechanisms against Oxidative Damage in Nucleic Acids Caused by Reactive Oxygen Species: From Mitochondria to Nuclei Ann. N.Y. Acad. Sci., April 1, 2004; 1011(1): 101 - 111. [Abstract] [Full Text] [PDF]
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I. Zurer, L. J. Hofseth, Y. Cohen, M. Xu-Welliver, S. P. Hussain, C. C. Harris, and V. Rotter The role of p53 in base excision repair following genotoxic stress Carcinogenesis, January 1, 2004; 25(1): 11 - 19. [Abstract] [Full Text] [PDF]
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 E. Speina, M. Zielinska, A. Barbin, D. Gackowski, J. Kowalewski, M. A. Graziewicz, J. A. Siedlecki, R. Olinski, and B. Tudek Decreased Repair Activities of 1,N6-Ethenoadenine and 3,N4-Ethenocytosine in Lung Adenocarcinoma Patients Cancer Res., August 1, 2003; 63(15): 4351 - 4357. [Abstract] [Full Text] [PDF]
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T. Arai, V. P. Kelly, K. Komoro, O. Minowa, T. Noda, and S. Nishimura Cell Proliferation in Liver of Mmh/Ogg1-deficient Mice Enhances Mutation Frequency because of the Presence of 8-Hydroxyguanine in DNA Cancer Res., July 15, 2003; 63(14): 4287 - 4292. [Abstract] [Full Text] [PDF]
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S. L. Habib, M. N. Phan, S. K. Patel, D. Li, T. J. Monks, and S. S. Lau Reduced constitutive 8-oxoguanine-DNA glycosylase expression and impaired induction following oxidative DNA damage in the tuberin deficient Eker rat Carcinogenesis, March 1, 2003; 24(3): 573 - 582. [Abstract] [Full Text] [PDF]
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 L. J. Hofseth, S.'i. Saito, S. P. Hussain, M. G. Espey, K. M. Miranda, Y. Araki, C. Jhappan, Y. Higashimoto, P. He, S. P. Linke, et al. Nitric oxide-induced cellular stress and p53 activation in chronic inflammation PNAS, January 7, 2003; 100(1): 143 - 148. [Abstract] [Full Text] [PDF]
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 J. Tuo, P. Jaruga, H. Rodriguez, M. Dizdaroglu, and V. A. Bohr The Cockayne Syndrome Group B Gene Product Is Involved in Cellular Repair of 8-Hydroxyadenine in DNA J. Biol. Chem., August 16, 2002; 277(34): 30832 - 30837. [Abstract] [Full Text] [PDF]
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N. Phoa and B. Epe Influence of nitric oxide on the generation and repair of oxidative DNA damage in mammalian cells Carcinogenesis, March 1, 2002; 23(3): 469 - 475. [Abstract] [Full Text] [PDF]
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