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Tumor Biology |
Center for Basic Research in Digestive Diseases [M. J., N. F. L., G. J. G], Division of Gastroenterology and Hepatology, Department of Laboratory Medicine and Pathology [L. J. B.], Mayo Clinic/Foundation/Medical School, Rochester, Minnesota 55905
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and tumor necrosis factor
) in three human cholangiocarcinoma cell lines. NO-dependent
DNA damage as assessed by the comet assay was demonstrated during
exposure of the three cholangiocarcinoma cell lines to cytokines.
Moreover, global DNA repair activity was inhibited by 70% by a
NO-dependent process after exposure of cells to cytokines. Our data
indicate that activation of iNOS and excess production of NO in
response to inflammatory cytokines cause DNA damage and inhibit DNA
repair proteins. NO inactivation of DNA repair enzymes may provide a
link between inflammation and the initiation, promotion, and/or
progression of cholangiocarcinoma. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NO3 is often generated in inflammatory conditions due to the induction of NOS in epithelial cells by inflammatory cytokines released from adjacent mononuclear cells (6, 7, 8, 9) . Because it is induced, this enzyme is referred to as iNOS to distinguish it from endothelial NOS and neuronal NOS (10) . iNOS is capable of generating relatively large amounts of NO compared to endothelial NOS and neuronal NOS (11) . NO produced in infected and inflamed tissues has been postulated to contribute to epithelial cell carcinogenesis by causing damage to DNA and proteins (12, 13, 14, 15) . Indeed, NO can directly oxidize DNA, resulting in mutagenic changes (16) . Furthermore, NO can nitrosylate thiol and tyrosine residues in susceptible proteins altering their function (17, 18, 19) . Although the ability of NO to directly damage DNA has been studied to a limited degree (20) , the role of protein nitrosylation in promoting potentially mutagenic changes in DNA has received far less attention. Thus, fundamental information on the effect of iNOS-generated NO on proteins that repair DNA damage is scientifically important.
DNA repair proteins are vital for the prevention of potential DNA mutations resulting from oxidative damage. Several distinct DNA repair proteins (21) are involved in selective repair pathways (22, 23, 24, 25) , some displaying narrow substrate specificity (base excision) and others characterized by a wide substrate range (nucleotide excision repair and mismatch repair). Oxidative DNA damage is predominantly repaired by base excision repair proteins; these distinct glycosylases recognize specific oxidative lesions and cleave the N-glycosidic bond, releasing the excised damaged base. The resulting abasic site can then be removed by an apurinic/apyrimidic endonuclease. Repair proteins are themselves potentially vulnerable to oxidative damage from NO because of their active site sulphydryl (26) , tyrosine, and/or phenol side chains. Some DNA repair enzymes (i.e., those that correct specific lesions such as O6-alkylguanine DNA alkyltransferase (27) and formamidopyrimidine-DNA glycosylase (28 , 29) , T4 DNA ligases (30) , and poly(ADP) ribose polymerase have in their active sites zinc finger motifs that may also be inactivated by NO nitrosylation of the thiol moieties of their cysteine-rich residues. Clearly, nitrosylation of these DNA repair proteins by excessive NO generation could compromise genomic stability. It therefore appears that the integrity of the cell may be challenged during exposure to high concentrations of NO not only by direct oxidative damage to DNA but also by potential NO-mediated disruption of DNA repair enzymes.
Based on this information, we formulated the central hypothesis that induction of NOS and subsequent NO production will result in oxidative DNA damage and inhibition of DNA repair enzymes. To begin to test this hypothesis, we addressed the following specific questions: (a) Is iNOS expressed in cholangiocarcinoma? (b) Does induction of iNOS expression result in the generation of NO from cultured cholangiocarcinoma cells? (c) Is the magnitude of NO generated by iNOS sufficient to cause DNA damage? and (d) Are global DNA repair processes inhibited by NO?
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture Medium and Technique.
Three human cholangiocarcinoma cell lines KMCH, KMBC, and WITT were
used for this study (31)
. For control studies, normal rat
cholangiocytes and RAW 267.4 mouse macrophages were also cultured. The
normal rat cholangiocytes were cultured as previously described by us
in detail (32)
. The RAW 267.4 and cholangiocarcinoma cell
lines were cultured in Dulbecco’s modified Eagles medium (Life
Technologies, Gaithersburg, MD) supplemented with 2 mM
L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 5% fetal bovine serum and maintained at 37°C with
5% CO2 and 95% humidity.
Measurement of NO Production by Chemiluminescence.
NO was measured in the culture media using a nitric oxide analyzer
(Sievers, Boulder, CO). Nitrite and nitrate present in the culture
medium (100 µl) was converted to NO by a saturated solution of
VCl3 in 0.8 M HCl, and the NO was
detected by a gas phase chemiluminescent reaction between NO and ozone
(33)
. Nitrite and nitrate concentrations were determined
by interpolation from known standards.
RT-PCR.
Total RNA was extracted from the cells using the TRIzol reagent (Life
Technologies, Grand Island, NY). The human iNOS primers were sense;
5'-CCCTTTACTTGACCTCCTAAC-3'; antisense; 5'-AAGGAATCATACAGGGAAGAC-3'.
After reverse transcription (6)
, PCR amplification was
performed by Taq polymerase (Perkin-Elmer, Branchburg, NJ)
using a programmed thermocycler (TwinBlock systems, Ericomp, San Diego,
CA) using the following conditions: (a) an initial
denaturation for 2 min at 94°C; and (b) 35 amplification
cycles (94°C for 1 min, 56.1°C for 1 min, and 72°C for 1 min).
PCR products were separated on a 1% agarose gel containing 0.5 µg/ml
ethidium bromide and photographed under UV
trans-illumination.
Western Blot Analysis.
Cytokine-stimulated and unstimulated human cholangiocarcinoma cells and
RAW 267.4 (ATCC, Bethesda, MD) mouse macrophage cells were harvested
and lysed by sonication in ice-cold lysis buffer containing 100
mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5
mM EGTA, 2 mM DTT, and protease inhibitors (5
mg/ml leupeptin, pepstatin, and chymostatin and 87 mg/ml
phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). Whole cell
lysates were boiled in Laemmli buffer and 40 µg/lane protein 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 Bio-Rad
TransBlot cell. The membrane was blocked with 5% nonfat dried milk in
TTBS [Tris 20 mM, Tween 0.05%, 0.5 M NaCl (pH 7)] for
1 h. The Primary antibody for iNOS (Transduction Laboratories,
Kensington, KY) was applied at a 1:2500 dilution for 2 h. The
membrane was washed three times in TTBS for 10 min each before applying
the secondary antibody (Transduction Laboratories, Kensington, KY) was
applied 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 commercially
enhanced chemiluminescence reagent (Amersham, Buckingshire, United
Kingdom) and exposed to photographic film.
Comet Assay.
The assay was performed as described in a protocol from Trevigen
(34)
. Cells were suspended in 0.5% (w/v) solution of low
melt agarose in PBS (pH7.4) at 37°C and immediately pipetted onto a
frosted microscope slide (Trevigen, Gaithersburg, MD). The agarose was
allowed to set for 10 min at 4°C and the slides were incubated in
lysis solution [2.5 M NaCl, 100 mM EDTA
(pH10), 10 mM Tris base, 1% sodium lauryl sarcosinate and
0.01% Triton X-100, Trevigen, Gaithersburg, MD] at 4°C to remove
cellular proteins for 1 h. This step leaves the DNA as distinct
nucleoids. After lysis, the slides were washed three times for 5 min
each in Fpg glycosylase buffer [100 mM Tris (pH7.5), 10
mM EDTA (pH 8.0), and 500 mM NaCl] and tapped
dry. The agarose-embedded cells were covered with either Fpg DNA
glycosylase (0.1 unit/gel; Trevigen, Gaithersburg, MD) or buffer alone
and incubated in a moist atmosphere at 37°C for 1 h. The slides
were immersed in prechilled denaturing buffer [0.3 M NaOH
and 0.001 M EDTA (pH 12.1)] for 30 min before
electrophoresis at 25 V for 30 min. The slides were then washed three
times in 0.4 M Tris-HCl (pH 7.5) buffer for 5 min each and
stained with SYBR green dye (1:10 dilution from concentrate;
Trevigen, Gaithersburg). Statistical evaluation was performed using NIH
image (Netscape Navigator) and Komet 3.0 Macro from Trevigen.
Repair Incorporation Assay.
DNA repair was assessed by determining the ability of cell extracts to
incorporate radiolabeled nucleotide into oxidatively damaged plasmid
DNA as previously described (25
, 35)
. The pBluescript II
KS (+) 2961-bp [pKS(+)] and the pKB-CMV 4518-bp plasmid substrates
were prepared by lysozyme-Triton X-100 method (25)
with
modifications. The oxidatively damaged pKS II (+) plasmid was prepared
by treating 0.1 mg/ml DNA with 10 µM methylene blue in
0.01 M sodium phosphate buffer (pH 7.4). The plasmid DNA
was kept on ice and exposed to visible light at a fluence of 117
W/m2 from a 100-W tungsten bulb for 3 min. This
exposure of 21 kilojoules/m2 visible light
in the presence of 10-µM methylene blue produces 1.6-Fpg
protein sites, primarily 8-oxodG (paper) on the 2.9-kbp plasmid
(25
, 35)
. The Fpg protein possesses 8-oxodG glycosylase
and lapurinic lyase activities (23)
. Oxidatively
damaged pKS II (+) and pKB-CMV (control) plasmids were further purified
on cesium chloride-ethidium bromide gradient centrifugation steps and
on one sucrose gradient step until none of the plasmids had a
detectable population of nicked molecules. Human cholangiocarcinoma
cells were harvested at a density of 
6 x 105 cells/ml. WCEs were prepared as described in
a previous study (25)
. Briefly, reactions
containing 300 ng each of supercoiled damaged pKS II (+) and undamaged
pKB-CMV, 100 µg of WCE protein in a volume of 50 µl with 44
mM HEPES-KOH (pH 7.9), 70 mM KCl, 7.5
mM MgCl2, 1.2 mM DTT, 0.5
mM EDTA, 2 mM ATP, 250 µg/ml BSA, 40
mM phosphocreatine, 50 µg/ml creatine phosphokinase, 50
µM each of dATP, dCTP, and dTTP, 5 µM dGTP
(Sigma, St. Lois, MO), and 1 µCi of
[-32P]dGTP (New England Nuclear Life
Science products, Boston, MA). The reactions were incubated at 30°C
for 2 h. The reaction was stopped by the addition of EDTA and then
treated with RNaseA followed by proteinase K. The plasmid DNA was
recovered by phenol-chloroform extraction and ethanol precipitation.
Plasmids were linearized with EcoRI endonuclease and
separated by 1% agarose gel electrophoresis containing 0.5 µg/ml
ethidium bromide. The gel was scanned to measure the intensity of
ethidium bromide fluorescence (MultiImage light Cabinet,
Ionnotech. Corp., San Leandro, CA). Fluorescence intensities of
linearized plasmid bands in comparison with known amounts of DNA were
used to determine DNA loading. The gel was dried under vacuum and
exposed to a storage screen (phosphoImager, Molecular Dynamics) for
18 h. Autoradiograms were analyzed using ImageQuant software
(Molecular Dynamics). Background corrected values of the radioactivity
incorporated for the damaged and undamaged plasmids were normalized for
the amount of DNA.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in all 18 human cholangiocarcinoma specimens. In this
limited sample, no relationship was observed between tumor grade and
intensity of iNOS staining (data not shown). Evidence for catalytic
activity of the expressed iNOS protein was identified by performing
immunohistochemistry for 3-nitrotyrosine, a reaction product of
peroxynitrite (formed from NO reacting with the superoxide anion) with
susceptible tyrosine residues. Similar to the immunohistochemistry for
iNOS, all 18 cholangiocarcinoma specimens revealed positive staining
for 3-nitrotyrosine. The presence of nitrotyrosine residues implies a
high level of activity of the expressed iNOS (36)
. In
comparison, the biliary epithelia from normal liver biopsies did not
stain either for iNOS or 3-nitrotyrosine. Thus, cholangiocarcinomas
appear to uniformly express iNOS and stain intensely for
3-nitrotyrosine, a marker of oxidative protein damage.
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. In contrast, when all of the three cell lines were incubated in the
presence of a mixture of inflammatory cytokines [human recombinant
IL-1ß (0.5 ng/ml), human recombinant TNF- (500 units/ml), and
human recombinant IFN- (100 units/ml)] known to increase iNOS
expression in other cell types (8)
, both iNOS mRNA and
protein were readily detectable. The identification of iNOS in the
cholangiocarcinoma specimens but only in cytokine-stimulated
cholangiocarcinoma cells in vitro suggests that
cholangiocarcinomas in vivo grow in a cytokine-rich milieu.
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. NO generation was increased in all of the three cell lines incubated
in the presence of stimulatory cytokines compared to controls. We
confirmed that iNOS was the source of the NO by incubating the cells in
the presence of inflammatory cytokines plus the iNOS inhibitor
L-NMMA (Fig. 3)
. Indeed, stimulated NO production was
completely suppressed by the addition of the competitive iNOS
inhibitor. Thus, iNOS expressed by the cholangiocarcinoma cells is
catalytically active.
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shows a representative image from the KMBC cell line. The control cells
(Fig. 4A)
demonstrate compact tight nucleoids. However, the
cells treated with stimulatory cytokines (Fig. 4B)
demonstrate a tail (hence the term comet) indicative of DNA damage. The
DNA damage was blocked with the iNOS inhibitor
L-NMMA (Fig. 4D)
. Although the
unmodified comet assay detects single-stranded and double-stranded DNA
damage, it does not identify oxidatively damaged DNA, the principle
form of DNA damage induced by NO. Therefore, Fpg protein, a
lesion-specific repair enzyme was incorporated in the assay. This
protein has a glycosylase as well as an apurinic-endonuclease activity
that recognizes and cleaves 8-oxodeoxyguanosine lesions, the most
common lesion formed by oxidative stress such as NO. If
8-oxodeoxyguanosine residues are present in the DNA, Fpg treatment will
cleave the DNA at these sights, resulting in single-strand breaks; the
increase in DNA-strand breaks would increase the amount of DNA moving
toward the anode in the comet assay (increased DNA in the comet tail).
All of the three cholangiocarcinoma cell lines demonstrated an increase
in comet tails following this modification of the comet assay (Fig. 4C)
. Statistical analysis of 75 comets (Fig. 4)
from each
treatment showed the extent of DNA damage. There was an average of
7.1% DNA damage in cytokine-stimulated cells that increased to 12% by
using the Fpg enzyme in the assay. The DNA damage in buffer alone
(control) and cytokine + L-NMMA-treated
cells was minimal and virtually identical. These data demonstrate that
the magnitude of NO generated by iNOS induction is sufficient to cause
single-stranded/double-stranded DNA damage as well as oxidative
lesions.
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. Control or unstimulated WCEs exhibited normal repair activity as
evidenced by the incorporation of the radiolabeled nucleotide during
repair of the damaged DNA plasmid substrate. As a negative control for
DNA repair, the WCEs were boiled to denature repair proteins and, as
expected, there was no incorporation of radioactivity. The repair
efficiency decreased by 76% in the WCEs from cells stimulated with
proinflammatory cytokines, relative to repair by the unstimulated cells
(Fig. 5)
. The repair activity was also tested in response to exogenous
exposure to NO from SNAP, a chemical that produces NO. The decrease in
repair efficiency in response to exogenous (SNAP) was virtually
identical to that of cells incubated in the presence of stimulatory
cytokines (Fig. 5)
. To establish that the decrease in overall repair
activity of the cholangiocarcinoma cells was in fact a direct effect of
NO production by cytokine stimulation of iNOS,
L-NMMA was added to the culture medium with the
cytokines and incubated for 24 h. Repair assay of the WCEs
demonstrated that L-NMMA returned the DNA repair
activity to basal levels despite cytokine stimulation. (Fig. 6)
. Thus, induced NO generation is capable of impairing the cells’ DNA
repair apparatus. Inhibition of DNA repair by NO may explain, in part,
the accumulation of damaged DNA during cytokine stimulation of
cholangiocarcinoma cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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iNOS expression with NO generation has been observed in several human malignancies in which chronic inflammation is a predisposing factor, including colon cancer (37 , 38) , hepatocellular cancer (39, 40, 41) , Barett’s esophagus (36 , 42) , and breast cancer (43) . Our findings add human cholangiocarcinomas to this group of inflammation-associated malignancies. Because iNOS was not expressed by cholangiocarcinoma cells in vitro in the absence of cytokines, it would appear that cholangiocarcinomas in vivo elicit a proinflammatory cytokine response sufficient to induce iNOS expression. Indeed, there is histological evidence of tumor-associated inflammatory cells, a potential source of inflammatory cytokines, in most cholangiocarcinoma specimens. The near universal expression of NO by cholangiocarcinoma suggests that it plays an important role in the biology of this cancer.
There are many potential mechanisms by which NO may be important in the initiation, promotion, and progression of this cancer. Our data suggest that NO could promote the accumulation of potential oncogenic mutations by inhibiting DNA repair enzymes. Inhibition of proapoptotic effector proteins by protein nitrosylation, such as caspase proteases, may also promote extended survival of malignant cells (44) . Indeed, nitrosylation of caspases would disable apoptotic pathways promoting cell survival despite DNA damage. Finally, NO may confer a survival advantage for cancer by serving as an angiogenesis factor (45 , 46) .
The induction of iNOS with NO generation could explain, in part, the link between chronic inflammation and cholangiocarcinoma (47) . Although the precise mechanisms by which chronic inflammation of the bile ducts increases the risk of malignant transformation of biliary epithelia is unclear, it is known that activation of phagocytes and subsequent cytokine released in inflamed tissue generate large quantities of NO. For example, increased synthesis of NO and endogenous formation of nitrite and nitrosamines are risk factors for cholangiocarcinoma in subjects infested with Opisthorchis Viverrini (48) . As demonstrated in our comet assay, NO generated by the induction of iNOS with inflammatory cytokines is sufficient to induce oxidative DNA damage. It is likely that the inhibition of the DNA repair mechanisms contributes to this DNA damage. These observations suggest that NO may play an important role in causing the oncogenic mutations that are important in the development of cholangiocarcinoma. However, the specific potential oncogenic mutations induced by NO remain obscure. Recently, p53 mutations have been identified in most cholangiocarcinomas associated with primary sclerosing cholangitis (49) . Whether NO can induce genetic alterations in p53 is unknown, but it would provide a mechanistic link between inflammation, NO formation, and the development of this malignancy.
Our data directly demonstrate the inhibition of the DNA repair machinery in a NO-dependent manner. NO may affect proteins by nitrosylation of tyrosine and cysteine residues. Although tyrosine nitrosylation of proteins has been amply documented as a marker of protein oxidation (50 , 51) , its effect on the catalytic functions of enzymes is obscure. In contrast, cysteine nitrosylation is known to directly inactivate enzymes (52 , 53) . Inhibition of DNA repair enzymes by sulfhydryl nitrosylation is the likely mechanism for the NO-dependent inhibition of global DNA repair. Indeed, the exposure of O6-alkylguanine DNA alkyltransferase to NO causes nitrosylation of its active thiol moiety inhibiting its activity (54 , 55) . DNA repair proteins with zinc finger motifs (56) such as formamidopyrimidine DNA-glycosylase (Fpg), which repairs 8-oxodeoxyguanine residues (29) , are also directly inhibited in the presence of aerobic NO (30) . Our studies significantly extend these previous biochemical observations by demonstrating inhibition of DNA repair activity by a NO-dependent manner in intact cells during exposure to inflammatory cytokines. These data demonstrate that the magnitude of NO generated by iNOS in these cells is sufficient to inhibit DNA repair processes. The specific oxidative base excision DNA repair enzymes nitrosylated in these cells was not elucidated but is presently being pursued in our laboratory.
In summary, data in the present study suggest the likely involvement of NO in the initiation, progression, and/or promotion of cholangiocarcinoma during inflammatory conditions of the bile ducts. NO and associated reactive oxygen species such as peroxynitrite modify DNA bases and result in direct DNA damage. Concomitantly, nitrosylation of key repair proteins inhibit the repair of the DNA alterations promoting the accumulation of potential oncogenic mutations important in the initiation and/or progression of this cancer. Based on this information, we speculate that iNOS inhibitors targeted to cholangiocytes could potentially have a chemopreventive role in patients with chronic inflammatory cholangiopathies, such as patients with primary sclerosing cholangitis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grants DK41876 (to
G. J. G.) and DK24031 (to N. F. L.) from the NIH
and grants from the American Liver Foundation (to M. J.), Cedar
Grove, NJ, the Gainey Foundation, St. Paul, MN, and 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: NO, nitric oxide;
Fpg, formamidopyrimidine DNA glycosylase; NOS, nitric oxide synthase;
iNOS, inducible nitric oxide synthase; L-NMMA,
NG-methyl-L-arginine acetate; SNAP,
S-nitroso-N-acetyl-penicillamine; RT-PCR, reverse transcription-PCR;
Fpg, foramidopyrimidine; WCE, whole cell extract; IL-1ß, interleukin
1ß; TNF-, tumor necrosis factor .
Received 7/23/99. Accepted 10/29/99.
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S. Landi, V. Moreno, L. Gioia-Patricola, E. Guino, M. Navarro, J. de Oca, G. Capella, and F. Canzian Association of Common Polymorphisms in Inflammatory Genes Interleukin (IL)6, IL8, Tumor Necrosis Factor {alpha}, NFKB1, and Peroxisome Proliferator-activated Receptor {gamma} with Colorectal Cancer , Cancer Res., July 1, 2003; 63(13): 3560 - 3566. [Abstract] [Full Text] [PDF]
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B. A. Narayanan, N. K. Narayanan, B. Simi, and B. S. Reddy Modulation of Inducible Nitric Oxide Synthase and Related Proinflammatory Genes by the Omega-3 Fatty Acid Docosahexaenoic Acid in Human Colon Cancer Cells Cancer Res., March 1, 2003; 63(5): 972 - 979. [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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N. J. Torok, H. Higuchi, S. Bronk, and G. J. Gores Nitric Oxide Inhibits Apoptosis Downstream of Cytochrome c Release by Nitrosylating Caspase 9 Cancer Res., March 1, 2002; 62(6): 1648 - 1653. [Abstract] [Full Text] [PDF]
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C. V. Rao, C. Indranie, B. Simi, P. T. Manning, J. R. Connor, and B. S. Reddy Chemopreventive Properties of a Selective Inducible Nitric Oxide Synthase Inhibitor in Colon Carcinogenesis, Administered Alone or in Combination with Celecoxib, a Selective Cyclooxygenase-2 Inhibitor Cancer Res., January 1, 2002; 62(1): 165 - 170. [Abstract] [Full Text] [PDF]
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M. Jaiswal, N. F. LaRusso, and G. J. Gores Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis Am J Physiol Gastrointest Liver Physiol, September 1, 2001; 281(3): G626 - G634. [Abstract] [Full Text] [PDF]
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M. Jaiswal, N. F. LaRusso, N. Nishioka, Y. Nakabeppu, and G. J. Gores Human Ogg1, a Protein Involved in the Repair of 8-Oxoguanine, Is Inhibited by Nitric Oxide Cancer Res., September 1, 2001; 61(17): 6388 - 6393. [Abstract] [Full Text] [PDF]
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F. Feger, H. Ferry-Dumazet, M. M. Matsuda, J. Bordenave, M. Dupouy, A. K. Nussler, M. Arock, L. Devevey, J. Nafziger, J.-J. Guillosson, et al. Role of Iron in Tumor Cell Protection from the Pro-Apoptotic Effect of Nitric Oxide Cancer Res., July 1, 2001; 61(13): 5289 - 5294. [Abstract] [Full Text] [PDF]
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D.T. Bau, J.R. Gurr, and K.Y. Jan Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision Carcinogenesis, May 1, 2001; 22(5): 709 - 716. [Abstract] [Full Text] [PDF]
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 Md. A. Rahman, D. K. Dhar, E. Yamaguchi, S. Maruyama, T. Sato, H. Hayashi, T. Ono, A. Yamanoi, H. Kohno, and N. Nagasue Coexpression of Inducible Nitric Oxide Synthase and COX-2 in Hepatocellular Carcinoma and Surrounding Liver: Possible Involvement of COX-2 in the Angiogenesis of Hepatitis C Virus-positive Cases Clin. Cancer Res., May 1, 2001; 7(5): 1325 - 1332. [Abstract] [Full Text]
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A. S. Fleisher, M. Esteller, N. Harpaz, A. Leytin, A. Rashid, Y. Xu, J. Liang, O. C. Stine, J. Yin, T.-T. Zou, et al. Microsatellite Instability in Inflammatory Bowel Disease-associated Neoplastic Lesions Is Associated with Hypermethylation and Diminished Expression of the DNA Mismatch Repair Gene, hMLH1 Cancer Res., September 1, 2000; 60(17): 4864 - 4868. [Abstract] [Full Text]
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