[CANCER RESEARCH 60, 184–190, January 1, 2000]

Inflammatory Cytokines Induce DNA damage and Inhibit DNA repair inCholangiocarcinoma Cells by a Nitric Oxide-dependent Mechanism1

Meeta Jaiswal, Nicholas F. LaRusso, Lawrence J. Burgart, and Gregory J. Gores2

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

ABSTRACT

Chronic infection and inflammation are risk factors for the develop-ment of cholangiocarcinoma, a highly malignant, generally fatal adeno-carcinoma originating from biliary epithelia. However, the link betweeninflammation and carcinogenesis in these disorders is obscure. Becausenitric oxide (NO) is generated in inflamed tissues by inducible nitric oxidesynthase (iNOS) and because DNA repair proteins are potentially suscep-tible to NO-mediated nitrosylation, we formulated the hypothesis thatinflammatory cytokines induce iNOS and sufficient NO to inhibit DNArepair enzymes leading to the development and progression of cholangio-carcinoma. iNOS and nitrotyrosine were demonstrated in 18/18 cholan-giocarcinoma specimens. Furthermore, iNOS and NO generation could beinduced in vitro by inflammatory cytokines (mixture of interleukin-1 b,IFN-g, and tumor necrosis factora) in three human cholangiocarcinomacell lines. NO-dependent DNA damage as assessed by the comet assay wasdemonstrated during exposure of the three cholangiocarcinoma cell linesto cytokines. Moreover, global DNA repair activity was inhibited by 70%by a NO-dependent process after exposure of cells to cytokines. Our dataindicate that activation of iNOS and excess production of NO in responseto inflammatory cytokines cause DNA damage and inhibit DNA repairproteins. NO inactivation of DNA repair enzymes may provide a linkbetween inflammation and the initiation, promotion, and/or progressionof cholangiocarcinoma.

INTRODUCTION

Cholangiocarcinoma is a malignant neoplasm originating fromcholangiocytes, the epithelial cells lining the bile ducts in the liver (1,2). Although it is well known that chronic inflammatory conditionsinvolving the bile ducts (e.g., primary sclerosing cholangitis,clonorchis sinensis infections, biliary stone disease, and Caroli’sdisease) predispose to the development of cholangiocarcinoma (3–5),the relationship between chronic inflammation and malignant trans-formation is unclear.

NO3 is often generated in inflammatory conditions due to theinduction of NOS in epithelial cells by inflammatory cytokines re-leased from adjacent mononuclear cells (6–9). Because it is induced,this enzyme is referred to as iNOS to distinguish it from endothelialNOS and neuronal NOS (10). iNOS is capable of generating relativelylarge amounts of NO compared to endothelial NOS and neuronal NOS(11). NO produced in infected and inflamed tissues has been postu-lated to contribute to epithelial cell carcinogenesis by causing damageto DNA and proteins (12–15). Indeed, NO can directly oxidize DNA,

resulting in mutagenic changes (16). Furthermore, NO can nitrosylatethiol and tyrosine residues in susceptible proteins altering their func-tion (17–19). Although the ability of NO to directly damage DNA hasbeen studied to a limited degree (20), the role of protein nitrosylationin promoting potentially mutagenic changes in DNA has received farless attention. Thus, fundamental information on the effect of iNOS-generated NO on proteins that repair DNA damage is scientificallyimportant.

DNA repair proteins are vital for the prevention of potential DNAmutations resulting from oxidative damage. Several distinct DNArepair proteins (21) are involved in selective repair pathways (22–25),some displaying narrow substrate specificity (base excision) andothers characterized by a wide substrate range (nucleotide excisionrepair and mismatch repair). Oxidative DNA damage is predomi-nantly repaired by base excision repair proteins; these distinct glyco-sylases recognize specific oxidative lesions and cleave the N-glyco-sidic bond, releasing the excised damaged base. The resulting abasicsite can then be removed by an apurinic/apyrimidic endonuclease.Repair proteins are themselves potentially vulnerable to oxidativedamage from NO because of their active site sulphydryl (26), tyrosine,and/or phenol side chains. Some DNA repair enzymes (i.e., those thatcorrect specific lesions such as O6-alkylguanine DNA alkyltransferase(27) and formamidopyrimidine-DNA glycosylase (28, 29), T4 DNAligases (30), and poly(ADP) ribose polymerase have in their activesites zinc finger motifs that may also be inactivated by NO nitrosy-lation of the thiol moieties of their cysteine-rich residues. Clearly,nitrosylation of these DNA repair proteins by excessive NO genera-tion could compromise genomic stability. It therefore appears that theintegrity of the cell may be challenged during exposure to highconcentrations of NO not only by direct oxidative damage to DNA butalso by potential NO-mediated disruption of DNA repair enzymes.

Based on this information, we formulated the central hypothesisthat induction of NOS and subsequent NO production will result inoxidative DNA damage and inhibition of DNA repair enzymes. Tobegin to test this hypothesis, we addressed the following specificquestions: (a) Is iNOS expressed in cholangiocarcinoma? (b) Doesinduction of iNOS expression result in the generation of NO fromcultured cholangiocarcinoma cells? (c) Is the magnitude of NO gen-erated by iNOS sufficient to cause DNA damage? and (d) Are globalDNA repair processes inhibited by NO?

MATERIALS AND METHODS

Immunohistochemistry. Paraffin-embedded normal human liver tissuesamples and liver tissue samples obtained from patients with cholangiocarci-noma were used for iNOS and 3-nitrotyrosine immunohistochemistry. Fivemicron tissue sections were deparaffinized twice in xylene for 10 min each andrehydrated in ethanol followed by water and PBS. Endogenous peroxidase wasblocked by immersion in 3% hydrogen peroxide in methanol for 20 min, andthe tissue was washed twice in PBS for 10 min each. Nonspecific binding wasblocked, and the sections were permealized by 0.3% Triton X-100, 0.2%normal goat serum, and 0.5% BSA in PBS buffer for 20 min. The tissuesections were drained well and then incubated with iNOS antibody (Transduc-tion Labs, Lexington, KY) at a concentration of 15mg/ml (1:500 dilution) for1 h at room temperature and with 3-nitrotyrosine antibody (Upstate Biotech-nology, Lake Placid, NY) at a concentration of 5mg/ml (1:450 dilution).

Received 7/23/99; accepted 10/29/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

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@mayo.edu.

3 The abbreviations used are: NO, nitric oxide; Fpg, formamidopyrimidine DNAglycosylase; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase;L-NMMA,NG-methyl-L-arginine acetate; SNAP, S-nitroso-N-acetyl-penicillamine; RT-PCR, reversetranscription-PCR; Fpg, foramidopyrimidine; WCE, whole cell extract; IL-1b, interleukin1b; TNF-a, tumor necrosis factora.

184

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

Control sections were incubated without a primary antibody. The sections werewashed twice for 5 min each in PBS. Immunostaining for iNOS and 3-nitro-tyrosine was detected with the Vectastain peroxidase kit (Vector labs, Burl-ingame, CA). The immunostain was developed with diaminobenzidine tetra-hydrochloride for 5 min. The sections were counterstained with hematoxylin,rehydrated in ethanol followed by xylene, and coverslipped. Immunohisto-chemical staining for iNOS was evaluated by light microscopy. Specific iNOSstaining was graded on a semiquantitative scale from 0 to 3 (0, none; 1, weak;2, intermediate; and 3, strong) in comparison to the grade of the cancer (1,well-differentiated; 2, moderately differentiated; 3, poorly differentiated; and4, anaplastic) by an experienced hepatopathologist.

Cell Culture Medium and Technique. Three human cholangiocarcinomacell lines KMCH, KMBC, and WITT were used for this study (31). For controlstudies, normal rat cholangiocytes and RAW 267.4 mouse macrophages werealso cultured. The normal rat cholangiocytes were cultured as previouslydescribed by us in detail (32). The RAW 267.4 and cholangiocarcinoma celllines were cultured in Dulbecco’s modified Eagles medium (Life Technolo-gies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 100 units/mlpenicillin, 100mg/ml streptomycin, and 5% fetal bovine serum and maintainedat 37°C with 5% CO2 and 95% humidity.

Measurement of NO Production by Chemiluminescence.NO was meas-ured in the culture media using a nitric oxide analyzer (Sievers, Boulder, CO).Nitrite and nitrate present in the culture medium (100ml) was converted to NOby a saturated solution of VCl3 in 0.8 M HCl, and the NO was detected by agas phase chemiluminescent reaction between NO and ozone (33). Nitrite andnitrate 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;59-CCCTTTACTTGACCTCCTAAC-39; antisense; 59-AAGGAATCATA-CAGGGAAGAC-39. After reverse transcription (6), PCR amplification wasperformed byTaq polymerase (Perkin-Elmer, Branchburg, NJ) using a pro-grammed thermocycler (TwinBlock systems, Ericomp, San Diego, CA) usingthe 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 1min). PCR products were separated on a 1% agarose gel containing 0.5mg/mlethidium bromide and photographed under UVtrans-illumination.

Western Blot Analysis. Cytokine-stimulated and unstimulated humancholangiocarcinoma cells and RAW 267.4 (ATCC, Bethesda, MD) mousemacrophage cells were harvested and lysed by sonication in ice-cold lysisbuffer 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 chy-mostatin and 87 mg/ml phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO).Whole cell lysates were boiled in Laemmli buffer and 40mg/lane proteinloaded on a 7.5% SDS-polyacrylamide gel and separated electrophoretically.The proteins were transferred to a nitrocellulose membrane (Schleicher andSchuell, Keene, NH) overnight at 90 mA in a Bio-Rad TransBlot cell. Themembrane 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 dilutionfor 2 h. The membrane was washed three times in TTBS for 10 min eachbefore applying the secondary antibody (Transduction Laboratories, Kensing-ton, KY) was applied at a 1:5000 dilution for 1 h. The blot was washed inTTBS four times for 10 min each. It was then incubated in commerciallyenhanced chemiluminescence reagent (Amersham, Buckingshire, UnitedKingdom) and exposed to photographic film.

Comet Assay.The assay was performed as described in a protocol fromTrevigen (34). Cells were suspended in 0.5% (w/v) solution of low meltagarose in PBS (pH7.4) at 37°C and immediately pipetted onto a frostedmicroscope slide (Trevigen, Gaithersburg, MD). The agarose was allowed toset for 10 min at 4°C and the slides were incubated in lysis solution [2.5M

NaCl, 100 mM EDTA (pH10), 10 mM Tris base, 1% sodium lauryl sarcosinateand 0.01% Triton X-100, Trevigen, Gaithersburg, MD] at 4°C to removecellular proteins for 1 h. This step leaves the DNA as distinct nucleoids. Afterlysis, the slides were washed three times for 5 min each in Fpg glycosylasebuffer [100 mM Tris (pH7.5), 10 mM EDTA (pH 8.0), and 500 mM NaCl] andtapped dry. The agarose-embedded cells were covered with either Fpg DNAglycosylase (0.1 unit/gel; Trevigen, Gaithersburg, MD) or buffer alone andincubated in a moist atmosphere at 37°C for 1 h. The slides were immersed inprechilled denaturing buffer [0.3M NaOH and 0.001M EDTA (pH 12.1)] for

30 min before electrophoresis at 25 V for 30 min. The slides were then washedthree times in 0.4M Tris-HCl (pH 7.5) buffer for 5 min each and stained withSYBR 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 determiningthe ability of cell extracts to incorporate radiolabeled nucleotide into oxida-tively damaged plasmid DNA as previously described (25, 35). The pBlue-script II KS (1) 2961-bp [pKS(1)] and the pKB-CMV 4518-bp plasmidsubstrates were prepared by lysozyme-Triton X-100 method (25) with modi-fications. The oxidatively damaged pKS II (1) plasmid was prepared bytreating 0.1 mg/ml DNA with 10mM methylene blue in 0.01M sodiumphosphate buffer (pH 7.4). The plasmid DNA was kept on ice and exposed tovisible 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-mM

methylene blue produces 1.6-Fpg protein sites, primarily 8-oxodG (paper) onthe 2.9-kbp plasmid (25, 35). The Fpg protein possesses 8-oxodG glycosylaseand lapurinic lyase activities (23). Oxidatively damaged pKS II (1) andpKB-CMV (control) plasmids were further purified on cesium chloride-ethidium bromide gradient centrifugation steps and on one sucrose gradientstep until none of the plasmids had a detectable population of nicked mole-cules. Human cholangiocarcinoma cells were harvested at a density of;6 3 105 cells/ml. WCEs were prepared as described in a previous study (25).Briefly, reactions containing 300 ng each of supercoiled damaged pKS II (1)and undamaged pKB-CMV, 100mg of WCE protein in a volume of 50ml with44 mM HEPES-KOH (pH 7.9), 70 mM KCl, 7.5 mM MgCl2, 1.2 mM DTT, 0.5mM EDTA, 2 mM ATP, 250mg/ml BSA, 40 mM phosphocreatine, 50mg/mlcreatine phosphokinase, 50mM each of dATP, dCTP, and dTTP, 5mM dGTP(Sigma, St. Lois, MO), and 1mCi of [a-32P]dGTP (New England Nuclear LifeScience 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 withRNaseA followed by proteinase K. The plasmid DNA was recovered byphenol-chloroform extraction and ethanol precipitation. Plasmids were linear-ized withEcoRI endonuclease and separated by 1% agarose gel electrophoresiscontaining 0.5mg/ml ethidium bromide. The gel was scanned to measure theintensity of ethidium bromide fluorescence (MultiImage light Cabinet, Ionno-tech. Corp., San Leandro, CA). Fluorescence intensities of linearized plasmidbands in comparison with known amounts of DNA were used to determineDNA loading. The gel was dried under vacuum and exposed to a storage screen(phosphoImager, Molecular Dynamics) for 18 h. Autoradiograms were ana-lyzed using ImageQuant software (Molecular Dynamics). Background cor-rected values of the radioactivity incorporated for the damaged and undamagedplasmids were normalized for the amount of DNA.

RESULTS

Is iNOS Expressed by Human Cholangiocarcinoma?To deter-mine whether iNOS is expressed by human cholangiocarcinomas, weperformed immunohistochemistry for iNOS in tissue specimens from18 patients with cholangiocarcinoma. There was intense staining foriNOS (Fig. 1D) in all 18 human cholangiocarcinoma specimens. Inthis limited sample, no relationship was observed between tumorgrade and intensity of iNOS staining (data not shown). Evidence forcatalytic activity of the expressed iNOS protein was identified byperforming immunohistochemistry for 3-nitrotyrosine, a reactionproduct of peroxynitrite (formed from NO reacting with the superox-ide anion) with susceptible tyrosine residues. Similar to the immuno-histochemistry for iNOS, all 18 cholangiocarcinoma specimens re-vealed positive staining for 3-nitrotyrosine. The presence ofnitrotyrosine residues implies a high level of activity of the expressediNOS (36). In comparison, the biliary epithelia from normal liverbiopsies did not stain either for iNOS or 3-nitrotyrosine. Thus, cholan-giocarcinomas appear to uniformly express iNOS and stain intenselyfor 3-nitrotyrosine, a marker of oxidative protein damage.

To more specifically demonstrate iNOS expression in humancholangiocarcinoma cells, we assayed for iNOS mRNA and proteinexpression using RT-PCR and immunoblot analysis in three human

185

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

cholangiocarcinoma cell lines. Cells cultured in the absence of stim-ulatory cytokines failed to express iNOS (Fig. 2). In contrast, when allof the three cell lines were incubated in the presence of a mixture ofinflammatory cytokines [human recombinant IL-1b (0.5 ng/ml), hu-man recombinant TNF-a (500 units/ml), and human recombinant

IFN-g (100 units/ml)] known to increase iNOS expression in othercell types (8), both iNOS mRNA and protein were readily detectable.The identification of iNOS in the cholangiocarcinoma specimens butonly in cytokine-stimulated cholangiocarcinoma cellsin vitro sug-gests that cholangiocarcinomasin vivogrow in a cytokine-rich milieu.

Do Cytokine-stimulated Cholangiocarcinoma Cells ProduceNO? Although the above studies demonstrate that cholangiocarci-noma expresses iNOS, the catalytic activity of the expressed proteinwas not ascertained. Therefore, we measured NO in the media ofcholangiocarcinoma cells incubated in the presence and absence ofstimulatory cytokines (Fig. 3). NO generation was increased in all ofthe three cell lines incubated in the presence of stimulatory cytokinescompared to controls. We confirmed that iNOS was the source of theNO by incubating the cells in the presence of inflammatory cytokinesplus the iNOS inhibitorL-NMMA (Fig. 3). Indeed, stimulated NOproduction was completely suppressed by the addition of the compet-itive iNOS inhibitor. Thus, iNOS expressed by the cholangiocarci-noma cells is catalytically active.

Does Stimulated NO Generation Result in Oxidative DNADamage?The alkaline comet assay provides a sensitive tool for thedetection of DNA damage (single-stranded breaks and alkali labilesites) at the single cell level. Fig. 4 shows a representative image fromthe KMBC cell line. The control cells (Fig. 4A) demonstrate compacttight nucleoids. However, the cells treated with stimulatory cytokines(Fig. 4B) demonstrate a tail (hence the term comet) indicative of DNAdamage. The DNA damage was blocked with the iNOS inhibitorL-NMMA (Fig. 4D). Although the unmodified comet assay detects

Fig. 1. Immunohistochemical analysis of iNOS anddetection of 3-nitrotyrosine in human liver biopsies. Theleft panel represents immunostaining without primaryantibody, and theright panel represents immuno-staining with the primary antibody. Normal liver tissue,specifically the bile duct epithelia (arrows), did notstain for either iNOS (B) or for 3-nitrotyrosine (F).There was intense staining (arrows) for iNOS (D) and3-nitrotyrosine (H) in the malignant biliary epithelia ofcholangiocarcinoma patients.

Fig. 2. Cytokines enhance expression of iNOS mRNA and protein in human cholan-giocarcinoma cell lines. Total RNA and protein was extracted from three cholangiocar-cinoma cell lines after 24-h stimulation with (1) and without (2; IL-1b:0.5ng/ml;IFN-g:100 units/ml, and TNF-a:500 units/ml) cytokines. iNOS mRNA expression isindicated by the 289-bp RT-PCR product from stimulated cells on electrophoresis. iNOSprotein expression was assayed by immunochemistry on 50mg of cell lysate. Theelectrophoretically transferred nitrocellulose membrane was immunoblotted with mono-clonal human iNOS antibody.b-actin was probed to monitor equal loading. iNOS isexpressed by cytokine-stimulated CC cells.

186

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

single-stranded and double-stranded DNA damage, it does not iden-tify oxidatively damaged DNA, the principle form of DNA damageinduced by NO. Therefore, Fpg protein, a lesion-specific repair en-zyme was incorporated in the assay. This protein has a glycosylase aswell as an apurinic-endonuclease activity that recognizes and cleaves8-oxodeoxyguanosine lesions, the most common lesion formed byoxidative stress such as NO. If 8-oxodeoxyguanosine residues are

present in the DNA, Fpg treatment will cleave the DNA at thesesights, resulting in single-strand breaks; the increase in DNA-strandbreaks would increase the amount of DNA moving toward the anodein the comet assay (increased DNA in the comet tail). All of the threecholangiocarcinoma cell lines demonstrated an increase in comet tailsfollowing this modification of the comet assay (Fig. 4C). Statisticalanalysis of 75 comets (Fig. 4) from each treatment showed the extentof DNA damage. There was an average of 7.1% DNA damage incytokine-stimulated cells that increased to 12% by using the Fpgenzyme in the assay. The DNA damage in buffer alone (control) andcytokine1 L-NMMA-treated cells was minimal and virtually identi-cal. These data demonstrate that the magnitude of NO generated byiNOS induction is sufficient to cause single-stranded/double-strandedDNA damage as well as oxidative lesions.

Does NO Inhibit the DNA Repair Machinery? AccumulativeDNA damage could result from: (a) inactivation of DNA repairmechanisms; (b) DNA damage in excess of the cells repair capacity;or (c) DNA damage that is incapable of being repaired. As an initialeffort to assess the mechanisms of accumulative DNA damage byiNOS stimulation and NO production, we used the DNA repairincorporation assay to assess DNA repair capacity (Fig. 5). Control orunstimulated WCEs exhibited normal repair activity as evidenced bythe incorporation of the radiolabeled nucleotide during repair of thedamaged DNA plasmid substrate. As a negative control for DNArepair, the WCEs were boiled to denature repair proteins and, asexpected, there was no incorporation of radioactivity. The repairefficiency decreased by;76% in the WCEs from cells stimulatedwith proinflammatory cytokines, relative to repair by the unstimulated

Fig. 3. Cytokines stimulate NO32/NO2

2 generation in human cholangiocarcinoma celllines. NO3

2/NO22 mM levels in the media of three human cholangiocarcinoma cell lines

were assayed 24 h after exposure to cytokines (IL-1b:0.5ng/ml; IFN-g:100 units/ml; andTNF-a:500 units/ml). Markedly elevated levels of NO3

2/NO22 were found in all stim-

ulated cultures (Cyt) when compared with unstimulated control (Ctrl). To determinewhether the source of NO3

2/NO22 was from iNOS, the CC cells were incubated in the

presence of cytokines and 0.03 mM L-NMMA, an inhibitor of iNOS. The inhibitor reducedthe detected levels of NO3

2/NO22 to control levels. Results are expressed as the mean of

three different experiments6 SEM.

Fig. 4. A, cytokine stimulation induces NO-dependent DNA damage. The extent of DNA damage incurred by the cholangiocarcinoma cells on exposure to NO was evaluated bysingle-cell gel electrophoresis using the comet assay. The cells were embedded onto slides with “low melt” agarose and lysed. Damaged single- and double-stranded DNA within thenucleus unwinds and is pulled toward the anode during alkaline electrophoresis, thereby giving a “comet”-like appearance. Representative photomicrographs of cells subjected to thecomet assay are shown (arrow headsdenote comet appearance of damaged DNA). The comet tails seen with cytokine stimulation (B) are further enhanced with incubation with oxidativelesion repair enzyme formamidopyrimidine glycosylase (C). Unstimulated (A) and cytokine1 L-NMMA-treated (D) cells showed intact spherical nuclei, indicating that the DNAdamage was induced by cytokine-stimulated NO via inducible NOS.B, the percentage of DNA in 75 comet tails was analyzed as an expression of extent of damage incurred inunstimulated (control), cytokine-stimulated (cytokine), cytokine1 Fpg enzyme, and cytokine1 L-NMMA, respectively.

187

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

cells (Fig. 5). The repair activity was also tested in response toexogenous 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 ofstimulatory cytokines (Fig. 5). To establish that the decrease in overallrepair activity of the cholangiocarcinoma cells was in fact a directeffect of NO production by cytokine stimulation of iNOS,L-NMMAwas added to the culture medium with the cytokines and incubated for24 h. Repair assay of the WCEs demonstrated thatL-NMMA returnedthe DNA repair activity to basal levels despite cytokine stimulation.(Fig. 6). Thus, induced NO generation is capable of impairing thecells’ DNA repair apparatus. Inhibition of DNA repair by NO mayexplain, in part, the accumulation of damaged DNA during cytokinestimulation of cholangiocarcinoma cells.

DISCUSSION

The original observations of this study relate to inflammation, NOproduction, DNA damage, and inhibition of DNA repair as relatedmechanisms for the development and/or progression of cholangiocar-cinoma. Our results directly demonstrate the following: (a) humancholangiocarcinomas express the iNOS protein; (b) proinflammatory

cytokines stimulate iNOS message and protein expression and theproduction of NO in cholangiocarcinoma cell lines; (c) the magnitudeof NO produced is sufficient to cause single-stranded, double-stranded, and oxidative DNA lesions in the malignant cell lines; and(d) stimulated NO generation is associated with impaired global DNArepair activity in the cholangiocarcinoma cell lines. These data sug-gest that NO generated in response to inflammation may initiatemalignant transformation of biliary epithelia and/or promote progres-sion of established cholangiocarcinoma. Each of these observations isdiscussed in greater detail below.

iNOS expression with NO generation has been observed in severalhuman malignancies in which chronic inflammation is a predisposingfactor, including colon cancer (37, 38), hepatocellular cancer (39–41), Barett’s esophagus (36, 42), and breast cancer (43). Our findingsadd human cholangiocarcinomas to this group of inflammation-asso-ciated malignancies. Because iNOS was not expressed by cholangio-carcinoma cellsin vitro in the absence of cytokines, it would appearthat cholangiocarcinomasin vivo elicit a proinflammatory cytokineresponse sufficient to induce iNOS expression. Indeed, there is his-tological evidence of tumor-associated inflammatory cells, a potentialsource of inflammatory cytokines, in most cholangiocarcinoma spec-imens. The near universal expression of NO by cholangiocarcinomasuggests that it plays an important role in the biology of this cancer.

There are many potential mechanisms by which NO may be im-portant in the initiation, promotion, and progression of this cancer.Our data suggest that NO could promote the accumulation of potentialoncogenic mutations by inhibiting DNA repair enzymes. Inhibition ofproapoptotic effector proteins by protein nitrosylation, such as caspaseproteases, may also promote extended survival of malignant cells(44). Indeed, nitrosylation of caspases would disable apoptotic path-ways promoting cell survival despite DNA damage. Finally, NO mayconfer a survival advantage for cancer by serving as an angiogenesisfactor (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 ofthe bile ducts increases the risk of malignant transformation of biliaryepithelia is unclear, it is known that activation of phagocytes andsubsequent cytokine released in inflamed tissue generate large quan-

Fig. 5. NO32/NO2

2 generated in response to inflam-matory cytokines, and SNAP inhibited DNA repair inhuman cholangiocarcinoma cells. Repair efficiency ofcholangiocarcinoma cell lines was analyzed under fourconditions: unstimulated [Ctrl (1)]; heat-inactivatedWCEs as negative control [Ctrl (2)]; and cytokine-stim-ulated (Cyt) and 0.3 mM SNAP, releasing endogenousand exogenous NO, respectively. Repair was evaluated asa percentage of relative repair incorporation of radiola-beled dGMP into photoactivated methylene blue-dam-aged plasmid DNA by three cholangiocarcinoma WCEs.

Fig. 6. DNA repair is normal during treatment of human cholangiocarcinoma cells withcytokines1 L-NMMA. To determine whether the decrease in repair incorporation ofdGMP by the stimulated cholangiocarcinoma cells was due to increased NO production byiNOS, the cells were treated with cytokines plus 0.03 mM iNOS inhibitor L-NMMA. Therepair efficiency as indicated by the incorporation of radiolabeled dGMP into photoacti-vated methylene blue-damaged plasmid DNA by the three CC WCEs was the same as inthe control (Ctrl), unstimulated cells.

188

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

tities of NO. For example, increased synthesis of NO and endogenousformation of nitrite and nitrosamines are risk factors for cholangio-carcinoma in subjects infested withOpisthorchis Viverrini(48). Asdemonstrated in our comet assay, NO generated by the induction ofiNOS with inflammatory cytokines is sufficient to induce oxidativeDNA damage. It is likely that the inhibition of the DNA repairmechanisms contributes to this DNA damage. These observationssuggest that NO may play an important role in causing the oncogenicmutations that are important in the development of cholangiocarci-noma. However, the specific potential oncogenic mutations inducedby NO remain obscure. Recently, p53 mutations have been identifiedin most cholangiocarcinomas associated with primary sclerosingcholangitis (49). Whether NO can induce genetic alterations in p53 isunknown, but it would provide a mechanistic link between inflam-mation, NO formation, and the development of this malignancy.

Our data directly demonstrate the inhibition of the DNA repairmachinery in a NO-dependent manner. NO may affect proteins bynitrosylation of tyrosine and cysteine residues. Although tyrosinenitrosylation of proteins has been amply documented as a marker ofprotein oxidation (50, 51), its effect on the catalytic functions ofenzymes is obscure. In contrast, cysteine nitrosylation is known todirectly inactivate enzymes (52, 53). Inhibition of DNA repair en-zymes by sulfhydryl nitrosylation is the likely mechanism for theNO-dependent inhibition of global DNA repair. Indeed, the exposureof O6-alkylguanine DNA alkyltransferase to NO causes nitrosylationof its active thiol moiety inhibiting its activity (54, 55). DNA repairproteins with zinc finger motifs (56) such as formamidopyrimidineDNA-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 observa-tions by demonstrating inhibition of DNA repair activity by a NO-dependent manner in intact cells during exposure to inflammatorycytokines. These data demonstrate that the magnitude of NO gener-ated by iNOS in these cells is sufficient to inhibit DNA repairprocesses. The specific oxidative base excision DNA repair enzymesnitrosylated in these cells was not elucidated but is presently beingpursued in our laboratory.

In summary, data in the present study suggest the likely involve-ment of NO in the initiation, progression, and/or promotion of cholan-giocarcinoma during inflammatory conditions of the bile ducts. NOand associated reactive oxygen species such as peroxynitrite modifyDNA bases and result in direct DNA damage. Concomitantly, nitrosy-lation of key repair proteins inhibit the repair of the DNA alterationspromoting the accumulation of potential oncogenic mutations impor-tant in the initiation and/or progression of this cancer. Based on thisinformation, we speculate that iNOS inhibitors targeted to cholangio-cytes could potentially have a chemopreventive role in patients withchronic inflammatory cholangiopathies, such as patients with primarysclerosing cholangitis.

ACKNOWLEDGMENTS

We gratefully acknowledge the secretarial assistance of Sara Erickson;technical assistance and advice of Steve Bronk in the comet assay; theintellectual and technical assistance of Dr. Virginia Miller in measuring NO;and Dr. Sum Lee in supplying us with benign cholangiocytes.

REFERENCES

1. Roberts, S. K., Ludwig, J., and LaRusso, N. F. The pathology of biliary epithelia.Gastroenterology,112: 269–279, 1997.

2. Watanapa, P. Cholangiocarcinoma in patients with opisthorchiasis. Br. J. Surg.,83:1062–1064, 1996.

3. LaRusso, N. F. Morphology, physiology and biochemistry of biliary epithelia. Toxi-col. Pathol.,24: 84–89, 1996.

4. Que, F. G., Gores, G. J., and LaRusso, N. F. Development and initial application ofan in vitro model of apoptosis in rodent cholangiocytes. Am. J. Physiol.,272:106–115, 1997.

5. Haswell-Elkins, M. R., Satarug, S., Tsuda, M., Mairiang, E., Esumi, H., Sithithaworn,P., Mairiang, P., Saitoh, M., Yongvanit, P., and Elkins, D. B. Liver like infection andcholangiocarcinoma: model of endogenous nitric oxide and extragastric nitrosation inhuman carcinogenesis. Mutat. Res.,305: 241–252, 1994.

6. Geller, D. A., Nussler, A. K., Di Silvio, M., Lowenstein, C. J., Shapiro, R. A., Wang,S. C., Simmons, R. L., and Billiar, T. R. Cytokines, endotoxin and glucocorticoidsregulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl.Acad. Sci. USA,90: 522–526, 1992.

7. Geller, D. A., Di Silvio, M., Nussler, A. K., Wang, S. C., Shapiro, R. A., Simmons,M. D., and Billiar, T. R. Nitric oxide synthase expression is induced in hepatocytesin vivo during hepatic inflammation. J. Surg. Res.,55: 427–432, 1993.

8. Nussler, A. K., Geller, D. A., Sweetland, M. A., Di Silvio, M., Billiar, T. R.,Madariaga, J. B., Simmons, R. L., and Lancaster, J. R. Induction of nitric oxidesynthesis and its reactions in cultured human and rat hepatocytes stimulated withcytokines plus lipopolysaccharides. Biochem. Biophys. Res. Commun.,194: 820–835, 1993.

9. Nussler, A. K., Liu, Z. Z., Di Silvio, M., Sweetland, M. A., Geller, D. A., Lancaster,J. R., Billiar, T. R., Freeswick, P. D., Lowenstein, C. L., and Simmons, R. L.Hepatocyte inducible nitric oxide synthesis is influencedin vitro by cell density.Am. J. Physiol.,266: C394–C401, 1994.

10. Marletta, M. A. Nitric oxide synthase: Aspects concerning structure and catalysis.Cell, 78: 927–930, 1994.

11. Michel, T., and Feron, O. Perspective series: Nitric oxide and nitric oxide synthases.J. Clin. Invest.,100: 2146–2152, 1997.

12. Tamir, S., Burney, S., and Tannenbaum, S. R. DNA damage by nitric oxide. Chem.Res. Toxicol.,9: 821–827, 1996.

13. Demple, B., and Harrison, L. Repair of oxidative damage to DNA: Enzymology andBiology. Annu. Rev. Biochem.,63: 915–948, 1994.

14. Stamler, J. S. Redox signaling: Nitrosylation and related target interactions of nitricoxide. Cell,78: 931–936, 1994.

15. Filep, J. G., Lapierre, C., Lachance, S., and Chan, J. S. D. Nitric oxide co-operateswith hydrogen peroxide in inducing DNA fragmentation and cell lysis in murinelymphoma cells. Biochem. J.,321: 897–901, 1997.

16. Wink, D. A., Kasprzak, K. S., Maragos, C. M., Elespuru, R. K., Misra, M., Dunams,T. M., Cebula, T. A., Koch, W. H., Andrews, A. W., Allen, J. S., and Keefer, L. K.DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science(Washington DC),254: 1001–1003, 1991.

17. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., andBeckman, J. S. Oeroxynitrite-mediated tyrosine nitrosation catalyzed by superoxidedismutase. Arch. Biochem. Biophys.,284: 431–437, 1992.

18. Kong, S. K., Yim, M. B., Stadtman, E. R., and Chock, P. B. Peroxynitrite disables thetyrosine phosphorylation regulatory mechanism: lymphocyte-specific tyrosine kinasefails to phosphorylate nitrated cdc2(6–20)NH2. Proc. Natl. Acad. Sci. USA,93:3377–3382, 1996.

19. Martin, B. L., Wu, D., Jakes, S., and Graves, D. J. Chemical influences on thespecificity of tyrosine phosphorylation. J. Biol. Chem.,265: 7108–7111, 1990.

20. Nguyen, T., Brunson, D., Crespi, C. L., Penman, B. W., Wishnok, J. S., andTannenbaum, S. R. DNA damage and mutation in human cells exposed to nitric oxidein vitro. Proc. Natl. Acad. Sci. USA,89: 3030–3034, 1992.

21. Meyers, L. C., and Verdine, G. L., DNA repair proteins. Curr. Opin. Struct. Biol.,4:51–59, 1994.

22. Sancar, A. DNA excision repair. Annu. Rev. Biochem.,65: 43–61, 1996.23. Wood, R. D. DNA repair in eukaryotes. Annu. Rev. Biochem.,65: 135–167, 1994.24. Friedberg, E. C. Relationship between DNA repair and transcription. Annu. Rev.

Biochem.,65: 15–42, 1994.25. Jaiswal, M., Lipinski, L. J., Bohr, V. A., and Mazur, S. J. Efficient in vitro repair of

7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple path-ways. Nucleic Acids Res.,26: 2184–2191, 1998.

26. Starke, D. W., Chen, Y., Bapna, C. P., Lesnefsky, E. J., and Mieyal, J. J. Sensitivityof protein sulphydryl repair enzymes to oxidative stress. Free Radic. Biol. Med.,23:373–384, 1997.

27. Laval, J., and Wink, D. A. Inhibition by nitric oxide of the repair protein O6-methylguanine-DNA methyltransferase. Carcinogenesis (Lond.),15: 443–447, 1994.

28. Wink, D. A., and Laval, J. The Fpg protei, a DNA repair enzyme, is inhibited by thebiomediator nitric oxidein vitro andin vivo. Carcinogenesis (Lond.),15: 2125–2129,1994.

29. O’Connor, T., Graves, R. V., Murcia, G., Castaing, B., and Laval, J. Fpg protein ofE. coli is a zinc finger protein whose cysteine residues have a structural and/orfunctional role. J. Biol. Chem.,268: 9063–9070, 1993.

30. Lindhal, T., and Barnes, D. E. Mammalian DNA ligases. Annu. Rev. Biochem.,61:251–281, 1992.

31. Harnois, D., Que, F. G., Celli, A., LaRusso, N. F., and Gores, G. J. Bcl-2 isoverexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line.Hepatology,26: 884–890, 1997.

32. Vroman, B., and LaRusso, N. F. Development and characterization of the polarizedprimary cultures of rat intrahepatic bile duct epithelial cells. Lab. Invest.,74:303–313, 1996.

33. Archer, S. Measurement of NO in biological models. FASEB J.,7: 349–360, 1993.34. Duthie, S. J., and McMillan, P. Uracil misincorporation in human DNA detected

using single gel electrophoresis. Carcinogenesis (Lond.),18: 1709–1714, 1997.35. Jaiswal, M. Repair of oxidative damage in DNA induced by photoactivated methylene

blue in human lymphoblastoid whole cell extracts (Thesis). Ann Arbor, MI: UM1Company, 1997.

189

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

36. Mirvish, S. S. Role of N-nitroso-compounds (NOC) and N-nitrosation in etiology ofgastric esophageal, nasopharyngeal and bladder cancer and contribution to cancer ofknown exposure to NOC. Cancer Lett.,93: 17–48, 1995.

37. Singer, I. E., Kwaka, D. W., Scott, S., Weidner, J. R., Mumford, R. A., Reihl, T. E.,and Stenson, W. F., Expression of inducible nitric oxide synthase and nitrotyrosine incolonic epithelium in inflammatory bowel disease. Gastroenterology,111: 877–885,1996.

38. Ambs, S., Merriam, W. G., Bennet, W. P., Felley-Bosco, E., Ogunfusika, M. O., Oser,S. M., Klein, S., Sheilds, D. G., Billiar, T. R., and Harris, C. C. Frequent NOS-2expression in human colon adenocarcinomas: Implication for tumor angiogenesis andcolon cancer progression. Cancer Res.,58: 334–341, 1998.

39. Majano, P. L., Garcia-Monzon, C., Lopez-Cabrera, M., Lara-Pezzi, E., Fernandez-Ruitz, E., Garcia-Iglesias, C., Borque, M. J., and Moreno-Ortero, R. Inducible nitricoxide synthase expression in chronic viral hepatitis: Evidence for a virus-inducedgene-up-regulation. J. Clin. Invest.,101: 1343–1352, 1998.

40. Garcia-Monzon, C., Moreno-Ortero, R. Pajares, J. M., Garcia-Sanchez, A., Lopez-Botet, M., de Landazuri, M. O., and Sanchez-Madrid, F. Expression of a novelactivation antigen on intrahepatic CD81 T lymphocytes in viral chronic activehepatitis. Gastroenterology,98: 1029–1035, 1990.

41. Hata, K., Van Theil, D. H., Herberman, R. B., and Whiteside, T. H. Phenotypic andfunctional characteristics of lymphocytes isolated from liver biopsy patients withactive liver disease. Hepatology,15: 816–823, 1992.

42. Wilson, K. T., Kalathur, S. F., Ramanujam, K. S., and Meltzer, S. J. Increasedexpression of inducible nitric oxide synthase and cycloxygenase-2 in Barett’s esoph-agus and associated adenocarcinoma. Cancer Res.,58: 2929–2934, 1998.

43. Thomsen, L. L., Miles, D. W., Happerfield, L., Bobrow, L. G., Knowles, R. G., andMoncada, S. Nitric oxide synthase activity in human breast cancer. Br. J. Cancer,72:41–44, 1995.

44. Mannick, J. B., Hausladen, A., Liu, L., Hess, D. T., Zeng, M., Miao, Q. X., Kane,L. S., Gow, A. J., and Stamler, J. S. Fas-induced caspase denitrosylation. Science(Washington DC),284: 651–654, 1999.

45. Chandrashekar, B., Melby, P. C., Troyer, D. A., Colston, J. T., and Freeman, G. L.Temporal expression of pro-inflammatory cytokines and inducible nitric oxide syn-

thase in experimental acute chagasic cardiomyopathy. Am. J. Pathol.,152: 925–934,1998.

46. Facchetti, F., Vermi, W., Fiorentini, S., Chilosi, M., Caruso, A., Duse, M.,Notarangelo, L. D., and Badolato, R. Expression of inducible nitric oxide synthase inhuman granulomas and histocytic reactions. Am. J. Pathol.,154: 145–152, 1999.

47. Ames, B. N., Gold, L. S., and Willet, W. C. The cause and prevention of cancer. Proc.Natl. Acad. Sci. USA,92: 5258–5265, 1995.

48. Srivatanakul, P., Ohshima, H., Khlat, M., Parkin, D. M., Sukaryodhin, S., Brouet, I.,and Bartsch, H. Endogenous nitrosamines and liver fluke as risk factors for cholan-giocarcinoma in Thailand. Int. J. Cancer,48: 821–825, 1991.

49. Rizzi, P. M., Ryder, S. D., Portman, B., Ramage, J. K., Naoumov, N. V., andWilliams, R. p53 protein overexpression in cholangiocarcinoma arising in primarysclerosing cholangitis. Gut,38: 265–268, 1996.

50. Padgett, C. M., and Whorton, A. R., Cellular responses to nitric oxide: Role of proteinS-thiolation/dethiolation. Arch. Biochem. Biophys.,358: 232–242, 1998.

51. Leeuwenburg, C., Hansen, P., Shaish, A., Holloszy, J. O., and Heinecke, J. W.Markers of protein oxidation by hydroxyl radical and reactive nitrogen species intissues of aging rats. Am. J. Physiol.,274: R453–R461, 1998.

52. Starke, D. W., Chen, Y., Bapna, C. P., Lesnefsky, E. J., and Mieyal, J. J. Sensitivityof protein sulphydryl repair enzymes to oxidative stress. Free Radic. Biol. Med.,23:373–384, 1997.

53. Laval, F., Wink, D. A., and Laval, J. A discussion of mechanisms of NO genotoxicity:implication of inhibition of DNA repair proteins. Rev. Physiol. Biochem. Pharmacol.,131: 175–191, 1997.

54. Ruiz, F., Corrales, E. J., Migueo, C., and Mato, J. M. Nitric oxide inactivates rathepatic methionine adenosyltransferasein vivo by nitrosylation. Hepatology,28:1051–1057, 1998.

55. Ling-Ling, C., Nakamura, T., Nakatsu, Y., Sakumi, K., and Hayakawa, H. Specificamino acid sequences required for O6-methylguanine-DNA methyltransferase activ-ity: analysis of three residues at or near the methyl acceptor site. Carcinogenesis(Lond.), 13: 837–843, 1992.

56. Schmeidescamp, M., and Klevit, R. E. Zinc finger diversity. Curr. Opin. Struct. Biol.,4: 28–35, 1994.

190

iNOS AND CHOLANGIOCARCINOMA

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

2000;60:184-190. Cancer Res   Meeta Jaiswal, Nicholas F. LaRusso, Lawrence J. Burgart, et al.   Oxide-dependent Mechanismrepair in Cholangiocarcinoma Cells by a Nitric Inflammatory Cytokines Induce DNA damage and Inhibit DNA

  Updated version

  http://cancerres.aacrjournals.org/content/60/1/184

Access the most recent version of this article at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/60/1/184.full#ref-list-1

This article cites 51 articles, 11 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/60/1/184.full#related-urls

This article has been cited by 42 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/60/1/184To request permission to re-use all or part of this article, use this link

Research. on April 11, 2020. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from