Inflammatory Cytokines Induce DNA damage and ... - Cancer Research [PDF]

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

Inflammatory Cytokines Induce DNA damage and Inhibit DNA repair in Cholangiocarcinoma 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 development of cholangiocarcinoma, a highly malignant, generally fatal adenocarcinoma originating from biliary epithelia. However, the link between inflammation and carcinogenesis in these disorders is obscure. Because nitric oxide (NO) is generated in inflamed tissues by inducible nitric oxide synthase (iNOS) and because DNA repair proteins are potentially susceptible to NO-mediated nitrosylation, we formulated the hypothesis that inflammatory cytokines induce iNOS and sufficient NO to inhibit DNA repair enzymes leading to the development and progression of cholangiocarcinoma. iNOS and nitrotyrosine were demonstrated in 18/18 cholangiocarcinoma specimens. Furthermore, iNOS and NO generation could be induced in vitro by inflammatory cytokines (mixture of interleukin-1␤, IFN-␥, 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 Cholangiocarcinoma is a malignant neoplasm originating from cholangiocytes, the epithelial cells lining the bile ducts in the liver (1, 2). Although it is well known that chronic inflammatory conditions involving the bile ducts (e.g., primary sclerosing cholangitis, clonorchis sinensis infections, biliary stone disease, and Caroli’s disease) predispose to the development of cholangiocarcinoma (3–5), the relationship between chronic inflammation and malignant transformation is unclear. 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 –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 (12–15). 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 (17–19). 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 iNOSgenerated 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 (22–25), 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? MATERIALS AND METHODS

Immunohistochemistry. Paraffin-embedded normal human liver tissue samples and liver tissue samples obtained from patients with cholangiocarcinoma were used for iNOS and 3-nitrotyrosine immunohistochemistry. Five micron tissue sections were deparaffinized twice in xylene for 10 min each and rehydrated in ethanol followed by water and PBS. Endogenous peroxidase was blocked by immersion in 3% hydrogen peroxide in methanol for 20 min, and the tissue was washed twice in PBS for 10 min each. Nonspecific binding was blocked, 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 tissue sections were drained well and then incubated with iNOS antibody (Transduction Labs, Lexington, KY) at a concentration of 15 ␮g/ml (1:500 dilution) for 1 h at room temperature and with 3-nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) at a concentration of 5 ␮g/ml (1:450 dilution). 184

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 marked advertisement in accordance with 18 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: [email protected]. 3 The abbreviations used are: NO, nitric oxide; Fpg, formamidopyrimidine DNA glycosylase; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; L-NMMA, G N -methyl-L-arginine acetate; SNAP, S-nitroso-N-acetyl-penicillamine; RT-PCR, reverse transcription-PCR; Fpg, foramidopyrimidine; WCE, whole cell extract; IL-1␤, interleukin 1␤; TNF-␣, tumor necrosis factor ␣.

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

Control sections were incubated without a primary antibody. The sections were 30 min before electrophoresis at 25 V for 30 min. The slides were then washed washed twice for 5 min each in PBS. Immunostaining for iNOS and 3-nitro- three times in 0.4 M Tris-HCl (pH 7.5) buffer for 5 min each and stained with tyrosine was detected with the Vectastain peroxidase kit (Vector labs, Burl- SYBR green dye (1:10 dilution from concentrate; Trevigen, Gaithersburg). ingame, CA). The immunostain was developed with diaminobenzidine tetra- Statistical evaluation was performed using NIH image (Netscape Navigator) hydrochloride for 5 min. The sections were counterstained with hematoxylin, and Komet 3.0 Macro from Trevigen. Repair Incorporation Assay. DNA repair was assessed by determining rehydrated in ethanol followed by xylene, and coverslipped. Immunohistochemical staining for iNOS was evaluated by light microscopy. Specific iNOS the ability of cell extracts to incorporate radiolabeled nucleotide into oxidatively damaged plasmid DNA as previously described (25, 35). The pBluestaining 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, script II KS (⫹) 2961-bp [pKS(⫹)] and the pKB-CMV 4518-bp plasmid well-differentiated; 2, moderately differentiated; 3, poorly differentiated; and substrates were prepared by lysozyme-Triton X-100 method (25) with modifications. The oxidatively damaged pKS II (⫹) plasmid was prepared by 4, anaplastic) by an experienced hepatopathologist. Cell Culture Medium and Technique. Three human cholangiocarcinoma treating 0.1 mg/ml DNA with 10 ␮M methylene blue in 0.01 M sodium cell lines KMCH, KMBC, and WITT were used for this study (31). For control phosphate buffer (pH 7.4). The plasmid DNA was kept on ice and exposed to studies, normal rat cholangiocytes and RAW 267.4 mouse macrophages were visible light at a fluence of 117 W/m2 from a 100-W tungsten bulb for 3 min. also cultured. The normal rat cholangiocytes were cultured as previously This exposure of 21 kilojoules/m2 visible light in the presence of 10-␮M described by us in detail (32). The RAW 267.4 and cholangiocarcinoma cell methylene blue produces 1.6-Fpg protein sites, primarily 8-oxodG (paper) on lines were cultured in Dulbecco’s modified Eagles medium (Life Technolo- the 2.9-kbp plasmid (25, 35). The Fpg protein possesses 8-oxodG glycosylase gies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 100 units/ml and lapurinic lyase activities (23). Oxidatively damaged pKS II (⫹) and pKB-CMV (control) plasmids were further purified on cesium chloridepenicillin, 100 ␮g/ml streptomycin, and 5% fetal bovine serum and maintained ethidium bromide gradient centrifugation steps and on one sucrose gradient at 37°C with 5% CO2 and 95% humidity. Measurement of NO Production by Chemiluminescence. NO was meas- step until none of the plasmids had a detectable population of nicked moleured in the culture media using a nitric oxide analyzer (Sievers, Boulder, CO). cules. Human cholangiocarcinoma cells were harvested at a density of ⬃6 ⫻ 105 cells/ml. WCEs were prepared as described in a previous study (25). Nitrite and nitrate present in the culture medium (100 ␮l) was converted to NO by a saturated solution of VCl3 in 0.8 M HCl, and the NO was detected by a Briefly, reactions containing 300 ng each of supercoiled damaged pKS II (⫹) gas phase chemiluminescent reaction between NO and ozone (33). Nitrite and and undamaged pKB-CMV, 100 ␮g of WCE protein in a volume of 50 ␮l with nitrate concentrations were determined by interpolation from known standards. 44 mM HEPES-KOH (pH 7.9), 70 mM KCl, 7.5 mM MgCl2, 1.2 mM DTT, 0.5 RT-PCR. Total RNA was extracted from the cells using the TRIzol reagent mM EDTA, 2 mM ATP, 250 ␮g/ml BSA, 40 mM phosphocreatine, 50 ␮g/ml (Life Technologies, Grand Island, NY). The human iNOS primers were sense; creatine phosphokinase, 50 ␮M each of dATP, dCTP, and dTTP, 5 ␮M dGTP 32 5⬘-CCCTTTACTTGACCTCCTAAC-3⬘; antisense; 5⬘-AAGGAATCATA- (Sigma, St. Lois, MO), and 1 ␮Ci of [␣- P]dGTP (New England Nuclear Life CAGGGAAGAC-3⬘. After reverse transcription (6), PCR amplification was Science products, Boston, MA). The reactions were incubated at 30°C for 2 h. performed by Taq polymerase (Perkin-Elmer, Branchburg, NJ) using a pro- The reaction was stopped by the addition of EDTA and then treated with grammed thermocycler (TwinBlock systems, Ericomp, San Diego, CA) using RNaseA followed by proteinase K. The plasmid DNA was recovered by phenol-chloroform extraction and ethanol precipitation. Plasmids were linearthe following conditions: (a) an initial denaturation for 2 min at 94°C; and (b) ized with EcoRI endonuclease and separated by 1% agarose gel electrophoresis 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 ␮g/ml containing 0.5 ␮g/ml ethidium bromide. The gel was scanned to measure the intensity of ethidium bromide fluorescence (MultiImage light Cabinet, Ionnoethidium bromide and photographed under UV trans-illumination. Western Blot Analysis. Cytokine-stimulated and unstimulated human tech. Corp., San Leandro, CA). Fluorescence intensities of linearized plasmid cholangiocarcinoma cells and RAW 267.4 (ATCC, Bethesda, MD) mouse bands in comparison with known amounts of DNA were used to determine macrophage cells were harvested and lysed by sonication in ice-cold lysis DNA loading. The gel was dried under vacuum and exposed to a storage screen buffer containing 100 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, (phosphoImager, Molecular Dynamics) for 18 h. Autoradiograms were ana2 mM DTT, and protease inhibitors (5 mg/ml leupeptin, pepstatin, and chy- lyzed using ImageQuant software (Molecular Dynamics). Background cormostatin and 87 mg/ml phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO). rected values of the radioactivity incorporated for the damaged and undamaged Whole cell lysates were boiled in Laemmli buffer and 40 ␮g/lane protein plasmids were normalized for the amount of DNA. loaded on a 7.5% SDS-polyacrylamide gel and separated electrophoretically. The proteins were transferred to a nitrocellulose membrane (Schleicher and RESULTS 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, Is iNOS Expressed by Human Cholangiocarcinoma? To deterTween 0.05%, 0.5 M NaCl (pH 7)] for 1 h. The Primary antibody for iNOS mine whether iNOS is expressed by human cholangiocarcinomas, we (Transduction Laboratories, Kensington, KY) was applied at a 1:2500 dilution performed immunohistochemistry for iNOS in tissue specimens from for 2 h. The membrane was washed three times in TTBS for 10 min each 18 patients with cholangiocarcinoma. There was intense staining for before applying the secondary antibody (Transduction Laboratories, KensingiNOS (Fig. 1D) in all 18 human cholangiocarcinoma specimens. In ton, KY) was applied at a 1:5000 dilution for 1 h. The blot was washed in this limited sample, no relationship was observed between tumor TTBS four times for 10 min each. It was then incubated in commercially enhanced chemiluminescence reagent (Amersham, Buckingshire, United grade and intensity of iNOS staining (data not shown). Evidence for catalytic activity of the expressed iNOS protein was identified by Kingdom) and exposed to photographic film. Comet Assay. The assay was performed as described in a protocol from performing immunohistochemistry for 3-nitrotyrosine, a reaction Trevigen (34). Cells were suspended in 0.5% (w/v) solution of low melt product of peroxynitrite (formed from NO reacting with the superoxagarose in PBS (pH7.4) at 37°C and immediately pipetted onto a frosted ide anion) with susceptible tyrosine residues. Similar to the immunomicroscope slide (Trevigen, Gaithersburg, MD). The agarose was allowed to histochemistry for iNOS, all 18 cholangiocarcinoma specimens reset for 10 min at 4°C and the slides were incubated in lysis solution [2.5 M vealed positive staining for 3-nitrotyrosine. The presence of NaCl, 100 mM EDTA (pH10), 10 mM Tris base, 1% sodium lauryl sarcosinate nitrotyrosine residues implies a high level of activity of the expressed and 0.01% Triton X-100, Trevigen, Gaithersburg, MD] at 4°C to remove iNOS (36). In comparison, the biliary epithelia from normal liver cellular proteins for 1 h. This step leaves the DNA as distinct nucleoids. After biopsies did not stain either for iNOS or 3-nitrotyrosine. Thus, cholanlysis, 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 giocarcinomas appear to uniformly express iNOS and stain intensely tapped dry. The agarose-embedded cells were covered with either Fpg DNA for 3-nitrotyrosine, a marker of oxidative protein damage. To more specifically demonstrate iNOS expression in human glycosylase (0.1 unit/gel; Trevigen, Gaithersburg, MD) or buffer alone and cholangiocarcinoma cells, we assayed for iNOS mRNA and protein 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 expression using RT-PCR and immunoblot analysis in three human 185

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

Fig. 1. Immunohistochemical analysis of iNOS and detection of 3-nitrotyrosine in human liver biopsies. The left panel represents immunostaining without primary antibody, and the right panel represents immunostaining with the primary antibody. Normal liver tissue, specifically the bile duct epithelia (arrows), did not stain for either iNOS (B) or for 3-nitrotyrosine (F). There was intense staining (arrows) for iNOS (D) and 3-nitrotyrosine (H) in the malignant biliary epithelia of cholangiocarcinoma patients.

cholangiocarcinoma cell lines. Cells cultured in the absence of stimulatory cytokines failed to express iNOS (Fig. 2). In contrast, when all of the three cell lines were incubated in the presence of a mixture of inflammatory cytokines [human recombinant IL-1␤ (0.5 ng/ml), human recombinant TNF-␣ (500 units/ml), and human recombinant

Fig. 2. Cytokines enhance expression of iNOS mRNA and protein in human cholangiocarcinoma cell lines. Total RNA and protein was extracted from three cholangiocarcinoma cell lines after 24-h stimulation with (⫹) and without (⫺; IL-1␤:0.5ng/ml; IFN-␥:100 units/ml, and TNF-␣:500 units/ml) cytokines. iNOS mRNA expression is indicated by the 289-bp RT-PCR product from stimulated cells on electrophoresis. iNOS protein expression was assayed by immunochemistry on 50 ␮g of cell lysate. The electrophoretically transferred nitrocellulose membrane was immunoblotted with monoclonal human iNOS antibody. ␤-actin was probed to monitor equal loading. iNOS is expressed by cytokine-stimulated CC cells.

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. Do Cytokine-stimulated Cholangiocarcinoma Cells Produce NO? Although the above studies demonstrate that cholangiocarcinoma expresses iNOS, the catalytic activity of the expressed protein was not ascertained. Therefore, we measured NO in the media of cholangiocarcinoma cells incubated in the presence and absence of stimulatory cytokines (Fig. 3). 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. Does Stimulated NO Generation Result in Oxidative DNA Damage? The alkaline comet assay provides a sensitive tool for the detection of DNA damage (single-stranded breaks and alkali labile sites) at the single cell level. Fig. 4 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

186

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

Fig. 3. Cytokines stimulate NO3⫺/NO2⫺ generation in human cholangiocarcinoma cell lines. NO3⫺/NO2⫺ mM levels in the media of three human cholangiocarcinoma cell lines were assayed 24 h after exposure to cytokines (IL-1␤:0.5ng/ml; IFN-␥:100 units/ml; and TNF-␣:500 units/ml). Markedly elevated levels of NO3⫺/NO2⫺ were found in all stimulated cultures (Cyt) when compared with unstimulated control (Ctrl). To determine whether the source of NO3⫺/NO2⫺ 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 reduced the detected levels of NO3⫺/NO2⫺ to control levels. Results are expressed as the mean of three different experiments ⫾ SEM.

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. Does NO Inhibit the DNA Repair Machinery? Accumulative DNA damage could result from: (a) inactivation of DNA repair mechanisms; (b) DNA damage in excess of the cells repair capacity; or (c) DNA damage that is incapable of being repaired. As an initial effort to assess the mechanisms of accumulative DNA damage by iNOS stimulation and NO production, we used the DNA repair incorporation assay to assess DNA repair capacity (Fig. 5). 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

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 by single-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 the nucleus unwinds and is pulled toward the anode during alkaline electrophoresis, thereby giving a “comet”-like appearance. Representative photomicrographs of cells subjected to the comet assay are shown (arrow heads denote comet appearance of damaged DNA). The comet tails seen with cytokine stimulation (B) are further enhanced with incubation with oxidative lesion repair enzyme formamidopyrimidine glycosylase (C). Unstimulated (A) and cytokine ⫹ L-NMMA-treated (D) cells showed intact spherical nuclei, indicating that the DNA damage 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 in unstimulated (control), cytokine-stimulated (cytokine), cytokine ⫹ Fpg enzyme, and cytokine ⫹ L-NMMA, respectively.

187

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

Fig. 5. NO3⫺/NO2⫺ generated in response to inflammatory cytokines, and SNAP inhibited DNA repair in human cholangiocarcinoma cells. Repair efficiency of cholangiocarcinoma cell lines was analyzed under four conditions: unstimulated [Ctrl (⫹)]; heat-inactivated WCEs as negative control [Ctrl (⫺)]; and cytokine-stimulated (Cyt) and 0.3 mM SNAP, releasing endogenous and exogenous NO, respectively. Repair was evaluated as a percentage of relative repair incorporation of radiolabeled dGMP into photoactivated methylene blue-damaged plasmid DNA by three cholangiocarcinoma WCEs.

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. DISCUSSION The original observations of this study relate to inflammation, NO production, DNA damage, and inhibition of DNA repair as related mechanisms for the development and/or progression of cholangiocarcinoma. Our results directly demonstrate the following: (a) human cholangiocarcinomas express the iNOS protein; (b) proinflammatory

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

cytokines stimulate iNOS message and protein expression and the production of NO in cholangiocarcinoma cell lines; (c) the magnitude of NO produced is sufficient to cause single-stranded, doublestranded, and oxidative DNA lesions in the malignant cell lines; and (d) stimulated NO generation is associated with impaired global DNA repair activity in the cholangiocarcinoma cell lines. These data suggest that NO generated in response to inflammation may initiate malignant transformation of biliary epithelia and/or promote progression of established cholangiocarcinoma. Each of these observations is discussed in greater detail below. 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 – 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 quan-

188

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

tities 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 NOdependent 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. ACKNOWLEDGMENTS We gratefully acknowledge the secretarial assistance of Sara Erickson; technical assistance and advice of Steve Bronk in the comet assay; the intellectual 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. Toxicol. Pathol., 24: 84 – 89, 1996.

4. Que, F. G., Gores, G. J., and LaRusso, N. F. Development and initial application of an 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 and cholangiocarcinoma: model of endogenous nitric oxide and extragastric nitrosation in human 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 glucocorticoids regulate 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 hepatocytes in 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 oxide synthesis and its reactions in cultured human and rat hepatocytes stimulated with cytokines 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 influenced in 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 and Biology. Annu. Rev. Biochem., 63: 915–948, 1994. 14. Stamler, J. S. Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell, 78: 931–936, 1994. 15. Filep, J. G., Lapierre, C., Lachance, S., and Chan, J. S. D. Nitric oxide co-operates with hydrogen peroxide in inducing DNA fragmentation and cell lysis in murine lymphoma 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., and Beckman, J. S. Oeroxynitrite-mediated tyrosine nitrosation catalyzed by superoxide dismutase. Arch. Biochem. Biophys., 284: 431– 437, 1992. 18. Kong, S. K., Yim, M. B., Stadtman, E. R., and Chock, P. B. Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: lymphocyte-specific tyrosine kinase fails 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 the specificity of tyrosine phosphorylation. J. Biol. Chem., 265: 7108 –7111, 1990. 20. Nguyen, T., Brunson, D., Crespi, C. L., Penman, B. W., Wishnok, J. S., and Tannenbaum, S. R. DNA damage and mutation in human cells exposed to nitric oxide in 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 pathways. Nucleic Acids Res., 26: 2184 –2191, 1998. 26. Starke, D. W., Chen, Y., Bapna, C. P., Lesnefsky, E. J., and Mieyal, J. J. Sensitivity of 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 O6methylguanine-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 the biomediator nitric oxide in vitro and in vivo. Carcinogenesis (Lond.), 15: 2125–2129, 1994. 29. O’Connor, T., Graves, R. V., Murcia, G., Castaing, B., and Laval, J. Fpg protein of E. coli is a zinc finger protein whose cysteine residues have a structural and/or functional 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 is overexpressed 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 polarized primary 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: UM1 Company, 1997.

189

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

iNOS AND CHOLANGIOCARCINOMA

36. Mirvish, S. S. Role of N-nitroso-compounds (NOC) and N-nitrosation in etiology of gastric esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known 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 in colonic 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-2 expression in human colon adenocarcinomas: Implication for tumor angiogenesis and colon cancer progression. Cancer Res., 58: 334 –341, 1998. 39. Majano, P. L., Garcia-Monzon, C., Lopez-Cabrera, M., Lara-Pezzi, E., FernandezRuitz, E., Garcia-Iglesias, C., Borque, M. J., and Moreno-Ortero, R. Inducible nitric oxide synthase expression in chronic viral hepatitis: Evidence for a virus-induced gene-up-regulation. J. Clin. Invest., 101: 1343–1352, 1998. 40. Garcia-Monzon, C., Moreno-Ortero, R. Pajares, J. M., Garcia-Sanchez, A., LopezBotet, M., de Landazuri, M. O., and Sanchez-Madrid, F. Expression of a novel activation antigen on intrahepatic CD8 ⫹ T lymphocytes in viral chronic active hepatitis. Gastroenterology, 98: 1029 –1035, 1990. 41. Hata, K., Van Theil, D. H., Herberman, R. B., and Whiteside, T. H. Phenotypic and functional characteristics of lymphocytes isolated from liver biopsy patients with active liver disease. Hepatology, 15: 816 – 823, 1992. 42. Wilson, K. T., Kalathur, S. F., Ramanujam, K. S., and Meltzer, S. J. Increased expression of inducible nitric oxide synthase and cycloxygenase-2 in Barett’s esophagus and associated adenocarcinoma. Cancer Res., 58: 2929 –2934, 1998. 43. Thomsen, L. L., Miles, D. W., Happerfield, L., Bobrow, L. G., Knowles, R. G., and Moncada, 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-

46.

47. 48.

49.

50. 51.

52.

53.

54.

55.

56.

thase in experimental acute chagasic cardiomyopathy. Am. J. Pathol., 152: 925–934, 1998. Facchetti, F., Vermi, W., Fiorentini, S., Chilosi, M., Caruso, A., Duse, M., Notarangelo, L. D., and Badolato, R. Expression of inducible nitric oxide synthase in human granulomas and histocytic reactions. Am. J. Pathol., 154: 145–152, 1999. 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. 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 cholangiocarcinoma in Thailand. Int. J. Cancer, 48: 821– 825, 1991. Rizzi, P. M., Ryder, S. D., Portman, B., Ramage, J. K., Naoumov, N. V., and Williams, R. p53 protein overexpression in cholangiocarcinoma arising in primary sclerosing cholangitis. Gut, 38: 265–268, 1996. Padgett, C. M., and Whorton, A. R., Cellular responses to nitric oxide: Role of protein S-thiolation/dethiolation. Arch. Biochem. Biophys., 358: 232–242, 1998. Leeuwenburg, C., Hansen, P., Shaish, A., Holloszy, J. O., and Heinecke, J. W. Markers of protein oxidation by hydroxyl radical and reactive nitrogen species in tissues of aging rats. Am. J. Physiol., 274: R453–R461, 1998. Starke, D. W., Chen, Y., Bapna, C. P., Lesnefsky, E. J., and Mieyal, J. J. Sensitivity of protein sulphydryl repair enzymes to oxidative stress. Free Radic. Biol. Med., 23: 373–384, 1997. 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. Ruiz, F., Corrales, E. J., Migueo, C., and Mato, J. M. Nitric oxide inactivates rat hepatic methionine adenosyltransferase in vivo by nitrosylation. Hepatology, 28: 1051–1057, 1998. Ling-Ling, C., Nakamura, T., Nakatsu, Y., Sakumi, K., and Hayakawa, H. Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analysis of three residues at or near the methyl acceptor site. Carcinogenesis (Lond.), 13: 837– 843, 1992. Schmeidescamp, M., and Klevit, R. E. Zinc finger diversity. Curr. Opin. Struct. Biol., 4: 28 –35, 1994.

190

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.

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

Updated version

Cited articles Citing articles

E-mail alerts Reprints and Subscriptions Permissions

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/60/1/184

This article cites 51 articles, 12 of which you can access for free at: http://cancerres.aacrjournals.org/content/60/1/184.full#ref-list-1 This article has been cited by 36 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/60/1/184.full#related-urls

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/60/1/184. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from cancerres.aacrjournals.org on January 26, 2018. © 2000 American Association for Cancer Research.