increased expression of inducible nitric oxide synthase...

ICANCER RESEARCH 58. 2929-2934. July 15. 1998]

Advances in Brief

Increased Expression of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 inBarrett's Esophagus and Associated Adenocarcinomas1

Keith T. Wilson,2 Sidong Fu, Kalathur S. Ramanujam, and Stephen J. Meltzer

Department of Medicine, Division of Gastroenterologe Â¡K.T. W., S. f., K. S. R., S. J. M.l, Greeiiebauni Cancer Center {K. T. W.. S. J. MJ. Graduate Program in Molecular andCell Biology Â¡S.J. M.], and Department of Pathology Â¡S.J.M.I. University of Maryland School of Medicine and Baltimore Veterans Affairs Maryland Health Care System.Baltimore. Mailand 2I20I

Abstract

Barrett's esophagus is a premalignant condition arising in response to

chronic reflux esophagitis. Inducible nitric oxide synthase (Â¡NOS;NOS-2)and cyclooxygenase-2 (COX-2) are mediators of inflammation and regulators of epithelial cell growth. Expression levels of Â¡NOSand COX-2 arehigh in colorectal adenomas and carcinomas, and COX-2 expression is

elevated in gastric cancers. To determine the involvement of Â¡NOSandCOX-2 in Barrett's-associated neoplasia, we measured expression of thesegenes in metaplastic Barrett's and esophageal adenocarcinomas. We detected elevated Â¡NOSand COX-2 niKNA levels in Barrett's mucosa com

pared with paired gastric control tissues in 16 of 21 (76%) and 17 of 21(80%) patients, respectively (/' < 0.001 for both genes). In esophageal

adenocarcinomas, Â¡NOSand COX-2 inKNA levels were increased in four

of five and five of five cases, respectively. Furthermore, in 10 of 10Barrett's patients, immunohistochemical staining for Â¡NOSand COX-2

expression was strongly positive and higher than in matched gastriccontrols. Increased COX-2 expression was confirmed by Western blotting.These findings support the hypothesis that Â¡NOSand COX-2 are involvedearly and often in Barrett's-associated neoplastic progression.

Introduction

Barrett's esophagus is the replacement of the normal squamous

esophageal epithelium by a specialized columnar lining in response tochronic reflux esophagitis (1). This condition is highly premalignant,being associated with a risk of esophageal adenocarcinoma up to125-fold greater than the general population (2, 3). The molecular

basis of this increased risk is now being elucidated. Generalizedgenetic instability, evidenced by aneuploidy and chromosomal abnormalities, has been shown to occur early in this process (4, 5). Loss ofheterozygosity, inactivation of tumor suppressor genes, and microsatellite instability have all been identified in Barrett's metaplasia and

associated esophageal adenocarcinomas, but the frequency of thesealterations in metaplastic mucosa is generally lower than in frankesophageal adenocarcinomas (6). In any case, much remains to belearned about the molecular pathogenesis of Barrett's-associated neo

plastic progression.iNOS3 is the enzyme that catalyzes the high output pathway of

production of NO from L-arginine, whereas COX-2 is the rate-limiting

enzyme for the high output production of prostanoids (prostaglandinsand thromboxanes) from arachidonic acid. Increased expression of

Received 4/3/98; accepted 6/1/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article musi therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this faci.

1Supported by NIH Grants K08-DK02469. ROI-CA67497. ROI-DK47717. and R01-

DK53620: the Robert and Sally D. Funderburg Award in Gastric Cancer Biology; theUniversity of Maryland School of Medicine; and the Office of Medical Research.Department of Veterans Affairs.

2 To whom requests for reprints should be addressed, at Baltimore Veterans Affairs

Medical Center. Room 5D-151. 10 North Greene Street. Baltimore. MD 21201. Phone:(410) 605-7000. ext. 5211; Fax: (410) 605-7914; E-mail: [email protected].

3 The abbreviations used are: iNOS. induciblc nitric oxide synthase; NO. nitric oxide:COX-2. cyclooxygenase-2: RT-PCR. reverse transcription PCR; Gl. gastrointestinal.

both iNOS and COX-2 has been demonstrated in intestinal inflammation, in both animal models (7-9) and human diseases (10, 11).

Furthermore, we and others have identified high levels of expressionof iNOS and COX-2 in Helicobacter pylori-associated gastritis ( 12,13). Finally, in in vitro studies, both iNOS and COX-2 have beenshown to have mutagenic (14-16) and tumorigenic (17-19) activity.

Recently, the importance of COX-2 in GI carcinogenesis has beenrecognized. Induction of COX-2 expression has been demonstrated in

human and rodent colorectal adenomas and carcinomas (20, 21) and,importantly, COX inhibition results in both regression of neoplasticpolyps and prevention of their development in individuals with familial adenomatous polyposis, as well as in the murine models of thisdisease, the min mouse and APC knockout mouse (21-23). In humans,gastric adenocarcinomas have shown increased COX-2 expressionwith no alteration in levels of COX-1, the constitutive form of

cyclooxygenase (24, 25). In contrast, the role of NO and iNOS inhuman GÃ¬neoplasia is more uncertain. Although NO can induce DNAdamage (14, 15) and angiogenesis (26), it has been shown to inhibitthe growth of epithelial tumor cells (26). NOS activity has beenreported to be decreased in colorectal adenomas and carcinomas (27).However, a recent comprehensive study by Ambs et al. (28) demonstrated increased Ca-independent iNOS activity and protein expres

sion in both colorectal adenomas and carcinomas.We and others have determined that iNOS expression in human GI

inflammation predominates in the epithelium, and that intestinal epithelial cell lines abundantly express iNOS with cytokine stimulation(10, 29, 30). Although COX-2 expression is abundant in lamina

propria mononuclear cells and fibroblasts, it is also readily induced inintestinal epithelial cells in vitro (31). Because Barrett's epithelium is

an intestinoid metaplasia that arises in response to chronic injury (acidreflux), we hypothesized that Barrett's mucosa would express in

creased levels of iNOS and COX-2. We also sought to determine

whether levels of these gene products would remain elevated inadenocarcinomas arising in Barrett's esophagus. In this report, we

demonstrate frequent overexpression of iNOS and COX-2 mRNA andprotein in both Barrett's metaplastic tissues and frank esophageal

adenocarcinomas relative to matching gastric control mucosa.

Materials and Methods

Tissue Samples. All tissues in this study were obtained from endoscopie-

biopsies or surgical resections from patients at either the University of Maryland or Baltimore Veterans Affairs Medical Centers. Twenty-one paired Barrett's esophagus and gastric body tissues were included in this study. All caseshad histologically confirmed specialized columnar epithelium of Barrett's

esophagus. In addition, five paired tissues from surgical resections for adenocarcinoma arising in known Barrett's mucosa and adjacent normal esophageal

tissue were studied. Tissues were snap-frozen in liquid nitrogen for the mRNAand immunoblotting analysis and fixed in 10% buffered formalin for immu-

nohistochemistry.RT-PCR Analysis for Â¡NOSand COX-2. Total RNA was isolated using

Trizol reagent (Life Technologies. Inc.. Gaithersburg. MD) and 2 /ig of RNA

2929

on May 12, 2018. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Â¡NOSAND COX-2 EXPRESSION IN BARRETT'S ESOPHAGUS

A M SBSBSB MSBSBSBSBSB

â€”¿�ÃŸ-actin-Â¡NOS

â€”¿�ÃŸ-actinâ€”¿�COX-2

B 0.9

0.8

0.7

:~ 0.6; e

CQ.Â£>

0.4-

0.2-

0.1

OStomach Barrett's Stomach Barrett's

MNTNTNTN

â€”¿�ÃŸ-actin-Â¡NOS

-COX-2â€”¿�ÃŸ-actin

Fig. 1. RT-PCR analysis of Â¡NOSand COX-2 mRNA expression in Barrett's esophagus tissues and in associated adenocarcinomas. A, representative gels of transcripts for Â¡NOS((op) and COX-2 (bottom). PCR product sizes in bp are Â¡NOS,237; COX-2, 305; and ÃŸ-actin,496. M. DNA markers; S, stomach; B, Barrett's mucosa. B, densitometry of Â¡NOSandCOX-2 transcripts, standardized to ÃŸ-actin,from control stomach and Barrett's mucosa for each patient. *** P < 0.001 versus stomach control by paired Student's / test. C, transcript

abundance of Â¡NOSand COX-2 in esophageal adenocarcinomas. PCR product sizes (in bp) Â¡NOS,237; COX-2, 756; and ÃŸ-actin,496. /V, normal esophagus; T, tumor.

from each sample were reverse transcribed using Superscript II RT (LifeTechnologies, Inc.) in a total reaction volume of 20 fu. Reverse-transcriptionproduct (cDNA; ! fj.\) was PCR-amplified with with Ampli-Taq DNA polym-erase (Perkin-Elmer/Applied Biosystems, Foster City, CA) and 0.5 pmol eachof Â¡NOSor COX-2 forward and reverse primers, with ÃŸ-actinprimers included

in the same multiplex PCR reaction as an internal control for efficiency of RTand amount of RNA. Each PCR cycle consisted of a denaturation step (94Â°C,1 min), an annealing step (60Â°C, 1 min), and an elongation step (72Â°C, 1.5

min). There were a total of 35 cycles, followed by an additional extension step(72Â°C,7 min). The primer sequences (F = forward, R = reverse primers) andPCR product sizes were as follows: iNOS, 5'-TCTTGGTCAAAGC TGT-GCTC-3' (F) and 5'-CATTGCCAAACGTACTGGTC-3' (R), 237 bp; two setsof COX-2 primers, 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' (F)and S'-GATCATCTCTGCCTGAGTATCTT-S' (R), 305 bp plus 5'-CAG-

CACTTCACGCATCAGTT-3' (F) and 5'-TCTGGTCAATGGAAGCCT-GT-3' (R), 756 bp; and, finally, ÃŸ-actin,5'-CCAGAGCAAGAGAGGTAT-CC-3' (F) and 5'-CTGTGGTGGTGAAGCTGTAG-3' (R), 436 bp. PCR prod

ucts were run on 2% agarose gels with 0.5 /ig/ml ethidium bromide, andstained bands were visualized under UV light, photographed, digitized with anAgfa Arcus II scanner, and band intensity quantitated using NIH Image version1.61.

Immunohistochemical Analysis. Sections (5 jn.m)cut from paraffin-em

bedded tissues were deparaffmized, and endogenous peroxidase activity wasquenched by incubation in 0.3% H2O2 in methanol for 30 min. Sections werethen microwaved in citrate buffer, pH 6.1, for 30 min for antigen retrieval.Nonspecific binding was blocked with 5% normal horse serum in PBS and thetissue was incubated with monoclonal antibodies to Â¡NOS(antimouse, 1:500dilution; Transduction Labs, Lexington, KY) or COX-2 (antirat, 1:1000 dilu-

2930




feÃŽ^P^Ã¯^VO'Al-' F

?Ã™

' j$>..i'~~^,â€¢¿�-â€¢-

s---*â€¢¿�'

\ ^*i

rflP- ^K ^S&Wiv Â¿^ 'y<Â»M

/

Â¿â€¢¿�.Â»'SÃ¬-

|U|Ã¯Ã¯ ^Â£|$sl â€¢¿� ^Â»Â».* ^_. ft fr* * *

-M Â».TÂ»

Â¿^ t J^lÃ‰- Ã•LiSÃŽ*51*^

Fig. 2. Immunohistochemical detection of Â¡NOS(A-D) and COX-2 (Â£-W)in tissues from gastric body (A and Â£").Barrett's esophagus (ÃŸand F), dysplasia (C and G), and carcinoma(D and //). Staining was performed on formalin fixed tissues using specific monoclonal antibodies detected with peroxidase-3.3'-diaminobenzidine (brownish pigment). iNOS stainingwas absent in the normal gastric body (A) and squamous epithelium (B) in contrast to the staining of the metaplastic appearing glands of the Barrett's mucosa (B) which was especiallyintense in areas of dysplasia (O and carcinoma (D). COX-2 protein is present at a low level in the normal gastric body (Â£")and strongly increased in the lamina propria of Barrett's

mucosa (F) and in the epithelium of the dysplastic (C) and malignant glands (ff). Tissues incubated with mouse IgG rather than the primary antibodies had absent staining (data notshown) verifying the specificity of the antibody staining.

tion; Transduction Labs) overnight at 4Â°Cin 5% normal horse serum. As

controls, slides were incubated in the presence of 5% normal horse serum aloneor with the same concentration of mouse IgG (Vector Labs. Burlingame, CA)as used for the primary antibody. Immunoreactivity was detected by the ABCMethod (Vectastain Elite ABC kit, Vector Labs). Slides were subsequentlyincubated with a biotinylated secondary horse antimouse IgG antibody at 1:200

for 30 min. This was detected using avidin conjugated to horseradish peroxi-dase. The color was then developed using 3.3'-diaminobenzidine. Mayer's

hematoxylin was then added as a counterstain for 5 min.Western Blot Analysis. Tissues were homogenized in Tris-HCI pH

1.4 buffer containing 0.5% Triton X-100 and protease inhibitor cocktail

(Boehringer Mannheim, Indianapolis. IN). Protein concentration of 14,000 g

2931



iNOS AND COX-2 EXPRESSION IN BARRETT'S ESOPHAGUS

SBSB NTNTNT

70 kDa â€”¿� â€”¿�COX-2

Fig. 3. Increased COX-2 protein levels (70 kDa band) by immunoblotting in representative paired tissues from control stomach (.VIand Barrett's mucosa (ÃŸ:Â¡eftpanel}, and from

normal esophagus (AOand esophagcal tumors (T. righi). Equal concentrations of protein (100 ^g) were loaded in each lane. The nonspecific bands below 70 kDa on the n^lii are dueto some degradation of protein in surgical resections compared with an absence of such bands in endoscopie biopsies, which are more rapidly frozen.

soluble supernatants from each sample was measured by the method ofBradford (32). Then 100 /ng of protein was loaded per lane, separated bySDS-PAGE under reducing conditions, and transferred onto Hybond-PVDF

membranes (Amersham Inc.. Arlington Heights. IL) by electroblotting. Thetransfer of protein and equal loading in all lanes was verified using reversiblestaining with Ponceau S. Membranes were blocked using 5% nonfat dry milk.COX-2 protein was detected by incubation of blots with a monoclonal antibody to a synthetic peptide from the human COX-2 sequence from CaymanChemical Company (Ann Arbor. MI ) at a dilution of 1:1(XX)overnight at 4Â°C,

followed by a sheep antimouse secondary antibody conjugated to horseradishperoxidase and determination of enhanced chemiluminescence (Amersham)using exposure to Kodak BioMax MR film.

Results

Increased Â¡NOSand COX-2 mRNA Expression in Barrett's

Esophagus and Associated Adenocarcinorna. Â¡NOSmRNA wasreadily detectable by RT-PCR in 16of21 (76.2%) of Barrett's tissues,

and COX-2 mRNA was similarly expressed in 17of21 (81%)of thesetissues. As shown in Fig. 1, in all positive cases Â¡NOSand COX-2

levels were increased above those in the gastric control tissues fromthe same patients, and in no case were gastric levels greater than thosein Barrett's mucosa (P = 0.0004 for Â¡NOSand P = 0.0003 forCOX-2; Fisher's exact test). Fig. \A demonstrates the increased iNOS

and COX-2 mRNA abundance in eight representative patient tissue

pairs. Gel densitometry of transcripts (standardized to constitutivelyexpressed ÃŸ-actin)illustrates the pattern of markedly increased Â¡NOS(P = 0.0002; Student's / test) and COX-2 (P < 0.0001) mRNA levelsin the Barrett's tissues compared with paired control stomach tissues.Two of the 21 patients with Barrett's esophagus had dysplasia present

and both were positive for both iNOS and COX-2. As shown in Fig.1C, increased iNOS and COX-2 mRNA expression also occurred inadenocarcinomas arising in Barrett's mucosa. Expression of both

genes was very low to absent in adjacent normal esophagus butreadily detectable in four of five patients for Â¡NOSand five of five forCOX-2.

We sought to compare the frequency of iNOS and COX-2 expression with that of another growth-related gene previously shown to beabundant in Barrett's esophagus, transforming growth factor-a (33).

The results were very similar: transforming growth factor-a mRNAexpression was increased in 17 of 21 cases (81.0%) of Barrett'smetaplasias and in 5 of 5 Barrett's-associated esophageal adenocar

cinomas versus matched gastric epithelium (data not shown). Incontrast, mRNA expression levels of the proinflammatory cytokinestumor necrosis factor-a, interleukin-8, and monocyte chemotacticprotein-1 were not increased in Barrett's tissues compared with gastric

controls (data not shown).Increased iNOS and COX-2 Protein Expression. To confirm the

expression of iNOS and COX-2 at the protein level and to assess theircellular sources, immunostaining for iNOS and COX-2 was per

formed in 10 of the patients, with the following pattern consistentlyobserved (Fig. 2). With iNOS, there was a lack of staining in thegastric body compared with intense staining of metaplastic Barrett's

glands (Fig. 1A and B). There was also no iNOS protein in adjacent

squamous epithelium (Fig. 2B). For COX-2 (Fig. IF), staining localized primarily to lamina propria mononuclear cells and myofibro-blasts, with no detectable staining of the Barrett's epithelium or

adjacent squamous esophageal epithelium. Gastric mucosa showedoccasional low-level COX-2 staining in the lamina propria (Fig. 2E),but of much less intensity than in the lamina propria of the Barrett's

tissue. Particularly intense epithelial iNOS staining was discovered inareas of esophageal dysplasia and adenocarcinoma (Fig. 2C and D). Indysplastic and malignant esophageal glands, COX-2 uniquely local

ized to the epithelium, in addition to the lamina propria mononuclearcell staining seen in gastric control and Barrett's tissues (Fig. 2G

and//).Because we observed COX-2 protein expression in the gastric body

by irnmunohistochemistry, we sought to confirm up-regulation ofCOX-2 in Barrett's tissues by Western blot analysis. Fig. 3 demonstrates increased COX-2 protein abundance in Barrett's metaplasias

relative to paired stomach, as well as increased COX-2 protein expression in Barrett's-associated carcinomas compared with adjacent

esophagus.

Discussion

These results suggest, for the first time, that iNOS and COX-2 areinvolved in Barrett's-associated esophageal neoplasia. Furthermore,

our data suggest that overexpression of these proteins constitutes anearly event in the esophageal neoplastic transformation process whichoccurs at the precancerous metaplastic stage. Overexpression of Â¡NOSand COX-2 was demonstrated in the great majority of metaplastic anddysplastic or cancerous Barrett's specimens, suggesting that this abnormality is involved in most cases of Barrett's lesions at diverse

stages of neoplastic development. Findings obtained at the mRNAlevel (by RT-PCR) were strongly corroborated by similar findings at

the protein level (by immunoblotting or irnmunohistochemistry).Our findings that iNOS and COX-2 were consistently up-regulated

in Barrett's tissues, in contrast to other pro-inflammatory cytokines,

suggest that this is a somewhat specific effect and not simply afunction of generalized inflammation, per se. Moreover, the persistence of increased iNOS and COX-2 expression in dysplasia and

carcinoma implies that induction of these genes may be necessary formaintenance of the malignant phenotype as well. The hypothesis thatiNOS and COX-2 are involved in human neoplastic transformation

has been well supported by data obtained in tumors of other organsystems. This evidence is particularly strong in the case of COX-2,where up-regulation has been demonstrated in colorÃ©ela!and gastric

cancers (20, 21, 24, 25). Furthermore, pharmacological inhibition ofCOX-2 activity has proven effective in reducing colonie polyp for

mation in humans and mouse models as well as in triggering polypregression (22, 23). Mice doubly null for the COX-2 and APC genesshow a marked reduction of polyp formation relative to APC-null

mice alone (21). In vitro data suggest that a key underlying mechanism for the proneoplastic effect of COX-2 is potent inhibition of

epithelial apoptosis coupled with stimulatory effects on epithelialproliferation (18, 34).

2932



Â¡NOSAND COX-2 EXPRESSION IN BARRETT'S ESOPHAGI'S

Our data implicate the involvement of iNOS in GI carcinogenesis.Previous studies have demonstrated increased iNOS expression inbreast and gynecological malignancies (35, 36), but only recently hasiNOS induction been demonstrated in colorectal cancer (28). We andothers have identified increased iNOS expression in H. pylori gastritis(12, 13, 37), but the present study is the first report, to our knowledge,of iNOS expression in upper GI tract dysplasia or carcinoma. NO hasbeen shown to produce DNA damage and mutation in cell-free sys

tems (14, 15), but specific findings have been difficult to demonstratein cultured cells.4 NO can induce wild-type 53 protein expression,

which then down-regulates iNOS transcription via a p53 binding site

in the iNOS promoter (38). This suggests that regulation of iNOSexpression may be one important effect of p53, and that NO may becapable of inducing p53 mutation with chronic stimulation (38, 39).Recently, peroxynitrite, the reaction product of NO and Superoxide,was shown to induce DNA strand breaks in gastric epithelial cellswhereas NO alone had no effect (40). In vivo, nitrosamines, which canbe formed from NO, have long been implicated in upper GI tractcarcinogenesis (41).

It should be noted that both NO and prostaglandins can haveimportant protective effects in the GÃ¬mucosa. Although NOS inhibition has been associated with amelioration of intestinal inflammationin several chemically-induced models, iNOS null mice develop moresevere acetic acid colitis than wild-type controls (42). Moreover,

recent data from one of our laboratories (K. T. W.) indicate thatselective iNOS inhibition worsens the enterocolitis of IL-IO null miceand acute colitis in rat models. Similarly, COX-2 inhibition exacerbates rat models of colitis (9). It is likely that some of the anti-

inflammatory effects of NO and prostaglandins may ultimately proveto be deleterious in the transformation to dysplasia and cancer, however. For example, inhibition of cytokine production by NO andprostaglandins (43, 44) and leukocyte adhesion to vascular endothe-

lium by NO (45) may reduce immune response to tumors. Similarly,induction of apoptosis by NO (39) may ultimately result in increasedepithelial turnover and proliferation as may the cytoprotective effectsof prostaglandins in the GI epithelium.

The localization of iNOS predominantly to the epithelium in theBarrett's tissues is consistent with published findings in human in

flammatory bowel disease (10) and supports a potential role for NO inthe regulation of epithelial cell growth. This role now seems morelikely, in light of the particularly intense iNOS staining that weobserved in dysplastic and malignant Barrett's epithelium. Interestingly, in metaplastic Barrett's mucosa, COX-2 expression was ob

served primarily in the lamina propria, consistent with our findings inintestinal inflammation and H. pylori gastritis; however, upon progression to dysplasia and carcinoma, a shift of staining to the epithelium was observed, suggesting that COX-2 overexpression in these

cells may constitute a relatively late event.Our findings in the present study have important clinical implica

tions. iNOS, COX-2, or both genes may prove valuable as biomarkersof cancer risk for metaplastic and dysplastic Barrett's lesions, as well

as prognostic indicators for frank adenocarcinomas. iNOS or COX-2

may form the basis for future novel therapeutic and/or preventivestrategies using techniques to inhibit these genes, such as molecularmimicry, pharmacological antagonism, or antisense gene therapy. Inaddition, our findings suggest that the involvement of iNOS andCOX-2 should be investigated in other tumor types and premalignant

lesions such as cervical dysplasia, oral leukoplakia, gastric intestinalmetaplasia, ulcerative colitic dysplasia, pancreatic hyperplasia, orearly premalignant skin lesions.

References

to.

14.

16.

19.

20

4 K. T. Wilson and S. J. Meltzer. unpublished observations.

22.

23.

24.

25

Reid. B. J.. and Weinstein, W. M. Barrett's esophagus and adenocarcinoma. Annu.

Rev. Med.. 38: 477-492. 1987.Bartlesman, J. F.. Hameeteman. W.. and Tytgat. G. N. Barrett's oesophagus. Eur. J.

Cancer Prev.. /: 323-325, 1992.

Williamson. W. A., Ellis. F. H.. Jr.. Gibb. S. P.. Shahian. D. M.. Aretz. H. T.. Heatley.G. J., and Watkins, E., Jr. Barrett's esophagus. Prevalence and incidence of adeno

carcinoma. Arch. Intern. Med., 151: 2212-2216, 1991.Rabinovitch. P. S.. Reid. B. J.. Haggitt. R. C.. Norwood, T. H., and Rubin. C. E.Progression to cancer in Barrett's esophagus is associated with genomic instability.

Lab. Invest., 60: 65-71. 1989.

Reid. B. J.. Blount, P. L.. Rubin. C. E., Levine, D. S.. Haggitt, R. C., and Rabinovitch.P. S. Flow-cytometric and histological progression to malignancy in Barrett's esoph

agus: prospective endoscopie surveillance of a cohort. Gastroenterology. 102: 1212-

1219. 1992.Souza. R. F.. and Meltzer, S. J. The molecular basis for carcinogenesis in metaplasticcolumnar-lined esophagus. Gastroenterol. Clin. N. Am., 26: 583-597. 1997.Miller. M. J. S., Thompson. J. H.. Zhang. X-J., Sadowskakrowicka, H.. Kakkis, J. L.,Munshi. U. K.. Sandoval. M., Rossi. J. L., Elobychildress, S.. Beckman. J. S., Ye,Y. Z.. Rodi, C. P., Manning. P. T., Currie, M. G.. and Clark, D. A. Role of induciblenitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis.Gastroenterology. 109: 1475-1483. 1995.Chang, A. D.. Ramanujam. K. S.. and Wilson. K. T. Co-expression of inducible nitricoxide synthase (iNOS), cyclooxygenase (COX-2). and TGF-/3 in rat models of colitis

(Abstract). Gastroenterology. 110: A881, 1996.Reuter. B. K.. Asfaha. S.. Buret. A.. Sharkey. K. A., and Wallace. J. L. Exacerbationof inflammation-associated colonie injury in rat through inhibition of cyclooxygen-ase-2. J. Clin. Invest.. 98: 2076-2085. 1996.Singer. I. I., Kawka, D. W.. Scott, S.. Weidner. J. R.. Mutnford, R. A.. Riehl, T. E.,and Stenson. W. F. Expression of inducible nitric oxide synthase and nitrotyrosine incolonie epithelium in inflammatory bowel disease. Gastroenterology. ///: 871-885.

1996.Fu. S., Ramanujam. K. S., Meltzer, S. J., and Wilson, K. T. Inducible nitric oxidesynthase (iNOS) and cyclooxygenase (COX-2) expression in ulcerative colitis andCrohn's disease. Gastroenterology. 110: A910. 1996.

Wong, A., Fu, S.. Varanasi. R. V., Ramanujam. K. S.. Fantry. G. T.. and Wilson. K. T.Expression of inducible nitric oxide synthase and cyclooxygenase-2 and modulation

by omeprazole in Helicohacler pylori gastritis. Gastroenterology. 112: A332. 1997.Mannick. E. F... Bravo. L. E.. Zarama. G.. Realpe. J. L., Zhang. X. J.. Ruiz. B.,Fontham, E. T.. Mera. R.. Miller, M. J., and Correa. P. Inducible nitric oxide synthase.nitrotyrosine. and apoptosis in Helicnbacler pylori gastritis: effect of antibiotics andantioxidants. Cancer Res., 56: 3238-3243. 1996.

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.

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. Nati. Acad. Sci. USA, 89: 3030-3034. 1992.Plummer. S. M.. Hall. M.. and Faux. S. P. Oxidation and genotoxicity of fecapen-taene-12 are potentiated by prostaglandin H synthase. Carcinogenesis (Lond.). 16:1023-1028. 1995.

Mordan. L. J.. Burnett. T. S.. Zhang. L. X.. Tom. J.. and Cooney, R. V. Inhibitors ofendogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H 10T1/2 fibroblasts. Carcinogenesis (Lond.). 14: 1555-1559, 1993.

Tsujii. M.. and DuBois, R. N. Alterations in cellular adhesion and apoptosis inepithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 83:493-501. 1995.

Boolbol. S. K.. Dannenberg, A. J.. Chadbum. A.. Martucci. C., Guo. X. J.. Ramonetti.J. T.. Abreu-Goris. M., Newmark, H. L., Lipkin. M. L.. DeCosse. J. J.. andBertagnolli. M. M. Cyclooxygenase-2 overexpression and tumor formation are

blocked by sulindac in a murine model of familial adenomatous polyposis. CancerRes., 56: 2556-2560. 1996.

Eberhart, C. E., Coffey. R. J.. Radhika, A.. Giardiello, F. M.. Ferrenbach. S.. andDuBois. R. N. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology. 707: 1183-1188. 1994.

Oshima. M.. Dinchuk. J. E.. Kargman, S. L., Oshima. H.. Hancock. B.. Kwong. E..Trzaskos, J. M.. Evans. J. F., and Taketo, M. M. Suppression of intestinal polyposisin Ape delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87:803-809. 1996.

Giardiello. F. M.. Hamilton. S. R., Krush, A. J., Piantadosi. S.. Hylind. L. M.. Celano.P.. Booker, S. V.. Robinson, C. R.. and Offerhaus. G. J. Treatment of colonie andrectal adenomas with sulindac in familial adenomatous polyposis. N. Engl. J. Med..328: 1313-1316. 1993.

Jacoby. R. F., Marshall. D. J., Newton. M. A., Novakovic, K., Tutsch. K., Cole, C. E.,Lubet. R. A.. Kelloff. G. J.. Verma. A.. Moser. A. R.. and Dove, W. F. Chemopre-

vention of spontaneous intestinal adenomas in the Ape Min mouse model by thenonsteroidal anti-inflammatory drug piroxicam. Cancer Res.. 56: 710-714. 1996.Soydan, A. S.. Gaffen, J. D., Weech, P. K., Tremblay, N. M.. Kargman. S.. O'Neill.

G.. Bennett. A., and Tavares. I. A. Cytosolic phospholipase A2, cyclo-oxygenases andarachidonate in human stomach tumours. Eur. J. Cancer. 33: 1508-1512. 1997.

Rislimaki. A.. Honkanen. N.. Jankala. H.. Sipponen. P.. and Harkonen. M. Expressionof cyclooxygenase-2 in human gastric carcinoma. Cancer Res., 57: 1276-1280. 1997.

2933




26. Jenkins, D. C., Charles, I. G., Thomsen, L. L., Moss, D. W., Holmes, L. S., Baylis,S.A., Rhodes, P., Westmore, K., Emson, P. C, and Moneada, S. Roles of nitric oxidein tumor growth. Proc. Nati. Acad. Sci. USA, 92: 4392-4396, 1995.

27. Moochhala, S., Chhatwal, V. J., Chan, S. T., Ngoi, S. S., Chia, Y. W., and Rauff, A.Nitric oxide synthase activity and expression in human colorectal cancer. Carcino-genesis (Lond.), 17: 1171-1174, 1996.

28. Ambs, S., Merriam, W. G., Bennett, W. P., Felley-Bosco, E., Ogunfusika, M. 0.,Oser, S. M., Klein, S., Shields, P. G., Billiar, T. R., and Harris, C. C. Frequent nitricoxide synthase-2 expression in human colon adenomas: implication for tumor angio-genesis and colon cancer progression. Cancer Res., 58: 334-341, 1998.

29. Ramanujam, K. S., Topczy, N., Fu, S., and Wilson, K. T. Expression and regulationof inducible nitric oxide synthase (Â¡NOS)in intestinal epithelial cells. Gastroenter-

ology, HO: A997, 1996.30. Sherman, P. A., Laubach, V. E., Reep, B. R., and Wood, E. R. Purification and cDNA

sequence of an inducible nitric oxide synthase from a human tumor cell line.Biochemistry, 32: 11600-11605, 1993.

31. DuBois, R. N., Tsujii, M., Bishop, P., Awad, J. A., Makita, K., and LaÃ±aban,A.Cloning and characterization of a growth factor-inducible cyclooxygenase gene fromrat intestinal epithelial cells. Am. J. Physiol., 266: G822-G827, 1994.

32. Bradford, M. M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.,72: 248-254, 1976.

33. Brito, M. J., Filipe, M. I., Linchan, J., and Jankowski, J. Association of transforminggrowth factor a (TGFa) and its precursors with malignant change in Barrett's

epithelium: biological and clinical variables. Int. J. Cancer, 60: 27-32, 1995.34. Sheng, H., Shao, J.. Morrow, J. D., Beauchamp, R. D., and DuBois, R. N. Modulation

of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells.Cancer Res., 58: 362-366, 1998.

35. Thomsen, L. L., Lawton, F. G., Knowles, R. G., Beesley, J. E., Riveros-Moreno, V.,and Moneada, S. Nitric oxide synthase activity in human gynecological cancer.Cancer Res., 54: 1352-1354, 1994.

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

37. Wilson, K. T., Ramanujam, K. S., Mobley, H. L., Musselman, R. F., James, S. P., andMeltzer, S. J. Helicobacter pylori stimulates inducible nitric oxide synthase expression and activity in a murine macrophage cell line. Gastroenterology, ///: 1524-

1533, 1996.38. Forrester, K., Ambs, S., Lupold, S. E.. Kapust, R. B., Spillare, E. A., Weinberg,

W. C., Felley-Bosco, E., Wang, X. W., Geller, D. A., Tzeng, E., Billiar, T. R., andHarris, C.C. Nitric oxide-induced p53 accumulation and regulation of inducible nitricoxide synthase expression by wild-type p53. Proc. Nati. Acad. Sci. USA. 93:2442-2447, 1996.

39. Brune, B., Gotz, C.. Messmer, U. K., Sandau, K., Hirvonen, M. R., and Lapetina,E. G. Superoxide formation and macrophage resistance to nitric oxide-mediatedapoptosis. J. Biol. Chem., 272: 7253-7258, 1997.

40. Kennedy, M., Denenberg, A. G., Szabo, C., and Salzman, A. L. Poly(ADP-ribose)synthetase activation mediates increased permeability induced by peroxynitrite inCaco-2BBe cells. Gastroenterology, 114: 510-518, 1998.

41. Bartsch, H., Ohshima, H., Pignatelli, B., and CalmÃ©is,S. Endogenously formedN-nitroso compounds and nitrosating agents in human cancer etiology. Pharmacoge-netics, 2: 272-277, 1992.

42. McCafferty, D. M., Mudgett, J. S., Swain, M. G., and Kubes, P. Inducible nitric oxidesynthase plays a critical role in resolving intestinal inflammation. Gastroenterology,112: 1022-1027, 1997.

43. Peng, H. B., Rajavashisth, T. B., Libby, P., and Liao, J. K. Nitric oxide inhibitsmacrophage-colony stimulating factor gene transcription in vascular endothelial cells.J. Biol. Chem., 270: 17050-17055, 1995.

44. Betz, M., and Fox, B. S. Prostaglandin E2 inhibits production of Thl lymphokines butnot of Th2 lymphokines. J. Immunol., 746: 108-113, 1991.

45. Kubes, P., Suzuki, M., and Granger, D. N. Nitric oxide: an endogenous modulator ofleukocyte adhesion. Proc. Nati. Acad. Sci. USA, 88: 4651-4655, 1991.

2934



1998;58:2929-2934. Cancer Res Keith T. Wilson, Sidong Fu, Kalathur S. Ramanujam, et al. AdenocarcinomasCyclooxygenase-2 in Barrett's Esophagus and Associated Increased Expression of Inducible Nitric Oxide Synthase and

Updated version

http://cancerres.aacrjournals.org/content/58/14/2929

Access the most recent version of this article at:

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

Subscriptions

Reprints and

[email protected] 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/58/14/2929To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



increased expression of inducible nitric oxide synthase...

Documents