FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum (v14,p1470)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MEI, J. M.
Right arrow Articles by PHANG, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MEI, J. M.
Right arrow Articles by PHANG, J. M.
(The FASEB Journal. 2000;14:1188-1201.)
© 2000 FASEB

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells

JAY M. MEI*,1,2, NORMAN G. HORD*,2,3, DOLORES F. WINTERSTEIN§, STEVEN P. DONALD* and JAMES M. PHANG*,1

* Metabolism and Cancer Susceptibility Section, Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute and
§ Intramural Research Support Program, SAIC-Frederick, NCI-FCRDC, Frederick, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc+/+). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-{gamma} and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused ß-catenin/LEF-1 DNA complex formation. Super-shift by anti-ß-catenin antibody confirmed the presence of ß-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic ß-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of ß-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and ß-catenin/LEF-1 DNA complex formation, we studied IMCE (ApcMin/+) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their ß-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher ß-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble ß-catenin and the formation of nuclear ß-catenin/LEF-1 DNA complex.—Mei, J. M., Hord, N. G., Winterstein, D. F., Donald, S. P., and Phang, J. M. Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.


Key Words: PGHS-2 • NOS II • nitric oxide • colonic epithelial cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
INCREASED EXPRESSION OF prostaglandin endoperoxide H synthase-2 (PGHS-2) is associated with inflammatory bowel diseases and colon cancer (1 2 3 4 5) . Moreover, high PGHS-2 expression in human colon cancer cells is closely associated with increased metastatic potential (6) . In contrast, PGHS-1, a constitutively expressed PGHS family member with high-sequence homology to PGHS-2, is not induced during colorectal neoplastic transformation, supporting the hypothesis that the inducible expression of the PGHS-2 isozyme may be important in the development of colon adenoma and cancer. Nonsteroid anti-inflammatory drugs that inhibit PGHS activity, such as aspirin and the newly developed selective PGHS-2 inhibitors, have well-documented preventive effects against precancerous colon polyp formation and subsequent malignant transformation (7 8 9) . Furthermore, it has been reported that intestinal polyp formation was dramatically reduced by ablation of the PGHS-2 gene (10) . Although PGHS-2 protein catalyzes the first step in the production of a host of bioactive arachidonate metabolites, the production of prostaglandin E2 (PGE2) is probably the most important in carcinogenesis. This prostanoid has been shown to have antiapoptotic and mitogenic activities that may enhance neoplastic progression (11 , 12) .

The role of the adenomatous polyposis coli gene (Apc) as a ‘gate-keeper’ in colonic carcinogenesis and its link with the downstream transcription regulator ß-catenin/Tcf-LEF complexes has been recently established (13 14 15 16 17 18) . It has been hypothesized, although not proven, that PGHS-2 may be one of the genes responsive to ß-catenin accumulation and possibly the ß-catenin/Tcf-LEF transcription pathway (19 , 20) . PGHS-2 mRNA and protein are highly overexpressed in human and mouse colonic adenomas compared with normal tissue (3 4 5) . However, evidence is mounting to support the contributory role for inducible nitric oxide synthase (NOS II) expression in colorectal tumorigenesis. Overexpression of NOS II frequently occurs in inflammatory bowel diseases that predispose the subjects to colon cancer (21 , 22) . Like PGHS-2, NOS II mRNA and protein are overexpressed in colonic adenomas compared with normal tissue (23) . These studies, taken together with in vitro studies of PGHS-2 catalysis by NO, link the production of NO to the regulation of PGHS-2 catalytic activity as well as transcriptional activation of the PGHS-2 gene. Indeed, NO-releasing drugs have been shown to either activate the catalytic function of PGHS-2 or transcription of the PGHS-2 gene, resulting in accumulation of PGHS-2 protein (24 25 26 27) . But, it has not yet been established whether NOS II and PGHS-2 expression are coincident, temporally distinct, or causally related biological events, and the possible mechanism(s) involved remain unknown.

To gain insight into the interconnection between PGHS-2 and NOS II and the potential mechanisms involved, we examined the expression of PGHS-2 and NOS II as well as the production of PGE2 and NO in the nontransformed and nontumorigenic murine colonic epithelial cells. These conditionally immortal cells are designated YAMC (Apc+/+) derived from a SV40LT antigen parental mouse; IMCE (ApcMin/+) are derived from the F1 hybrids resulting from the mating of ApcMin/+ and SV40LT antigen transgenic mice. This pair of nontransformed murine colon epithelial cell lines of similar genetic background includes one that carries the ApcMin/+ mutation and consequently expresses higher ß-catenin levels. Because both YAMC and IMCE express the heat-labile SV40LT antigen that allows them to proliferate at 33°C, they revert to a nontransformed phenotype at the restrictive temperature of 39°C, at which the proliferation of these cells ceases (28 29 30) . The epithelial nature of these cells was demonstrated by staining with anti-keratin antisera. The genotype and expression of adematous polyposis coli (APC) protein of these cell types have been confirmed by allele-specific polymerase chain reaction and by western immunoblotting, respectively (28 , 29) . Furthermore, these cells have been used to demonstrate that the ApcMin/+ mutation in IMCE cells can cooperate with stably transduced oncogenic ras to produce the transformed, tumorigenic phenotype (e.g., growth in soft agar; tumor formation in athymic mice) (31) . In contrast to most transformed cells or cell lines derived from colon tumors, these cells have undetectable (YAMC) or little (IMCE) constitutive PGHS-2 expression by northern and Western blot analysis. On stimulation with IFN-{gamma}/LPS or NO donors at 39°C, they express high levels of PGHS-2 and generate PGE2. This stimulation by LPS and IFN-{gamma} or NO mimics the exposure of colonic epithelial cells to products of bacterial metabolism and immune cells in vivo. Data from the present study suggest that the presence of NO caused by IFN-{gamma}/LPS stimulation or NO-releasing drugs may play an important contributory role in PGHS-2 overexpression in these conditionally immortalized, nontumor-derived, colonic epithelial cells under nontransforming conditions. Furthermore, this overexpression of PGHS-2 is associated, either directly or indirectly, with the accumulation of ß-catenin in the cytoplasm and nucleus, and possibly the formation of the ß-catenin/LEF-1 DNA complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Anti-NOS II, anti-PGHS-2, anti-ß-catenin, anti-E-cadherin, and anti-mouse immunoglobulin G (IgG) horseradish peroxidase monoclonal antibodies were purchased from Transduction (Lexington, Ky.). Anti-ß-catenin polyclonal antibody was from Sigma (St. Louis, Mo.). Anti-actin monoclonal antibody was from Boehringer Mannheim (Indianapolis, Ind.). Murine cDNA PGHS-2 probe was purchased from Oxford Biomedical (Oxford, Mich.). For cell culture, the following products were used and purchased from their respective sources: RPMI 1640 media and mouse IFN-{gamma} from Life Technologies-BRL (Grand Island, N.Y.); neonatal calf serum from Gemini (Calabasas, Calif.), ITS+ from Collaborative (Bedford, Mass.). An ELISA (enzyme-linked immunosorbent assay) test kit for the quantitative analysis of PGE2 was purchased from Cayman (Ann Arbor, Mich.). NOS II inhibitors S-methylisothiourea sulfate (SMT), and L-N6-(1-iminoethyl) lysine (L-NIL) and NO donors (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) and S-nitroso-N-acetylpenicillamine (SNAP) were purchased from Calbiochem (San Diego, Calif.). Bovine hemoglobin was purchased from Sigma and then prepared by dissolving in H2O and reacting with 1 mol excess of sodium dithionite, followed by purification with gel-chromatography (Sephadex G 25, Pharmacia, Piscataway, N.J.). All other chemicals and reagents were purchased from Sigma unless indicated otherwise.

Cell culture
Experiments were carried out using the conditionally immortalized murine colonic epithelial cell YAMC (young adult mouse colon, gift of Dr. Robert Whitehead at Ludwig Institute for Cancer Research, Melbourne, Australia). Both YAMC and IMCE cells express the heat-labile SV40 large T antigen with an IFN-{gamma}-inducible promoter (28 , 29 , 31) . The temperature-sensitive SV40 large T antigen with IFN-{gamma}-inducible promoter is active only at 33°C. It becomes inactivated and nonfunctional once cells are transferred to 39°C. All cells were grown on 75 cm2 culture flask coated with type I collagen (5 µg/cm2) in RPMI 1640 media supplemented with 5% neonatal calf serum, ITS+ (insulin 6.25 µg/ml, transferrin 6.25 µg/ml, selenious acid 6.25 ng/ml, linoleic acid 5.35 mg/ml, and bovine serum albumin 1.25 mg/ml), 5 IU/ml of murine IFN-{gamma}, 100,000 IU/l penicillin and 100 mg/l streptomycin. They were cultured under transforming (permissive) conditions in a 33°C incubator with 5% CO2 plus all the aforementioned supplements in the media. All cells were then transferred on attaining confluency into a 39°C incubator under nontransforming (nonpermissive) conditions in serum-free and IFN-{gamma}-free media for 72 h before each experiment. For NOS II and PGHS-2 induction by inflammatory stimuli, cells in the treatment groups were stimulated by incubation in media containing LPS (1 µg/ml) and IFN-{gamma} (100 IU/ml) for the desired period of time as indicated in each experiment.

Nitrite assay
Culture media from treated cells were incubated for 60 min at 25°C in the presence of 3.5 mU/ml of nitrate reductase and 0.18 nM NADPH to reduce nitrate to nitrite. Nitrite levels were then measured in culture media by adding equal volumes of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride, 2.5% phosphoric acid) and absorbance was read at 550 nm with a microplate reader (Molecular, Sunnyvale, Calif.). Sodium nitrite was used as the standard.

Analysis of PGE2
An ELISA test kit (Cayman) for the quantitative analysis of PGE2 was used that operates on the basis of competition between the enzyme conjugate and PGE2 in the sample for a limited number of binding sites on the monoclonal antibody-coated plate. The sample (culture media from treated cells) or standard solution was first added to the microplate. Next, the diluted enzyme conjugate was added, and the mixture was shaken and incubated at room temperature for 1 h. The plate was then washed, removing all unbound material. The bound enzyme conjugate was detected by the addition of K-Blue Substrate, which generated color intensity after 30 min. Quantitative test results were obtained by measuring and comparing the absorbance reading of the sample wells against the standards with a microplate reader at 650 nm. The extent of color development was inversely proportional to the amount of PGE2 in the sample or standard.

Northern blotting
As described previously (30) , total RNA was prepared from YAMC cells by the Trizol method (Life Technologies-BRL). We electrophoresed 10 µg RNA on a 1% agarose/6.66% formaldehyde gel at 25V for 18 h with 1x MOPS buffer containing 0.2 M MOPS (pH 7.0), 0.08 M sodium acetate, and 0.01 M ethylenediaminetetraacetate (EDTA) (pH 8.0). The RNA was transferred onto Nytran (Schleicher & Schuell, Keene, N.H.) membrane in 20x SSC overnight. The murine cDNA of PGHS-2 (1.9 kb) probe was radiolabeled by random priming using Pharmacia Labeling Beads (dCTP) (Pharmacia). The probe was then hybridized to the RNA blot using 2 x 106 cpm/ml in ExpressHyb (Clontech, Palo Alto, Calif.) hybridization solution at 65°C for 1 h. Blots were washed at a stringency of 50°C in 0.2x SSC/0.1% sodium dodecyl sulfate (SDS).

Western blotting
Cells were washed twice with cold phosphate-buffered saline (PBS) and harvested under either denaturing conditions by scraping in boiling 2x Laemmli sample buffer (Bio-Rad, Hercules, Calif.) or nondenaturing conditions by using a RIPA Buffer Set (Boehringer Mannheim). For total cell lysates under denaturing conditions, samples were heated at boiling temperature for an additional 5 min. Homogenates were then prepared in the Laemmli sample buffer by sonication (1 min each). After centrifugation at 2000 g for 5 min, the supernatants were used as the protein source. To make protein preparations that contain only soluble cytosolic fractions, cells were lysed in RIPA buffer by a micro-homogenizer (Sonifier, Branson, Danbury, Conn.) under nondenaturing conditions at 4°C. The homogenized soluble supernatant was then prepared by centrifugation at 100,000 g for 30 min at 4°C. The protein concentration was determined by the BCA method (Pierce, Rockford, Ill.). Electrophoresis samples were prepared by mixing the respective protein preparations with 2x Laemmli sample buffer. To each well of SDS-polyacrylamide gel, 15–30 µl of cell lysate (~15–30 µg total protein) was applied and electrophoresed in 0.75 mm thick 7.5% Tris-glycine gel (Bio-Rad). Transfer to nitrocellulose membrane was done using a semi-dry blotter (Bio-Rad) at 15V for 30 min. Blots were then probed with the respective primary antibodies at the manufacturers’ suggested dilution, followed by a secondary anti-mouse IgG antibody conjugated to horseradish peroxidase (1:2000). Detection was done using an ECL kit (Amersham, Arlington Heights, Ill.). Blots were routinely stripped by Encore Blot Stripping Kit (Novus, San Diego, Calif.) and reprobed with anti-actin monoclonal antibody to serve as controls.

Preparation of nuclear extracts and electrophoretic mobility shift assays
Nuclear extracts were prepared from YAMC cells according to the method by Dignam et al. with modifications (30 , 32) . Cells were rinsed once with cold PBS, followed by trypsinization. After centrifugation at 1000 g for 5 min, they were resuspended in 5 pellet volumes of hypotonic buffer containing 0.2 mM PMSF and 0.5 mM DTT. They were then chilled on ice for 10 min, followed by lysis with a PT 3000 Polytron (Brinkmann, Littau, Switzerland) for 30 s and centrifuged at 4000 g for 15 min. The pellet was resuspended in 0.5 pellet volumes of low-salt buffer. An equal volume of high-salt buffer was added dropwise to the gently stirred suspension. The nuclear extracts were subjected to centrifugation at 16,000 g for 30 min, followed by dialysis overnight. We added 5 µg nuclear protein to a 20 µl reaction mix containing 300 ng poly dI-dC; binding buffer (10 mM Hepes, pH 7.6; 60 µM KCl; 1 mM EDTA; 1 mM DTT; 12% glycerol); with or without double-stranded mouse LEF-1 oligonucleotide (Life Technologies-BRL), CACCCTTTGAAGCTC with 5' overhang, as a specific competitor. Samples were incubated on ice for 10 min. Then, LEF-1 oligonucleotide, radio-labeled using T4 kinase (Life Technologies-BRL) and {gamma}32P ATP (NEN, Boston, Mass.), was added at 1.5–2 x 104 cpm per reaction and incubated at RT for 30 min. DNA loading dye (Quality Biological, Gaithersburg, Md.) was added to stop the reaction. Samples were run on a 4% polyacrylamide (37.5:1) (Protogel, National Diagnostics, Atlanta, Ga.) gel at 189V for 2.5 h in 0.5x TBE running buffer. Gels were dried and exposed to XAR-5 film (Kodak). For super-shift studies, 3–5 µg nuclear lysate was mixed in a 20 µl reaction mixture as described for electrophoretic mobility shift assays (EMSA) and incubated on ice for 10 min. Antibodies, 12 µg polyclonal anti-ß-catenin and 500 ng monoclonal anti-E-cadherin, or preimmune IgG, were then added to the respective reaction tubes. Reactions were incubated on ice for 15 min. 32P-labeled murine LEF-1 oligonucleotide probe was added at 1.5–2 x 104 cpm per reaction and incubated at RT for 30 min.

Immunocytochemical staining
Cells grown on Lab-Tec chambered coverslips (Nunc, Naperville, Ill.) were fixed in 95% Ethanol at room temperature for 10 min and air-dried. Immunocytochemical staining was performed following the protocol from a Vectastain ABC kit (Vector, Burlingame, Calif.). Coverslips were rinsed in TBS for 10 min before being blocked in 2% nonimmune mouse serum for 20 min. Negative control coverslips were kept in PBS without exposure to the primary antibody, monoclonal mouse anti-ß-catenin antibody (Transduction), whereas the remaining coverslips were incubated for 30 min with the primary antibody at 1:50 dilution. The coverslips were then incubated with 0.5% mouse biotinylated secondary antibody for 30 min and stained with biotin-avidin reagents (Vectorstain Elite ABC kit, Vector) for 30 min, followed by 3,3'-diaminobenzidine tetrahydrochloride staining for 5 min. The coverslips were then counterstained with Gill’s hematoxylin for 6 s.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of PGHS-2 and NOS II induced by inflammatory stimuli IFN-{gamma} and LPS
The expression of PGHS-2 and NOS II was assessed by Western blot in YAMC cells in response to IFN-{gamma} (100 IU/ml) and LPS (1 µg/ml), the classic inflammatory stimuli for PGHS-2 and NOS II induction (Fig. 1A ). Actin served as a control. We found that not only PGHS-2 was induced, as previously reported (30) , but also NOS II was induced by the same stimulation. To ascertain the functionality of these proteins, their enzymatic products PGE2 and nitrite (the stable derivative of NO) were measured (Fig. 1B ). Furthermore, the generation of PGE2 and NO was inhibited by the respective inhibitors of PGHS-2 (sulindac sulfide) and NOS II (L-NIL and SMT), demonstrating the specific functional activity of the induced PGHS-2 and NOS II.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Expression of PGHS-2 and NOS II as well as production of PGE2 and nitrite in YAMC cells. YAMC were cultured in nonpermissive conditions for 72 h before each experiment throughout this study. The nonpermissive conditions are defined as incubation at 39°C in the absence of serum, ITS+, and IFN-{gamma}. A) Expression of PGHS-2 and NOS II induced by IFN-{gamma} and LPS. YAMC cells were stimulated with IFN-{gamma} (100 IU/ml) and LPS (1 µg/ml) for 24 h. PGHS-2 and NOS II proteins were analyzed by Western blot with respective monoclonal antibodies. The same blot was then stripped and probed for actin as a control. This is a representative blot from at least five experiments. B) The generation of PGE2 and nitrite in response to IFN-{gamma} (100 IU) and LPS (1 µg/ml). The culture media from cells treated with IFN-{gamma} and LPS were collected for PGE2 and nitrite measurement. Specific inhibitors of PGHS-2 and NOS II, sulindac sulfide (5 µM) and L-NIL (10 µM) or SMT (10 µM), respectively, were used to co-treat the cells and to block the production of PGE2 and nitrite. Data are the mean ± SD of triplicate determinations and were analyzed by one-way ANOVA with the Duncan test. *P<0.01, compared basal levels with the induced production of PGE2 or nitrite. **P<0.01, compared the induced production of PGE2 or nitrite in the absence and presence of respective inhibitors.

Attenuation of PGHS-2 expression and PGE2 generation by selective NOS II inhibitors
The close interconnection between NO and the enzymatic activity of PGHS-2 has been reported in the literature (24 25 26 27) . NO has been found to stimulate the generation of PGE2 through the activation of PGHS-2 activity. To examine this effect of endogenously produced NO in YAMC, we tested two selective NOS II inhibitors, L-NIL and SMT, on the expression of PGHS-2 and the generation of PGE2 induced by IFN-{gamma} and LPS. We found that the inducible expression of PGHS-2 was greatly reduced by both L-NIL and SMT (Fig. 2A, B ). As expected, L-NIL and SMT also significantly decreased PGE2 generation by blocking endogenous NO production (Fig. 2C ). This experiment not only confirmed that NO increases the enzymatic activity of PGHS-2 but also implied that NO may play a role in regulating the expression of PGHS-2.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Inhibition of PGHS-2 expression and PGE2 generation by L-NIL and SMT. YAMC cells were stimulated with IFN-{gamma} (100 IU/ml) and LPS (1 µg/ml) for 24 h in the absence or presence of specific NOS II inhibitors, L-NIL (10 µM) and SMT (10 µM). Culture media were collected for PGE2 analysis while total cell lysates were prepared for Western blot. Attenuation of PGHS-2 expression by L-NIL (A) and SMT (B). Total cell lysates were analyzed by Western blot using the monoclonal anti-PGHS-2 antibody. The same blot was then stripped and reprobed for actin as a control. This is a representative blot from at least three experiments. C) Inhibition of PGE2 generation by L-NIL and SMT. Data are the mean ± SD of triplicate determinations and were analyzed by one-way ANOVA with the Duncan test. *P<0.01 compared basal levels with the induced production of PGE2. **P<0.01, compared the induced production of PGE2 in the absence and presence of respective inhibitors.

Induction of PGHS-2 by NO-releasing compounds NOR-1 and SNAP
The above findings suggest that NO may exert an induction effect on PGHS-2 overexpression in response to the stimulation of IFN-{gamma} and LPS. In an attempt to determine whether exogenous NO could generate the same effect, NO donors, NOR-1 and SNAP, were used at several different concentrations to treat these cells. The expression of PGHS-2 was then examined by northern and western blotting (Figs. 3 , 4 ). In addition, the enzymatic activity of PGHS-2 was studied by measuring PGE2 production (Fig. 5 ). As demonstrated in these experiments, both NOR-1 and SNAP induced PGHS-2 mRNA expression in YAMC cells in a time-dependent manner (Fig. 3) . NOR-1 and SNAP also caused a dose-dependent induction of PGHS-2 protein (Fig. 4) . These effects were specifically caused by NO released spontaneously from these two chemically distinct compounds as they were abolished in the presence of hemoglobin, a potent NO scavenger (Figs. 4 , 5) . These experiments demonstrated that NO, either endogenously produced or exogenously supplied, can greatly enhance the expression of PGHS-2 in colonic epithelial cells.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 3. Induction of PGHS-2 mRNA by NO donors, NOR-1 and SNAP. A) YAMC cells were treated with either NOR-1 (5 µM) or SNAP (10 µM) for various periods of time (0, 0.5, 2, and 5 h). Total RNA was extracted and analyzed by Northern blot. PGHS-2 (4.3 kb) was probed and found to be induced in a time-dependent fashion. B) The same blot with ethidium bromide stained total RNA was used for loading control. This is a representative blot from three experiments.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. Expression of PGHS-2 protein induced by NO donors, NOR-1 and SNAP. YAMC cells were treated with either NOR-1 or SNAP for 24 h at various concentrations before cells were harvested. The total cell lysates were then prepared and analyzed by Western blot. A) NOR-1 induced expression of PGHS-2 in a dose-dependent fashion. The expression of PGHS-2 protein was analyzed by Western blot in response to NOR-1 treatment at concentrations of 0, 1, 5, and 10 µM. As a positive control, LPS plus IFN-{gamma} (L+I) was used to induce PGHS-2 expression. Hemoglobin (Hb) (1 mM), an avid scavenger of NO, was used to block the NOR-1 (5 µM) effect. The same blot was then stripped and probed for actin as a control. This is a representative blot from three experiments. B) SNAP induced expression of PGHS-2 in a dose-dependent fashion. The expression of PGHS-2 protein was analyzed by Western blot in response to SNAP treatment at concentrations of 0, 5, 10, and 20 µM. As a positive control, LPS plus IFN-{gamma} (L+I) was used to induce PGHS-2 expression. Hemoglobin (Hb) (1 mM) was used to block SNAP (10 µM) effect. The same blot was then stripped and probed for actin as a control. This is a representative blot from three experiments.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 5. Generation of PGE2 in response to NO donors, NOR-1 or SNAP. YAMC cells were stimulated as in Fig. 4 with either NOR-1 (5 µM) or SNAP (10 µM) for 24 h. The culture media were collected and analyzed for PGE2 generation. Hemoglobin (Hb) (1 mM) was used to block the effect of either NOR-1 or SNAP. Data are the mean ± SD of triplicate determinations and were analyzed by one-way ANOVA with the Duncan test. *P<0.01 compared basal levels with the induced production of PGE2 by either NOR-1 or SNAP. **P<0.01 compared the induced production of PGE2 by NO donors with or without concurrent treatment of hemoglobin.

Formation of DNA/ß-catenin/LEF-1 complex induced by NO-releasing compounds NOR-1 and SNAP
It has been suggested that the promoter region of the PGHS-2 gene contains Tcf-LEF binding sites. Therefore, PGHS-2 has been proposed to be under the control of the ß-catenin/Tcf-LEF complex, although definitive proof is still lacking (19) . We found that the mouse PGHS-2 gene promoter does indeed contain potential binding sites of the consensus sequence for Tcf-LEF (see Discussion). We set out to explore the possibility that the expression of PGHS-2 by NO donors in these colonic epithelial cells may be associated with, directly or indirectly, the ß-catenin/Tcf-LEF DNA complex formation. Unlike cell lines derived from colon tumors that often express constitutively high levels of ß-catenin/Tcf-LEF activity, the nontransformed and nontumorigenic YAMC cells express very little ß-catenin/LEF-1 complex when cultured under nontransforming conditions (see Materials and Methods). As shown in Fig. 6 , the formation of the ß-catenin/LEF-1 complex was greatly enhanced by two NO donors, NOR-1 and SNAP. Furthermore, the amount of ß-catenin/LEF-1 complex induced by both NOR-1 (1, 5, and 10 µM) and SNAP (5, 10, and 20 µM) appeared to be concentration-dependent.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 6. Analysis by electrophoretic mobility shift assay (EMSA) of the formation of DNA/ß-catenin/LEF-1 transcription complex by NO donors, NOR-1 or SNAP. The formation of DNA/ß-catenin/LEF-1 complex was greatly enhanced by a 2 h treatment with either NOR-1 (0, 1, 5, 10 µM) or SNAP (0, 5, 10, 20 µM) in a concentration-dependent manner. This is a representative blot from at least five different experiments.

Time-dependence of DNA/ß-catenin/LEF-1 complex formation induced by NO-releasing compounds NOR-1 and SNAP
To further confirm that NO released by NOR-1 and SNAP specifically caused DNA/ß-catenin/LEF-1 complex formation in YAMC cells, a time course study was carried out. Cells were treated with either NOR-1 (5 µM) or SNAP (10 µM) for 10, 30, 60, and 120 min (Figs. 7A, B ). The formation of ß-catenin/LEF-1 DNA complex induced by NOR-1 and SNAP occurred in a time-dependent fashion.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 7. Time course of DNA/ß-catenin/LEF-1 complex formation induced by NOR-1 or SNAP. YAMC cells were treated with either (A) NOR-1 (5 µM) or (B) SNAP (10 µM) for various periods of time (0, 10, 30, 60, 120 min). DNA/ß-catenin/LEF-1 complex formation was induced by either NOR-1 or SNAP in a time-dependent fashion. Unlabeled LEF-1 probe was used as a competitor and effectively competed away the DNA/ß-catenin/LEF-1 complex. This is a representative blot from at least three different experiments.

Super-shifting by anti-ß-catenin antibody of the DNA/ß-catenin/LEF-1 complexes induced by NOR-1 or SNAP
The suggested participation of ß-catenin in the DNA binding complex was confirmed by a series of super-shift assays using an anti-ß-catenin antibody (Figs. 8A, B ). Cells were first treated with either NOR-1 (5 µM) or SNAP (10 µM) for 30 min before nuclear extracts were harvested. Nonspecific antibodies, namely, rabbit preimmune IgG and anti-E-cadherin antibody, were used as controls in the super-shift assays and did not cause any super-shifting of the bands. This study directly demonstrates the presence of ß-catenin in the DNA binding complex in conjunction with LEF-1.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 8. Super-shifting by anti-ß-catenin antibody of the DNA/ß-Catenin/LEF-1 complexes induced by NOR-1 or SNAP. The suggested participation of ß-catenin in the DNA binding complex was confirmed by super-shift assays using a polyclonal anti-ß-catenin antibody. Cells were first treated with either NOR-1 (5 µM) (A) or SNAP (10 µM) (B) for 30 min before nuclear extracts were collected and analyzed by EMSA. The anti-ß-catenin antibody caused a concentration-dependent increase of the amount of super-shift with the following dilutions from stock: 1:12.5, 1:25, and 1:50. Nonspecific antibodies, namely rabbit pre-immune IgG and anti-E-cadherin antibody, were used as controls in the super-shift assays and did not cause any super-shifting of the complex. This is a representative blot from at least four different experiments.

Increase of free ß-catenin in the soluble cytosolic fraction in response to NOR-1 or SNAP treatment
The localization of ß-catenin is normally associated with the transmembrane glycoprotein E-cadherin. The resting ß-catenin level remains very low because of the constant degradation by APC and GSK-3ß in conjunction with Axin (34 , 35) . We attempted to explore the source of the increased nuclear ß-catenin present in the DNA binding complex. The amount of total ß-catenin in whole cell lysate was examined and did not change after various treatments with NO donors, NOR-1 (5 µM) and SNAP (10 µM) (Fig. 9A ). In contrast, the amount of free ß-catenin in the soluble cytosolic fraction showed a dramatic increase from nondetectable in control cells to easily appreciated levels in cells treated with NO donors for 2 h (Fig. 9B ). Although this finding does not reveal the mechanism responsible for the increase in the free cytoplasmic pool of ß-catenin, it implies that NO donors may facilitate the nuclear ß-catenin/LEF-1 complex formation by increasing the amount of free ß-catenin available for its nuclear translocation.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 9. Analysis by Western blot of the redistribution of ß-catenin into a soluble cell fraction in response to NO donors, NOR-1 or SNAP. YAMC cells were stimulated with either NOR-1 (5 µM) or SNAP (10 µM) for 2 h. Cells were then harvested and fractionated into either total cell lysate or soluble fraction preparation. ß-catenin was analyzed by Western blot. A) ß-catenin was detected by Western blot in the total cell lysate and remained constant. We applied 30 µg of total protein into each lane of the pre-cast Bio-Rad 7.5% Tris-HCl gels. ß-catenin was detected by an anti-ß-catenin monoclonal antibody. The same blot was then stripped and reprobed with actin as a control. This is a representative blot from at least three different experiments. B) ß-catenin was only detected in the soluble fraction from cells treated for 2 h with either NOR-1 (5 µM) or SNAP (10 µM), but not that from the untreated cells. The same blot was then stripped and reprobed for actin as a control. This is a representative blot from at least three different experiments.

Subcellular localization and redistribution of ß-catenin
ß-Catenin was primarily localized in the membrane in YAMC cells after culturing under nontransforming condition for 48 h, presumably bound to the transmembrane protein E-cadherin (Fig. 10A ). There was very little, if any, staining of ß-catenin in the nucleus and cytoplasm. However, pronounced nuclear staining of ß-catenin was detected when cells were exposed to NOR-1 (5 µM) overnight (16 h) (Fig. 10B ), indicating a redistribution and nuclear translocation of subcellular ß-catenin in response to NO treatment. Corroborating the findings demonstrated in Fig. 9A, B , immunocytochemical studies showed the increase of ß-catenin in cytoplasm (Fig. 10B ). Similar findings were observed in the redistribution of ß-catenin with SNAP treatment (data not shown).



View larger version (133K):
[in this window]
[in a new window]
  		Figure 10. Demonstration by immunohistochemical staining of the relocalization of ß-catenin from the membrane to the cytosol and nucleus. A) ß-catenin was primarily localized in the membranes of the cell-to-cell borders in YAMC cells that were cultured in nontransforming conditions for 72 h, As a control, no positive staining was observed in sections when the primary anti-ß-catenin antibody was not applied (data not shown). B) Relocalization of ß-catenin in YAMC cells. Cytosolic and nuclear staining for ß-catenin was prominently observed after treatment with NOR-1 (5 µM) for 16 h. x400.

Differential formation of ß-catenin/LEF-1 DNA binding complex and induction of PGHS-2 mRNA and protein as well as production of PGE2 in YAMC and IMCE cells contrasting in Apc genotype
Although the findings with NO treatment suggested a link between PGHS-2 expression and the formation of ß-catenin/LEF-1 DNA binding complex, the effects of NO on PGHS-2 may be mediated by distinct NO-activated pathways. To implicate the participation of ß-catenin in the NO effect on PGHS-2, we compared YAMC and IMCE cells differing in ß-catenin degradation because of their genetic difference in Apc. We found that IMCE cells expressed markedly higher ß-catenin/LEF-1 DNA binding complex than YAMC cells (Fig. 11A ). The difference in ß-catenin/LEF-1 DNA complex between these two cell lines and the corresponding differential expression of PGHS-2 mRNA and protein were both amplified after treatment with SNAP (Fig. 11B, C ). Furthermore, PGE2 production in IMCE was significantly higher than that in YAMC cells (Fig. 11D ). Although this is not definitive proof that ß-catenin/LEF DNA complex is directly responsible for the expression of PGHS-2, it strongly indicates that they are associated either directly or indirectly.



View larger version (57K):
[in this window]
[in a new window]
  		Figure 11. Differential formation of ß-catenin/LEF-1 DNA binding complex and induction of PGHS-2 mRNA and protein as well as production of PGE2 in YAMC and IMCE cells. A) Analysis by EMSA of the formation of DNA/ß-catenin/LEF-1 DNA binding complex by NO donor SNAP. As shown in panel A, the formation of DNA/ß-catenin/LEF-1 complex was greatly enhanced by SNAP (5–20 µM) in a concentration-dependent manner. The specificity of the complex was demonstrated by competition with unlabeled LEF-1 probe. B) Induction of PGHS-2 mRNA by NO donor SNAP. YAMC and IMCE cells were treated with SNAP (10 µM) for various periods of time (0, 0.5, 2, and 5 h). Total RNA was extracted and analyzed by Northern blot. PGHS-2 (4.3 kb) was probed and found to be induced in a time-dependent fashion. The same blot with ethidium bromide-stained total RNA was used for loading control. C) Expression of PGHS-2 protein induced by NO donor SNAP (5–20 µM). SNAP was used at several different concentrations to treat YAMC and IMCE cells for 24 h. The total cell lysates were then prepared and analyzed by Western blot. As demonstrated in panel C, SNAP induced differential PGHS-2 expression in YAMC and IMCE cells in a dose-dependent manner. These effects were specifically caused by NO released spontaneously from SNAP because they were abolished in the presence of hemoglobin (1 mM), a potent NO scavenger. D) YAMC and IMCE cells were stimulated with SNAP (10 µM) for 24 h. The culture media were collected and analyzed for PGE2 generation. Data are the mean ± SD of triplicate determinations and were analyzed by one-way ANOVA with the Duncan test. As demonstrated in this figure, SNAP induced PGE2 generation in YAMC and IMCE cells, which was blocked by hemoglobin (1 mM), a specific NO scavenger. *P<0.01, compared induced levels by SNAP (10 µM) with basal levels of PGE2 within the same genotype group. **P<0.01, compared induced levels of PGE2 caused by SNAP between the two genotype groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The conditionally immortal murine colonic epithelial cells have been used to elucidate the role of NO in the overexpression of PGHS-2. These nontransformed and nontumorigenic cells provide an excellent model system in which to study the early events in colorectal carcinogenesis, such as the inducible expression of PGHS-2 and NOS II. They have an average life span of ~10 days under nontransforming conditions at 39°C when the temperature-sensitive SV40 large T antigen becomes inactive and nonfunctional (28 29 30) . Using the classical stimuli for NOS II and PGHS-2, IFN-{gamma} and LPS, we used these cells to investigate the association between NO generation and PGHS-2 expression as well as the possible roles of soluble free ß-catenin accumulation and ß-catenin/Tcf-LEF complex formation. It is important to note that the effect of IFN-{gamma} and LPS during the treatment is unrelated to the IFN-{gamma} inducible promoter activity because the temperature-sensitive mutant SV40 large T antigen is inactivated and remains nonfunctional at 39°C during the experiment (data not shown) (28 29 30) . The link between PGHS-2 and NO was suggested by the finding that selective NOS II inhibitors, L-NIL and SMT, greatly reduced the overproduction of PGE2 induced by IFN-{gamma} and LPS under the above-described conditions (Fig. 2C ). It is unlikely that these two chemically distinct selective NOS inhibitors nonspecifically inhibited the activity of PGHS-2. Instead, it was likely that perturbations of NO generation modulated PGHS-2 catalytic activity as previously reported (24 25 26 27) . However, to our surprise, we found that both L-NIL and SMT decreased the expression of PGHS-2 induced by IFN-{gamma} and LPS (Fig. 2A , 2B ). To confirm this apparent connection between endogenous NO and PGHS-2 expression, we used two NO-releasing compounds, NOR-1 and SNAP, which do not share any structural similarity; both released NO spontaneously when dissolved in aqueous solution. We found that not only did NOR-1 and SNAP increase PGE2 production (Fig. 5) , they also induced the expression of PGHS-2 at both mRNA and protein levels in YAMC cells (Figs. 3 , 4) . When these cells were co-treated with hemoglobin, an avid scavenger of NO, the induction of PGHS-2 and generation of PGE2 by NO was abolished, indicating the specificity of NO-induced PGHS-2 expression (Figs. 4 , 5) . Furthermore, the induction of PGHS-2 by either NOR-1 or SNAP appeared both time and concentration dependent (Figs. 3 , 4) . To explore the possible role of the ß-catenin/Tcf-LEF pathway in PGHS-2 induction, electrophoretic mobility shift assays were carried out. We found that NOR-1 and SNAP increased DNA binding ß-catenin/LEF-1 complex formation in these cells (Figs. 6 , 7 , 11 A) and unlabeled probe competed away this complex (Fig. 7A, B ; Fig. 11A ). The amount of the ß-catenin/LEF-1 complex formed in response to NO donors, either NOR-1 or SNAP, increased with either dose of the drugs applied or duration of exposure (Figs. 6 , 7 , 11A ). The participation of ß-catenin in the DNA binding complex was confirmed by super-shifting with an anti-ß-catenin antibody, but not by either preimmune IgG or the anti-E-cadherin antibody (Fig. 8) . Further studies indicate that NO may promote ß-catenin/LEF-1 DNA binding complex formation by increasing the level of free soluble ß-catenin but not the total amount of whole cell ß-catenin (Fig. 9) . This finding is corroborated by immunocytochemistry studies showing the redistribution of ß-catenin into the nucleus and cytoplasm (Fig. 10) .

Our demonstration that NO is involved in the accumulation of ß-catenin and activation of the ß-catenin/LEF-1 DNA binding in nontransformed mouse colonic epithelial cells may help elucidate the early molecular events during colorectal carcinogenesis. Several investigators have emphasized the importance of Apc mutations and their effects on ß-catenin in the early phase of colorectal tumor development (13 14 15 16 17 18 , 35 36 37) . From transfection studies in cultured cells and analysis of normal and neoplastic human colorectal tissues, a model has emerged (19 , 20 , 34 , 35) . APC, along with interacting proteins GSK-3ß and Axin, actively degrades ß-catenin and maintains cytoplasmic ß-catenin at very low levels. Direct or indirect disruption of the degradation process resulting in accumulation of cytoplasmic ß-catenin leads to the formation of a transcriptionally active ß-catenin/Tcf-LEF complex (38) . These activities have been associated with the transformation of normal colonic epithelium to adenomas and adenocarcinomas (40 41 42 43) . Although the genes that are controlled by the ß-catenin/Tcf-LEF pathway have not been fully elucidated, the role of ß-catenin in the early stage of colon cancer development is now well demonstrated (35 36 37 38 , 43 44 45) . Thus, our demonstration that NO may activate the ß-catenin/LEF-1 complex may be of considerable importance.

Although the free ß-catenin is being kept at a very low level in the cytoplasm and nucleus as a result of APC-mediated degradation, an abundant amount of ß-catenin is normally associated with the transmembrane protein E-cadherin. The E-cadherin–bound ß-catenin is also called the ‘insoluble’ ß-catenin because it is present in the cytoplasm and nucleus at very low or undetectable levels (Figs. 9 , 10) . It has been reported, however, that the dissociation of ß-catenin from E-cadherin can increase free soluble ß-catenin in the cytoplasm. As a matter of fact, the association of ß-catenin and E-cadherin can be affected by a variety of signals directly or indirectly (46 47 48) . Our data, as demonstrated in Figs. 9 and 10 , suggest that NO may be able to facilitate such dissociation and increase free ß-catenin in the cytoplasm and nucleus. The morphological changes of NOR-1-treated cells (Fig. 10) , namely, the enlargement as well as swelling and blebbing of the cells, could be because of the dissociation of the ß-catenin and E-cadherin complex at the membrane and the subsequent disruption of the adherens junctions at the cell–cell border (49 50) . But, it remains unknown how NO works to dissociate ß-catenin from the membrane-bound cadherins.

It is clearly demonstrated that NO is involved in the release and/or accumulation of ß-catenin into the cytosol and the formation of ß-catenin/LEF-1 DNA binding complex in the nucleus. But, the association of ß-catenin either directly or indirectly to the expression of PGHS-2 required additional evidence. The use of an oncogenic ß-catenin construct in transfection studies was attractive, but we were unsuccessful in transfecting these nontransformed, nonmalignant colonic epithelial cells with the constructs we tried. Therefore, we took advantage of cells differing in Apc genotype. IMCE cells, with their truncated, nonfunctional APC, are defective in their degradation of ß-catenin. Thus, one would expect higher levels of ß-catenin under resting and stimulated conditions. Our finding that IMCE expressed markedly higher ß-catenin/LEF-1 DNA binding complex formation than YAMC is a manifestation of the genotypic difference between these two cell lines (Fig. 11A ). The amplification of the difference after treatment with NO is consistent with the defective degradation machinery for ß-catenin in IMCE cells. The differential formation of ß-catenin/LEF-1 DNA binding complex corresponds to the differential expression of PGHS-2 mRNA and protein as well as the generation of PGE2 (Fig. 11B ,C ,D ). Although NO may also cause PGHS-2 expression and ß-catenin/LEF-1 DNA complex formation by other independent pathways, our data suggests that these two events are functionally connected.

Both PGHS-2 and NOS II genes share a common regulatory pathway in their transcriptional expression; both are under the regulatory control of NF{kappa}B mediated response, either through inflammatory stimulation or hypoxia (51 52 53) . Recently, it has been suggested that the up-regulation of PGHS-2 in colon polyp and adenoma as well as adenocarcinoma may be a result of activation of the ß-catenin/Tcf-LEF pathway (19) . Although the evidence is still inconclusive regarding the precise role of ß-catenin/Tcf-LEF, it clearly indicates that Apc through the Wnt-signaling pathway may be involved in the expression of PGHS-2 (20) . We found that the promoter region of murine PGHS-2 gene (Accession Number s82456, GenBank) does contain several potential binding sites for LEF-1 with close homology (seven out of nine bases as compared with the murine LEF-1 consensus sequence CCTTTGAAG). Two of these tentative LEF-1 binding sites are identical to the consensus sequence within the seven core bases at the center (positions -1963 to -1955: ACTTTGAAT; -2974 to -2966: TCTTTGAAT). Although further studies are needed to confirm that the ß-catenin/Tcf-LEF heterodimeric complex directly modulates transcriptional activation of the PGHS-2 gene, data presented in this paper support that ß-catenin/LEF-1 is at least indirectly involved in this process. Furthermore, this study indicates that PGHS-2 expression may be regulated by NO, presumably through the Apc/ß-catenin pathway, a feature that may represent a level of integration in epithelial cells. Although, in circulating immune cells, NF{kappa}B is known as the primary transcriptional regulator for inflammatory response, the overexpression of PGHS-2 that is often seen in precancerous adenomas as well as malignant carcinomas, may be the result of both pathways. This may help explain the findings reported in animal studies that NOS II inhibitors are potent chemopreventive agents against cancer (54 55 56 57) .

The expression of both NOS II and PGHS-2 has been associated with vascular injury, inflammation, and cellular proliferation in pathological colonic conditions (58 59 60 61 62) . More specifically, in addition to its widespread detection in various malignancies, NOS II is known to be highly expressed in invasive inflammatory bowel diseases and precancerous colonic adenomas as well as carcinomas (21 22 23) . NO can cause DNA damage directly or indirectly through the formation of carcinogenic compounds in the body (59 , 60) . Selective NOS II inhibitors have been demonstrated to exert anti-tumor and chemopreventive effects in various animal models (54 55 56 57) . NOS II expression is relatively decreased in colon adenocarcinomas compared with adenomas, indicating that NO may contribute to colon cancer progression at the transition of colon adenoma to carcinoma in situ (23) . This evidence for a temporally distinct expression pattern in humans shows that the role of NOS II in colon tumor progression may be stage-specific.

The role of PGHS-2 in the cellular inflammatory response and carcinogenesis also has been established. Increased PGHS-2 expression is closely correlated with the malignancies of the gastrointestinal tract, colonic adenoma and carcinoma in particular (3 4 5) . Aspirin, a nonselective inhibitor of both PGHS-1 and PGHS-2, has shown a preventive effect against polyp formation in FAP patients. Recently, the newly developed selective PGHS-2 inhibitors have shown potent chemopreventive effects against colon tumor growth (7 8 9) . The chemopreventive activity of these drugs can be explained, at least partially, by the finding that PGHS-2 and its eicosanoid products have potent anti-apoptotic, mitogenic, and proliferative effects (11 , 12 , 62) .

Our data indicate that one of the consequences associated with elevated NOS II expression in murine colon epithelial cells is the NO-associated induction of PGHS-2 and accumulation of PGE2. Evidence presented in this study supports the notion that these events may be causally linked. Furthermore, they are probably associated with, albeit indirectly, the accumulation of free soluble ß-catenin in the cytoplasm and the formation of ß-catenin/Tcf-LEF DNA binding complex in the nucleus. This is the first direct demonstration in nontransformed and nontumorigenic colonic epithelial cells that the induction of NOS II activity and subsequent NO generation contributes to PGHS-2 overexpression and PGE2 accumulation in response to inflammatory stimuli. Our study suggests that the expression of NOS II and the presence of NO under these pathological conditions may contribute to the overexpression of PGHS-2 that has been frequently observed in colonic epithelium of precancerous adenomas as well as adenocarcinomas. Taken together, these findings lend biological plausibility to the hypothesis that NOS II-derived NO may play an important role in the early stages of colorectal carcinogenesis.


   ACKNOWLEDGMENTS
 
The authors acknowledge the National Cancer Institute for allocation of computing time and staff support at the Frederick Biomedical Supercomputing Center of the Frederick Cancer Research and Development Center.


   FOOTNOTES
 
1 Correspondence: Metabolism and Cancer Susceptibility Section, Basic Research Laboratory, Division of Basic Sciences, Rm 12–90, Bldg. 560, National Cancer Institute, Frederick, MD 21702, USA. E-mail: mei{at}mail.ncifcrf.gov; phang{at}mail.ncifcrf.gov

2 These authors contributed equally to this work.

3 Present address: Department of Food Science and Human Nutrition, Michigan State University, 2100 Anthony Hall, East Lansing, MI 48824-1225.

Received for publication August 3, 1999. Revised for publication November 18, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Singer, I. I., Kawka, D. W., Schloemann, S., Tessner, T., Riehl, T., Stenson, W. F. (1998) Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease. Gastroenterology 115,297-306[Medline]
  2. Hendel, J., Nielsen, O. H. (1997) Expression of cyclooxygenase-2 mRNA in active inflammatory bowel disease. Am. J. Gastroenterol. 92,1170-1173[Medline]
  3. Kutchera, W., Jones, D. A., Matsunami, N., Groden, J., McIntyre, T. M., Zimmerman, G. A., White, R. L., Prescott, S. M. (1996) Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc. Natl. Acad. Sci. USA 93,4816-4820[Abstract/Free Full Text]
  4. DuBois, R. N., Radhika, A., Reddy, B. S., Entingh, A. J. (1996) Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 110,1259-1262[Medline]
  5. Williams, C. S., Luongo, C., Radhika, A., Zhang, T., Lamps, L. W., Nanney, L. B., Beachamp, R. D., DuBois, R. N. (1996) Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology 111,1134-1140[Medline]
  6. Tsujii, M., Kawano, S., DuBois, R. N. (1997) Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc. Natl. Acad. Sci. USA 94,3336-3340[Abstract/Free Full Text]
  7. 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., Dove, W. F. (1996) Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam. Cancer Res 56,710-714[Abstract/Free Full Text]
  8. Boolbol, S. K., Dannerberg, A. J., Chadburn, A., Martucci, C., Guo, X., Ramonetti, J. T., Abreu-Goris, M., Newmark, H. L., Lipkin, M. L., DeCosse, J. J., Bertagnolli, M. M. (1996) Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 56,2556-2560[Abstract/Free Full Text]
  9. Subbaramaiah, K., Zakim, D., Weksler, B. B., Dannenberg, A. J. (1997) Inhibition of cyclooxygenase: a novel approach to cancer prevention. Proc. Soc. Exp. Biol. Med. 216,201-210[Abstract]
  10. Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trzaskos, J. M., Evans, J. F., Taketo, M. M. (1996) Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase-2. Cell 87,803-809[Medline]
  11. Tsujii, M., DuBois, R. N. (1995) Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 83,493-501[Medline]
  12. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., DuBois, R. N. (1998) Bcl-2 expression by prostaglandin E2 in modulation of apoptosis human colon cancer cells. Cancer Res 58,362-366[Abstract/Free Full Text]
  13. Kinzler, K. W., Vogelstein, B. (1996) Lessons from hereditary colorectal cancer. Cell 87,159-170[Medline]
  14. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Josyln, G., Stevens, J., Spirio, L., Robertson, M., Sargeant, L., Krapcho, K., Wolff, E., Burt, R., Hughes, J. P., Warrington, J., McPherson, J. P., Wasmuth, J., Paslier, D. L., Abderrahim, H., Cohen, D., Leppert, M., White, R. (1991) Identification and characterization of the familial adenomatous polyposis coli gene. Cell 66,589-600[Medline]
  15. Oshima, M., Oshima, H., Kitagawa, K., Kobayashi, M., Itakura, C., Taketo, M. (1995) Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc. Natl. Acad. Sci. USA 92,4482-4486[Abstract/Free Full Text]
  16. Moser, A. R., Luongo, C., Gould, K. A., McNeley, M. K., Shoemaker, A. R., Dove, W. F. (1995) ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur. J. Cancer 31,1061-1066
  17. Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B., Polakis, P. (1995) Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. Proc. Natl. Acad. Sci. USA 92,3046-3050[Abstract/Free Full Text]
  18. Morin, P. J., Sparks, A. B., Krinek, V., Barker, N., Clevers, H., Vogelstein, B., Kinzler, K. W. (1997) Activation of beta-cateni-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275,1787-1790[Abstract/Free Full Text]
  19. Prescott, S. M., White, R. L. (1996) Self-promotion? Intimate connections between APC and prostaglandin H synthase-2. Cell 87,783-786[Medline]
  20. Howe, L. R., Subbaramaiah, K., Chung, W. J., Dannenberg, A. J., Brown, A. M. C. (1999) Transcriptional activation of cyclooxygenase-2 in Wnt-1-transformed mouse mammary epithelial cells. Cancer Res 59,1572-1577[Abstract/Free Full Text]
  21. ter Steege, J., Buurman, W., Arends, J. W., Forget, P. (1997) Presence of inducible nitric oxide synthase, nitrotyrosine, CD68, and CD14 in the small intestine in celiac disease. Lab. Invest. 77,29-36[Medline]
  22. Singer, I. I., Kawka, D. W., Scott, S., Weidner, J. R., Mumford, R. A., Riehl, T. E., Stenson, W. F. (1996) Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 111,871-885[Medline]
  23. Ambs, S., Merriam, W. G., Bennett, W. P., Felley-Bosco, E., Ogunfusika, M. O., Oser, S., Klein, S., Shields, P. G., Billiar, T. R., Harris, C. C. (1998) Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res 58,334-341[Abstract/Free Full Text]
  24. Salvemini, D., Misko, T. P., Masferrer, J. L., Seibert, K., Currie, M. G., Needleman, P. (1993) Nitric oxide activates cyclooxygenase enzymes. Proc. Natl. Acad. Sci, USA 90,7240-7244[Abstract/Free Full Text]
  25. von Knethen, A., Brune, B. (1997) Cyclooxygenase-2: an essential regulator of NO-mediated apoptosis. FASEB J 11,887-895[Abstract]
  26. Landino, L. M., Crews, B. C., Timmons, M. D., Morrows, J. D., Marnett, L. J. (1996) Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc. Natl. Acad. Sci. USA 93,15069-15074[Abstract/Free Full Text]
  27. Salvemini, D. (1997) Regulation of cyclooxygenase enzymes by nitric oxide. Cell. Mol. Life Sci. 53,576-582[Medline]
  28. Whitehead, R. H., Joseph, J. L. (1994) Derivation of conditionally immortalized cell lines containing the Min mutation from the normal colonic mucosa and other tissues of an "Immortomouse"/Min hybrid. Epith. Cell Biol. 3,119-125[Medline]
  29. Whitehead, R. H., VanEeden, P. E., Nobel, M. D., Ataliotis, P., Jat, P. S. (1993) Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc. Natl. Acad. Sci. USA 90,587-591[Abstract/Free Full Text]
  30. Mei, J. M., Hord, N. G., Winterstein, D. F., Donald, S. P., Phang, J. M. (1999) Differential expression of prostaglandin endoperoxide H synthase-2 and formation of activated beta-catenin-LEF-1 transcription complex in mouse colonic epithelial cells contrasting in Apc. Carcinogenesis 20,737-740[Abstract/Free Full Text]
  31. D’Abaco, G. M., Whitehead, R. H., Burgess, A. W. (1996) Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas. Mol. Cell. Biol. 3,884-891
  32. Dignam, J. D., Lebovitz, R. M., Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11,1475-1489[Abstract/Free Full Text]
  33. Sakanaka, C., Weiss, J. B., Williams, L. T. (1998) Bridging of beta-catenin and glycogen synthase kinase-3 beta by axin and inhibition of beta-catenin-mediated transcription. Proc. Natl. Acad. Sci. USA 95,3020-3023[Abstract/Free Full Text]
  34. Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. H., Mas