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[Cancer Research 60, 6326-6331, November 15, 2000]
© 2000 American Association for Cancer Research

Biochemistry and Biophysics

Nitric Oxide Synthase Is Induced in Tumor Promoter-sensitive, but not Tumor Promoter-resistant, JB6 Mouse Epidermal Cells Cocultured with Interferon- {gamma}-stimulated RAW 264.7 Cells: The Role of Tumor Necrosis Factor-{alpha}1

Akira Murakami, Kyuichi Kawabata, Teruaki Koshiba, Guanxin Gao, Yoshimasa Nakamura, Koichi Koshimizu and Hajime Ohigashi2

Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493 [A. M., K. K., T. K., K. K.], and Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 [G. G., Y. N., H. O.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-{gamma} for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-{gamma}-treated P+ or P- subclones. Interestingly, when IFN-{gamma}-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8 ± 3.6 µM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0 ± 2.3 µM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-{gamma}-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß. The addition of IFN-{gamma}-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-{alpha}-specific but not IL-1ß-specific antibody. When combined with IFN-{gamma}, the recombinant TNF-{alpha} (1–100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1ß (1–100 ng/ml) did not. These results led us to conclude that IFN-{gamma}-treated RAW 264.7 cells release TNF-{alpha} to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-{alpha} via a paracrine loop to induce epithelial iNOS protein expression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO), a gaseous free radical, is a short-lived molecule that has a multitude of functions in some biological phenomena (1) . NO rapidly and nonenzymatically reacts with superoxide anion to form peroxynitrite anion, a highly toxic molecule causing a wide range of DNA and protein modifications (2) . Conversely, there are a large number of both experimental and clinical reports showing expression of iNOS3 in the tissues of chronic inflammatory diseases, including cancer. For instance, iNOS protein has been induced in premalignant and malignant, but not normal, clinical specimens from such organs as the stomach (3) , colon (4) , lungs (5) , esophagus (6) , prostate (7) , and duodena (8) .

A considerable fraction of the carcinogenic process involves chronic inflammation, particularly in the postinitiation stage. Upon infection with microorganisms, one of the rapid immune responses is leukocyte infiltration and activation, which produces reactive oxygen and nitrogen intermediates, prostaglandins, leukotrienes, and proinflammatory cytokines. Prolonged and excessive leukocyte activation without homeostatic regulation leads to oxidative damage to DNA and to repetitive cell death with compensatory cell division and mutation (9 , 10) . Thus, leukocyte activation plays a number of essential roles in the carcinogenic processes. On the other hand, little is known regarding the biochemical interactions between infiltrated leukocytes and neighboring epithelial cells, where dormant tumor cells originate and then acquire malignancy after repetitive cycles of cell death, regeneration, and tissue remodeling.

A mouse epidermal cell line, JB6, has been widely used to study tumor-promoting factors (11, 12, 13, 14, 15) , antitumor promoters (16, 17, 18, 19) , and the mechanisms of tumor-promoting actions (20, 21, 22) . The experimental strength of this model lies in the establishment of several subclones that can be divided into two distinctive variants, transformation-sensitive (P+) subclones that undergo anchorage-independent transformation in response to tumor promoters and transformation-resistant (P-) variants that do not (23) . Both P+ and P- subclones are recognized to exhibit preneoplastic phenotypes, but only the P+ cells transform into tumor cell (Tx) phenotype in response to tumor promoters, such as phorbol esters or TNF-{alpha} (23) .

In the present study, we attempted to gain insight into the cellular interactions between cocultured activated mouse macrophage RAW 264.7 cells and either JB6 P+ or P- cells. As a result, iNOS protein, constitutively not detectable in either the P+ or P- subclones, was highly up-regulated in P+ but not P- subclones when cocultured with IFN-{gamma}-stimulated RAW264.7 cells. Furthermore, this expression was mediated via macrophage-released TNF-{alpha}. The role of macrophages in epithelial iNOS expression is discussed.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Cells.
DMEM and FBS were purchased from Life Technologies, Inc. (Grand Island, NY). IFN-{gamma} was purchased from Genzyme (Cambridge, MA). All other chemicals were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan), unless specified otherwise. Recombinant mouse TNF-{alpha} and IL-1ß were obtained from Chemicon International, Inc. (Temecula, CA). JB6 subclones were purchased from American Type Culture Collection. RAW 264.7 cells were kindly donated by Ohtsuka Pharmaceutical Co. Ltd. (Ohtsu, Japan).

Monoculture.
RAW 264.7 cells (5 x 105 ) and JB6 (P+ or P-) cells (5 x 105 ) were exclusively preincubated on a membrane culture insert (pore size, 0.45 µm; diameter, 24 mm; Becton Dickinson Labware, Franklin Lakes, NJ) and a 6-well plate (diameter, 35 mm; Becton Dickinson Labware), respectively, in 5 ml of 10% FBS-supplied DMEM for 24 h. After washing the cells with PBS twice, IFN-{gamma} (0 or 100 units/ml) was added to each cell line and incubated in 5 ml of DMEM without FBS or phenol red. After a 24-h incubation, the supernatant thus obtained was used for measuring NO2-, TNF-{alpha}, or IL-1ß. Residual cells were subjected to protein determination and Western blotting. These methods are described below. Each experiment was done in triplicate, and the data are mean ± SD.

Coculture.
A membrane culture insert, upon which RAW 264.7 cells (5 x 105 ) were preincubated in 5 ml of 10% FBS-supplied DMEM for 24 h, was placed in a 6-well plate where JB6 P+ or P- cells (5 x 105 ) were also preincubated under the same conditions. After washing each cell line with PBS twice, IFN-{gamma} (0 or 100 units/ml) was added to the cells, followed by incubation in DMEM without FBS or phenol red. After a 24-h incubation, the supernatant thus obtained was used for measuring NO2-. Residual cells were subjected to protein determination and Western blotting. These methods are described below. Each experiment was done in triplicate, and the data are shown as mean ± SD.

Addition of Activated RAW264.7 to Cell-conditioned Media.
JB6 P+ cells (1 x 105 ) were preincubated in 1 ml of 10% FBS-supplied DMEM for 24 h in a 24-well plate. Cells were then treated with 0 or 100 units/ml of IFN-{gamma} for 12 h as negative and positive controls, respectively. Additionally, the medium from RAW264.7 cells (1 x 105 ) on membrane culture inserts (pore size, 0.45 µm; diameter, 9 mm), cultured for 12 h with IFN-{gamma} (0 or 100 units/ml), anti-TNF-{alpha} (0 or 20 µg/ml), anti-IL-1ß antibody (0 or 20 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and normal goat IgG (0 or 20 µg/ml; Genzyme), were added to JB6 P+ cells (1 x 105 ) that had been preincubated for 24 h independently in a 24-well plate. After a 12 h-incubation with IFN-{gamma} (0 or 100 units/ml) in DMEM, without FBS or phenol red, the concentrations of NO2- as well as the protein amounts were obtained as described below. Each experiment was done in triplicate, and the data are shown as mean ± SD.

Western Blotting.
A boiling lysis solution (1% SDS, 1 mM sodium vanadate, and 10 mM Tris buffer, pH 7.4) was added to the cells, which were then scraped off from the dish, sonicated, and boiled for 10 min. Ten-µg proteins were separated on 10% polyacrylamide gels and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore, MA). After blocking, the membranes were incubated with a primary antibody, either rabbit antimouse iNOS (1:1000 dilution; Affinity Bioreagents, Inc., Golden, CO) or rabbit polyclonal anti-ß-actin antibody (1:1000 dilution; Biochemical Technologies, Stoughton, MA), and then with the respectively corresponding secondary antibodies, antirabbit IgG (1:1000 dilution; Dako, Glostrup, Denmark) or peroxidase-conjugated rabbit antigoat IgG (1:1000 dilution; Dako). The blots were developed using an enhanced chemiluminescence detection kit (Amersham Life Science, Buckinghamshire, United Kingdom). After each development, the antibodies were stripped, and the blots were successively reprobed with each primary antibody. The levels of iNOS, semiquantified using an NIH Image, were corrected using those of ß-actin as an internal standard. Each experiment was done independently in duplicate twice, and the data are shown as mean ± SD.

Addition of TNF-{alpha} and IL-1ß to JB6 P+ Cells.
JB6 P+ cells (1 x 105 ) were preincubated in a 24-well plate in 1 ml of 10% FBS-supplied DMEM. After washing the cells with PBS twice, recombinant TNF-{alpha} (0, 1, 10, or 100 ng/ml), IL-1ß (0, 1, 10, or 100 ng/ml), and IFN-{gamma} (0 or 100 units/ml) were added to the cells and then incubated in DMEM, which was free of both phenol red and FBS. After 24 h, the NO2- concentrations as well as the protein amounts were measured as described below. Each experiment was done in triplicate, and the data are shown as mean ± SD.

NO2- Determination.
The concentrations of NO2- in the medium were determined by a Griess assay, as reported previously (24) , and the medium supernatants were subjected to the assay without dilution.

Cytokine Determination.
The supernatants from the medium at 0, 3, 6, 12, and 24 h after monoculture were subjected to measurement of TNF-{alpha} and IL-1ß concentrations using a commercial experimental kit (Endogen, Inc., Woburn, MA), according to the protocol of the manufacturer. The media supernatants were subjected to the assay without dilution.

Protein Determination.
Protein concentrations were determined using a DC Protein Assay kit (Bio-Rad laboratories, Hercules, CA), with BSA used as the standard.

Cytotoxicity Determination.
Cytotoxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, as reported previously (25) .

Statistical Analyses.
The statistical significance of differences between groups in each assay was assessed by a Student’s t test (two-sided) that assumed unequal variance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
iNOS Protein Expression in RAW264.7 and JB6 P+/- Cells after Monoculture and Coculture.
Stimulation of mouse RAW264.7 macrophages with IFN-{gamma} (100 units/ml) for 24 h led to a dramatic increase in iNOS protein expression (59-fold) and NO2- formation (6.4 ± 0.5 nmol/ml/mg protein, 13-fold; Fig. 1Citation ). In contrast, iNOS expression was not observed in either JB6 P+ or P- cells, with or without IFN-{gamma} treatment. We then investigated the cellular interactions between stimulated RAW264.7 cells and either P+ or P- cells separated by a transwell (pore size, 0.45 µm) in which the secreted factors and medium components can pass through, but the cells themselves cannot. We noted that IFN-{gamma} treatment markedly induced iNOS protein expression (55-fold) in JB6 P+ but not P- cells cocultured with IFN-{gamma} treated RAW 264.7 cells (Fig. 2A)Citation . Accordingly, the NO2- concentration observed in the medium from the RAW/P+ coculture (30.8 ± 3.6 µM) was significantly higher than the sum of the concentrations (20.0 ± 2.3 µM;p < 0.01) added from each cell line monoculture (Fig. 2B)Citation . On the other hand, the RAW/P- coculture generated no such significant synergistic effect (observed, 18.5 ± 4.6 µM; calculated, 17.9 ± 3.6 µM; Fig. 2BCitation ). The levels of iNOS expression in RAW264.7 cells were constant after both monoculture and coculture, with either P+ or P- subclones (Figs. 1Citation and 2A)Citation . No notable cytotoxicity, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was observed in the experiments.



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  		Fig. 1. iNOS protein is induced in IFN-{gamma}-stimulated RAW264.7 but not JB P+/- cells under monoculture. RAW 264.7 cells and JB6 (P+ or P-) cells were separately preincubated on a membrane culture insert and in a 6-well plate, respectively, in 5 ml of 10% FBS-supplied DMEM for 24 h. After washing the cells, IFN-{gamma} (0 or 100 units/ml) was added to and then incubated in 5 ml of DMEM without FBS or phenol red. After a 24-h-incubation, the supernatant thus obtained and the cells were used for measuring NO2- and Western blotting, respectively, as described in "Materials and Methods." n, nonstimulated; s, stimulated with IFN-{gamma}. Each experiment was done in triplicate, and the data are shown as means; bars, SD. *, P < 0.001 versus negative control in Student’s t test.

 


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  		Fig. 2. iNOS protein is induced in P+ but not P- cells cocultured with IFN-{gamma}-stimulated RAW264.7. RAW 264.7 cells were preincubated in 5 ml of 10% FBS-supplied DMEM on a membrane culture insert for 24 h and then placed in a 6-well plate, where JB6 P+ or P- cells were also preincubated in the same conditions. After washing each cell line, IFN-{gamma} (0 or 100 units/ml) was added to the cells, followed by incubation in DMEM without FBS or phenol red. After a 24-h incubation, the cells and supernatants thus obtained were used for Western blotting (A) and measuring NO2- (B), respectively, as described in "Materials and Methods." B: {square}, NO2- concentrations that were added by those shown in each monoculture experiment (columns 1, 3, 5, and 7); {blacksquare}, observed NO2- concentrations in coculture experiments (columns 2 and 4, RAW264.7/JB6P+ coculture; columns 6 and 8, RAW264.7/JB6P- coculture). Columns 1, 2, 5, and 6, nonstimulated; columns 3, 4, 7, and 8, stimulated with IFN-{gamma}. Each experiment was done in triplicate, and the data are shown as means; bars, SD. *, P < 0.001 versus nonstimulated cells (A); *, P < 0.05 versus calculated concentration ({square}) under IFN-{gamma} stimulation (B) in Student’s t test.

 
Secretion of IL-1ß and TNF-{alpha} from RAW 264.7 Cells.
IFN-{gamma}-stimulated RAW264.7 cells are known to release a variety of proinflammatory cytokines. We have previously performed time course studies that measured the secretion of IL-1ß and TNF-{alpha} from these cells after being treated with IFN-{gamma} (100 units/ml). As shown in Fig. 3Citation A, IL-1ß was slightly but significantly released after a 24-h incubation (0.3 ± 0.1 ng/ml/mg protein; P < 0.05), as compared with nonstimulated cells that released no detectable IL-1ß over a 24-h period. In contrast, a dramatic TNF-{alpha} release was observed from 12–24 h (6.9–12.5 nmol/ml/mg protein; P < 0.05 versus control), whereas the nonstimulated cells also spontaneously released TNF-{alpha} (3.8–7.6 nmol/ml/mg protein from 12–24 h) time dependently.



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  		Fig. 3. Proinflammatory cytokines are released from IFN-{gamma}-stimulated RAW264.7 cells. RAW 264.7 cells were preincubated on a membrane culture insert in 5 ml of 10% FBS-supplied DMEM for 24 h. After washing the cells, IFN-{gamma} (0 or 100 units/ml) was added, and the cells were incubated in 5 ml of DMEM without FBS or phenol red. The supernatants from medium after 0, 3, 6, 12, and 24 h of incubation were subjected to the measurement of IL-1ß (A) and TNF-{alpha} (B) concentrations using a commercial experimental kit. {circ} and •, data of nonstimulated and stimulated cells, respectively. Each experiment was done in triplicate, and the data are shown as means; bars, SD. *, P < 0.05 versus nonstimulated cells in Student’s t test (A and B).

 
Effect of Activated RAW 264.7 Cell-conditioned Media on NO2- Production in JB6 P+ Cells.
To confirm that a soluble biochemical factor was released from activated RAW264.7 cells and passed through the transwell to induce iNOS expression in P+ cells, we added conditioned media from 12-h-stimulated P+ cells or RAW264.7 cells to the P+ subclones and then incubated them with IFN-{gamma} for another 12 h. As shown in Fig. 4Citation , neither conditioned medium from IFN-{gamma}-stimulated-P+ cells nor that from nonstimulated RAW 264.7 cells showed any significant increase in NO2- concentration in the media of JB6 P+ subclones, as compared with that observed in the media of P+ cells stimulated by IFN-{gamma} for 24 h. In contrast, the 12-h-IFN-{gamma}-stimulated RAW 264.7 cell-conditioned medium notably enhanced NO2- generation 2.2-fold (P < 0.001). Furthermore, we believe that it is extremely important to note that the specific anti-TNF-{alpha} antibody, but not the anti-IL1-ß antibody or nonspecific IgG, diminished NO2- formation by 84% (P < 0.001).



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  		Fig. 4. Activated RAW264.7 cell-conditioned medium induced NO2- formation in JB6 P+ cells. JB6 P+ cells in a 24-well plate were preincubated in 1 ml of 10% FBS-supplied DMEM for 24 h. Cells were then treated with 0 or 100 units/ml of IFN-{gamma} for 24 h as negative and positive controls, respectively. Additionally, the media from RAW264.7 cells on membrane culture inserts cultured for 12 h with IFN-{gamma} (0 or 100 units/ml), anti-TNF-{alpha} (0 or 20 µg/ml), anti-IL-1ß antibody (0 or 20 µg/ml), and normal goat IgG (0 or 20 µg/ml) were added to JB6 P+ cells that had been preincubated for 24 h independently in a 24-well plate. After a 12-h incubation, with or without IFN-{gamma} in DMEM, the concentrations of NO2- as well as the protein amounts were obtained as described in "Materials and Methods." IFN/P+-med., medium from IFN-{gamma}-stimulated P+ cells. Each experiment was done in triplicate, and the data are shown as means; bars, SD. *, P < 0.001 versus positive control; **, P < 0.001 versus IFN/RAW-med.

 
Effect of Recombinant TNF-{alpha} and IL-1ß on NO2- Production in JB6 P+ Cells.
Treatment of JB6 P+ cells with IFN-{gamma} (100 units/ml) for 24 h led to NO2- accumulation (1.2 ± 0.2 nmol/ml/mg protein; Fig. 5Citation ). The recombinant TNF-{alpha} (1–100 ng/ml), together with IFN-{gamma}, markedly enhanced NO2- production 1.8–3.8-fold (P < 0.05) as compared with the positive control, whereas IL-1ß (1–100 ng/ml) showed no significant synergy with IFN-{gamma}. On the other hand, TNF-{alpha} (1–100 ng/ml), but not IL-1ß, also potentiated NO2- production by 1.5–6.3-fold over the negative control (P < 0.05) in the absence of IFN-{gamma}. The enhancing effects caused by a combination of TNF-{alpha} and IL-ß, with or without IFN-{gamma}, on NO2- formation were almost comparable with those by TNF-{alpha} alone. No notable cytotoxicity was observed in the experiments.



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  		Fig. 5. Enhancing effect of recombinant TNF-{alpha}, but not IL-1ß, on NO2- production in JB6 P+ cells. JB6 P+ cells were preincubated in a 24-well plate in 1 ml of 10% FBS-supplied DMEM. After washing the cells, recombinant TNF-{alpha} (0, 1, 10, or 100 ng/ml), IL-1ß (0, 1, 10, or 100 ng/ml), and IFN-{gamma} (0 or 100 units/ml) were added to the cells and then incubated in DMEM that was free of both phenol red and FBS. After 24 h, the NO2- concentrations, and protein amounts were measured as described in "Materials and Methods." Each experiment was done in triplicate, and the data are shown as means; bars, SD. *, P < 0.05; **, P < 0.02; ***, P < 0.001 versus corresponding NO2- concentrations generated in P+ cells without TNF-{alpha} and/or IL-1ß treatment.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides experimental evidence that IFN-{gamma}-stimulated RAW264.7 cells secrete TNF-{alpha} via a paracrine loop to induce iNOS expression in JB6 P+ but not P- cells. Although not in a statistically significant manner, the RAW 264.7 cell-conditioned medium with added PBS slightly enhanced NO2- production (Fig. 4)Citation . We assume that this was attributable to the effect of the spontaneously released TNF-{alpha} (Fig. 3B)Citation . The TNF-{alpha} concentration after a 12-h stimulation with IFN-{gamma} (1.4 ± 0.3 ng/ml; Fig. 3BCitation ) was within the range of that in medium of a separate experiment that had recombinant TNF-{alpha} added (1–100 ng/ml; Fig. 5Citation ). On the other hand, the possibility of iNOS protein induction by TNF-{alpha} via an autocrine loop from P+ cells can be ruled out, because the stimulated P+ cell-conditioned medium did not significantly enhance iNOS induction. IFN-{gamma} synergistically enhanced the levels of iNOS induction by TNF-{alpha}. A similar synergism between IFN-{gamma} and TNF-{alpha} has been observed previously by Chan et al. (26) and other groups. We also investigated the role of another proinflammatory cytokine, IL-1ß, with iNOS expression in P+ cells, because it was demonstrated previously to induce iNOS expression in murine lung epithelial cells with a combination of both IFN-{gamma} and TNF-{alpha} (27) . However, our present experiments with antibody neutralization (Fig. 4)Citation and recombinant IL-1ß addition (Fig. 5)Citation excluded the involvement of IL-1ß, even as a synergist, from the iNOS-expressing pathways in P+ cells. iNOS protein was expressed in JB6 P+ cells when cocultured not only with IFN-{gamma}-stimulated RAW 264.7 cells but also with those treated with lipopolysaccharide alone and a combination of IFN-{gamma} and lipopolysaccharide.4

TNF-{alpha} has been reported to induce the activation of an iNOS gene transcriptional factor, NF-{kappa}B, via the function of an endosomal acidic sphingomyelinase, the product of which, ceramide, induces degradation of an NF-{kappa}B inhibitor (28) . The iNOS induction by TNF-{alpha} may also be mediated via the Jun NH2-terminal kinase and stress-activated protein kinase signal transduction pathways (29) . In addition, TNF-{alpha} induces transformation of JB6 cells via the Jun NH2-terminal kinase (30) and NF-{kappa}B pathways (21) . COX-2 is another essential inducer of inflammation, and we have found recently that the expression level of COX-2 protein in JB6 P+ but not P- cells increased by 2-fold as compared with the control when cocultured with IFN-{gamma}-stimulated RAW264.7 cells.4 Therefore, the dual induction of iNOS and COX-2 by TNF-{alpha} might play some important roles in the transformation pathways of JB6 cells. The observed lack of NF-{kappa}B activity in P- subclones5 can provide a rationale for the contrastive susceptibility between P+ and P- cells to the TNF-{alpha}-induced iNOS expression. On the other hand, it is notable that TNF-{alpha} and/or IFN-{gamma} did not induce iNOS expression in promoter-resistant P- variants,4 suggesting that cytokine-induced iNOS expression is selective for the epithelial cells in which transformation-related, transcriptional factors, such as activator protein-1 or NF-{kappa}B, are active.

Thus far, noticeable differences in cellular phenotypes between the JB6 P+ and P- subclones have been reported to be involved with the activity of such transcriptional factors as activator protein-1 (31, 32, 33, 34) , arachidonic acid metabolism (35) , oxidation sensitivity (36) , expression of the tissue inhibitor of matrix metalloproteinases (37, 38, 39, 40) , and phospholipid metabolism (41) . The present study shows, for the first time, that there is a substantial difference between these two subclones in their susceptibility to TNF-{alpha}-induced iNOS expression.

Studies with immunohistochemical staining to this point have localized iNOS expression to epithelial cells (3, 4, 5, 6, 7, 8 , 42) , monocytes/macrophages (5 , 43, 44, 45) , joints (46) , and endothelial and smooth muscle cells (47) . Although certain bacteria are known to directly induce epithelial iNOS expression in vitro (48 , 49) , an alternative route for inducing epithelial iNOS expression through interactions between inflammatory leukocytes and epithelial cells might be plausible. The present results lead to the hypothesis that NF-{kappa}B-active epithelial cells themselves express iNOS protein using neighboring macrophage-secreted TNF-{alpha}. Although activated macrophage-induced epithelial iNOS expression remains to be demonstrated in vivo, some coculture studies have shown iNOS expression in colon epithelial cells with T lymphocytes (50) , airway epithelial cells with lung mononuclear cells (51) , and retinal pigment epithelial cells with activated T cells (52) . These findings may support our hypothesis regarding the mechanisms of epithelial iNOS expression. It is important to stress that the above-mentioned three reports did not specifically address which cellular properties are a prerequisite for iNOS expression. As described above, whereas both JB6 P+ and P- subclones exhibit a preneoplastic phenotype, the presence or absence of NF-{kappa}B activity readily differentiates these two variants. In fact, the constitutive activation of NF-{kappa}B is one of the conspicuous cellular properties involved with malignancy and tumorigenicity (53 , 54) . Along a similar line, a coculture of macrophages with the L929 fibrosarcoma cell line showed synergistic NO generation (55) , further suggesting that a neoplastic phenotype is required for epithelial iNOS expression.

The process of clonal expansion of initiated cells requires time, because it involves repetitive cell death, regeneration, and the remodeling of neighboring normal cells. During these repeated steps, inflammation plays a pivotal role in many organs of potential carcinogenesis, such as the stomach and colon. Upon infection with organisms like a virus or bacteria, one of the rapid immune responses is the infiltration and activation of monocytes/macrophages and neutrophils. Neutrophil-mediated inflammatory phenomena have been reported to be the hydroxylation, nitration, and chlorination of protein (56) and DNA (57) by reactive oxygen and nitrogen intermediates, which induce alterations of cellular properties including the acquisition of malignant phenotypes. Although these biochemical modifications may be relevant to the carcinogenic process, it is possible to imagine that neutrophil-derived free radicals have much shorter life spans, limiting their possibility of access to the target molecules of epithelial cells through the extracellular matrix. With this in mind, it can be supposed that iNOS expression in neoplastic cells may cause more severe oxidative injuries to themselves than by intercellular oxidative insult, i.e., endogenously generated NO and its more reactive metabolites, such as peroxynitrite, would rapidly and effectively react with the intracellular components.

In conclusion, the present results lead us to hypothesize that NF-{kappa}B-active epithelial cells induce endogenous iNOS expression through TNF-{alpha} that is secreted via a paracrine loop from activated macrophages. Moreover, this event may lead to more efficient and substantial oxidative injuries to the cells themselves.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Japan Society for the Promotion of Science (to Y. N.) and the Program for Promotion of Basic Research Activities for Innovative Biosciences (to A. M., K. K., H. O.). Back

2 To whom requests for reprints should be addressed, at Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Phone: 81-75-753-6281; Fax: 81-75-753-6284; E-mail: ohigashi{at}kais.kyoto-u.ac.jp Back

3 The abbreviations used are: iNOS, inducible nitric oxide synthase; P+, promotion-sensitive; P-, promotion-resistant; NO2-, nitrite; TNF, tumor necrosis factor; IL, interleukin; FBS, fetal bovine serum; NF-{kappa}B, nuclear factor-{kappa}B; COX, cyclooxygenase. Back

4 A. Murakami, K. Kawabata, and H. Ohigashi, unpublished data. Back

5 T. Hsu and N. H. Colburn, unpublished data. Back

Received 5/16/00. Accepted 9/13/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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