Cytokines differentially regulate the synthesis of prostanoid and nitric oxide mediators in tumorigenic versus non-tumorigenic mouse lung epithelial cell lines

Received August 26, 2004; revised and accepted February 22, 2005

Lori D. Dwyer-Nield *, Mary C. Srebernak, Bradley S. Barrett, Jinhee Ahn, Pippa Cosper, Amy M. Meyer 1, Lori R. Kisley, Alison K. Bauer 1, David C. Thompson and Alvin M. Malkinson

Department of Pharmaceutical Sciences and 1 Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262, USA

* To whom correspondence should be addressed. Tel: +1 303 315 6769; Fax: +1 303 315 6281; Email: Lori.Nield{at}uchsc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studies using transgenic and knockout mice have demonstrated that particular cytokines influence lung tumor growth and identified prostaglandin E2 (PGE2), prostacyclin (PGI2) and nitric oxide (NO) as critical mediators of this process. PGE2 and NO were pro-tumorigenic while PGI2 was antitumorigenic. We describe herein an in vitro experimental approach to examine interactions among cytokines, prostaglandins (PGs) and NO. PGE2, PGI2, and NO levels were assayed in culture media from non-tumorigenic mouse lung epithelial cell lines, their spontaneous transformants and mouse lung tumor-derived cell lines, before or after exposure to the cytokines TNF{alpha}, IFN{gamma} and IL1ß, alone and in combination. More PGE2 than PGI2 was produced by neoplastic cells, while the opposite was observed in non-tumorigenic lines. Cytokine exposure magnified the extent of these differential concentrations. The PGE2 to PGI2 ratio was also greater in chemically-induced mouse lung tumors than in adjacent tissue or control lungs, supporting the physiological relevance of this in vitro model. Expression of PG biosynthetic enzymes in these cell lines correlated with production of the corresponding PGs. Cytokine treatment enhanced NO production by inducing the inflammation-associated biosynthetic enzyme, inducible NO synthase (iNOS), but this did not correlate with the neoplastic status of cells. Inhibition of iNOS or cyclooxygenase 2 activity using aminoguanidine or NS-398 respectively, demonstrated that NO did not affect PG production nor did PGs influence NO production. Since lack of iNOS inhibits mouse lung tumor formation, we propose that this is independent of any modulation of PG synthesis in epithelial cells. The similar normal/neoplastic trends in PGE2 to PGI2 ratios both in vitro and in vivo, together with an amplification of this difference upon cytokine exposure, are consistent with the hypothesis that cytokines released during inflammation exacerbate differences in the behavior of neoplastic and normal lung cells.

Abbreviations: COX, cyclooxygenase; COX-2, cyclooxygenase 2; cPLA2, cytosolic phospholipase A2; HRP, horse radish peroxidase; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PGI2, prostacyclin; PGIS, prostacyclin synthase; PGs, prostaglandins; PGE2, prostaglandins E2; PGES, prostaglandin E2 synthase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epithelial cells in inflamed tissue are not passive bystanders but active contributors to the inflammatory milieu (1). The lungs constitute the largest epithelial area in the body and receive the entire cardiac output from the pulmonary artery. As a result, the lungs must adapt to continual exposure to inhaled substances from the external environment and to substances circulating in the bloodstream. How the pulmonary epithelium interfaces with inflammatory mediators secreted by resident and infiltrating inflammatory cells as part of the healing aspects of acute inflammation or in pathologies caused by chronic inflammation is currently unclear. Pulmonary innate immunity is a first line of antimicrobial and antiparticulate defense. Chronic inflammatory states where this protective function goes awry result in obstructive diseases such as bronchitis, emphysema and asthma. Dysregulation of inflammation predisposes to lung cancer, as shown by epidemiologic (2) and familial clustering (3) studies and by pharmacologic (4) and genetic (5,6) experiments in mouse models.

Mechanisms by which inflammation encourages lung tumor growth are being intensely studied (7,8). Inflammatory cell infiltration into the lungs correlates with poor prognosis of lung cancer patients (9). Linkage analysis of complex traits in mice have identified inflammation-related loci that affect susceptibility to lung cancer (10). Treatment of mice with anti-inflammatory drugs prevents carcinogen-induced lung tumor formation (4), and mice that overexpress anti-inflammatory genes (5) or are null for pro-inflammatory genes (6) have decreased lung tumor susceptibility. The coordinated activities of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and 5-lipoxygenase can achieve a complete inflammatory response (7). In vitro models using human lung cell lines derived from tumors demonstrate how the products of these enzymes affect cell physiology (11), proliferation (12) and cell death (12). The physiological consequences of cancer-related alterations in prostaglandin (PG) production are not fully understood. Upon synthesis, PGs released from cells act as autocrine or paracrine signals affecting resident mast cells, histocytes, endothelial cells, infiltrating inflammatory cells and fibroblasts (13). Regulatory roles for PGs in tumorigenesis include stimulating angiogenesis (14) and cellular invasiveness (15), immmunosuppression (16) and inhibition of apoptosis (17), each a hallmark of tumorigenesis (18). Glucocorticoids reduce inflammation through many distinct activities, including decreasing PG production (19). The non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin (20) and sulindac (21), inhibit COX activity and reduce mouse lung tumor formation, although treatment with celecoxib, a COX-2 specific inhibitor, did not (22). By inhibiting COX activity, NSAIDs reduce the formation of both prostaglandin E2 (PGE2) and prostacyclin (PGI2). However, they may also modulate PPAR, NF{kappa}B, and MAPK activities independent of COX activity, making interpretation of these results difficult (23).

Gene targeting of hormones and enzymes contributing to inflammation in mice have helped to define their roles in initiating and/or promoting lung tumor growth. For example, haploinsufficiency of the pro-inflammatory cytokine, TNF{alpha}, lowers lung tumor multiplicity (24), as does genetic deletion of each of the following proteins: cPLA2 which releases arachidonate from membrane phospholipids (25), PGE2 receptor 2 (EP2) which activates signaling pathways after binding PGE2, and iNOS which produces large amounts of nitric oxide (NO) during chronic inflammation (6). Conversely, overexpression of prostacyclin synthase (PGIS) in the alveolar type 2 and bronchiolar Clara cells that serve as the cellular precursors of mouse lung tumors decreases tumor formation (5). In the development of mouse lung tumors, therefore, TNF{alpha}, PGE2 and NO may be postulated to be pro-tumorigenic while PGI2 is antitumorigenic. PGI2 has anti-inflammatory properties in the lungs (26), while PGE2 is typically pro-inflammatory (27,28). One of the many actions of NO is to either up-modulate or downmodulate cyclooxygenase (COX) activity (29), while PGs can affect iNOS activity (30).

PGIS expression was targeted to lung epithelial cells (5), but the cellular sources (macrophages, epithelial cells, etc.) for the other inflammatory mediators are not apparent. Using immunohistochemistry, both the tumor parenchyma and peritumoral macrophages express cPLA2 (25), COX-1 and COX-2 (31), microsomal prostaglandin E2 synthase (PGES) (25) and iNOS (6). We used a panel of non-tumorigenic and neoplastic mouse lung epithelial cell lines with well characterized molecular and proliferative properties (32) to examine basal production of PGE2 and PGI2 and induction of these PGs, in response to cytokine prominent in pulmonary inflammation. ‘Cytomix’, a mixture of the inflammatory cytokines TNF{alpha}, IFN{gamma} and IL1ß, induces NO and PG production both clinically and in cell culture (3335). We therefore investigated whether TNF{alpha}, IFN{gamma} and IL1ß affect PG production in these cell lines, and if NO contributes to tumorigenesis by influencing the synthesis of PGE2 and PGI2. We find that PGE2 and PGI2 production differ between normal and neoplastic cells, and these cytokines amplify this distinction. Cytokines induce the synthesis of COX and iNOS, but pharmacologic inhibition of these enzymes shows no cross-talk between these inflammatory pathways.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
We examined two non-tumorigenic cell lines, their respective spontaneous transformants and four tumor-derived lines that have been described in detail (32). C10 and E10 cells were originally derived from a normal BALB/c mouse lung explant and are non-tumorigenic, contact-inhibited and had alveolar type 2 cell features at early passage (36). A5 and E9 are the respective spontaneous neoplastic transformants of C10 and E10 (36). These four cell lines were maintained in CMRL 1066 medium. LM1 and LM2 cells were derived from solid and papillary urethane-induced A/J mouse lung tumors, respectively, and were maintained in MEM{alpha} (32). The 82–132 cells were derived from an ethylnitrosourea, transplacentally-induced, solid lung tumor in a Swiss–Webster mouse and cultured in DMEM (37). PCC4 cells were derived from a spontaneous BALB/c papillary tumor and were cultured in McCoy's media (32). All cell lines were maintained in their respective media (Invitrogen, Carlsbad, CA) and supplemented with 10% fetal bovine serum (FBS; Gemini Bioproducts, Woodland, CA), 100 U/ml penicillin, 100 µg/ml streptomycin and 100 µg/ml amphotericin B (Invitrogen), and grown in a humidified atmosphere of 5% CO2 in air at 37°C. Cells were passaged twice weekly by trypsinization, and, unless otherwise stated, harvested 1–2 days after reaching confluence.

PGE2 and PGI2 determination
Media samples (1 ml) were collected prior to cell harvest and stored at –80°C. ELISA immunoassays (Cayman Chemical, Ann Arbor, MI) for PGE2 and 6-keto PGF1{alpha} [the stable metabolite of PGI2 (38)] were performed in duplicate according to manufacturer's instructions. Samples were diluted so that values were in the linear portion of standard curves.

Cytokine administration
Cells were grown (3 plates/condition) to confluence, serum-deprived for 24 h and treated with serum-free media containing 10 U/ng mouse IL1ß, 10 U/ng mouse TNF{alpha} or 15 U/ng mouse IFN{gamma} (Biosource, Camarillo, CA), pairs of cytokines or a combination of all three for 24 h. Control samples were serum-deprived for 24 h and then incubated with serum-free media for an additional 24 h.

Tissue PGE2/PGI2 determination
A/J mice [obtained at 4–6 weeks of age from Jackson Laboratories (Bar Harbor, ME] were housed on hardwood bedding with 12 h light/dark cycle and given water and food (Teklad) ad libitum. At 6–8 weeks of age, mice were injected with urethane (1 mg/g body wt in 0.9% saline, i.p.). Control mice received an equivalent volume of vehicle. A/J mice produce ~30 adenomas ranging from 0.6 to 1.2 mm in diameter after several months using this protocol (39,40). Mice were sacrificed by lethal IP phenobarbital injection intraperitoneally, 24 weeks after urethane treatment. Lungs were excised, and tumor and uninvolved lung tissue separated. Uninvolved (non-tumor tissue from tumor-bearing lungs) and control (lung tissue from age-matched naive mice) samples were weighed and placed in 4 volumes (w/v) of methanol. All tumors from each mouse were counted and pooled, and this pooled sample was weighed and immersed into 200 µl methanol. After Dounce-homogenization and centrifugation at 16000 g to remove tissue debris, supernatants were removed for analysis of PGE2 and PGI2 by ELISA (see previous section). PGE2 and PGI2 concentrations were expressed as pg/mg tissue.

Sample preparation for immunoblotting
Cultured cells were harvested by scraping, washed with 0.9% NaCl, resuspended in homogenization buffer [20 mM HEPES, pH 7.5, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 10% glycerol, 5 µg/ml aprotinin, 10 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride], disrupted by sonication, and centrifuged at 16000 g for 25 min. The resulting soluble fractions were used for cPLA2 and iNOS immunoblotting. The particulate fractions were resuspended in the above buffer +0.1% NP-40 and used for COX-1 and COX-2 immunoblotting. Whole cell homogenates (used for mPGES and PGIS immunoblotting) were prepared from cells (3 plates/cell line) harvested by scraping cells into 0.9% NaCl. Cells were collected by centrifugation at 2000 g and lysed in 15 mM Tris base pH 7.5, 2 mM EDTA, 20% glycerol, 50 mM ß-mercaptoethanol, 0.1% Triton X-100, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1 mM sodium vanadate and 1 mM sodium fluoride. Protein concentrations in each sample were determined by the method of Lowry et al. (41), and samples were mixed 1:1 with 2x sample loading buffer (100 mM Tris pH 6.8, 0.4% sodium dodecyl sulfate, 2% ß-mercaptoethanol, 20% glycerol and 0.3% pyronine Y).

cPLA2, COX-1, COX-2, PGES, PGIS and iNOS immunoblotting
One hundred micrograms of soluble (cPLA2, iNOS), particulate (COX-1, COX-2) or whole cell homogenate (mPGES, PGIS) protein/lane was applied to either 10% SDS-PAGE (cPLA2, COX-1, COX-2, PGIS, and iNOS) or 15% SDS–PAGE (mPGES). Separated proteins were transferred onto an Immobilon-P PVDF membrane (Millipore, Bedford, MA).

Immunoblot analysis of cPLA2 was performed as described previously (25). In brief, membranes incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at a 1:300 dilution in blocking solution, then incubated with goat anti-mouse alkaline phosphatase secondary antibody (Bio-Rad, Hercules, CA; 1:3000 dilution) for 1 h. cPLA2 bands were visualized by incubation with Immun-Star chemiluminescent substrate (Bio-Rad, Hercules, CA) followed by exposure to CL-XPosure X-ray film (Pierce, Rockford, IL). The 85 kDa cPLA2 protein migrates at ~100 kDa. Protein bands were quantified by densitometry using Un-Scan-It software (Silk Scientific Corporation, Orem, UT). To confirm even protein loading of the gels, the membranes were stained with 0.1% Ponceau S (Fisher Biotech, Fair Lawn, NJ) in 5% acetic acid. We were unable to identify a house-keeping gene that was expressed equally in all eight cell lines; quantification of a single protein product for determining equal loading was therefore not feasible.

COX-1 and COX-2 protein immunoblotting was performed as previously described (31); the respective antibodies used are not cross-reactive (31). In brief, membranes were incubated with COX-1 or COX-2 primary antibody (Santa Cruz; 1:800 dilution), washed and incubated with horseradish peroxidase (HRP)-labeled donkey anti-goat secondary antibody (Santa Cruz Biotechnology; 1:20000 dilution). After treatment with Super Signal West HRP-substrate (Pierce Biotechnology; Rockford, IL), membranes were exposed to X-ray film and the resulting 72 kDa bands were quantified as above.

The mPGES protein was detected using a 1:500 dilution of a rabbit polyclonal primary antibody (Cayman Chemical, Ann Arbor, MI). Primary antibody incubation was followed by incubation with a 1:20000 dilution of HRP-conjugated anti-rabbit secondary antibody (Transduction Lab, Lexington, KY). After exposure to Super Signal West HRP-substrate, membranes were exposed to X-ray film and the resulting 14 kDa bands quantified as above.

PGIS protein was detected using a 1:1000 dilution of a rabbit polyclonanl antibody directed against mouse PGIS (kindly provided by Dr David DeWitt, Michigan State University, East Lansing, MI). Immunoblots were incubated overnight in primary antibody followed by 1 h incubation with 1:20000 dilution of HRP-conjugated, anti-rabbit secondary antibody (Santa Cruz Biotechnology). After exposure to Super Signal West reagent, membranes were exposed to X-ray film and the resulting 49 kDa bands were quantified as above. The 56 kDa protein migrates at 49 kDa on a 10% acrylamide gel.

The enzyme iNOS was detected and quantified in the soluble protein fraction using a modification of Kisley et al. (6). Membranes were incubated with a rabbit polyclonal antibody to iNOS (Santa Cruz; 1:1000 dilution) followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology; 1:20000 dilution). Blots were incubated with Super Signal West reagents and the resulting 110 kDa band quantified as above.

NO determination
NO production by cells was estimated by measurement of its nitrite and nitrate metabolites by a colorimetric assay/LDH method (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's instructions. Media was collected prior to cell harvest and frozen at –80°C until assayed. Samples were diluted so that values were in the linear portion of the standard curve.

Inhibitor treatment
The cell-permeant, COX-2 specific inhibitor, NS-398 (Sigma, St Louis, MO), was freshly prepared in dimethyl sulfoxide, diluted into serum-free media immediately before use and added to cells at final concentrations of 0.01, 0.1 and 1.0 µM. The iNOS inhibitor aminoguanidine (AMG; Sigma) was prepared fresh in the appropriate serum-free media and added to cells at a final concentration of 1 mM, a concentration shown previously to inhibit NO production in these cells (42). Both inhibitors were added to cells upon serum deprivation.

Statistical analysis
Experimental results are represented in graphs as means ± SEM. Data were analyzed by one-way ANOVA followed by Newman–Kuels post hoc test for comparisons of multiple data sets and by Student's t-test for comparison of two data sets using GraphPad Prism version 3.20 for Windows (San Diego, CA), with statistical significance at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Basal and cytokine-induced PGE2 and PGI2 production
PGE2 and 6-keto PGF1{alpha} (the stable metabolite of PGI2) were assayed in the media of untreated cells or of cells exposed to ‘cytomix’, a mixture of TNF{alpha}, IFN{gamma} and IL1ß clinically used to treat certain pulmonary diseases (33) (Table I). All six tumorigenic cell lines basally produced orders of magnitude more PGE2 than the two non-tumorigenic lines (C10 and E10), while the opposite was true for 6-keto PGF1{alpha}. In C10 cells, both PGs were highly induced by cytokines, with PGE2 slightly more than PGI2. Media content of PGI2 but not PGE2 increased in E10 cells exposed to cytokines. PG production by the A5 and E9 spontaneous transformants and tumor-derived 82–132 cells were not affected by cytokines. Media concentrations of PGE2 were induced in three tumor-derived lines (LM1, LM2 and PCC4) by cytokines while PGI2 production was induced only in LM2 and PCC4 cells. Both cell line selectivities and differential capacities for PGE2 and PGI2 production in response to cytokine exposure were thus demonstrated.


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Table I. PGE2 and PGI2 accumulation in the media of control and cytokine-treated mouse lung epithelial cell lines

 
When the ratio of PGE2 to PGI2 accumulation was calculated, C10 and E10 cells had ratios <1 while this ratio was >1 in neoplastic cells (Table II). Cytokine treatment significantly amplified this ratio in three of the six tumorigenic cell lines but not in C10 or E10 cells. The ratios for C10 and E10 cells are 300-fold less than their respective spontaneous transformants (A5 and E9), whose PG levels are unchanged by cytokine treatment. The PGE2 to PGI2 ratio in these transformants is similar to three of the tumor-derived lines (82–132, LM2 and PCC4) in the absence of cytokines and remains similar to 82–132 cells treated with cytokines (since this line is also unaffected by cytokines). Untreated and cytokine-induced LM1 cells and cytokine-treated LM2 and PCC4 cells have the highest PGE2 to PGI2 ratios. This increased PGE2 to PGI2 ratio in neoplastic versus non-neoplastic cells is consistent with that determined using human lung cell lines (5,12).


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Table II. Basal and cytokine-induced PGE2 to PGI2 ratios in lung epithelial cell lines

 
Steady-state PGE2 and PGI2 concentrations in extracts prepared from age-matched, untreated control mouse lung, chemically-induced murine lung tumors, as well as uninvolved (normal-appearing tissue from tumor-bearing lungs) lung tissue were determined (Figure 1). Extracts prepared from control tissue contained more PGI2 than PGE2. Both PGs increased in neoplastic lungs but to different extents. The PGE2 to PGI2 ratio in lung tissue from control mice was <1, similar to that found in the media of non-tumorigenic C10 and E10 cell lines, while the higher ratio in tumor extracts is analogous to that observed in neoplastic cell lines (Table II). Uninvolved tissue contained more of both PGs than control tissue, suggestive of an inflammation associated with the presence of neoplastic tissue.



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Fig. 1. PGE2 and PGI2 concentrations in extracts from normal and neoplastic lung tissue. PGE2 (white bars) and PGI2 (black bars; as measured by its stable metabolite, 6-keto PGF1{alpha}) contents in lungs from age-matched control mice (N = 10), non-tumor bearing lung tissue from mice with lung tumors (uninvolved; N = 15), and lung tumor tissue (tumors pooled from each mouse; N = 15). The PGE2 to PGI2 ratio of each sample is indicated in italics. *P < 0.01 versus both control and uninvolved tissue. Data represent mean ± SEM of the combined results of two independent experiments.

 
Enzymes in the PG pathway
To determine whether PGs produced by cell lines reflect expression of the relevant biosynthetic enzymes, we examined basal expression of mPGES and PGIS by immunoblot analysis. The mPGES content was highest in LM1 cells and lowest in the non-tumorigenic lines, with the other lines exhibiting intermediate amounts (Figure 2A). Plotting densitometry from the mPGES immunoblot against PGE2 levels in each cell line yielded a linear correlation (R2 = 0.95; Figure 2B). PGIS expression correlates with the amount of PGI2 detected in the culture media (Figure 2C and D), with C10 and E10 cells expressing the most PGIS and producing the most PGI2 (R2 = 0.99). The data are consistent with PGE2 and PGI2 production in these cell lines being dependent on the intracellular concentrations of their respective synthases.



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Fig. 2. Immunoblot analysis of mPGES and PGIS expression in mouse lung epithelial cell lines. (A) mPGES immunoblot; (B) comparison of basal mPGES protein expression with basal PGE2 levels (determined in media from the cells used for A, 3 points/cell line); R2 = 0.95. (C) PGIS immunoblot; (D) comparison of basal PGIS protein expression with basal PGF1{alpha} levels (determined in media from the cells used for C, 3 points/cell line). R2 = 0.94. Blots are representative of two independent experiments.

 
To determine the relative inductive potencies of TNF{alpha}, IFN{gamma} and IL1ß, induction of expression of PG pathway enzymes from single and pairwise cytokine addition was compared in C10 and LM2 cells, since these cell lines were both responsive and represented a non-neoplastic or neoplastic cell line, respectively (Figure 3). No individual cytokine significantly increased PGE2 production in C10 cells. The combination of TNF{alpha} and IL1ß increased PGE2 production, while TNF{alpha} plus IFN{gamma} was as potent as ‘cytomix’. TNF{alpha} plus IFN{gamma} raised PGI2 levels in C10 cells as effectively as ‘cytomix’. In contrast, IFN{gamma} alone induced PGE2 levels in LM2 cells as much as pairwise cytokine combinations. Either TNF{alpha} or IFN{gamma} individually induced PGI2 levels in LM2 cells; addition of IL1ß to either cytokine did not elevate PGI2 production any further. As in C10 cells, the TNF{alpha}–IFN{gamma} pair was as potent as ‘cytomix’ for inducing both PGs in LM2 cells. We conclude that IL1ß is not required for stimulation of PGE2 and PGI2 production in either cell line, although it augments PGE2 synthesis slightly by each of the other cytokines in LM2 cells. The combination of TNF{alpha} plus IFN{gamma} is synergistic for inducing PGE2 and PGI2 production in C10 cells and for PGE2 production in LM2 cells. The induction of PGI2 by TNF{alpha} and IFN{gamma} was additive in LM2 cells.



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Fig. 3. Fold induction of PGE2 and PGI2 production by individual or pairs of cytokines. Media samples were collected from C10 (white bars) and LM2 (black bars) cells just prior to cell harvest and analyzed for PGE2 (left) and 6-keto PGF1{alpha} (PGI2, right). *P < 0.05 versus control. Graph is representative of two independent experiments.

 
We analyzed both basal and cytokine-induced expression of enzymes upstream from mPGES and PGIS (namely cPLA2, COX-1 and COX-2) to determine whether their expression pattern was similar to that of mPGES or PGIS among the cell lines. The relative basal cPLA2 contents among cell lines (Figure 4) were similar to PGIS, with the exception that LM1 cells expressed cPLA2 but not PGIS. LM1 cells express the most COX-1 and COX-2 (Figure 4) as well as mPGES (Figure 2). Non-neoplastic C10 and E10 cells basally express COX-1 and COX-2 protein, but mPGES is undetectable in these cells.



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Fig. 4. Basal cPLA2, COX-1 and COX-2 expression in mouse lung epithelial cell lines. Samples used in Figure 2 were immunoblotted for cPLA2, COX-1, and COX-2 protein. Immunoblots are representative of three independent experiments containing three unique samples of each cell line which yielded similar results.

 
The induction of cPLA2, COX-1, COX-2, mPGES and PGIS by cytokines is shown in Table III. cPLA2 protein was induced in C10, LM1 and LM2 cells by ~50%. COX-1, considered to be constitutively expressed in many biological systems, was induced several-fold by cytokine treatment in LM2 and PCC4 cells. COX-2 expression was induced by cytokines in all lines except for the spontaneous transformants (A5 and E9) and 82–132 cells. The level of mPGES was increased in LM1 and LM2 cells, and PGIS only in C10 cells. No enzyme was induced by cytokines in A5, E9, and 82–132 cells.


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Table III. Expression of enzymes in the PG pathway after cytokine exposurea

 
Lack of cross-talk between PG and NO pathways
Deletion of the iNOS structural gene greatly reduces lung tumor formation in mice (6). Since NO and PGs have been reported to influence each other's biosynthesis in many cellular systems (30), we determined whether this also applied to these mouse lung epithelial cell lines. After assaying iNOS expression in these cell lines with and without cytokine exposure, we applied inhibitors of iNOS or COX-2 enzymatic activity to examine possible interactions between these two pathways and determine if modulating PG synthesis in epithelial cells is part of the mechanism by which NO promotes tumorigenesis. The iNOS protein was not reproducibly detected in any cell line under basal conditions (Figure 5A). Cytokine administration increased iNOS protein in C10, E10, LM1 and LM2 cells, with a resultant rise of media NO content (Figure 5B). Little iNOS protein was detected in A5 cells even though NO was detected in media from these cells. NO may be produced by the other NOS enzymes, the presence or absence of which has not been assessed in these cells.



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Fig. 5. iNOS expression (A) and NO production (B) in mouse lung epithelial cell lines in response to cytokine treatment. Cells treated with TNF{alpha}, IFN{gamma} and IL1ß were immunoblotted for iNOS protein and media taken from control and cytokine-treated cells was analyzed for NO content by estimating the stable metabolites of NO, nitrite and nitrate. iNOS protein was not detected in the absence of cytokines. The data is representative of three independent experiments each comprised duplicate or triplicate samples. Bars represent mean ± SEM of data pooled from three independent experiments. *P < 0.02 versus media from untreated controls. Fold-induction of NO production is indicated under the graph for all cell lines except for E10 which did not produce NO in the absence of cytokines.

 
To examine possible relationships between the NO and PG biosynthetic pathways in these cell lines, we compared the abilities of individual and combinations of cytokines to induce COX-1, COX-2 and iNOS in C10 and LM2 cells (Figure 6A–C). IL1ß by itself did not induce biosynthesis of any of these enzymes but augmented induction by both TNF{alpha} and IFN{gamma}. While no cytokine pair induced COX-1 expression in C10 cells, each pairwise combination significantly stimulated COX-1 expression in LM2 cells. Both TNF{alpha} and IFN{gamma} were necessary to induce COX-2 in C10 cells, while either TNF{alpha} or IFN{gamma} individually elevated COX-2 protein in LM2 cells. IFN{gamma} by itself and in combination with any other cytokine induced iNOS in C10 cells, while either IFN{gamma} or TNF{alpha} alone induced iNOS in LM2 cells. Thus, LM2 cells appeared more responsive than C10 cells to the induction of both COX-2 and iNOS by individual cytokines. TNF{alpha}/IFN{gamma} induced COX and iNOS proteins to the same extent as cytomix in both cell lines.



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Fig. 6. Induction of COX-1, COX-2, and iNOS proteins by individual or combinations of cytokines. Cell samples were immunoblotted for COX-1 (A), COX-2 (B) and iNOS (C). Fold-induction was calculated by densitometry; comparisons were treated groups versus control. Data represent mean ± SEM from three independent samples/condition in C10 (white bars) and LM2 (black bars) cells treated with TNF{alpha}, IFN{gamma} and/or IL1ß. Representative of three independent experiments. *P < 0.01 versus control.

 
As another means of assessing the similarities and differences in the induction of COX-2 and iNOS, we compared the time course of increased iNOS expression following cytokine exposure in C10 cells with that for COX-2 (Figure 7). COX-2 protein began to rise within 1 h after cytokine addition to C10 cells, but iNOS protein was not detectable until after 8 h. This correlates with our earlier study where iNOS mRNA in E10 cells began to increase 4 h after cytokine addition and NO levels rose 24 h later (42). Elevation of COX-2 protein prior to a rise in iNOS protein is inconsistent with a role for NO (derived from iNOS) in COX-2 induction.



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Fig. 7. Time course of iNOS and COX-2 induction by cytokines. C10 cells were treated with cytomix and harvested at 0 (control), 1, 4, 8 and 24 h after treatment (three independent samples/time point). iNOS (upper panel) and COX-2 (lower panel) protein expression was analyzed by immunoblot. This is representative of two independent experiments.

 
To test whether inhibiting COX-2 activity affects iNOS catalytic activity, C10 and LM2 cells were incubated with three doses of NS-398, a cell-permeant, COX-2-specific inhibitor, prior to TNF{alpha}/IFN{gamma} addition. NS-398 inhibited COX activity at even the lowest dose used, as determined by the media contents of PGE2 and PGI2 (Figure 8A and B). This suggests that COX-1, as well as COX-2, was inhibited by NS-398 or COX-1 activity did not produce PGs in these cells. NS-398 did not affect NO production in either cell line, indicating that COX activity does not affect iNOS activity in this system (Figure 8). NS-398 had no effect on COX-1, COX-2, or iNOS expression, as determined by immunoblot analysis (data not shown).



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Fig. 8. Effect of NS-398 pretreatment on TNF{alpha}/IFN{gamma}-induction of PGE2, PGI2 and NO production in C10 and LM2 cells. Media from C10 (white bars) and LM2 (black bars) were analyzed for PGE2 (A), PGI2 (B) and NO (C) content. Data represent mean ± SEM from three independent samples from each treatment group. *P < 0.001 versus control and NS-398 only samples. Representative of each of two independent experiments.

 
To determine if NO production affects PG production, C10 and LM2 cells were treated with aminoguanidine (AMG), a cell-permeant NOS inhibitor (43). AMG significantly inhibited iNOS activity (Figure 9A), but had no effect on cytokine-mediated induction of PGE2 or PGI2 (Figure 9B and C). AMG did not perturb the expression of COX-1, COX-2 or iNOS proteins, as determined by immunoblot analysis (data not shown). These results indicate a lack of cross-talk interaction between the NO and PG biosynthetic pathways in these cultured murine lung cells.



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Fig. 9. Effect of AMG pretreatment on TNF{alpha}/IFN{gamma}-induction of NO, PGE2 and PGI2 production in C10 and LM2 cells. Media from C10 (white bars) and LM2 (black bars) were analyzed for NO (A), PGE2 (B) and PGI2 (C) production. Data represent the mean ± SEM of three independent samples/treatment group. *P < 0.01 versus untreated and AMG-treated control samples. Representative of each of two independent experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We initiated studies of non-neoplastic and neoplastic mouse lung epithelial cells to investigate PG and NO inflammatory pathways that may contribute to lung tumor development in vivo (5,6). Basal PG and NO production from a panel of eight mouse lung epithelial cell lines demonstrated that neoplastic cells produced at least an order of magnitude more PGE2 than non-neoplastic cells. Conversely, non-neoplastic cells produced more PGI2 than the neoplastic cells. The cellular contents of mPGES and PGIS correlated with the extent of PGE2 and PGI2 production, respectively, implying that the intracellular concentrations of these two enzymes is generally rate limiting. Indeed, when PGIS or mPGES is transgenically overexpressed in mouse lungs, pulmonary levels of the respective PGs are increased (5,44). Pulmonary PGE2 is pro-inflammatory (27,28) while PGI2 is anti-inflammatory (26), consistent with the inflammation marked by macrophage infiltration in tissue adjacent to these lung tumors (25,31). PGE2 to PGI2 ratios in mouse cell lines were consistent with those observed in mouse tissue and human lung cell lines and tissue (45). Neoplastic tissue and tumor-derived cells produce predominantly PGE2, while normal tissue and untransformed cells predominantly synthesize PGI2. Both the stroma and tumor parenchyma can produce PGs. Since mice that are null for one of the PGE2 receptors (46) or that overexpress PGIS (5) develop fewer tumors than their wild-type littermates, this PGE2 to PGI2 ratio may influence the ability of the host to resist tumor development.

Although NSAIDs are potent chemopreventive agents for some human cancers, the differential regulation of PG synthesis in normal and neoplastic mouse lung cells suggests that a more targeted approach may increase efficacy. Inhibition of mPGES and antagonism of specific PGE2 receptors emerge as potential chemopreventive and chemotherapeutic targets, since PGE2 production appears to be a key mediator of chronic inflammation. Stable PGI2 agonists would be predicted to be efficacious in preventing lung tumor progression. Preclinical and clinical studies are currently underway to examine the effect of the stable PGI2 analog, Iloprost, in both mouse and human lung tumorigenesis (47).

We used several mouse lung epithelial cell lines in these studies because diversity in PG metabolism was previously observed among human lung tumor cell lines (12,45). Cell lines derived from human AC and large cell carcinomas varied over 100-fold in constitutive PGE2 production, while only 1 of these 15 cell lines produced PGI2 (45). The concentration of PGIS is greatly diminished in human lung cancer (48) and is not detectable in any neoplastic mouse lung cell line. The cell line used, whether herein non-tumorigenic or neoplastic, appears to be either resistant or sensitive to cytokine exposure, with regard to enhanced PG production. The only exception is E10, where the cells do not make more PGE2 in response to TNF{alpha}/INF{gamma} treatment while they make nearly twice as much PGI2. Since one of the two non-tumorigenic lines and three of six neoplastic lines make more PGE2 in response to cytokines, cytokine sensitivity may not be a normal versus neoplastic property. Rather, the relative contents of the two PGs distinguish non-tumorigenic from neoplastic cells and tissues. In these mouse lung cell lines, application of both TNF{alpha} and IFN{gamma} maximally stimulated PG and NO production as well as inducing COX-1, COX-2 and iNOS expression. In the human lung tumor-derived cell line, A549, cytomix also stimulates NO and PGE2 production, but, in contrast to our studies, IL1ß was required for full induction (49). Although TNF{alpha}/IFN{gamma} induced expression of cPLA2, COX-1, COX-2, mPGES, PGIS and iNOS in at least one mouse lung cell line, there were few similarities in the pattern of induction of these enzymes across the entire cell line panel. Several aspects of Table III are noteworthy. LM2 and PCC4, both derived from papillary tumors with a probable bronchiolar Clara cell origin, are a valuable resource for mechanistic investigations of COX-1 regulation. We measured COX-1 protein and not mRNA levels, and protein content can be regulated by many processes (e.g. changes in relative rates of protein translation and/or mRNA and protein stability) that are independent of transcription. This adds to the complexities of COX-1 responses to environmental stimuli, since the number of enzyme molecules may be regulated at many levels. Cell lines in which cPLA2, COX-1 and COX-2 enzymes were most highly induced by cytokines are also inducible for production of the PGE2 and PGI2 products of this pathway (Table I). All aspects of PG metabolism except for PGIS increased in LM2 cells after cytokine exposure. Except for the lack of COX-1 induction in C10 cells, and cPLA2 and mPGES induction in PCC4 cells, PG biosynthesis is sensitive to cytokines in these cell lines.

iNOS protein is overexpressed in human lung cancer, AC in particular (50), and lung cancer patients exhale higher levels of NO than healthy individuals (51). Mice null for iNOS expression develop 83% fewer tumors than their wild-type littermates (6). NO stimulates angiogenesis (52), blood flow (53) and vascular permeability (54), and VEGF production is decreased in tumors from mice that null for iNOS (6), so this may be the mechanism by which iNOS exerts tumor promoting activity.

The cPLA2 content correlated inversely with proliferative rate, since C10, E10 and LM1 doubling times are longer than those of the other five cell lines (32). The intracellular concentrations of COX-1, COX-2, mPGES and PGIS do not correlate with cellular proliferation rates. In human non-small cell lung cancer cell lines, cPLA2 and COX-2 expression increases in cells containing Kras activating mutations (12). Kras mutational status is not, however, associated with enzyme content or inducibility in mouse lung cell lines. Cell lines containing at least one mutant Kras allele (E9, A5, LM2 and PCC4) do not express more cPLA2 or COX-2 than cells expressing only wild-type Kras [C10, E10, LM1 and 82–132 (55)]. The iNOS induction and NO production following cytokine exposure were not related to either Kras mutational status or growth rate.

Cross-talk between the NO and PG inflammatory pathways has been described in several cell types (29). In inflammatory cells in particular, NO and PGs influence the synthesis of the other mediator, i.e. NO can inhibit or stimulate COX activity and PGE2 stimulates iNOS activity. Inhibition of iNOS activity in A549 cells (56), murine macrophages (30) and other cell types inhibits cytokine-induced COX-2 expression. Since TNF{alpha}/IFN{gamma} treatment activated both pathways in these cells, interactions between the two pathways were examined. The time-course of COX-2 and iNOS protein induction demonstrated that COX-2 expression rose several hours before iNOS protein was detected, implying that NO derived from iNOS is unlikely to be responsible for increased COX-2 expression. Specific inhibitors of COX-2 and iNOS activity prevented the TNF{alpha}/IFN{gamma}-induced increases in both PG and NO accumulation, respectively, but no cross-talk was observed. Similarly, in a rat model of LPS-induced inflammation, treatment of animals with either a selective iNOS or COX-2 inhibitor did not affect the induction of the other enzyme (57). In this rat study, NO and PGE2 production were each inhibited by the anti-inflammatory steroid, dexamethasone, implying that a common upstream element controls both NO and PG production. There is an NF{kappa}B response element in each of the COX-2 (58), iNOS (59) and TNF{alpha} (60) promoters. Endotoxin-mediated phosphorylation of I{kappa}B releases NF{kappa}B which in turn upregulates transcription of COX-2 (61), iNOS (62) and TNF{alpha} (63). Glucocorticoids repress NF{kappa}B-induced transactivation and this is thought to be one of the pathways by which glucocorticoids inhibit inflammation (64). Since both the NO and PG pathways are induced by treating mouse lung epithelial cell with TNF{alpha} and IFN{gamma}, we also postulate a common element that regulates PG and NO synthesis in these cells. NO regulation of PG production does not occur in cultured mouse lung cells, but NO may affect PG synthesis in peritumoral inflammatory cells, and cross-talk between the two pathways may occur intracellularly and intercellularly within the tumor stroma.

Local PG production changes during an inflammatory response (65). A single PG may transduce different signals depending on the receptor to which it binds and the specific signal transduction cascades coupled to that receptor (66). Mammary tumors appear in the absence of carcinogens by mere overexpression of COX-2 targeted to the mammary epithelium (67). Similarly, colon cells are transformed by transfection with the structural gene for mPGES (68). However, COX-2 overexpression inhibits skin tumorigenesis (69), and NSAIDs increase skin tumor multiplicity (69). Interactions between cytokine signaling pathways and PGs are also complex. Production of PGs in mouse and human AC lines is stimulated by cytokines (70), and PGs modulate cytokine synthesis in lymphocytes (16). PGs thus regulate tumor development in an organ-specific manner.

Our in vitro model examines the regulation of PG and NO synthesis in normal and neoplastic epithelial cells in response to pro-inflammatory cytokines. In these cells, COX and iNOS pathways operate independently of each other but may have a common upstream regulator. In addition, the relative production of PGE2 and PGI2 in mouse lung cells and tissues reflects their state of transformation. Identification of this regulator and molecular clarification of the roles of these PGs in mouse lung tumor formation will enhance the design of rational chemoprevention protocols for use in high risk populations.


    Acknowledgments
 
The authors would like to thank Drs Laura Zerbe, Raphael Nemenoff and Pamela Rice, and Greg Hurteau and Katherine Peebles for their insightful comments during the preparation of this paper. This work was funded by CA33497 and CA96133.

Conflict of Interest Statement: None declared.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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