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[Cancer Research 63, 766-770, February 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Abnormal Interleukin 10R{alpha} Expression Contributes to the Maintenance of Elevated Cyclooxygenase-2 in Non-Small Cell Lung Cancer Cells1

Nathalie Heuze-Vourc’h, Li Zhu, Kostyantyn Krysan, Raj K. Batra, Sherven Sharma and Steven M. Dubinett2

University of California at Los Angeles Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, David Geffen School of Medicine at University of California at Los Angeles, and the Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90095


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Non-small cell lung cancer (NSCLC) cells are known to constitutively overexpress cyclooxygenase (COX)-2. Tumor COX-2-dependent production of PGE2 triggers the synthesis of lymphocyte and macrophage interleukin (IL)-10 that, in turn, is known to potently suppress COX-2 in normal cells. Thus, we investigated the capacity of IL-10 to down-regulate COX-2 expression in NSCLC cells. Western blotting and ELISA analyses revealed that IL-10 did not affect COX-2 expression and subsequent PGE2 production in NSCLC cells. Although normal human bronchial epithelial cells expressed both intracellular and membrane IL-10R{alpha}, NSCLC cells only expressed intracellular but not cell surface membrane IL-10R{alpha}. Unresponsiveness of COX-2 to IL-10 is due to the deficiency of IL-10R{alpha} on the surface of NSCLC cells. Our findings highlight a novel mechanism contributing to maintenance of elevated COX-2 and PGE2 in the lung tumor environment.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
COX3 catalyzes the biosynthesis of PGs such as PGE2 and thromboxane from free arachidonic acid (1) . Two COX isoforms have been identified: a constitutively expressed enzyme COX-1 and an inducible isoenzyme COX-2. Overexpression of COX-2 has been detected in numerous tumors, including NSCLC (2 , 3) . However, the mechanisms that regulate the expression of COX-2 in lung cancer are not completely understood. Studies indicate that COX-2 expression is induced in cancer cells at different levels in response to growth factors, cytokines, tumor promoters, and mutational events, and several signaling pathways have been implicated (2 , 4, 5, 6) . Type 2 cytokines (IL-4, IL-10, and IL-13) have been reported to be negative regulators of COX-2 expression and PGs production in normal and tumor cells (7, 8, 9, 10) . For example, IL-10 decreases induced COX-2 expression as well as PGE2 synthesis in astrocytes, chorion trophoblast cells, and monocytes (8, 9, 10) . In the lung tumor environment IL-10, which has been implicated in tumor-mediated immunosuppression, is the predominant type 2 cytokine. Indeed, IL-10 is produced by lung tumor cells themselves as well as by host immune cells such as monocytes and lymphocytes in response to tumor COX-2-dependent production of PGE2 (3 , 11 , 12) . Because tumor-derived PGE2 triggers the synthesis of IL-10 that is known as a potent suppressor of COX-2, we investigated the ability of IL-10 to regulate COX-2 expression in NSCLC cells. Our results show that IL-10 does not regulate COX-2 in NSCLC because of lack of tumor cell surface expression of IL-10R{alpha}. Deficiency of the IL-10-mediated COX-2 regulatory feedback loop in NSCLC cells may contribute to COX-2 overexpression and maintenance of high level PGE2 in the lung cancer microenvironment.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Human Cell Lines and Cell Culture.
Clonetics NHBE cells were obtained from BioWhittaker (Walkersville, MD). Human large cell lung carcinoma H460 was obtained from the National Cancer Institute (Bethesda, MD). Lung adenocarcinoma line A549 and THP-1 human monoblastic leukemia cell line were obtained from American Type Culture Collection (Manassas, VA). The human squamous cell carcinoma line RH2 was established in our laboratory (11) . The tumor cells were grown at 37°C in an atmosphere of 5% CO2 in RPMI 1640 (Mediatech, Inc., Herndon, VA) supplemented with 10% FBS (Gemini Bio-Products, Woodland, CA), 100 units/ml penicillin/streptomycin and 2 mM glutamine. The NHBE cells were grown in the same conditions in bronchial epithelium cell growth media (BioWhittaker) supplemented with 5.2 µg/ml bovine pituitary extract, 5 µg/ml insulin, 0.5 pg/ml human recombinant epidermal growth factor, 0.5 µg/ml hydrocortisone and epinephrine, 10 µg/ml transferrin, 0.1 pg/ml retinoic acid, 6.5 pg/ml triiodothyronine, 50 µg/ml gentamicin, and 50 pg/ml amphotericin-B.

For COX-2 assays, tumor cells (0.5 x 106 NSCLC cells in a 6-well plate or 0.5 x 106 THP-1 cells/ml in a 12-well plate) cultured in complete medium were pretreated with different amounts of IL-10 (Peprotech, Inc., Rocky Hill, NY) before stimulation with IL-1ß (280 units/ml; BD Biosciences PharMingen, San Diego, CA) or LPS (1 µg/ml; Sigma Chemical Co., St. Louis, MO) for 24 h.

Semiquantitative RT-PCR Analysis of IL-10R Gene Expression.
Total RNA was isolated from each cell type using RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions. Total RNA (1.5 µg) was reverse transcribed using 200 units of SuperScript II RNase H Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA) following the manufacturer’s instructions. A total of 3.3 µl of the cDNA was amplified by PCR using TaqDNA Polymerase (Invitrogen Corp). Reactions were performed under the following conditions: IL-10R{alpha}, 35 cycles of denaturation at 94°C for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 20 s; and IL-10Rß, 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s and extension at 72°C for 40 s. The following primers were used: IL-10R{alpha} sense primer 5'-ATGCTGCCGTGCCTCGTAGTGC-3'; IL-10R{alpha} antisense primer 5'-ACTCTGGCCCG GTAGCCATTGC-3' as previously described (13) ; IL-10Rß sense primer 5'-CAAGATAAATGCATGAATAC-3'; and IL-10Rß antisense primer 5'-GAAAGGAGAAAAACAGAAG-3'. The ß-actin gene expression was used as an internal control for the quality of RNA specimens used as templates and as a standard to evaluate IL-10R expression in the different cell lines.

Western Blotting Analysis of COX-2 and IL-10R{alpha} Expression.
Tumor cells were cultured for 24 h in different conditions, washed in PBS, and then lysed at 4°C for 20 min in lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1x complete protease inhibitor mixture (Roche Diagnostics Corp., Indianapolis, IN)]. The protein concentration in the cell lysates was determined using a bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL). Cell lysate proteins were separated on a 7.5 or 10% SDS-PAGE and transferred on polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The proteins were immunodetected with anti-human COX-2 monoclonal antibody (Cayman Chemical Company, Ann Arbor, MI) or antihuman IL10-R{alpha} polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membranes were developed using an enhanced chemiluminescence-plus detection system (Amersham Biosciences, Piscataway, NJ). Equal protein loading was confirmed by immunodetecting the membranes with anti-actin antibody. Ramos cell lysate (Santa Cruz Biotechnology, Inc.) was used as positive control to evaluate IL10-R{alpha} protein expression.

COX-2 Synthesis Analysis by EIA.
The human COX-2 monoclonal EIA kit was obtained from IBL Co., Ltd. (Gunma, Japan), and COX-2 EIA was performed according to the manufacturer’s instructions. Briefly, COX-2 standard protein or 15–30 µg of total protein from tumor cells were added to each well of an EIA plate precoated with an anti-hCOX-2 monoclonal antibody. After 1 h of incubation at 37°C, the plate was washed seven times, and an horseradish peroxidase-conjugated anti-hCOX-2 polyclonal antibody was added to each well. After 30 min of incubation at 4°C, the plate was washed, and the 3,3',5,5'-tetramethylbenzidine substrate buffer was added to each well. Reactions were stopped by adding sulfuric acid solution, and the absorbance was read at 450 nm in a microtiter plate spectrophotometer.

PGE2 Analysis by EIA.
The PGE2 monoclonal EIA was obtained from Cayman Chemical Company and performed as described previously (3) .

Flow Cytometry Analysis of IL-10R.
Cells cultured in complete medium for 24 h were recovered, washed with PBS 1x, and incubated in PBS 1x + 2% FBS (fluorescence-activated cell sorting buffer) containing phycoerythrin-human IL-10R{alpha} monoclonal antibody (BD Biosciences PharMingen) or human IL-10Rß polyclonal antibody (R&D Systems) for 30 min at room temperature. When unlabelled antibodies were used, a second incubation with a FITC-conjugated (Fab')2 fragment rabbit antigoat IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was performed. Matched isotype immunoglobulin were used in control samples. After the last washing step, cells were resuspended in 500 µl of fluorescence-activated cell sorting buffer containing propidium iodide (1 µg/ml; Sigma Chemical Co.) or 7-amino-actinomycin D (1 µg/ml; Calbiochem, San Diego, CA) for dead cell exclusion.

To detect intracellular IL-10R{alpha} expression, cells were fixed and permeabilized with 0.2% Tween 20 prior the staining. Cells were analyzed by a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA).

Statistical Analysis.
The unpaired two-tailed Student’s t test was used to compare differences in NSCLC COX-2 expression and P = 0.05 were considered significant.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
IL-10 Does not Regulate COX-2 in NSCLC Cells.
To evaluate the ability of IL-10 to regulate COX-2 in NSCLC, we performed in vitro assays using three different NSCLC cell lines (A549, H460, and RH2) and the monocyte cell line THP-1 as a positive control. IL-1ß was used to increase COX-2 expression in NSCLC cells, and THP-1 cells were stimulated with LPS as described previously (3 , 10 , 14) .

As shown in Fig. 1ACitation , in the absence of stimuli, NSCLC and THP-1 cell lines produced either undetectable or low levels of COX-2. Addition of IL-10 alone did not affect constitutive COX-2 production in the different cell lines. As anticipated from previous reports, after stimulation with IL-1ß or LPS, both NSCLC and THP-1 cells produced higher levels of COX-2 (3 , 14) . In THP-1 cells, treatment with IL-10 (>=10 ng/ml) markedly inhibited LPS-induced COX-2 production (Fig. 1A)Citation , demonstrating that the regulatory effect of exogenous IL-10 was intact in this monocyte cell line. Consistent with these findings for COX-2 levels, IL-10 potently down-regulated PGE2 production in THP-1 cells (Fig. 1C)Citation . In contrast, addition of IL-10 did not decrease IL-1ß-induced COX-2 overexpression in the three NSCLC cell lines as shown by Western blotting (Fig. 1A)Citation . These results were supported by quantification of COX-2 production in NSCLC cell lines using ELISA, which demonstrated that IL-10 did not have any significant effect on either constitutive or IL-1ß-induced COX-2 expression (Fig. 1B)Citation . As shown in Fig. 1CCitation , PGE2 levels in the culture medium of NSCLC remained unchanged when IL-10 was added, indicating that COX-2-dependent production of PGE2 is not affected by IL-10. Taken together, these findings suggest that in contrast to monocytes, COX-2 expression in NSCLC cells is not decreased by exogenous IL-10.



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  		Fig. 1. Regulation of COX-2 by IL-10 in human NSCLC lines. IL-1ß or LPS were used to increase COX-2 in NSCLC or THP-1 cells, respectively. Cells were cultured for 24 h with different amounts of IL-10 and lysates were prepared. Expression of COX-2 was assessed by Western blot analysis with anti-human COX-2 antibody (A; 20 µg of total proteins for NSCLC and 50 µg for THP-1) or by EIA (B). COX-2-dependent production of PGE2 was analyzed by EIA (C) in the cell culture medium. Concentration of IL-10 is expressed in ng/ml. The results are expressed as ratio of control value and are representative of at least three independent experiments for NSCLC cells and two independent experiments for THP-1. *, P < 0.002 compared with IL-1ß-treated cells.

 
IL-10R Expression in NSCLC Cells.
Cellular responses to IL-10 are mediated through its receptor (IL-10R) composed of two cell surface subunits: an IL-10 binding chain IL-10R{alpha}, which is abundantly expressed in hematopoietic cells, and an accessory subunit IL-10Rß, constitutively expressed in most cells and tissues (9) .

To assess whether unresponsiveness of NSCLC cells to IL-10 results from absence of expression of either one or both IL-10R subunits, total RNA was purified from the different cell lines, and IL-10R{alpha} and IL-10Rß mRNA expression profiles were analyzed by RT-PCR using ß-actin as an internal control (Fig. 2A)Citation . Both IL-10R{alpha} and IL-10Rß mRNAs were detected in THP-1 and NSCLC cell lines under our experimental conditions (Fig. 2A)Citation . Similar results were obtained when IL-10R mRNA expression was analyzed in nine other NSCLC cell lines (data not shown), indicating that IL-10R{alpha} and IL-10Rß are expressed in all of the NSCLC cell lines tested. Whereas both THP-1 and NSCLC cells showed abundant IL-10Rß mRNA expression, IL-10R{alpha} mRNA expression was relatively low in NSCLC compared with the monocyte cell line.



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  		Fig. 2. IL-10R expression in human NSCLC cell lines. A, IL-10R{alpha} and IL-10Rß mRNA expression using RT-PCR. Lane M, 100-pb molecular weight marker; Lane 1, A549; Lane 2, H460; Lane 3, RH2; Lane 4, positive control (THP-1); and Lane 5, negative control (RT-PCR mixture without RNA samples). B, production of IL-10R{alpha} in NSCLC cells. Proteins (75 µg) from cell lysates were immunodetected with antihuman IL-10R{alpha} polyclonal antibody. Lane 1, positive control (Ramos cell lysate); Lane 2, A549; Lane 3, H460; Lane 4, RH2; and Lane 5, THP-1.

 
To determine whether the protein synthesis of IL-10R{alpha} correlates with its mRNA expression, cell lysates from THP-1 and NSCLC cell lines were prepared and analyzed by Western blot with an anti-human IL-10R{alpha} polyclonal antibody. Consistent with the results obtained for mRNA expression by RT-PCR, we found that IL-10R{alpha} protein was expressed in both monocyte and NSCLC cell lines (Fig. 2B)Citation . Differences observed in the apparent molecular weight of IL-10R{alpha} and immunodetection of several bands in the protein from different cell lines extracts may be because of heterogeneity of protein glycosylation.

Intracellular and Surface Expression of IL-10R{alpha} and IL-10Rß in Human Lung Cells.
Colocalization of both IL-10R{alpha} and IL-10Rß on the cell surface is required for cellular responses to IL-10 (9 , 15) . Thus, to gain insight into the mechanisms underlying the unresponsiveness of NSCLC cells to IL-10, cell localization of both IL-10R subunits was examined in NSCLC and THP-1 cell lines by flow cytometry. As shown in Fig. 3Citation , whereas THP-1 cells displayed cell membrane expression of both IL-10R subunits, only IL-10Rß was detected on the surface of NSCLC cells. Consistent with their unresponsiveness to IL-10, NSCLC displayed no surface expression of IL-10R{alpha} (Fig. 3)Citation . However, the intracellular IL-10R{alpha} protein was found in permeabilized cells (Fig. 3)Citation , supporting the results obtained by Western blot (Fig. 2B)Citation . Additional analysis of IL-10R expression in three other NSCLC cell lines by flow cytometry confirmed the lack of cell surface IL-10R{alpha} (data not shown).



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  		Fig. 3. Intracellular and membrane surface expression of IL-10R in human NSCLC lines and THP-1 cells by flow cytometry. Surface expression of IL-10R was evaluated on alive cells, whereas internal staining was performed on fixed and permeabilized cells. Histograms represent the relative fluorescence intensity obtained with no stained cells (dotted histogram), cells stained with control IgG (dashed histogram), or with IL-10R antibodies (filled histogram). Results are representative of at least three independent experiments.

 
To determine whether the deficit of IL-10R{alpha} surface expression is present in nontransformed lung epithelial cells, we examined its expression in NHBE cells. Interestingly, IL-10R{alpha} was detected both on the surface and intracellularly in NHBE cells (Fig. 4A)Citation , whereas IL-10R{alpha} surface expression in A549 remained undetectable when the cells were cultured in the enriched medium required for NHBE growth (Fig. 4B)Citation .



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  		Fig. 4. IL-10R{alpha} expression in normal and tumor lung cells. A, intracellular and membrane expression of IL-10R{alpha} in normal lung cells (NHBE). Surface expression of IL-10R{alpha} was evaluated on viable cells, whereas internal staining was performed on fixed and permeabilized cells. B, surface expression of IL-10R{alpha} in A549 cells cultured in RPMI 1640 supplemented with 10% FBS (left histogram) or in the same medium as NHBE cells (right histogram) for 24 h. Histograms represent the relative fluorescence intensity obtained with no stained cells (dotted histogram), cells stained with control IgG (dashed histogram), or with IL-10R{alpha} antibody (filled histogram). Results are representative of at least three independent experiments.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Studies have shown that COX-2 is overexpressed in numerous epithelial derived tumors, including NSCLC (2 , 3) . Tumor COX-2 overexpression has been implicated in enhanced angiogenesis, apoptosis resistance, invasion, and suppression of antitumor immunity (16, 17, 18, 19) . Thus, based on its importance in regulating the malignant phenotype, investigations have focused on delineating the mechanisms mediating tumor COX-2 overexpression.

A variety of compounds, growth factors, cytokines, or tumor promoters have been revealed to up-regulate COX-2 expression at the transcriptional level (2 , 5) . For example, these include transforming growth factor ß, IL-1ß, and epidermal growth factor, which are all highly represented in the lung tumor microenvironment. Mutational events have been also implicated to contribute to transcriptional up-regulation of COX-2 in cancer cells (4 , 6) . Moreover, basal transcription has been shown altered in murine lung cancer (20) . Modulation of COX-2 protein levels can also be achieved via posttranscriptional mechanisms involving 3' regulatory elements and translational effects regulating the rate of protein synthesis and/or degradation (2 , 5 , 6) . As previously reported in certain type of cells, IL-1ß-dependent regulation of COX-2 expression can involve both transcriptional mechanisms and posttranscriptional mRNA stability (21) . On the other hand, type 2 cytokines, including IL-4, IL-10 and IL-13, have been reported to suppress either LPS- or IL-1ß-induced COX-2 expression in various cell types, thereby inhibiting PGE2 synthesis (7, 8, 9) . Indeed, in normal cells such as monocytes and epithelial or dendritic cells, the COX-2-dependent production of PGE2 potently up-regulates IL-10 production by both lymphocytes and macrophages within the inflammatory microenvironment (22 , 23) . Increased levels of IL-10, in turn, have the capacity to potently inhibit induced COX-2 expression and subsequent PGE2 production in normal cells. This autocrine/paracrine regulatory loop in normal cells has been suggested to serve as an important checkpoint in the balance of PGE2-mediated regulation of Th1/Th2 cytokine homeostasis in normal host immune responses (23) . In the lung tumor environment, IL-10 is the predominant type 2 cytokine, expressed by either lung tumor cells or host immune cells, and this induction occurs, in part, through a COX-2-dependent PGE2-mediated mechanisms (3 , 11 , 12) . Moreover, clinical studies have reported that both IL-10 and COX-2 overexpression are correlated with poor prognosis of NSCLC (13 , 24) . In this study, we have shown that IL-10 effectively down-regulates both COX-2 and PGE2 production in the THP-1 monocyte cell line consistent with previous studies in normal cells (8, 9, 10) . In contrast, we found that IL-10 has no effect on either constitutive or induced COX-2 expression and COX-2-dependent PGE2 production in NSCLC cells. This result indicates that NSCLC COX-2 expression is not inhibited by IL-10 in the regulatory feedback loop operative in normal cells. Thus, defective functioning of this homeostatic mechanism could result in maintenance of high-level COX-2 expression and PGE2 synthesis as well as heightened IL-10 production in lung cancer. The persistent tumor COX-2 elevation may cause alteration of immune responses, enhanced angiogenesis, apoptosis resistance, and invasion, leading to the promotion of tumorigenesis.

Loss or decrease of receptor expression is a common mechanism associated with abrogation of response to certain ligand in human cancer (25 , 26) . IL-10 exerts its action through a heterodimeric membrane receptor formed by a binding subunit IL-10R{alpha} and an accessory chain IL-10Rß. Although IL-10Rß is constitutively expressed in most cells and tissues, IL-10R{alpha} expression is often inducible and largely restricted to hematopoietic cells. In this study, we found that all of the NSCLC cell lines tested express mRNA for both IL-10R subunits, suggesting that NSCLC unresponsiveness to IL-10 is not a consequence of a transcriptional defect inhibiting IL-10R mRNA expression. This finding regarding IL-10R{alpha} mRNA expression in NSCLC cells is consistent with a recent report showing that >95% of NSCLC tissue samples expressed IL-10R{alpha} at the RNA level (13) . In contrast Naruke et al. (27) , who analyzed xenografts of NSCLC by RT-PCR, only detected IL-10R{alpha} mRNA in 18% of the samples. These apparently discrepant observations may be because of the conditions used to amplify IL-10R{alpha} mRNA. Indeed, our studies showed that the number of RT-PCR cycles seems to be a critical parameter for IL-10R{alpha} detection.

Because IL-10R{alpha} mRNA expression was found to be relatively low in NSCLC cells compared with the monocyte cell line, we analyzed its expression at the protein level. Consistent with the results obtained for mRNA expression by RT-PCR, we found that IL-10R{alpha} protein was expressed in both monocyte and NSCLC cell lines. However, qualitatively, our results indicate that there does not seem to be a direct correlation between IL-10R{alpha} transcript level and protein generation in the different cell lines tested (Fig. 2)Citation . Potential explanations for these results include differences in posttranscriptional regulation (2 , 5 , 6) .

Known mechanisms underlying cell resistance to ligand effect, in normal and pathological conditions, include synthesis of nonfunctional receptor and alteration of receptor trafficking. In particular, recent studies have shown that unresponsiveness to IL- 10 may be the consequence of either synthesis of an IL-10R-truncated protein in colon epithelial cells or reduced membrane IL-10R{alpha} expression in mature dendritic cells (7 , 28) . In the current study, we found that IL-10Rß was expressed on the surface membrane of NSCLC cells but IL-10R{alpha} was not. The lack of IL-10R{alpha} on the cell membrane was neither because of the cell growth conditions as a monolayer nor to the presence of serum (data not shown).

Cell surface expression of IL-10R{alpha} is a critical factor in cellular response to IL-10, and its colocalization with IL-10Rß on the plasma membrane localization is required for optimal IL-10 signaling (9 , 15) . Therefore, lack of cell surface IL-10R{alpha} may explain the defect of COX-2 down-regulation by IL-10 in lung tumor cells. Detection of intracellular protein in NSCLC suggests that deficiency of membrane IL-10R{alpha} expression is unlikely the result of a translation defect but may instead be secondary to posttranslational events. These posttranscriptional mechanisms may result in either alteration of receptor trafficking from intracellular vesicles to the membrane or synthesis of a soluble form of IL-10R{alpha}. However, there have been no reports of detection of soluble IL-10R{alpha} in vivo (9) . In the case of deregulation of IL-10R{alpha} trafficking, our preliminary studies suggest that it is neither because of the alteration of the cell polarity nor to the presence of any factors in the culture medium. Studies are currently under way to determine the mechanisms underlying alteration of IL-10R{alpha} cell surface trafficking; these findings may be valuable from a diagnostic and therapeutic standpoint.

In contrast to our findings in lung cancer cells, we found that IL-10R{alpha} is expressed on the surface of normal lung epithelial cells. Recent reports showed aberrant chemokine receptor expression and disruption of transforming growth factor ß receptor, TßIIR, signaling in malignant hematopoietic cells compared with normal cells (25 , 26) . Therefore, the mechanism involved in membrane IL-10R{alpha} deficiency may be attributed to the malignant transformation of the lung epithelial cells. Additional experiments are required to confirm this hypothesis.

In conclusion, our results provide evidence that NSCLC responsiveness to IL-10 may be regulated through modulation of cell surface IL-10R{alpha} expression. This is the first report of abnormal IL-10R cell surface expression as a contributor to the maintenance of elevated COX-2 expression in human cancer. A more complete understanding of the regulatory pathways leading to the up-regulation and maintenance of tumor COX-2 may assist in developing therapeutics or chemoprevention targeting COX-2 in lung cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Felicita Baratelli for critical review of the manuscript.


   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 This work was supported by the UCLA Specialized Program of Research Excellence in Lung Cancer, NIH Grants 1P50 CA90388, RO1 CA 71818, and RO1 CA85686, Research Enhancement Award Program in Cancer Gene Medicine, and by Merit Review Research Funds from the Department of Veterans Affairs. Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility that is supported by NIH awards CA-16042 and AI-28697, by the Jonsson Cancer Center, the UCLA AIDS Institute, and David Geffen School of Medicine. Back

2 To whom requests for reprints should be addressed, at Lung Cancer Research Program, Division of Pulmonary and Critical Care Medicine, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, 37-131 CHS, Los Angeles, CA 90095. E-mail: sdubinett{at}mednet.ucla.edu Back

3 The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; PGE2, prostaglandin E2; IL, interleukin; IL-10R, interleukin 10 receptor; NSCLC, non-small cell lung cancer; NHBE, normal human bronchial epithelial; LPS, lipopolysaccharide; RT-PCR, reverse transcription-PCR; FBS, fetal bovine serum; EIA, enzyme immunoassay. Back

Received 11/ 7/02. Accepted 1/ 3/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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