Carcinogenesis

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Carcinogenesis, Vol. 21, No. 4, 543-550, April 2000
© 2000 Oxford University Press

Accelerated Papers

High cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) contents in mouse lung tumors

Alison K. Bauer, Lori D. Dwyer-Nield¹ and Alvin M. Malkinson^1,2

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

	Abstract

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Mouse lung tumorigenesis is a convenient model for examining all stages of lung adenocarcinoma (AC) progression. Because enhanced cyclooxygenase 2 (COX-2) expression has been observed in advanced human AC, we investigated the intracellular concentrations of the two cyclooxygenases, cyclooxygenase 1 (COX-1) and COX-2, at different times after carcinogen administration to A/J mice. The concentrations of both proteins were much higher in urethane-induced adenomas and carcinomas compared with control A/J mouse lung tissue (P < 0.03 and P < 0.01 in adenomas and AC, respectively, for COX-1; P < 0.003 and P < 0.004 in adenomas and AC, respectively, for COX-2). Small benign tumors that arose spontaneously in 13-month-old mice also stained for COX-1 and COX-2, showing that this elevated enzyme content does not depend on chemical induction. COX-1 and COX-2 immunostaining was observed in normal bronchiolar and alveolar epithelia, alveolar macrophages and bronchiolar smooth muscle. This is the first report of the cellular distribution of COX-1 and COX-2 in murine lungs and the first in any species to demonstrate their co-localization. COX content in isolated bronchiolar Clara cells, a putative cell of tumor origin, was equal to that found in tumors, suggesting that the high enzyme content in neoplasms is due to their proportionally high concentration of these tumor precursor cells. Different patterns of COX-1 and COX-2 expression were observed in tumors of different growth patterns; only occasional small foci stained in solid adenomas, while most cells in papillary adenomas were immunoreactive. This staining pattern was also seen in adenocarcinomas, but some of the papillary portions also included focally stained and unstained regions. The continued expression during neoplastic progression of these specialized enzymes present in normal cells of tumor origin suggests their function in maintenance of the neoplastic state.

Abbreviations: AC, adenocarcinoma; COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs; NSCLC, non-small cell lung carcinoma; PG, prostaglandin.

	Introduction

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Lung cancer will cause 180 000 deaths in the USA in 1999 and is the single greatest cause of cancer mortality (1). About 75% of lung cancer is classified as non-small cell lung carcinoma (NSCLC). Adenocarcinoma (AC), a secretory tumor, is the most common NSCLC type among smokers and the only form of lung cancer that non-smokers develop. AC incidence has increased alarmingly in both smokers and non-smokers in recent decades (2). The peripheral nature of AC makes early detection by sputum cytology difficult and the disease is seldom diagnosed until advanced stages yield clinical symptoms; unfortunately, advanced AC is therapeutically intractable (3). Biomarkers are needed for early detection and a more complete biochemical characterization of pre-malignant lesions would help to identify new sites for chemopreventive strategies that inhibit further neoplastic progression.

Epidemiological studies (4,5) have shown that non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin significantly reduce the risk of colorectal, esophageal, gastric, lung and breast cancers. NSAIDs inhibit prostaglandin endoperoxide synthase (cyclooxygenase, COX) activity. This enzyme introduces two molecules of O₂ into arachidonic acid to form prostaglandin (PG) G₂, which is then reduced to PGH₂. PGH₂ is converted to PGE₂, PGF₂, PGI₂ and thromboxanes by separate enzymes. The two genes that encode the different isoforms of COX, called COX-1 and COX-2, are over 60% homologous (6). COX-2, but not COX-1, concentrations are elevated in the epithelial cells within human colorectal (7), esophageal (8), head and neck (9) and lung (10–12) cancers. Although COX-1 is considered a housekeeping gene in most tissues (13), COX-1 knockout mice have a greatly diminished inflammatory response following injury (6). COX-2 is an inducible, immediate early gene that functions in inflammation, ovulation and carcinogenesis, however, COX-2 knockout mice respond normally to inflammatory stimuli (6). These recent findings using knockout strategies suggest that the respective physiological roles of COX-1 and COX-2 need further clarification (14).

Mouse lung carcinogenesis provides an experimental means of investigating early stages of AC development. Mice develop AC spontaneously and in response to environmental exposures; these tumors are similar to human AC in their histopathology, cells of tumor origin and alterations in the structure and expression of oncogenes and tumor suppressor genes (15,16). We have investigated COX-1 and COX-2 expression in early, intermediate and late stages of tumorigenesis and herein describe these findings.

	Materials and methods

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Carcinogen treatment, sample preparation for immunoblotting and immunohistochemistry
Six-week-old male A/J mice obtained from Jackson Laboratories (Bar Harbor, ME) were injected i.p. with 1 mg/g urethane dissolved in 0.9% NaCl or with the saline vehicle, as described (17), and the mice were killed 6, 9 and 12 weeks and 7 and 11 months after injection. Lungs from each time point were fixed for immunohistochemistry and the larger tumors obtained at the later times were dissected for use in immunoblots as described (18). Normal appearing lung tissue from tumor-bearing mice (uninvolved tissue) and age-matched, vehicle-treated mice were used as controls. For protein isolation, these neoplastic and normal tissues were homogenized in 5 v/w 20 mM HEPES, 10% glycerol, pH 7.5, buffer containing a mixture of protease inhibitors (2 mM EDTA, 2 mM EGTA, 5 µg/ml aprotinin and 10 µM leupeptin); particulate fractions were prepared by centrifugation (16 000 g for 30 min) and used for immunoblotting. Protein concentrations were assayed by the method of Lowry et al. (19). For immunohistochemistry, the lungs were inflated with 10% formalin, dehydrated, embedded in paraffin and 4 µm tissue sections cut.

Clara cell isolation
The lungs of 6-week-old A/J mice were digested with elastase (Worthington Biochemical, Freehold, NJ) and detached Clara cells separated from macrophages by adherence to an IgG-coated (Sigma, St Louis, MO) plate (20). The Clara cell purity was >80% and the cells were >95% viable (20,21). The cells were harvested for protein as above and used for immunoblotting.

Immunoblot analysis
One hundred micrograms of protein were applied to a 10% polyacrylamide gel, electrophoresed and transferred to Immobilon-polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 3% milk in 15 mM Tris, pH 7.4, 150 mM NaCl and subsequently incubated with a 1:1000 dilution of a specific COX-1 or COX-2 goat polyclonal antibody (Santa Cruz, Santa Cruz, CA) overnight at 4°C. A 1:20 000 dilution of a secondary antibody (horseradish peroxidase-conjugated rabbit anti-goat antibody; Sigma) was applied, followed by visualization by chemiluminescence (NEN, Boston, MA). Purified ovine COX-1 and COX-2 antigens (Cayman Chemical, Ann Arbor, MI) were used to demonstrate a lack of cross-reactivity. The COX-1 antibody reacts on western blots with the COX-1 antigen, but not with COX-2. The COX-2 antibody reacts with COX-2 antigen but not with COX-1. Immunoreactive proteins on western blots were quantified with a Quantity One densitometer (Bio-Rad, Hercules, CA). Different protein concentrations applied to the gels were used to construct standard curves so that quantitation was done within the linear range of protein staining. Ponceau staining of proteins transferred to the membrane confirmed equal protein loading. A Student t-test was done to determine statistical significance of the detected proteins.

Immunohistochemical staining
Four micrometer sections were rehydrated and endogenous peroxidase activity was inhibited with 3% H₂O₂, followed by antigen retrieval. Slides were then blocked with 10% rabbit serum (Vector Laboratories, Burlingame, CA). Primary COX-1 and COX-2 antibodies were added at a 1:2000 dilution and incubated overnight at 4°C. Secondary antibody (biotin-conjugated rabbit anti-goat; Vector) was applied, followed by addition of a peroxidase-conjugated streptavidin tertiary antibody complex (Vector) and 3,3-diaminobenzidine as the peroxidase substrate (Sigma) for visualization. At least 30 different adenomas, 50 different carcinomas and three different control lungs were examined using 3 sections/sample. Statistical analysis was done on the different staining patterns by a modified ² test that more stringently tests significance (22).

	Results

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Expression of COX-1 and COX-2 in normal and neoplastic mouse lung
Immunoblot analysis demonstrated that the contents of both COX-1 (Figure 1) and COX-2 (Figure 2) were elevated in adenomas and carcinomas compared with extracts from uninvolved lung tissue. The amounts of both proteins increased significantly compared with uninvolved controls: P < 0.04 and P < 0.01 in adenomas and AC, respectively, for COX-1; P < 0.003 and P < 0.004 for adenomas and AC, respectively, for COX-2.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Immunoblotting of COX-1 in adenomas and carcinomas in A/J mice. Benign and malignant tumors were dissected 7 and 11 months after urethane injection, respectively. (A) Immunoblot analysis. Each lane represents a tissue or tumor extract from a different mouse. Lanes 1–4, uninvolved (UN) tissue from adenoma-containing lungs; lanes 5–7, benign (BEN) adenomas; lanes 8–10, uninvolved (UN) tissue from AC-containing lungs; lanes 11–14, malignant (MAL) AC. This study was repeated two additional times. (B) Quantitation of protein content based on densitometry of the samples shown in (A) and other experimental replicates; means ± SEM. Percentage of control is based on uninvolved tissue from adenoma-containing lungs. UN, n = 8; BEN, n = 5; MAL, n = 6. *P < 0.03; +P < 0.01.

 

View larger version (22K): [in this window] [in a new window]   Fig. 2. Expression of COX-2 in adenomas and carcinomas in A/J mice. Benign and malignant tumors were dissected 7 and 11 months after urethane, respectively. (A) Immunoblot analysis. Each lane represents a tissue or tumor extract from a different mouse. Lanes 1–2, uninvolved (UN) tissue from adenoma-containing lungs; lanes 3–5, benign (BEN) adenomas; lanes 6 and 10, uninvolved (UN) tissue from AC-containing lungs; lanes 7–9, malignant (MAL) AC. This study was repeated two additional times. (B) Quantitation of protein content based on densitometry of the samples shown in (A) and other experimental replicates; means ± SEM. Percentage of control is based on uninvolved tissue from adenoma-containing lungs. UN, n = 6; BEN, n = 5; MAL, n = 6. *P < 0.003; +P < 0.004.

 
The lung contains over 40 different cell types, of which 17 are epithelial (23); determining which cells express COX-1 and COX-2 is crucial to understanding the basis of their elevated concentrations in tumor samples and how this may affect tumorigenesis. COX-1 immunostaining was observed in the bronchiolar and alveolar epithelia, alveolar macrophages and in the smooth muscle surrounding the bronchiolar epithelium (Figure 3A). In the non-tumor portions of the tumor-bearing lungs, the staining was the same as in the untreated control lungs. Immunohistochemical analysis supported the immunoblotting results. We observed hyperplasias containing fewer than eight cells and adenomas that ranged from small (<0.5 mm in diameter) to medium (1–2 mm) in size 6–12 weeks after urethane administration. Cytoplasmic staining of COX-1 was evident in the tumor epithelial cells of all these hyperplasias and adenomas (Figure 3B).

View larger version (146K): [in this window] [in a new window]   Fig. 3. Immunohistochemical staining of COX-1. (A) Control lung demonstrating positive staining in the bronchiolar epithelium (Clara cells), in the smooth muscle underlying this epithelium and in the alveolar epithelium. The inset shows staining in the bronchiolar (black arrowhead) and alveolar epithelium (red arrowhead). (B) Small adenomas adjacent to a bronchiole 6 weeks following urethane administration. The inset shows increased cellularity of epithelial cells. (C) Focal staining of epithelial cells within a solid adenoma 7 months following urethane treatment. (D) Uniform staining of epithelial cells in a papillary adenoma 7 months following urethane treatment. (E) Focal staining of epithelial cells within a papillary AC 11 months following urethane treatment. (F) Blocking peptide used to show specificity of binding; solid adenoma 7 months after urethane. The large images are at 100x magnification and the bar is 5 µm. The black arrowheads mark the locations of the insets. The insets are 200x and the bar is 2.5 µm. Hemotoxylin was used as the counterstain.

 
COX-1 staining was also seen in larger (>2 mm) tumors 7 and 11 months following urethane treatment (Figure 3C–E). These tumors displayed either of two distinct growth patterns: solid tumors that appear as a mass of cuboidal cells (Figure 3C) and papillary tumors (Figure 3D) that grow as finger-like projections of cuboidal and/or columnar cells (24,25). Seventy-two percent of the adenomas at 7 months following urethane were solid while papillary adenomas comprised the rest, as previously observed (25). Table I quantifies the different staining patterns observed in solid and papillary tumors. Focal staining occurred in 87% of solid tumors, wherein several neighboring cells within small discrete portions of the tumors immunostained while most cells in other areas of the tumor remained unstained (Figure 3C). A much greater proportion of the cells in papillary adenomas were immunoreactive and the staining was widespread and uniform within most of the tumor (Figure 3D).

View this table: [in this window] [in a new window]   Table I. COX-1 staining in tumors obtained 7 and 11 months after urethane administration

 
Nuclear atypia and uneven tumor margins characteristic of invading satellite cells were seen as the tumors progressed to malignancy. Fifty-three percent of the AC displayed a solid growth pattern, 32% were papillary and 15% had a mixed pathology with both solid and papillary regions within the same tumor (Table I). A reduction in the percentage of solid tumors with a corresponding increase in papillary tumors during progression was observed, consistent with earlier observations (26). The solid areas in carcinomas exhibited focal COX-1 staining that was indistinguishable from the solid adenomas (data not shown). Papillary portions of carcinomas were more variable; 23% displayed the same uniform staining pattern of the papillary adenomas (data not shown), but 44% stained more focally like the solid tumors (Figure 3E) and 33% had no staining in most of the carcinoma.

Occasional hyperplasias and small adenomas were seen in the lungs from untreated 13-month-old mice, this being the same age as those bearing AC. COX-1 stained positively in these spontaneous lesions (Figure 4A), indicating that COX-1 expression of sufficient quantity to be detectable by immunohistochemistry is not dependent on carcinogen treatment. Lack of staining following the application of a blocking peptide for the COX-1 antibody demonstrated immunospecificity (Figure 3F).

View larger version (103K): [in this window] [in a new window]   Fig. 4. Immunohistochemical staining for COX-1 and COX-2 in spontaneous tumors appearing in untreated mice at 13 months of age. (A) COX-1. The inset shows epithelial cells staining in an area of increased cellularity. (B) COX-2. The inset is as in (A). The large images are at 100x magnification and the bar is 5 µm. The black arrowheads are the locations of the insets. The insets are 200x and the bar is 2.5 µm. Hemotoxylin was used as the counterstain.

 
The cellular localization of COX-2 in control lungs (Figure 5A) as well as the staining pattern in urethane-induced tumors (Figure 5B–E) and spontaneously arising tumors (Figure 4B) were very similar to those observed for COX-1. Table II summarizes the staining patterns of the solid and papillary tumors for COX-2. As with COX-1, the staining of COX-2 was more extensive in papillary adenomas than in solid adenomas. Eighty-two percent of the solid adenomas stained focally and 55% of papillary adenomas stained uniformly for COX-2. Analogous to the greater heterogeneity observed with COX-1 staining in papillary portions of AC, 39% of the papillary portions stained uniformly for COX-2, 39% demonstrated immunoreactive focal patches (Figure 5E) and the remainder had large unstained regions.

View larger version (151K): [in this window] [in a new window]   Fig. 5. Immunohistochemical staining of COX-2. (A) Control lung demonstrating positive staining in the bronchiolar epithelium (Clara cells), in the smooth muscle underlying this epithelium and in the alveolar epithelium. The inset shows staining in the bronchiolar (black arrowhead) and alveolar epithelium (red arrowhead). (B) Small adenomas adjacent to a bronchiole 6 weeks following urethane administration. The inset shows increased cellularity of epithelial cells. (C) Focal staining of epithelial cells within a solid adenoma 7 months following urethane treatment. (D) Uniform staining of epithelial cells in a papillary adenoma 7 months following urethane treatment. (E) Focal staining of epithelial cells within a papillary AC 11 months following urethane treatment. (F) Blocking peptide used to show specificity of binding; solid adenoma 7 months after urethane. The large images are at 100x magnification and the bar is 5 µm. The black arrowheads indicate the locations of the insets. The insets are 200x and the bar is 2.5 µm. Hemotoxylin was used as the counterstain.

 

View this table: [in this window] [in a new window]   Table II. COX-2 staining in tumors obtained 7 and 11 months after urethane administration

 
Because of the similarity in these experimental observations on COX-1 and COX-2, we confirmed the specificity of the antibodies used in several ways. First, the COX-1 and COX-2 antibodies were tested for cross-reactivity using purified antigens as described in Materials and methods; no cross-reactivity was found. Secondly, RAW 264.7, a macrophage cell line that contains only COX-2 (27), demonstrated a band with the COX-2 antibody but did not cross-react with the COX-1 antibody. Some of the macrophages that surround the tumors stained positively for COX-1 and COX-2, while others did not (data not shown). Macrophages surrounding tumors can display different phenotypes, both tumor-enhancing and tumoricidal (28); this functional heterogeneity may be related to a differential expression of the COX enzymes. Thirdly, the expression of COX-1 and COX-2 diverged when mouse lung epithelial cell lines were examined in their basal, cytokine-induced, proliferating and quiescent states (A.Bauer, L.Dwyer-Nield, J.Ahn, and A.Malkinson, manuscript in preparation).

Expression of COX-1 and COX-2 in isolated Clara cells
Because the immunohistochemical studies (Figures 3–5) demonstrated expression of COX-1 and COX-2 in bronchiolar non-ciliated Clara cells and type 2 pneumocytes, we tested whether the elevated COX-1 and COX-2 expression in pulmonary neoplasms relative to control lungs reflected high concentrations of at least one of the presumed cells of tumor origin within these tumors (24–26,29–31). Primary Clara cells can be readily isolated from mouse lungs with a high degree (>80%) of purity (20,21). The COX-1 and COX-2 contents in Clara cell isolates were approximately equal to those found in adenomas and carcinomas, which is much greater than that present in unfractionated whole lung extracts (Figure 6). Therefore, the observed increase in COX content in tumors relative to whole lung may be due to the high content of Clara and type 2 cells present within tumors.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Expression of COX-1 and COX-2 in isolated Clara cells. (A) COX-1 immunoblot: lanes 1–2, whole lung homogenate from untreated mice (n = 2); lane 3, Clara cell isolate from an untreated mouse; lane 4, malignant AC obtained 11 months after urethane administration; lane 5, benign adenoma obtained 7 months after urethane. (B) COX-2 immunoblot: lanes 1–2, whole lung homogenate from untreated mice (n = 2); lane 3, Clara cell isolate from an untreated mouse; lane 4, benign adenoma obtained 7 months after urethane; lane 5, malignant AC obtained 11 months after urethane administration. Aliquots of 100 µg of protein were loaded per well.

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
We observed that mouse lung tumors ranging in size from small adenomas to large AC, as well as hyperplastic foci, contain more of both COX-1 and COX-2 than equivalent amounts of protein prepared from whole lung extracts (Figures 1, 2 and 6). The continued high level of expression of these enzymes from hyperplastic foci into advanced malignancy suggests that eicosanoids play important roles in tumorigenesis. PG synthesis is elevated in human lung tumors and lung cancer cell lines and PGE₂ levels were high in well-differentiated AC in lung cancer patients (32). The highest levels of PG were produced by cell lines derived from AC and large cell undifferentiated carcinoma, compared with other types of cancer (33). Various anti-inflammatory drugs inhibit mouse lung tumorigenesis. This includes NSAIDs such as indomethacin, sulindac and aspirin, that reduce 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induction of mouse lung tumors by up to 60% (34,35), and natural and synthetic glucocorticoids such as corticosterone (36) and budesonide (37), that reduce urethane and benzo[a]pyrene-induced tumorigenesis by 26 and 80%, respectively. NSAIDs directly inhibit the activities of both COX enzymes while glucocorticoids lower the availability of the arachidonate substrate of COX. In other systems, eicosanoid metabolites have been shown to be mitogenic (38), apoptotic (39) and angiogenic (40) and NSAIDs inhibited the anchorage-dependent growth of some NSCLC cell lines (41).

Both cell types of probable tumor origin, the bronchiolar Clara cells and type 2 pneumocytes, normally contain high concentrations of these enzymes (Figures 3 and 5) and constitute the greatest proportion of the tumor epithelial cell population (24–26,29–31). Isolated Clara cells contain amounts of COX-1 and COX-2 similar to the tumor extracts (Figure 6). COX-1 and COX-2 therefore cannot strictly be considered as biomarkers, since neoplastic tissue is not expressing proteins absent from the precursor cells of origin. However, because >90% of human AC are peripheral in location (42), elevated COX-1 and COX-2 contents in biopsy specimens resected on the basis of morphological dysplasia could help confirm a probable Clara or type 2 cell origin of these samples.

We found that not only COX-2 but also COX-1 is highly expressed in mouse lung tumors (Figures 1 and 2). In contrast, only high COX-2 concentrations have been reported in human lung cancers (10–12), with the exception of one paper describing COX-1 staining in NSCLC biopsy specimens (43). Analogous to our findings in mice, COX-2 is elevated in some atypical adenomatous hyperplasias in human specimens (11,12), which may be precursor lesions of AC (42). COX-2 staining was encountered more commonly in human AC than in other types of human lung cancer (11). COX-2 is also expressed in rat lung tumors (44). COX-1 was not expressed within human breast tumor epithelial cells, but the surrounding stroma and macrophages contained high amounts of COX-1 (27); few breast tumors (5%) expressed COX-2. Thus, in breast cancer, COX-1 is the predominant neoplastic marker. NSAIDs decrease breast cancer incidence (5). This, together with the observation that immortalized endothelial cells transfected with COX-1 become tumorigenic (45), implies that COX-1 does not function merely as a housekeeping gene.

We found staining of both COX-1 and COX-2 in the bronchiolar and alveolar epithelia, bronchiolar smooth muscle and alveolar macrophages. This is the first examination of the cellular location of COX-1 and COX-2 in mouse lung. The high concentrations of both COX enzymes in the tumors and their very similar cellular localization within the lung makes studies on the co-regulation of these two different gene products intriguing. In rats, COX-1 was expressed in bronchiolar epithelium and smooth muscle, alveolar macrophages, endothelial cells of large arteries and vascular smooth muscle cells of large veins (46). Rat COX-2 was expressed to a lesser extent than COX-1 in bronchial epithelium and alveolar macrophages, to the same extent in bronchial smooth muscle and even more highly in macrophage-like cells directly below the bronchial epithelium and in the surrounding connective tissue (46). Neither COX-1 nor COX-2 was expressed in rat alveolar epithelium. In contrast, other investigators found COX-2 in chemically induced rat lung tumors but not in non-neoplastic lung tissue, with negligible amounts detected in spontaneous tumors; COX-1 was not studied (44). In humans, COX-2 expression was observed in bronchial epithelium, alveolar type 1 and 2 pneumocytes and in smooth muscle, vascular endothelial and inflammatory mononuclear cells (13). COX-1 and COX-2 staining was seen in NSCLC cells, but there was no mention of staining in normal lung tissue (43); this is the only paper which addressed COX-1 staining in human lung tissue. Thus, the three species display some differences and the two reports on rat lung differed from each other. Generally, human COX-2 staining was similar to that in mouse, mouse COX-1 co-localized with mouse COX-2 and one report of COX-1 staining in human NSCLC supports our findings in mouse.

A greater proportion of the epithelial cells in papillary adenomas immunostained than did epithelial cells in solid adenomas (Figures 3–5). Whether this distinction is in any way causal to the generation of these distinct growth patterns is unknown. Papillary tumors may have a greater propensity to progress to malignancy than do solid tumors (26); the presumed increased eicosanoid production in papillary adenomas, resulting from more cells within the tumor expressing COX-1 and COX-2, might contribute to this progression. The more variable COX expression in papillary portions of AC contrasts with the more uniform expression in papillary adenomas, however, so the nature of such a role is unclear. Eicosanoids may have a more significant role in the growth of benign papillary adenomas than in malignant tumors or this heterogeneous staining in papillary AC may merely reflect the greater genetic instability and consequent erratic changes in gene expression found in carcinomas. Morphological and biochemical characteristics support the hypothesis that solid adenomas originate from type 2 alveolar pneumocytes (25,29,47). Type 2 pneumocytes are stem cells of the alveolar compartment and can differentiate to replace type 1 pneumocytes lost to injury (48). The origin of papillary adenomas is more controversial. Clara cells, the non-ciliated epithelial cells of the terminal bronchioles which can differentiate into ciliated cells, may constitute the cell type of papillary tumor origin (24,25,31). We observed Clara cell hyperplasias in the current studies (data not shown), as has been observed previously (31). However, markers characteristic of a type 2 cell phenotype have also been noted in papillary tumors (30,47). Clara and type 2 cells have high concentrations of enzymes that catalyze xenobiotic metabolism (49), so it may not be surprising that they also contain COX enzymes that initiate production of autocrine and paracrine factors which mediate environmental responsiveness.

	Acknowledgments

 
We thank Drs Dennis Ahnen and Pamela Rice for reviewing the manuscript, Dr Wilbur Franklin for assistance with lung pathology and Dr Deborah Hall for statistical assistance. This work was supported by Predoctoral Training Program in Pharmacology GM 07635 and by USPHS grant CA 33497.

	Notes

 
² To whom correspondence should be addressed Email: al.malkinson{at}uchsc.edu

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Received August 10, 1999; revised November 10, 1999; accepted November 22, 1999.

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