In Vitro Cell. Dev. Biol. 31:379-386. May 1995 © 1995 Societyfor In VitroBiology 1071-2690/95 $05.00+ 0.00

EXPRESSION OF AIRWAY SECRETORY EPITHELIAL FUNCTIONS B Y L U N G CARCINOMA CELLS WALTER E. FINKBEINER, LORNA T. ZLOCK, SUZANNE D. CARRIER, SUSAN Y. CHUN, LAWRENCE WATI', and ALBERT CHOW

Department of Pathology and Cardiovascular Research Institute, Universityof California, San Francisco, California 94143-0506 (Received 7 April 1994; accepted 5 October 1994)

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The ceils expressed some uhrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a n u m b e r of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.

Key words: traebeobronchial; mucus; differentiation. INTRODUCTION The underlying growth abnormalities of neoplastic cells are often associated with functional derangements. However, carcinoma cell lines, particularly several lines derived from human colonic carcinomas, are useful as models for studying epithelial cell functions (1,17,27,31). Although numerous lung carcinoma cell lines are available (3), they have been used infrequently for the study of normal airway biology, and for the most part it is not known whether these cell lines retain differentiated functions. To screen for the retention of airway secretory cell properties, we screened 12 non-small cell lung carcinoma cell lines for morphologic evidence of secretory epithelial differentiation, airway secretory cell products, spontaneous secretion of high molecular weight glycoconjugates, and mediatorinduced release of high molecular weight glycoconjugates. Our studies identified considerable heterogeneity among the cell lines. No cell lines maintained a high degree of differentiation for all the parameters studied. However, we identified several cell lines exhibiting some differentiated features of airway secretory epithelium, and these may prove useful for studies of specific aspects of airway cell biology. MATERIALS AND METHODS

Materials. Unless indicated otherwise, chemicals and reagents were obtained from Sigma Chemical, St. Louis, MO. Cell culture. Media and antibiotics were obtained from the Cell Culture Facility of the University of California, San Francisco. Fetal bovine serum lots were tested by the Cell Culture Facility for ability to support colony formation and growth of selected standard cell lines. Carcinoma cell lines representing the major histopathologic subtypes of non-small cell lung carcinoma were obtained from the American Type Tissue Collection (Rockville, MD). The cell lines, their histopathologic type, and reference(s) to their establishment and initial characterization are provided in Table 1. Cells were seeded at 2 × 104 per cm 2 into T-75 culture flasks (Coming Glass Works,

Coming, NY) for metabolic labeling studies or on tissue culture chamber slides (Nunc, Naperville, IL) for immunocytochemistry. Culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 nutrient medium (DF12) supplemented with 10% fetal bovine serum and gentamicin (50 lag/ml). Cultures were incubated at 37 ° C in a humidified atmosphere containing 95% air:5% CO2. Medium was changed every three days. Ultrastructure. Electron microscopy was performed on cells grown on tissue culture plastic or glass cover slips. Unless otherwise noted, processing was performed at room temperature. For fixation, culture medium was replaced with a solution of 2.5% glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.4). After 1 h, ceils were postfixed for 1 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4), followed by staining with 0.5% uranyl acetate in 0.05 M sodium maleate (pH 5.2) and dehydration. Cells grown on tissue culture vessels were removed by scraping and transferred into Beem capsules (Ted Pella, Redding, CA) where they were infiltrated with Polybed 812 (Polysciences, Warrington, PA). Ceils grown on glass cover slips were embedded by inverting Polybed 812-containing Beem capsules directly onto infiltrated monolayers. After polymerization at 60 ° C, the hardened resin cylinders containing the cell monolayers were separated from the cover slips by immersion in liquid nitrogen. Semithin (0.5 p.m) sections were cut with glass knives on an uhramicrotome, mounted on microscope slides, and stained with toluidine blue for examination with a light microscope. The sections were cut in two planes, parallel and perpendicular to the cell layers. Specific areas were selected, and thin sections were cut with a diamond knife and mounted on grids. These sections were stained with uranyl acetate and lead citrate before examination in a JEOL IOOS electron microscopelmmunocytochemistry. Cell cultures were washed 3 times with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde, 45% acetone, 1.4 mM sodium phosphate, and 7.35 mM potassium phosphate (pH 6.6). Immunocytochemistry with a panel of monoclonal antibodies directed against human airway secretions was performed with a modification of a biotin-avidin procedure (9). Sections of normal human tracheal tissue were used as positive controls. Negative controls consisted of irrelevant monoclonal antibody and omission of primary and secondary antibody. Metabolic radiolabeling and secretoryphysiology. After cells reached confluence, Naz[3~S}O4at 7.5 p_Ci/cm2 (carrier-free, specific activity 43 Ci/mg, ICN Radiochemicals, Inc., Irvine, CA) was added. After 24 h, medium was 379

380

FINKBEINER ET AL. TABLE 1

LUNG CARCINOMACELL LINES AND THEIR HISTOPATHOLOGIC TYPE Cell Line

A427 A549 CALU-1 CALU-3 CALU-6 NCI-H441 NCI-H460 NCI-H520 NCI-H596 NCI-H661 Sk-Lu-] Sk-Mes-1

HistopathologicType

Adenocarcinoma Adenocarcinoma Squamous cell carcinoma Adenocarcinoma Poorly differentiated adenoearcinoma Papillary adenocarcinoma Large cell carcinoma Poorly differentiated squamous cell carcinoma Adenosquamous carcinoma Large cell c~reinoma Poorly differentiated carcinoma Squamous cell carcinoma

Reference

(14) (14, 22) (12) (11) (28) (5) (2) (2.21) (2) (2) (13) (12)

removed and flasks were washed with PBS. Serum-free medium (SFM) was added to each flask, and every 30 min for 210 rain this medium was collected and replaced. At 180 rain, flasks, which would later receive 8-bromo-adenosine 3',5'-cyclic monophosphate (8-br-cAMP, 10 -:~ M), received SFM containing the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX, 10 z M). At 210 min. SFM containing experimental drugs was added. Drugs tested included bethanechol (10 s M), phenylephrine (10 -~' M), l-isoproterenol (10 ~ M), bradykinin (10 -~ M), histamine (10 -~ M), ATP (10 4 M), 8br-cAMP (10 ~/14) + IBMX(10 s M), and calcium ionophore A23187 (10 -~ M; Calbioehem, La Jolla, CA). At 240 rain, incubation was terminated and samples were dialyzed [Spectrapor membrane tubing molecular weight (MW) cutoff 12 to 14 kDa; Fisher Scientific, Pittsburgh, PAl against distilled water containing sodium azide (10 mg/ml). After addition of scintillation fluid (Hydrofluor; National Diagnostics, Sommelwille,NJ), the samples were counted on a beta scintillation counter (LS7500; Beckman Instruments, Ir~ine, CA). The degree of stimulation-induced secretion was evaluated by comparing the release of nondialyzable radiolabel (cpm) for the periods after (sample collected at 240 min) and immediately preceding (sample collected at 210 min) drug exposure. To control for baseline fluctuations among flasks, a relative secretory rate (RSR) was obtained by dividing the secretory rate obtained from flasks receiving drugs by the secretory rate obtained from control flasks not receiving drugs. A RSR significantly greater than 1.0 indicated the presence of a secretory response. Significance (P < 0.05) of pharmacologic effects was determined using Dunnett's test (7). We compared the viability of control and drug-treated cultures by measuring the concentration of lactate dehydrogenase in the 240 rain samples using a commercially available assay kit (Sigma). Gelfiltration chromatography. Medium collected from cultures after the 24-h period of metabolic radiolabeling as described above was dialyzed (Spectra/por membrane tubing, MW cutoff 12 to 14 kDa) against 1 mM EDTA, 0.02% sodium azide, and 0.5 mM phenylmethylsulfonyl fluoride. The material was lyophilized, dissolved in column buffer consisting of 0.1 M sodium acetate (pH 6.0) containing 4 M guanidine chloride and 0.5% Chaps and applied to a Sepharose CL-4B column (1.5 X 155 em). Fractions (3 ml) were collected and an aliquot of each fraction was counted for radioactivity. RESULTS

Uhrastructure. Examination of cells grown on tissue culture plastic or glass cover slips revealed identical findings. All cell lines were composed of poorly differentiated cells which showed some evidence of epithelial differentiation but minimal evidence of an exocrine cell phenotype (Fig.. 1). Cell nuclei were enlarged and pleomorphic. A549, Calu-1, NCI-H441, NCI-H460, NCI-H520, Sk-Lu-1, and SkMes-1 cells displayed prominent and sometimes multiple nuclei. The cytoplasm of all cell types contained scattered free ribosomes, some rough endoplasmic reticulum, rare Golgi apparatuses, and scattered

mitochondria. Pools of electron-lucent material were identified in Calu-3, NCI-H460, NCI-H520, and NCI-H661 ceils. Intermediated filaments were pronounced in A549, NCI-H441 and NCI-H596 cells. Scattered cytoplasmic vacuoles and myelin figures were present in A427, A549, Calu-1, NC1-596, and Sk-Lu-1 cells. Most cell lines showed a paucity of membrane specializations. All cell lines demonstrated variable numbers of microvilli on their apically oriented membranes. The lateral membranes of A549, Calu-1, Calu-3, NCIH596, and NCI-H661 displayed prominent membrane foldings with Calu-3 and NCI-H596 cells showing slightly more complex interdigitations of neighboring membranes. NCI-460, NCI-H520, and SKMes-1 cells had rare membrane projections between cells. The remainder of the cell lines (A427, Calu-6, NCI-H441, and Sk-Lu-1) lacked these projections. However, Sk-Lu-I cells had cytoplasmic projections or blebs between adjacent cells. Cell junctions were also rudimentary in most cells. However, A427, A549, Calu-3, NCI-H441 and to a lesser extent, NCI-H520 and NCI-H661 cells were attached by desmosomes. Calu-3. NCI-H441, and NCI-H460 cells had tight junctions, intermediate junctions, and desmosomes arranged in typical epithelial junctional complexes. These were better developed in the Calu-3 cells (Fig. 2). lmmunocytochemistry. In general, staining of the cell lines was heterogeneous with most cell lines expressing antigens associated with each of the airway secretory cell types (Table 2). Only Calu-3 cells expressed antigens associated with both goblet and serous gland cells (B3F10 and B8C3). NCI-H596 cells failed to express antigens that are exclusively associated with mucous gland cells (A8E4, BIF8, B1DT, B5E9) or serous gland cells (A2E7, A3B7, A10F5, B1D8, B7E5). However, they did express some of the antigens typically expressed by goblet cells. Secretoryphysiology. With the exception of ATP and calcium ionophore A23187, the agents tested were not generally effective at increasing release of high molecular weight glyconjugates (Table 3). ct-adrenergic and [3-adrenergic stimulation with phenylephrine and isoproterenol, respectively, failed to stimulate secretion in any of the cell lines. The cholinergic drug, bethanechol, had a mild stinmlatory effect on NCI-H520 cells. Mediators known to act by increasing intracellular calcium, bradykinin and histamine, did have a secretogogue effect on some cells. Thus, bradykinin caused release of radiolabel from A549 and NC1-H596 cells whereas histamine stimulated secretion in Calu-1 and NCI-H441 cells. Purinergic receptor stimulation with ATP released radiolabel from six of the cell lines including A549, Calu-1, Calu-3, NCI-H441, NCI-H596, and Sk-Lu-1 ceils. Bypassing receptors with the cAMP analogue 8-bromo-cAMP failed to stimulate secretion by any of the cell lines. However, in all cell lines, directly elevating intracellular calcium with the calcium ionophore A23187 caused significant stimulation of radiolabel release. In all cell lines and for all secretory agonists, the concentration of lactate dehydrogenase in the 240-min samples did not differ from that of control cultures. Gelfihration chromatography. The molecular weight of secretions in which :~zSwas incorporated varied among the cell lines (Fig. 3). Calu-3, Sk-Mesl, and NCI-H441 had notable incorporation of the radiolabel in distinct peaks, which eluted in both the excluded volume of the colume (->2 × 106 Da) as well as the included volume. A549, NCI-H460, NCI-H520, NCI-H596, and Sk-Lu-1 cell lines typically had rather large and broad peaks in the included volume. Three cell lines (A427, Calu-1, and Calu-6) incorporated radiolabel within material that was not distinctly resolved by Sepharose CI-4B

FUNCTIONAL CHARACTERIZATION NON-SMALL CELL LUNG CARCINOMA CELL LINES

~:;

~~ -

,~,:: ," ry;~

F~G. 1. Electron photomicrographs of non-small cell carcinoma cell lines. Orientation of sectioning was parallel to the cell sheets. A, A427; B, A549; C, CALU-1; D, CALU-3: E, CALU-6; F, NCI-H441.

381

382

FINKBEINER ET AL.

,.

.4

)

'.

.,

~

FIG. 1. (continued) Electron photomicrographs of non-small cell carcinoma cell lines. Orientation of sectioning was parallel to the cell sheets. G, NCI-H460; H, NCI-H520; 1, NCI-H596; J, NC1-H661; K, Sk-Lu-1; L, Sk-Mes-1. B a r = 1 p,m.

FUNCTIONALCHARACTERIZATIONNON-SMALLCELL LUNG CARCINOMACELL LINES

FIG. 2. Electron photonficrographof Calu-3 cells. A junctional complex connects the lateral membranes of adjacent cells. B a r = 1 ~tm.

but which eluted in broad peaks beginning at the void volume and extending well into the excluded volume. Finally, one cell line, NCtH661, had a single broad, excluded volume peak. DISCUSSION The establishment of cell lines from neoplastic tissues has been motivated primarily by the desire to study neoplastic conditions in vitro. However, if differentiated functions are preserved, such cell lines may become useful tools for the study of normal cellular functions. In an earlier study of 12 non-small cell carcinoma cell lines, we established that some continued to synthesize a number of molecules such as MUC2 (Calu-3, Calu-6, NCI-H460, NCI-H596, and A549), lysozyme (Calu-3, NCI-H441, NCI-H520, and NC1-H661), and lactoferrin (Calu-1, Calu-3, NCI-H460, and NCI-H661) that are normally secreted by surface epithelial goblet or submucosal gland ceils of the airway (10). In this study, we tested these same cell lines for expression of other features of secretory cell differentiation, including the presence of intact stimulus-secretion coupling mechanisms. Using electron microscopy, we failed to identify cell lines that contained secretory granules of the goblet/mucous cell (electron lucent) or serous cell (electron dense) type. However, 4 of the 12 cell lines did contain electron lucent material within their cytoplasm. The inability of these cell lines to package secretions in typical membrane-bound secretory granules may be due to the method of cell culture, because we have induced the formation of membrane-bound, electron lucent secretory granules in Calu-3 cells when they are grown on semi-permeable filter supports at an air-liquid interface (25). Immunocytochemistry revealed that these lung carcinoma cell lines do retain a number of airway secretory cell products which are recognized by a panel of cell-specific antibody markers. However, expression was heterogeneous, the only exception being NCI-H596 ceils which demonstrated a profile similar to normal goblet cells. Stimulus-secretion coupling mechanisms were more frequently than not lost in these cell lines. This may be due to loss of receptors, disruption of the intracellular messenger pathways, or other abnormalities present in the secretory pathway of these cells. It is possible that higher, non-physiologic concentrations of the agonists tested

383

could result in the release of radiolabel fiom some of these cells; however, our aim was the identification of differentiated secretory epithelial function. Despite this, several cell lines seem to maintain some of the known pathways of regulated mucus secretion. Thus, cell lines were identified which released high molecular weight glycoconjugates after stimulation with bethanechol, bradykinin, histamine, and ATP. We did not identify any cell lines that maintained either a-adrenergic or ~-adrenergic stimulus-secretion coupling measures at the drug concentrations tested. As described above, several patterns of incorporation of radiolabel were identified. The pattern of a distinct void volume and one or more included volume peaks as was seen with Calu-3, NCI-H441, and Sk-Mes-1 cells is similar to what has been described following analysis of human sputum (8,16) or secretions obtained from cell and organ cultures of haman airways (4,24,29). The void volume material obtained from primary cultures of ai~'ay surface epithelial cells or submucosal mucous gland cells consists primarily of mucin glycoproteins (6,18), however, the void volume fractions obtained from a cell line of bovine tracheal gland serous cells consists primarily of proteoglycans (23). Thus, additional analyses of the high molecular weight material generated by the lung carcinoma cell lines is necessary to determine their suitability as models for studying the biosynthesis of specific chemical components of respiratory mucus. Although loss of differentiated secretory function could be due to the neoplastic nature of the lung carcinoma cell lines, a contributing factor could be a deficient cell culture environment. That the latter may play an important role is indicated by additional studies of the Calu-3 cell line. This cell line, when grown on semi-permeable membranes at an air-liquid interface, forms a cell sheet containing scattered cells exhibiting well-formed, electron lucent secretory granules typical of the airway epithelial goblet cells or submucosal gland cells. Grown in this manner, the cells are attached by tight junctions and the cell sheets thus formed have a resistance of approximately 100 ~ ' c m 2 and a baseline short circuit current of approximately 35 pA/ cm2(25). Interestingly, our studies of the electrical properties of Calu3 cells demonstrated that these cells have abundant amounts of the cystic fibrosis transmembrane conductance regulator (CFTR) and that the short-circuit current present in cultures of Calu-3 cells was due to C1 secretion. Additionally, baseline short-circuit cmTent was stimulated by elevation of cAMP levels, either by stimulation of [3-adrenergic receptors or via direct elevation of intracellular cAMP by cAMP analogues, This contrasts with the findings of the current study in which isoproterenol had no effect on mucus secretion by Calu-3 cells. Furthermore, this was not due to differences in the methods of cell culture between the two studies because isoproterenot given to Calu-3 cells grown at an air-liquid interface still fails to stimulate the release of high molecular weight glycoconjugates (Finkbeiner and Zlock, unpublished data). However, additional studies are necessary to characterize the material secreted by Calu-3 cells and to investigate the relationship of CI secretion and mucus secretion in these cells. In summary, maintenance of regulated mucus secretion has been studied in 12 non-small cell lung carcinoma cell lines. Several of these cell lines may be useful in studies of specific pathways of regulated mucus secretion. Some of these cell lines may also be excellent models for studying the molecular biosynthesis of airway secretory products. As with any cells in culture, manipulation of the culture environment in which these carcinoma cells are grown could yield cells maintaining higher levels of differentiation. Finally, since

384

F I N K B E I N E R ET AL.

TABLE 2 IMMUNOCYTOCHEMICAL STAINING OF NON-SMALL CELL LUNG CARCINOMA CELL LINES WITH MOMOCLONAL ANTIBODIES THAT R E C O G N I Z E D A N T I G E N S P R E S E N T IN A I R W A Y S E C R E T O R Y C E L L S Ab

A427

A549

CAI,U-I

CALU-3

CALU-6

NCIH441

NCIH460

NC[H520

NCI H596

NCIH661

Sk-Lu-I

Sk-Mes-I

+

+

(+) (+) (+)

+

+

(+) (+) +

+

+

+

-

-

-

+

+ -

+ + + + +

+

+

G.M.S A1D3 A1FI1 A10G5 B2B8 B4F7 B8E]O

+

+

+

+

+ + +

+

-

+

+

+

.

+

+

+

+

+

+

-

-

+

+

-

+

+ -

+ + + + + + -

+ + + + + + + + + (+) + (+)

+ (+) + + +

(+) . + + + (+) + + .

+ -

+ + + + -

-

-

(+) (+ )

B1F8 ABE4 B1D7 B5E9

. + -

. + (+) (+)

S A2E7 A3B7 A10F5 B1D8 B7E5

+ + -

+ + + + +

. +

.

.

+ .

-

.

.

.

.

+

.

.

+

+

-

+

+

G.M AIEll A2F3 A3Gll A6D8 B3Dll B3E8 B3F2 B4Cll B5D5 B5D7 B6E8 B6G6

+ + -

+ . + -

+

+

.

.

+

+ . -

.

.

.

.

.

.

.

.

.

.

. .

.

+ + +

. -

.

.

+

.

. .

. (+)

.

+ +

+ . + .

.

-

.

.

.

.

.

.

.

.

.

.

G.S B3F10 B8C3

m

m

m

M

.

.

.

.

+ (+)

+ + (+)

+ . .

+ + + + +

+ + + + +

+ + + + +

Key: G = goblet cell; M = m u c o u s cell; S = serous cell. +

.

.

+

.

+

.

. .

. .

(+) + + + +

+ . .

. -

-

.

.

.

+ + + + +

.

+ + + + +

.

. -

.

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.

. + + + +

+ + + +

= positive; ( + ) = p o s i t i v e in s u b p o p u l a t i o n of cells; -

TABLE

+ + +

+

+ +

= negative.

3

E F F E C T OF M U C O U S S E C R E T O G O G U E S ON N O N - S M A L L C E L L L U N G C A R C I N O M A C E L L L I N E S ~ BCH

A427 A549 CALU-1 CALU-3 CALU-6 NCI-H441 NCI-H460 NCI-H520 NCI-H596 NCI-H661 Sk-Lu-1 Sk-Mes-1

1.2 0.9 1.1 0.9 1.1 1.3 1.0 1.4 1.1 0.9 1.1 0.9

+ ± ± ± ± _+ ± ± ± + ± +_

0.3 0.2 0.2 0.1 0.1 0.3 0.1 0.1' 0.2 0.2 0.1 0.1

PE

1.3 0.9 1.0 1.0 0.9 1.4 1.1 1.1 1.4 1.1 1.0 0.9

± +_ ± ± ± + _+ _+ ± +_ +_ ±

ISO

0.4 0.2 0.1 0.1 0.2 0.3 0.1 0.2 0.6 0.1 0.1 0.1

1.1 0.7 1.1 1.2 0.9 1.6 1.0 1.0 1.1 1.2 1.0 0.9

± ± ± +_ + +_ ± ± ± _+ + ±

BK

0.2 0.1 0.3 0.3 0.1 0.3 0.1 0.2 0.4 0.1 0.2 0.1

0_9 3.6 1.2 1.2 1.0 1.2 1.2 1.3 2.6 1.0 1.2 0.9

+ + + + ± + ± _+ _+ + + ±

HIST

0.1 1A b 0.3 0.2 0.3 0.2 0.2 0.2 0.7 ~' 0.2 0.2 0.1

0.9 1.2 3.4 1.0 1.1 2.0 1.0 1.1 1.6 1.0 1.4 1.1

_+ ± ± _+ ± ± +_ _+ ± ± ± ±

0.1 1 0.2 ~' 0.1 0.3 0.3 ~' 0.2 0.1 0.7 0.2 0.2 0.2

8BrAMP/ ]BMX

ATP

1.4 11.2 11.5 1.4 1.2 3.8 0.9 0.9 3.0 1.4 1.6 1.5

+ ± _+ ± ± ± ± _+ + ± ± ±

0.3 4.7 ~' 6.4 b 0.2 L 0.2 1.ff ~ 0.1 0.2 0.5" 0.4 0.6 ~ 0.5

0.9 0.9 1.2 1.0 0.8 1.4 1.0 1.1 0.9 1.1 1.2 0.9

+_ ± ± ± + ± _+ _+ _+ _+ _+ +-

0.1 2 0.5 0.2 0.2 0.4 0.2 0.1 0.3 0.2 0.2 0.2

A23187

2,1 6.8 2.9 3.3 2.9 3.7 1.7 2.1 5.3 5.4 3.0 2.8

± ± ± + ± ± + ± ± ± ± ±

0.5 ~ 3.1 b 0.5 b 0.4 b 0.8 t' 0.9 ~ 0.2 b 0.6 ~' 0.6 b 2.9 b 0,7 b 1.P'

BethanechoL 10 -z M (BCH); p h e n y l e p h r i n e , 10 ~ M (PE); isoproterenol 10 -~ M (ISO); b r a d y k i n i n , 10 -z M (BK): h i s t a m i n e , 10 -3 M (HIST), A d e n o s i n e t r i p h o s p h a t e (ATP), 8-bromo-cAMP, 10 -~ M (8-Br-cAMP) + IBMX, 10 -3 M (IBMX), and c a l c i u m ionophore A 2 3 1 8 7 , 10 z M. Data are expressed as relative secretory rate _+ SD (n >~ 5). b p < 0.01; ~ P < 0.05.

FUNCTIONAL CHARACTERIZATION NON-SMAI,L CELL LUNG CARCINOMA CELL LINES 10

10 ,

to.

A427

Vo

385

v,

A549 Vo

Vt

4

t

8-

Calu-1 Vo

Vt

t

t

84 4

e-I

6-

4 4-

4,

2-

2,

0-

44

0 0

20

40

60

80

100

10

0

x

E O. ~)

go

,~

:1 o

40

6O

gO

1oo

4o

gO

gO

too

40

gO

go

100

80

100

NCI-H441 8-

6-

6,

6.

I

t

20

1o

Calu-6 8.

4.

p

2.

2,

...-=

00

20

40

go

80

O. 40

loo

10-

6O

8O

loo

20

10.

10

NCI-H460

oO 03

go

8

4.

0

~

I0,

Calu-3

o2,

2o

NCI-H596

NCI-H520

8-

~4

6-

64

0

0," 40

go

gO

o

100

40

20

6O

8O

100

0 10

10,

10

NCI-H661

Sk-Mes-1

Sk-Lu-1

8,

8.

6,

6.

6

4,

4

2

2.

,

0

20

40

60

-

l

go

-

-

0

1

I00

0

,-. 20

,

40

.

,

gO

.

0

,

80

~00

0

,

i

40

6O

Fraction Number FIG. 3. Sepharose C1-4B (1.5 X 155 cm) gel filtration chromatography of metabolically radiolabel, spontaneously released material. V and V, refer to the elution positions of blue dextran and (~S]Q, respectively.

386

FINKBEINER ET AL.

these cells can be serially propagated in culture, selection of differentiated subpopulations by cloning (19) or induction of differentiation with chemicals (1,15,20,26,30) might yield lung carcinoma cell lines displaying phenotypes more closely resembling those of normal airway epithelial cells.

ACKNOWLEDGMENTS We thank Jonathan H. Widdicombe, D. Phil., for a critical reading of the manuscript. This study was supported by funds provided by the Cigarette and Tobacco Surtax Fund of the State of California through the TobaccoRelated Disease Research Program grant RT446 and the Cystic Fibrosis Foundation.

REFERENCES 1. Augeron, C.; Laboisse, C. L. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res. 44:3961-3969" 1984. 2. Banks-Schlegal, S. P.; Gazdar, A. F.; Harris, C. C. Intermediate filament and cross-linked envelope expression in human lung tumor cell lines. Cancer Res. 45:1187-1197; 1985. 3. Bepler, G.; Koehler, A.; Kiefer, P., et al. Characterization of the state of differentiation of six newly established human non-small-cell lung cancer lines. Differentiation 37:158-171; 1988. 4. Boat, T. F.: Cheng, P.-W.; Iyer, R. N., et ah Human respiratory tract secretions. Arch. Biochem. Biophys. 177:95-104; 1976. 5. Brower, M.; Carney, D. N.; Oie, H. K., etal. Growth of cell lines and clinical specimens of human non-small cell lung cancer in serumfree defined medium. Cancer Res. 46:798-806; 1986. 6. Culp, D. J.; Latchney, L. R. Mucinlike glycoproteins from eat tracheal gland cells in primary culture. Am. J. Physiol. 265:L260-L269; 1993. 7. Dunnett, C. W. New tables for multiple comparisons with a control. Biometrics 20:482-191; 1964. 8. Feldhoff, P. A.: Bhavanandan, V. P.; Davidson, E. A. Purification, properties, and analysis of human asthmatic bronchial mucin. Biochemistry 18:2430-2435; 1979. 9. Finkbeiner, W. E.; Basbaum, C. B. Monoclonal antibodies directed against human airway secretions: loealization and characterization of antigens. Am. J. Pathol. 131:290-297; 1988. 10. Finkbeiner, W. E.; Carrier, S. D.; Teresi, C. E. Reverse transcriptionpolymerase chain reaction (RT-PCR) phenotypic analysis of cell cultures of human tracheal epithelium, tracheobronchial glands and lung carcinomas. Am. J. Respir. Cell. Mol. Biol. 9:547-556: 1993. 11. Fogh, J.; Fogh, J. M.; Orfeo, T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. JNC158:209214: 1977. 12. Fogh, J.: Trempe, G. New human tumor cell lines. In: Fogh, J., ed. Human tmnor cells in vivo. New York: Plenum Press; 1975:115-159. 13. Fogh, J.; Wright, W. C.; Loveless, J. D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. JNCI 58:209214; 1977.

14. Giard, D. J.; Aaronson, S. A.; Todaro, G. J., et ab In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. JNC1 51:1417-1423; 1973. 15. Hagar, J. C.; Gold, D. V.; Barbosa, J. A., etal. N,N-dimethylformamideinduced modulation of organ- and tumor-associated markers in cultured human colon carcinoma cells. JNCI 64:439M46; 1980. 16. Havez, R.; Roussel, P. Bronchial mucus: physical and biochemical features. In: Weiss, E. B.; Segal, M. S, eds. Bronchial asthma: mechanisms and therapeutics. Boston: Little, Brown and Co.; 1976: 409-122, 17. Huet, C.: Sahuquillo-Merino, C.; Coudrier, E.. et al. Absorptive and mueus-secreting subelones isolated from a multipotent intestinal cell line (HT-29) provide new models for ceil polarity and terminal differentiation. J. Cell Biol. 105:345-357; 1987. 18. Kim, K. C. Biochemistry and pharmacology of mucin-like glycoproteins produced by cultured airway epithelial ceils. Exp, Lung Res. 17:533545; 1991. 19. Kuan, S.-F.; Byrd, J. C.; Basbaum, C. B., et al. Characterization of quantitative mucin variants from a human colon cancer cell line. Cancer Res. 47:5715-5724; 1987. 20. Lever, J. E. Inducers of mammalian celt differentiation stimulate dome formation in a differentiated kidney epithelial cell line (MDCK). Proc, Natl. Acad. Sci. USA 76:1323-1327; 1979. 21. Levitt, M. L.; Gazdar. A. D.: Lie, H. K., et al. Cross-linked enveloperelated markers for squamous differentiation in human lung cancer cell lines. Caneer Res. 50:120-128; 1990. 22. Lieber, M.; Smith, B.: Szakal, A., el al. A eontinuous tumor-cell line from a hmnan lung carcinoma with properties of Type II alveolar epithelial <'ells. Int. ,1. Cancer 17:62-70; 1976. 23. Paul, A.: Pieard, J.; Mergey, M., et al. Glycoconjugates secreted by bovine tracheal serous ('ells in culture. Arch. Biochem. Biophys. 260:75-84; 1988. 24. Shelhamer, J. H.; Marom, Z.; Logun, C., el ah Human respiratory mucous glyeoproteins. Exp. Lung Res. 7:149-162; 1984. 25. Shen, B.-Q.; Finkbeiner, W. E.; Wine, J. J., et al. Calu-3: a human airway epithelial cell line which shows cAMP-dependent CI secretion. Am. J. Physiol. 266:Lg93-L501; 1994. 26. Tarella, C.; Ferrero, D.; GaBo, E., etal. Induction of differentiation of HL-60 cells by dimethyl sulfoxide: evidence for a stochastic model not linked to cell division cycle. Cancer Res. 42:445-449; 1982. 27. Weymer, A.; Huott, P.; Wilson, L.. etal. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J. Clin. Invest. 76:1828-1836: 1985. 28. Wright, W. C.; Daniels, W. P.: Fogh, J. Distinction of seventy-one cultured human tumor cell lines by polymorphic enzyme analysis. JNCI 66:239-247; 1981. 29. Wu. R.: Martin. W. R.; Robinson, C. B., et ah Expression of mucin synthesis and secretion in human traeheobronchial epithelial cells grown in culture. Am. J. Respir. Cell Mol. Biol. 3:467-478; 1990. 30. Yang, P.-C.; Luh, K.-T.; Wu, R., et al. Characterization of the muein differentiation in human lung adenocarcinoma cell lines. Am. J. Respir. Cell Mol. Biol. 7:161-171; 1992. 31. Zweibanm. A.; Pinto, M.; Chevalier. G.. etal. Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT29 selected for growth in sugar-free medium and its inhibition by glucose. J. Cell. Physiol. 122:21-29; 1985.