Peripheral Airway Cell Differentiation in Human Lung ...cancerres.aacrjournals.org/content/canres/50/17/5481.full.pdf · PERIPHERAL AIRWAY CELL DIFFERENTIATION Table 1 Expression

(CANCER RESEARCH 50, 5481-5487. September 1. 1990]

Peripheral Airway Cell Differentiation in Human Lung Cancer Cell LinesAdi F. Gazdar,1 R. Ilona Linnoila, Yukio Kurita, Herbert K. Oie, James L. Mulshine, Jean C. Clark, and

Jeffrey A. WhitsettNCI-Navy Medical Oncology Branch, National Cancer Institute and Naval Hospital, Bethesda, Maryland 20814 [A. F. G., R. 1. L., Y. K., H. K. O., J. L. M.J, andPediatrics INeonatologo Division, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 [J. C. C., J. A. W.]

ABSTRACT

Clara cells and type II pneumocytes are the progenitor cells of thebronchioles and alveoli, respectively. These peripheral airway cells (PAC)contain characteristic cytoplasmic structures and express surfactant associated proteins. PAC cell markers are expressed by many pulmonaryadenocarcinomas having papillary and/or lepidic growth patterns, whichare characteristics of the bronchioloalveolar and papillary subtypes. Weinvestigated the expression of PAC markers in a panel of 41 lung cancercell lines. Ultrastructural studies demonstrated the presence of cytoplasmic structures characteristic of Clara cells or of type II pneumocytesin 9 of 34 (26%) non-small cell lung cancer cell lines, including 7 of 17(41%) adenocarcinomas, one squamous cell carcinoma, and one large cellcarcinoma. Of interest, the cytoplasmic structures were present in 5 of 6(83%) cell lines initiated from papillolepidic adenocarcinomas. In addition, we examined the lines for expression of the surfactant associatedproteins SP-A, SP-B, and SP-C. Eight of the nine cell lines containingcytoplasmic inclusions characteristic of PAC cells also expressed proteinand/or RNA of SP-A, the major surfactant associated protein. Five ofthese lines expressed SP-B RNA (either constitutively or after dexa-methasone induction), while a single line expressed SP-C only afterdexamethasone induction. None of six small cell lung cancer cell linesexamined expressed any of the PAC markers. Thus, PAC markers areexpressed frequently (but not exclusively) in pulmonary adenocarcinomacell lines, especially in those initiated from tumors having papillolepidicgrowth patterns. The establishment and identification of multiple celllines expressing PAC features provide an important new resource forbiological and preclinical therapeutic studies.

INTRODUCTION

The World Health Organization classification of lung cancersincludes four major types (squamous cell, large cell, small cell,and adenocarcinomas) as well as several subtypes and raretumors (1). While squamous cell carcinoma has long beenregarded as the commonest type occurring worldwide, severalreports have noted an apparent increase in the incidence ofadenocarcinoma in some countries including the United States(2-6). The WHO classification includes only light microscopicexamination. A study that combined light and ultrastructuralmethodologies indicated that dual adenosquamous differentiation was the commonest form of lung carcinoma (7).

The WHO classification of adenocarcinomas consists of foursubtypes, including acinar (gland forming), solid with mucin,bronchioloalveolar, and papillary (1). It is presumed that acinarcarcinomas arise more centrally from bronchi, while solidadenocarcinomas represent relatively poorly differentiated tumors. BAC2 and papillary carcinomas constitute about one-half

of all pulmonary adenocarcinomas (6). Both of these subtypeshave overlapping pathological features and share common his-togenetic origins. BAC tumors, especially when in an invasiveor metastatic mode, cannot always be differentiated from pap-

Received1/24/90;revised6/1/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.1The abbreviations used are: BAC, bronchioloalveolar carcinoma; PAC, pe

ripheral airway cells; cDNA, complementary DNA; NSCLC, non-small cell lungcarcinoma; SCLC, small cell lung carcinoma.

illary carcinomas (6). The cellular origins of both of thesesubtypes (in humans and rodents) are heterogeneous and include mucin secreting cells, Clara cells, and type II pneumocytes(8-16). For these reasons, we prefer to regard BAC and papillary tumors as a single entity and refer to them as papillolepidictumors, after their main histopathological features.

Because Clara and type II pneumocytes are the progenitorcells of the bronchioles and alveoli, respectively, we refer tothem as peripheral airway cells. Each of these cell types containscharacteristic cytoplasmic secretory structures. The Clara cellshave dense granules in the apical cytoplasm (17), while type IIpneumocytes contain multi hiniellar bodies (18). The functionof the Clara cell secretions have not been determined, while themultilamellar bodies of type II cells represent the storage siteof surfactant.

The major function of type II pneumocytes is the productionof pulmonary surfactant, which exerts a detergent-like actionessential for maintaining the patency of the alveoli (for reviewsee Ref. 19). While phospholipids comprise the major component of surfactant, several surfactant associated proteins havebeen identified which confer important physical properties tothe phospholipids and which are necessary for function. Themost abundant of these proteins is a M, 28,000-36,000 glyco-protein known as SP-A (previously referred to as SAP-35) (20-22). The cDNA for human SP-A codes for a smaller polypep-tide, which undergoes extensive posttranslational modification.Recently, two other surfactant proteins, SP-B and SP-C, havebeen identified (23). They are M, 5,000-14,000 hydrophobicproteins and are components of active surfactant extracts utilized for the therapy of newborn infants suffering from hyalinemembrane disease. The functions of Clara cells are not as welldefined. They may include secretion of surfactant-like substances, water and salt transport, and mixed-function oxidation(24-26).

In this report, we describe the expression of morphological,biochemical, and molecular properties of PAC by several members of a panel of human lung carcinoma cell lines establishedby us. Most of the cell lines expressing PAC features werederived from adenocarcinomas, especially those having papillolepidic growth patterns.

MATERIALS AND METHODS

Cell Lines. The lung adenocarcinoma cell line A549 (27) was obtainedfrom the American Type Culture Collection, Rockville, MD. All othercell lines were established by us. Cell lines were established frompathologically proved primary or metastatic lung carcinomas as described previously (28). Solid tumors were finely minced with scissorsand disassociated into small aggregates by pipeting. Malignant effusionswere collected, pelleted, washed, and resuspended in growth medium.Hemorrhagic effusions also underwent a Ficoll gradient separationprocedure in order to decrease the number of RBC. Approximately1-5 x IO6cells were seeded into 25-cm2 flasks. While multiple types of

media were utilized, cell lines were usually initiated, established, andmaintained either in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (RIO) or ACL-4, a serum-free medium wehave devised for the selective culture of NSCLC and other tumors (28-

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PERIPHERAL AIRWAY CELL DIFFERENTIATION

Table 1 Expression of PAC markers in lung cancer cell linesForty cell lines were examined for ultrastructural evidence of PAC differentiation. Of these 37 were examined for SP-A protein expression and 30 were examined

for SP RNAs (including all the lines containing cytoplasmic granules characteristic of PAC cells).

CytoplasmicgranulesTumor

typeAdenocarcinoma

(all)BACPapillaryOtherSquamous

cellcarcinomaLargecellcarcinomaAdenosquamous

carcinomaMucoepidermoidcarcinomaCarcinoidMesothcliomaSmall

cellcarcinomaTotalClara

cell4/17Â°2/22/4Â°0/110/31/5Â°0/30/10/30/20/65/40TypeII4/17Â°0/22/4Â°2/111/31/5Â°0/30/10/30/20/66/40SP-Aprotein5/152/23/40/90/20/60/20/10/20/30/65/37SP-ARNA7/152/23/42/91/20/30/30/1NT*NT0/68/30SP-BRNA5/15*0/23/42/90/20/30/30/1NTNT0/65/30SP-CRNA1/15*0/21/40/90/20/30/30/1NTNT0/61/30Total7/172/23/42/111/31/50/30/10/30/30/69/41

"'One papillary and one large cell carcinoma cell line contained both types of granules (see Table 2).* Constitutive or dexamethasone induced expression (see Table 2).c NT, not tested.

Table 2 Lung cancer cell lines expressing PAC cell lines

CelllineNCI-H226NCI-H322NCI-H358NCI-H441-4NC1-H726NC1-H820NCI-H920NCI-HI

334NC1-H1404Cytoplasmic

SP-A protein SP-Agranules (ng/mg)RNAType

11ClaraClaraClara

>typeIIType11TypeIITypeIIClara

>type11Clara052791700460001202

+C"2

+C1-t-C2

+C1

+C2+C1

+CAbsent1

+CSPA-B

RNAAbsentAbsentAbsent1

+I1

+C1+I1

+CAbsent1

+CSPA-C

RNAAbsentAbsentAbsentAbsentAbsent1

+IAbsentAbsentAbsent

Â°1+, weak expression; 2+, strong expression; C, constitutive expression; I,

dexamethasone induced expression.

30). Initial cell passages were performed whenever vigorous tumor cellgrowth was observed. Established cell lines were passaged weekly.Anchorage independent cultures were passaged by transfer of floatingmulticellular aggregates. Anchorage dependent cultures were passagedat subconfluence after trypsinization. If stromal cell growth was notedin initial cultures, differential trypsinization (31) was used to obtain apure tumor cell population. RPMI 1640. trypsin, and sera were obtained from Grand Island Biological Co., Grand Island, NY. Growthfactors used in ACL-4 medium were obtained from Sigma ChemicalCo., St. Louis, MO, and Collaborative Research, Bedford, MA. Cultures were maintained in humidified incubators at 37Â°Cin an atmos

phere of 5% CO2 and 95% air.Morphological Studies. Initial diagnostic materials (pathological and

cytological) were reviewed and compared to the appearances of thecorresponding cell culture and xenograft. Saccomanno fluid fixed cy-tospin preparations of floating cultures and trypsinized adherent clturesand paraffin embedded sections of xenografts were stained with hema-toxylin-eosin, Alcian blue, and mucicarmine.

For ultrastructural studies, cell pellets were fixed in 2.5% glutaral-

dehyde, postfixed in 1% osmium tetroxide, and stained with 1% uranylacetate, and thin sections were examined by an electron microscope.

Culture Characterization. Mycoplasma contamination was tested forby the use of a rRNA hybridization method (Gen-Probe, San Diego,CA). Cell homogenates were tested for the human forms of the following enzymes by starch gel (32) using the Authentikit system (CorningScience Products, East Walpole, MA): purine nucleoside phosphorylase(EC 2.4.2.1); glucose-6-phosphate dehydrogenase (EC 1.1.1.49); pep-tidase B (EC 3.4.11.4); and lÃ¡clatedehydrogenase (EC 1.1.1.27). Tu-morigenicity was tested for by inoculation of approximately 5 x IO6

cells s.c. into the flanks of male athymic nude mice of BALB/c background.

Enzyme-linked Immunosorbent Assay for SP-A. SP-A protein wasmeasured by an enzyme-linked immunosorbent assay as describedpreviously (33). This assay utilizes a goat anti-human SP-A IgG and arabbit anti-human SP-A in a capture assay. Lysates from tumor celllines were harvested and frozen at â€”20Â°Cuntil analyzed.

Northern Blot Analyses for Surfactant Protein RNAs. RNA wasextracted by the method of Chirgwin et al. (34). For Northern blotanalysis, three identical gels (1.2% agarose, 7% formaldehyde) wererun, loading 20 Â¿igof total RNA per lane. After electrophoresing, theRNA was transferred to Nytran filters (Schleicher and Scimeli. Keene,NH). Filters were baked at 80Â°Cfor 2 h prior to hybridization asdescribed previously (35). Probes used for hybridization (-1 to 5 x IO6cpm/ml) were partial cDNAs for SP-A (probe 7-1, a 0.9-kilobase clone);SP-B (probe 4-3, a 1.8-kilobase clone); and SP-C (probe 2-1, a 0.8-kilobase cDNA clone) (36-38). Probes were isolated from a Xgtllexpression library generated from adult human lung polyadenylatedRNA. The cDNA probes were labeled with |Â«-"P]dCTP using a nick-

translation kit.Dexamethasone Induction of Surfactant Associated Proteins. All cell

lines were tested for SP-A, SP-B, and SP-C RNA while cultured intheir usual maintenance medium (RIO or ACL-4) (constitutive expression). In addition, cell lines constitutively expressing SP-A RNA were

Table 3 Origin of lung cancer cell lines expressing PAC markers

CelllineNCI-H226NCI-H322NC1-H358NCI-H441-4NCI-H726NCI-H820NCI-H920NCI-HI

334NC1-H1404Pathological

diagnosis(see

text)BACBACPapillaryAdenoca.PapillaryAdenoca.Large

cellPapillaryTumor

sitePleuralef.Â°LungLungPericard.

ef.Pleuralef.LNLN1

piilm.ilLNAge(yr)952995753445648SexMMMMFMMMMCulture

dateMarch

1980Feb.1981Aug.1981May1982Apr.1984Sept.1984Dec.1984Feb.1986Dec.

1986Prior

therapyNoneNoneNoneNoneNoneChemo.NoneNoneNoneCulturemediumRIORIORIORIOACL-4ACL-4ACL-4RIOACL-4CulturegrowthAttachedAttachedAttachedAttachedFloatingFloatingFloatingAttachedFloatingTumorigenicityYesYesYesYesNTYesYesNTYesXenografthistologySquamousAdenoca.Adenosq.PapillaryPapillaryAdenoca.Papillary

" Pleural ef.. pleura! effusion; Pericard. fl., pericardial effusion; LN. lymph node; epidural. epidural mass; chemo., patient received prior chemotherapy; Adenoca.,

adenocarcinoma; adenosq. adcnosquamous carcinoma: NT. not tested.

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b

dFig. 1. Comparison of tumor, culture, and xenograft morphologies of cell line NCI-H820. a, malignant pleural effusion, demonstrating papillary clusters of

malignant cells; l>.supraclavicular lymph node from same patient, with metastatic adenocarcinoma (most of the lymph node consisted of undifferentiated metastatictumor, with foci of papillary carcinoma (as illustrated)); c. cell line NCI-H820. initiated from lymph node metastasis, demonstrating floating clusters of tumor cellsforming papillary structures; (/. xenograft morphology, cell line NCI-H820. demonstrating tubulopapillary morphology.

tested for expression of SP-B and SP-C RNA after growth in thepresence of dexamethasone (induced expression). Because ACL-4 medium contains hydrocortisone (S x 10 " M), cells maintained in that

medium were cultured for at least 2 days in the ACL-4 medium lackinghydrocortisone and then in fresh medium lacking hydrocortisone butcontaining dexamethasone. Dexamethasone (Sigma) was prepared as a10 /Â¿Mstock in 95% ethanol and diluted to the stated concentrations inhydrocortisone-free medium.

RESULTS

Selection of Cell Lines. While we have initiated more than 75NSCLC cell lines since 1977, 29 were available at the initiationof these studies in 1985. An additional 6 adenocarcinoma celllines, initiated at a subsequent time point, also were studied. Inaddition to the 35 NSCLC lines, six SCLC cell lines wererandomly selected from a panel of approximately 200 lines (39).All of the cell lines studied expressed human forms of theenzymes analyzed, and they were free of Mycoplasma contamination.

Expression of PAC Markers in Lung Cancer Lines. We examined our panel of 41 lung cancer cell lines for ultrastructural,biochemical, or molecular evidence of PAC differentiation.While not all cell lines were examined by all techniques (Table1), all cell lines identified as containing ultrastructural evidenceof PAC differentiation were examined for SP protein and RNA

expression. One or more markers were present in 9 of 35 (26%)NSCLC lines (Tables 1 and 2). Multiple markers were presentin 7 of 17 (41%) adenocarcinomas (including 3 of 4 papillarycarcinomas and 2 of 2 BACs), 1 of 6 large cell and 1 of 3squamous cell carcinomas. The latter cell line, NCI-H226, wasinitiated from a malignant pleural effusion with an originalcytological diagnosis of "tumor cells, possibly mesothelioma."

However, examination of the cell line demonstrated multiplebiochemical and ultrastructural features of squamous cell carcinoma (40). The large cell carcinoma line (which only hadultrastructural evidence of PAC differentiation) was derivedfrom a tumor that lacked morphological evidence of differentiation. However, the corresponding cell culture contained agland forming subpopulation. PAC markers were not detectedin carcinoids, mesotheliomas, adenosquamous, mucoepider-moid, or SCLC lines.

Properties of Cell Lines Expressing PAC Markers. The originand properties of nine cell lines expressing one or more PACmarkers are presented in Tables 2 and 3. Two cell lines wereinitiated from primary tumors, and seven were from mÃ©tastases.They had been cultured for periods of 2 to 8 years whencharacterized. All but one of the patients were males, and onlyone patient had received therapy prior to culture. The Fivecultures that had been initiated in serum containing RIO medium demonstrated substrate attachment and had an epithelioid

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Fig. 2. Ultraslructural appearances of celllines expressing PAC features, a, ultrastructural appearances of cell line NCI-H820. Thecells demonstrated polarity, with microvillibeing present on the apical surfaces. Manymultilamellar bodies are present in the apicalcytoplasm. Because of fixation artifact, thephospholipid contents of many of the bodieshave been dissolved. Original magnification.x7,050. Insel, a relatively well preserved multilamellar body. A,a multilamellar body in theprocess of being extruded from the apical cellsurface of cell line NCI-H726. Original magnification. X 22.400.

i

â€¢â€¢'-sfM .

*Â£Â£\ â€¢'â€¢

morphology. The four adenocarcinoma cultures that had beeninitiated in defined ACL-4 medium (which lacks attachmentfactors) lacked substrate attachment. They formed papillary(Fig. 1) or inter- and intracellular gland-like structures, thelumina of which were distended with fluid, not mucin. Adenocarcinoma cells demonstrated polarity, with microvilli beingpresent on the apical surfaces, and they were joined by tightjunctions and desmosomes. All 5 cell lines tested were tumori-genie in athymic nude mice. In general, xenograft histologyresembled that of the original tumor (Fig. 1). However, thexenograft histology of one BAC cell line was adeno-squamous cell carcinoma.

Ultrastructural Evidence of PAC Differentiation. All 9 celllines expressing PAC markers demonstrated cytoplasmic granules characteristic of Clara or type II pneumocytes (Fig. 2).Both of these types of granules were located in the apicalcytoplasm. Clara cell granules consisted of round, oval, orirregularly shaped membrane bound structures (about 0.3 urnin diameter) with finely granular contents of variable electrondensity. The type II cell inclusions consisted of larger, oval

membrane bound structures having whorled or lamellar osmio-philic structures. The contents of many granules appeared tohave been partially or completely dissolved by fixation. Onoccasion, the limiting membrane of the lamellar bodies wasobserved to be fused with the cell membrane, and the contentswere extruded into the extracellular space (Fig. 2). Clara celland type II cell granules were each observed in three cell lines,while two cell lines contained predominantly Clara cell granules, with a subpopulation of cells containing type II cell granules.

Expression of Surfactant Proteins. SP-A RNA was detectedin 8 of the 9 PAC cell lines, and in 5 of these SP-A protein wasalso demonstrated (Table 2; Fig. 3). SP-B RNA (constitutiveor induced) was detected in 5 of the lines demonstrating SP-ARNA. In a single cell line, NCI-H820, SP-C RNA was detected.When this cell line was maintained in hydrocortisone containing ACL-4 medium, a faint band was occasionally visible onNorthern blots. However, SP-C RNA could be readily detectedafter dexamethasone induction. In NCI-H820 cells, expressionof SP-A, SP-B, and SP-C RNAs was increased after dexameth-

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8cr>I

CDO

co LOIII

CMCMCOI

00 CO CO 00 LOLO TÃ• Tt CO CMco i- i- oo i-11 I I I

CDCO CMCM CMI I

CD

reported to express features of type II cells (27). While multi-lamellar cytoplasmic structures were readily found on ultra-structural examination, neither constitutive nor dexamethasoneinduced expression of SP-A protein or SP-A, SP-B, or SP-CRNAs were detected.

SP-A

SP-B -2.0kb

Fig. 3. Northern blots, demonstrating constitutive expression of SP-A andSP-B RNA by lung cancer cell lines. Top, SP-A RNA (2.20-kilobase band)expressed by cell lines NCI-H920 (adenocarcinoma), NCI-H441 (BAC), NCI-H358 (BAC), and NCI-H226 (squamous cell carcinoma); bottom, fainter 2.0-kilobase (*e) band, representing SP-B expression, present in cell lines NCI-H920andNCI-H441.

SP-A

SP-B

SP-C â€¢

0 0.1 1.0 10 50 100

Dexamethasone (nM)Fig. 4. Northern blots of cell line NCI-H820, derived from a papillary ade

nocarcinoma, demonstrating dose dependent dexamethasone induction of surfactant protein RNAs SP-A, SP-B, and SP-C. The cell line was cultured in steroidfree medium for 48 h and then exposed to varying concentrations of dexamethasone for 4 h. kh. kilobases.

asonÃ©stimulation in a dose dependent manner (Fig. 4).Properties of A549 Cells. In addition to our cell lines, we

examined A549 cells, a pulmonary adenocarcinoma cell line

DISCUSSION

â€”2 2 Kb ^e fÂ°un(ievidence of PAC differentiation in 9 lung cancercell lines, 7 of which were adenocarcinomas. Five of the adenocarcinoma lines were initiated from tumors that had papil-lolepidic growth features. All of the 9 cell lines had ultrastructural characteristics of Clara cells or type II pneumocytes and8 of them expressed SP-A RNA constitutively. In addition, fivelines expressed SP-B constitutively or after dexamethasonestimulation, while a single line expressed SP-C RNA only afterdexamethasone stimulation. We have previously describedexpression of surfactant proteins by two of the PAC cell linesdescribed in this report, NCI-H441-4 and NCI-H820 (35, 41,42).

Surfactant proteins were expressed by lung cancer cell linesdemonstrating ultrastructural features consistent with type IIpneumocytes or Clara cells. On occasion, features of both celltypes were present in the same lines. These findings have beendescribed previously in human tumors (43, 44). While Claracells express a characteristic M, 10,000 protein (45), normaland neoplastic Clara cells also may express surfactant proteinsindependently of surfactant phospholipids (26, 46). Our studyand others discussed above indicate the interrelationship between Clara and type II pneumocyte cells and suggest that PACtumors arise from bronchiolar or alveolar cells capable ofmultipotent differentiation. In addition, PAC differentiationmay, occasionally, be present in more centrally arising NSCLC

â€”22 kb tumors. We did not detect evidence of PAC differentiation inSCLC cell lines. In another study, we failed to demonstrate SP-A by immunohistochemical techniques in SCLC tumors or inadenocarcinomas of nonpulmonary origin (46).

Previous reports of established cell lines that express PACfeatures are very limited. A recent report in the Chinese literature describes lamellar bodies in a subline of a pulmonarycarcinoma culture, but not in the parent cell line (47). Multi-lamellar bodies have also been described in a cell line establishedfrom the lung of a sheep with jaagsiekte (ovine pulmonaryadenomatosis) (48). Jaagsiekte is a virall y induced ovine diseasecharacterized by hyperplastic or tumorous proliferation of typeII pneumocytes. We also studied A549 cells, a human lungcarcinoma cell line widely believed to be of BAC origin. TheA549 line was described by Giard and associates in 1973 asbeing initiated from a tumor designated simply as "lung carci-

â€”10 kb noma" (49). Xenografts of the cell line contained acinar struc

tures. In 1976 Lieber et al. (27) described ultrastructural features and phospholipid formation characteristic of type II pneumocytes in A549 cells and claimed that the line was initiatedfrom an "alveolar cell carcinoma." However, their illustration

(and description) of the tumor histology is that of a glandforming acinar carcinoma with mucin formation. Other published reports have suggested that A549 cells do not expressbiochemical properties characteristic of type II cells (50, 51).In addition, TGF-/3 induces goblet cell differentiation in A549cells (52). While our studies confirmed previous reports thatA549 cells contained cytoplasmic lamellar structures (27), thecells failed to express surfactant protein or RNAs. The ultra-structural appearances of Clara and type II granules are notcompletely diagnostic, and other cell types may contain artifac-

-2.0kb

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tual or real inclusions with similar morphologies (53, 54). Thus,we regard only those cell lines that express both ultrastructuraland molecular or biochemical markers as having unequivocalevidence of PAC differentiation. Using this criterion, eight ofour cell lines appear to be the first continuous lines of anyspecies that demonstrate definite evidence of PAC differentiation.

While most of our cell lines expressing PAC features wereinitiated from papillolepidic tumors, these tumors form a heterogeneous group, as discussed previously. In addition, someother NSCLC lung tumors may express markers of PAC differentiation (43, 50, 55). Also, mÃ©tastasesfrom non-lung carcinomas may have papillolepidic growth features (44, 56, 57).Thus, papillolepidic morphology and expression of PAC markers are suggestive, but not diagnostic, of PAC histogenesis.

Our PAC cell lines may be useful for several types of studiesincluding: (a) the physiology of surfactant and its proteins (41,42); (b) the biology of Clara cells and their role in xenobioticmetabolism; (c) in vitro testing of new therapeutic modalitiesspecifically directed at PAC cells, such as 4-ipomeanol (58);and (d) preparation of human-derived materials potentiallyuseful for the treatment of the respiratory distress syndrome ofthe newborn and other surfactant deficient states (21, 59). Theestablishment of multiple human cell lines manifesting unequivocal evidence of PAC differentiation provides a major newresource for biological and preclinical therapeutic studies.

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1990;50:5481-5487. Cancer Res Adi F. Gazdar, R. Ilona Linnoila, Yukio Kurita, et al. Cell LinesPeripheral Airway Cell Differentiation in Human Lung Cancer

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