Vol. 7, 1149-1156, September 1996 Cell Growth & DifferentIatIon 1149

Cell-Substratum Interactions Mediate Oncogene-inducedPhenotype of Lung Cancer Cells1

Linda F. Barr,2 Susan E. Campbell,Margaret B. Penno, Douglas W. Ball, andSteven B. BaylinDepartment of MedIcine, DivisIon of Pulmonary and Critical CareMedicine [L. F. B.] and Division of Endocrinology ED.W. B., S. B. B],Department of Oncology [L. F. B., S. C., S. B. B.], and Center forMedical Genetics [M. B. P.], Johns Hopkins School of MedicIne,BaltImore, Maryland 21205

Abstract

In vlvo and In vitro studies have linked small cell lungcancer (SCLC) and non-small cell lung cancer (NSCLC)cells along a differentiation continuum. The transitionof a SCLC toward a NSCLC phenotype is modeled Inculture by the simultaneous overexpresslon of myc and“as genes in cultured SCLC cells. A major phenotyplcdistinction between SCLC and NSCLC In culture Is thatSCLC cells usually grow In floating aggregates,whereas NSCLC cells and myc- plus ras-expressingSCLC cells grow as adherent spreading monolayerslike other epithellal cells. The present studies examinehow myc, ras, cell aggregation, and attachment tolaminin may interact to modulate transitions betweenthe SCLC and NSCLC phenotypes. We find that myc-expressing SCLC cells, which normally grow asanchorage-independent cells in plastic flasks, willadhere to laminin and exhibit an epithellal morphology.

In this setting, the cells express both NSCLC and SCLCmarkers, thus resembling a tumor type previouslytermed NSCLC with neuroendocrine features.

Anchorage-dependent SCLC cells simultaneouslyexpressing the myc family and an exogenous ras

oncogene move further toward the NSCLC phenotypethan the above myc-expressing cells. However, forcedsuspension of such cells restores the expression ofneuroendocrine SCLC features. These studies indicate

that cell environment, as much as gene expressionevents, profoundly affects aspects of the SCLC cellphenotype.

Received 12/28/95; revised 6/13/96; accepted 6/29/96.The costs of publication of this article were defrayed In part by thepayment of page charges. This article must therefore be hereby markedadvertisement In accordance with 18 U.S.C. Section 1734 solely to mdi-cate thIs fact.1 This work was supported by NIH Grants Ku CA01685 (L. F. B.), ROlCA48081 (S. B. B.), and P50 CA58184 (M. B. P.) and by a grant from theFrancis Family Foundation (L F. B.).2 To whom requests for reprints should be addressed, at Tumor Biology,Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD21231 . Phone: (410) 955-8506; Fax: (410) 614-9884.

Introduction

SCLC3 cells can evolve to display the differentiation charac-teristics of NSCLC both in vivo and in vitro (1 , 2). Also, bothin culture and in patients, cells simultaneously expressingfeatures of all the lung cancer subtypes have been wellcharacterized (3-7). Elucidating the genetic and biologicalevents that control the evolution of SCLC and NSCLC alonga differentiation continuum may help in understanding lungtumor progression and responses to therapy.

A significant difference between SCLC and NSCLC cells inculture is their aggregation and adherence profiles. SCLC

cells usually grow in floating aggregates with varying degreesof homotypic adhesion, whereas NSCLC cells, like otherepithelial cells, attach and spread on tissue culture surfaces.

The purpose of the present studies is to define the contn-bution of myc and ras gene expression events and cellularadherence to the phenotype manifested by lung cancer cellsalong the above SCLC-to-NSCLC differentiation continuum.We have determined that cell-substratum interaction has aprofound effect on the differentiation status of cells simulta-neously expressing c- or N-myc and v-Ha-ras oncogenes.The results suggest that lung cancer phenotypes may reflectinteractions between genetic alterations and cellular interac-tion with the immediate environment.

ResultsOverexpresslon of myc and ras Genes Affect Endocrineand Epithelial Features of SCLC Cells. For most of thesestudies, we have used the classic SCLC cell line 209, whichhas typical SCLC-associated neuroendocnne features (8), asexemplified in the present study by growth in tight floatingaggregates in culture (Fig. IA) and expression of transcriptsof constant markers for SCLC, such as hASH-i (Fig. 2; Ref.9), and small amounts of N-CAM (data not shown; Ref. 10).The 209 cells do not express PKC-�3 transcripts (Fig. 2; Ref.I 1), markers of NSCLC differentiation, such as TGF-a (Fig. 2;Ref. 12), or the cell surface hyaluronan-binding protein CD44(data not shown; Ref. 13), and they express only smallamounts of TGF-�3 (Fig. 2; Refs. 12 and 14).

As in previous studies (1 5), we found that the transfectionof c-myc into the 209 cell line led to looser cellular aggre-gates (Fig. 1C) and decreased the doubling time (Fig. 3) butdid not affect the expression of the characteristic SCLCmarkers dopa decarboxylase and bombesin (1 5). However,in the present study, we find the appearance of a NSCLC-

3 The abbrevIations used are; SCLC, small cell lung cancer; NSCLC,non-small cell lung cancer; 209, NCI H209 cell line; 249, NCI H249 cellline; hASH, human achaete-scute homologue; TOF, transforming growthfactor; PKC, protein kinase C; N-CAM, neural cell adhesion molecule;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hydron, poly(2-hydroxyethyl methacrylate); MiT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide assay.

plastic lamlnin hydren

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Fig. 1 . Morphology of myc- and ras-expressing 209 cells manipulatedfor substratum interaction. Parent 209 (A), 209 ras (B), and 209 myc (C)cells grow in suspended aggregates, whereas 209 myc-ras cells (0) growin adherent monolayers in the plastic flasks. The 209 cells adhere tolaminin-coated flasks as aggregates, with epithelial-appearing cells at theperiphery growing out onto the matrix (E). The 209 ras cells disperse onthe laminin surface, but individual cells maintain rounded morphology (F).The 209 myc cells on laminin have some epithelial morphology (G). The209 myc-ras cells on laminin have extensive epithelial morphology (H). The209 myc-ras cells forced into suspension by coating the plastic flasksurface with hydron (I) appear indistinguishable from suspended 209 myccells. Culture conditions are described in “Materials and Methods.” Phasecontrast micrographs: A-E, x200; G-l, x400.

1150 Cell-Substratum Interactions in Lung Cancer Cells

.

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associated transcript, TGF-c� (Fig. 2), in these c-myc-trans-

fected cells, which still expressed low levels of the neuroen-

docrine marker hASH-i (Fig. 2). These findings support the

placement of these c-myc-expressing cells along a SCLC-

to-NSCLC differentiation continuum. Finally, as we reported

previously, c-myc expression in these cells results in high

levels of PKC-p transcript expression (Fig. 2; Ref. 11).

We have previously found that the effect of v-Ha-ras ex-

pression in SCLC cells is to increase neuroendocrine differ-

entiation, modulated in part by the simultaneous expression

of c- or N-myc (1 6). The present studies extend these ob-

servations. For the 209 cells, v-Ha-ras expression increases

the manifestations of the SCLC phenotype, as exemplified

by an increasing tightness of the cell aggregates (Fig. 1B)

and a slight increase in the transcript levels of the neuroen-

docrine marker hASH-i (Fig. 2). However, expression of

another neuroendocrine marker, N-CAM (data not shown),

and the growth rate (Fig. 3) are unchanged.

The 209 cells simultaneously expressing both exogenous

c-myc and v-Ha-ras genes manifest the anchorage-depen-

dent monolayer growth typical of most NSCLC cell lines (Fig.

1). Consistent with this increased epithelial differentiation,

these 209 myc-ras cells express very little of the neuroen-

docrine marker hASH (Fig. 2), although expression of the

N-CAM protein does not change (data not shown). Further-

more, these cells express the epithelial marker TGF-cr, al-

though in lower amounts than the 209 myc cells (Fig. 2).

Finally, these adherent 209 myc-ras cells express increased

TGF-p (Fig. 2), a molecule that may function in the cell-matrix

pathway (reviewed in Ref. 1 7). It must be noted, however,

that these 209 myc-ras cells do not manifest all of the typical

features of epithelial cells, in that individual cells do not

spread extensively on the flask surface but, rather, maintain

a somewhat rounded shape (Fig. 1D). Additionally, these

cells do not express the NSCLC-assoeiated cell surface

protein CD44 (data not shown), and there is no change in

doubling time compared with the suspended 209 myc cells

(Fig. 3).

Effects of Manipulating Cell-Substratum Interactionson the Phenotype of SCLC Cells. The epithelial differenti-

ation characteristics of cells are profoundly influenced by the

substratum environment in which they grow (for review, seeRef. 1 8). A principal molecule with which such cells interact

is laminin, a major component of the basement membrane.

Adherence of cells to laminin has previously been shown to

alter gene expression for lung cancer cells (1 9, 20). There-

fore, we investigated the effects of laminin adherence on

each of the gene insertion events under study.

The parent 209 cells attach very poorly to laminin. Most of

these cells are tightly aggregated and remain anchorage

independent. Only a few cells on the outside of the aggre-

gates are less tightly bound. These cells have a spindle

shape and appear to interact with the laminin (Fig. 1E). Ex-

posure of SCLC cells to laminin has no effect on any of the

SCLC or NSCLC markers examined for these cells (Fig. 2).

When 209 cells express an infected v-Ha-ras gene (209 ras

cells), the attachment to laminin is increased substantially.

However, the cells do not spread like typical epithelial cells,

and a rounded shape is maintained (Fig. iF). Furthermore,

these cells exhibit little marker changes from those ex-

pressed by the suspended parent cells (Fig. 2).

In contrast to the above cells, the 209 myc cells develop a

much more typical epithelial morphology when exposed to

laminin. The cells have a more flattened shape and consid-

erable spreading activity (Fig. 1 G). In addition, these cells

maintain expression of both epithelial (TGF-a) and neuroen-

docrine (hASH) markers (Fig. 2). Importantly, the expression

of the exogenous c-myc is not altered by these maneuvers

(data not shown). Thus, these cells then resemble the phe-

notype of the NSCLC cells with neuroendoerine features,

which has been well characterized in culture (3) and in pa-

tients (4-7).The most striking effect of a laminin substrate on morphol-

ogy was seen for the 209 myc-ras cells. In contrast to the

rounded appearance of these cells when adherent to plastic,

209 myc-ras cells flatten and develop cytoplasmic extension

processes on laminin (Fig. 1H). However, with the exception

Cell Growth & Differentiation 1151

Fig. 2. Phenotypic marker expressi� of myc-and ras-expressing 209 cells manipulated for sub-stratum interaction. Northern blots of polyadeny-lated RNA were sequentially stripped and re-probed, and band density was measured, asdescribed in “Materials and Methods.” A, repre-sentative experiment of two independent experi-ments. B, cumulative data of levels of expressionof the 2.8-kb transcript of hASH, the 4.8-kb tran-script of TGF-a, the 3.6-kb transcript of PKC-f3,and the 5-kb transcript of TGF-�. Graphs showthe average of two experiments. V axis units arearbitrary and represent transcript densitometriescorrected for loading by dividing by those ofGAPDH transcripts and normalized by dividingthese values by those for the densities for thetranscripts of either 209 (in the case of hASH andTGF-f3) or 209 myc (in the case of TGF-a andPKC-f3) on the same blot. Bars, SD.

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of increases in the expression of PKC-� transcripts, laminin

growth does not elicit changes in the expression of the other

phenotypie markers examined in the 209 myc-ras cells (Fig.

2) and does not alter the expression of the exogenous e-myc

(data not shown). Furthermore, the cells do not demonstrate

changes in growth rates (data not shown). Thus, the epithelial

morphology, the expression of the epithelial cell marker

(TGF-a), and the decreased expression of the neuroendo-

crine marker (hASH-i ) suggest that the expression of oneo-

genie ras and myc, in conjunction with laminin-adherent

growth, has positioned these cells further toward a NSCLC

classification when examined in the context of a SCLC-to-

NSCLC phenotypic continuum.

To further understand the influence of cell-cell and cell-

matrix interaction on the cultured lung cancer cell pheno-

type, we next examined the effect of inducing normally ad-

herent lung cancer cells to grow in suspension using

surfaces coated with hydron [poly(2-hydroxyethyl methacry-

late) plasticizing agent]. For these studies, we used the 209

myc-ras cells, because as detailed above, these cells fully

manifest features of transition from the SCLC to the NSCLC

phenotype. For some of these studies, we also studied an-

other classic SCLC cell line, 249, which expresses the same

neuroendocrine features outlined for parent 209 cells, includ-

ing anchorage-independent growth. These cells, which have

an endogenously amplified and overexpressed N-myc gene,

also acquire epithelial characteristics, including adherence to

plastic, and expression of some NSCLC markers, such as

TGF-a, when transformed with v-Ha-ras (21).

Prevention of surface adhesion of 209 myc-ras cells first

restores many features of the SCLC morphology, including

growth as floating aggregates, which are indistinguishable

from 209 myc cells (Fig. 1 , C and I). Second, these floating

209 myc-ras cells have an increased doubling time, closer to

that of the parental 209 cells (Fig. 3). Third, abrogation of

adhesion of 209 myc-ras cells increases the expression of

transcripts for the neuroendoerine factor hASH comparedwith levels in these cells when adherent to plastic or laminin.

Fourth, levels of TGF-p and PKC-p transcripts are returned

to a pattern identical to that of the 209 myc cells (Fig. 2).

Finally, this maneuver does not alter the expression of the

exogenous c-myc (data not shown). Thus, prevention of an-

ehorage-dependent 209 myc-ras cells returns the cells to the

marker pattern more typical of the 209 parent and 209 myc

cells.

Like the 209 myc-ras cells, the 249 ras cells growing on

plastic have decreased neuroendoerine and increased epi-

thelial features when adherent to plastic (Fig. 4; Ref. 21).

Thus, ras infection of 249 cells decreases the expression of

two neuroendocrine markers, the hASH-i transcript and N-

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1152 Cell-Substratum Interactions in Lung Cancer Cells

a Fig. 3. Growth of myc- and ras-expressing209 cells manipulated for substratum interac-tion. Growth curves were performed as de-scribed in “Materials and Methods.” Shown arethe mean values of three independent experi-ments, each performed in triplicate. Bars,SE. MiT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyftetrazolium bromide.

CAM (Fig. 4). As was seen for the 209 myc-ras cells, forcedsuspension of these cells restores features of the endocrineSCLC phenotype. The parental 249 SCLC morphology isrestored (Fig. 4), and the levels of the hASH-i transcript andN-CAM move toward those seen in parent 249 cells (Fig. 4).No changes were seen in any of the other phenotypic mark-ers examined, and these maneuvers do not alter the expres-sion of the endogenous N-myc or exogenous v-Ha-ras genes

(data not shown). These results indicate that the cells understudy have an inherent plasticity for movement along theSCLC-to-NSCLC continuum, depending on interactions be-tween genetic alterations and cell substratum interaction.

Given the above results, we wondered about the plasticityof established NSCLC lines, which have never manifestedany SCLC features during the life history of the culture. Suchcells generally express considerable levels of endogenousc-myc and have multiple NSCLC markers (22-24). We thusmanipulated squamous carcinoma line Ui 752 (Fig. 5A) toinhibit interaction with the flask surface. In this setting, float-

ing aggregates of loosely adherent cells will form (Fig. 5B).Suspension inhibits the growth of this NSCLC line (Fig. 5), as

has been demonstrated for other normally adherent cells(25), although viability is maintained, as demonstrated byHoechst and trypan blue staining (data not shown). Forcedsuspension of Ui 752 cells does not lead to the expression ofthe neuroendocrine marker hASH-i or gastrin-releasing pep-tide, nor does it affect the substantial expression of theendogenous c-myc gene that is present in this cell line (datanot shown). Suspension does increase the expression of theSCLC marker N-CAM, but only for cells that have beenfloating for several weeks (Fig. 5D).

Discussion

Our present studies indicate that cell-matrix interactionsmodulate features that characterize and distinguish SCLCand NSCLC cells in culture, including those that are conse-quent to engineered myc and ras gene expression. Our find-ings underscore the collaborative effects of myc, ras, and cell

environment in determining the phenotype of neoplasticcells.

Overexpression of a c-myc gene in 209 SCLC cells in-duces the expression of some markers, such as TGF-a, thatare typical of NSCLC cells while allowing retention, to avariable degree, of neuroendocrine markers. Unlike typicalNSCLC cells, these cells do not acquire anchorage depend-ency. However, when exposed to the cell-matrix moleculelaminin, the 209 myc cells have an increased affinity forattachment compared with parent 209 cells. In this setting,

the cells now appear more epithelial and resemble the phe-notype of NSCLC cells with neuroendocrine features, havingmixed features of each of the lung cancer phenotypes.

In contrast to overexpression of the c-myc gene, 209 cellsexpressing an exogenous ras oncogene do not acquireNSCLC markers and actually express both SCLC and neu-roendocnne features. These cells also have an increased

ability to adhere to laminin but retain a SCLC marker profilein this environment.

Expression of the ras oncogene in SCLC cells that alreadyoverexpress either the c-myc (209 myc cells) or N-myc (249cells) oncogenes results in acquisition of anchorage depend-ency and either the appearance of (249 cells; Ref. 21) or theretention of (209 myc cells) NSCLC markers such as TGF-a.Also, these cells have a decrement in neuroendocrine mark-ers, such as hASH (both the 209 and 249 cells) and N-CAM(249 cells), in the present study, a result that is consistentwith previous studies of these cells for other markers (21).

This NSCLC marker profile is retained when these myc- andms-expressing cells are exposed to laminin and appear evenmore epithelial in morphology. Thus, the most complete tran-sition from a SCLC to a NSCLC phenotype in our modelsystem results from a combination of myc and ras oncogene

expression and adherence to the cell substratum. The criticalrole of adherence to the substratum for this phenotype isoutlined in the experiments in which we denied both 209myc-ras cells and 249 ras cells access to the flask surface. Inthis setting, levels of neuroendocnne markers (hASH and/or

1’.

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GAPDH � �. #{149}cf

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Fig. 4. Effect of substratum manipulation on pheno-typic consequences of ras expression in 249 cells. The249 parental cells (A) grow in suspended aggregates,whereas 249 ras cells (B) grow in adherent monolayersin plastic flasks. The 249 ras cells forced into suspen-sion by growth over hydron (C) look like parental 249cells. Phase-contrast micrographs; x200. D, hASHtranscript expression: representative Northem blot ofthree separate experiments. Northem blots of polya-denylated RNA were sequentially stripped and rep-robed, and band density was measured, as describedin “Materials and Methods.” E, cumulative data oflevels of expression of the 2.8-kb transcript of hASH.The graph shows the average of three separate exper-iments. Y axis units are arbitrary and represent tran-script densitometries corrected for loading by dividingby those of GAPDH transcripts. F, N-CAM expressiondetermined by fluorescence-activated cell sortinganalysis. Y axis, percentage of cells positive for fluo-rescence, measured as described in “Materials andMethods.” Shown are the mean values of three inde-pendent experiments, each performed in triplicate.Bars, SE.

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Cell Growth & Differentiation 1153

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N-CAM expression) were increased, and the cells, thus re-

sumed more of their SCLC programming. In essence, this

maneuver stressed the effects on the cells of the myc gene

and lessened those of the ras gene when both oneogenes

were concordantly overexpressed. Similar to our observa-

tions, others have found altered neuroendocrine marker ex-

pression among subelones of a SCLC cell line (NCI H446)that displayed varied substratum adhesion profiles (26). Fi-nally, it is important to note that abrogation of surface ad-

hesion of a typical NSCLC cell line not basally programmed

to express SCLC features (Ui 752) did not, acutely, result in

the appearance of neuroendoerine markers. Suspension did

increase the expression of the SCLC marker N-CAM, but

only for cells that had been floating for several weeks. This is

compatible with cell selection for this trait and is similar to the

findings of others for the expression of another SCLC

marker, the growth factor gastnn-releasing peptide, in a

NSCLC line grown in serum-free media (27).There are at least three important mechanisms that may be

implicated by these results. First, the interaction of cells with

a surface affects the cell shape, and this may alter growth(25) or cell differentiation characteristics (28-30). This

change in geometry may directly affect gene expression

through altered configuration of the nuclear matrix (31). See-

ond, interaction with laminin may elicit signaling through

integrin pathways, leading to modification of gene expres-

sion events through focal adhesion kinase or other signaling

pathways (reviewed in Ref. 32). Furthermore, surface inter-

action may also alter cell polarity, which is of particular

importance for epithelial cell function. Third, increased cell-

surface interaction implies decreased cell-cell interaction.This may affect the local concentration of paracrine sub-

stances, as has been demonstrated for TGF-a (33, 34). In

addition, the cell response to growth factors may be affected

by alterations in second messengers. Indeed, one of the

most markedly affected transcripts in our study, both in

response to genetic changes (e-myc and myc-ras overex-pression) and the presence or absence of substratum adhe-

sion (209 myc-ras cells), was for the PKC-j3 gene.In addition to the biology inherent to our results, the ther-

apeutie implications of these studies merit consideration.

Cells with altered differentiation characteristics within the

same tumor may respond differently to treatment. Indeed,

earlier studies showed the correlation of neuroendoerine dif-

ferentiation of SCLC lines with ‘y-radiation sensitivity (35, 36).

Furthermore, SCLC cells attached to either plastic flasks or

laminin were found to be more resistant to a variety of agents

used to treat SCLC than the floating lines from which they

were derived (36, 37), and the adhesion of SCLC cell lines to

fibronectin led to a decreased sensitivity to an inhibitor of

polyamine biosynthesis, difluromethylornithine (38). Thus,

the phenotypie features addressed in our present studies,

which are shown to be modulated by the interaction of myc,

ras, and cellular environment, are potentially major determi-

nants of the response of lung cancer to therapeutic interven-

tion.

The ability to generalize these effects of cell environment

on activating gene expression events and lung cancer tumor

C

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1154 Cell-Substratum Interactions in Lung Cancer Cells

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Fig. 5. Effect of substratum manipulation on the phenotype of Ui 752 cells. The Ui 752 parental cells (A) grow as adherent monolayers, whereas cellsforced into suspension by coating cell culture flask surfaces with hydron (B) lead to floating aggregates of viable Ui 752 cells. Phase contrast micrographs;x200. C, growth of Ui 752 in plastic and hydron-treated cell culture flasks. The graph shows the average of three separate experiments, each performedin triplicate, as described in “Materials and Methods.” Bars, SE. D, N-CAM expression determined by fluorescence-activated cell sorting analysis. The cellsanalyzed were grown either as adherent cells in plastic cell culture flasks (Ui 752) or as suspended cells for 1 week (acute hydron) or more than 6 weeks(>6 wk hydron) in flasks with hydron coating the surface. y axis, percentage of cells positive for N-CAM fluorescence, which was measured as describedin “Materials and Methods.” Shown are the mean values of three independent experiments, each performed in triplicate. Bars, SE.

biology is tempered by the number of cell lines investigated

in these initial studies. However, our present results present

a useful paradigm for future studies. In addition, threethemes in the literature support a broader application of

these current results. First, the modification of gene expres-

sion events by cell-matrix interactions is previously de-

scribed for both lung cancer cells (19, 20, 36, 37) and other

epithelial cells (reviewed in Ref. 18), including those of neu-

roendocrine gene markers (26). Second, multiple cell lines

exhibit transitions from a SCLC to a NSCLC phenotype (1 , 2)and vice versa (27). Third, the downstream effectors of on-

cogene activation and substratum receptor occupancy may

converge, suggesting an interdependence of these path-

ways (32). Because changing environmental interactions are

important components of cell population derivation during

tumorigenesis and the appearance of metastases, these

findings have implications for both understanding the biology

of, and designing the therapies for, lung cancer.

Materials and MethodsCell Culture. The NCI H209 (209) and NCI H249 (249) cells are estab-lished SCLC cell lines (8, 39). The 209 myc cell line, expressing a full-length exogenous c-myc gene behind its own intact promoters, was agenerous gift from Bruce Johnson (National Cancer Institute-Navy Med-ical Oncology Branch, Bethesda Naval Hospital, Bethesda, MD; Ref. 15)

and grows in neomycin-selective media containing 0.4 mg/mI G41 8 (Sig-ma Chemical Co.). The 209 ras, 209 myc-ras, and 249 ras cell lines were

created by infection of the 209, 209 myc, and 249 cells, respectively, with

the 1504A pseudotype of v-Ha-ras, as described previously (21 , 40). The

expression of the exogenous ras was verified by Northem hybridization(probe P2135; Oncor, Gaithersburg, MD) for all of these cell lines. U1752is a squamous cell lung cancer cell line described previously (41). All of

these cells were grown in RPMI 1640 with 9.5% fetal bovine serum(Sigma), 100 units/mI penicillin, and 100 pg/mI streptomycin, in 37”Cincubators containing 5% CO2.

Culture plates or flasks precoated with laminin were obtained from

Collaborative Research (Bedford, MA). The number of209 cells induced to

adhere to laminin was maximal by day 7 (data not shown), and this time

point was used for all studies. Poly(2-hydroxyethyl methacrylate)(hydron)

(Sigma P3932) was used as a plasticizing agent to abrogate cell-to-flaskadhesion (42). It was constituted as a 102 (w/v) stock solution in 95%

ethanol, and 0.3 or 3 ml were used to coat each well of a 24-well plate ora 75-cm2 flask surface, respectively. The coated surfaces were air dried

for 24 h and then rinsed with PBS or RPMI 1640 prior to use. The day 7

time point was used for the hydron studies as well, unless otherwise

indicated.

Growth Curves. Growth was measured by the (MTT)-assay (Sigma).Forthis assay, all cells were grown in phenol red-free RPMI 1640 (Sigma).

On day - 1 , log phase cells were triturated to one to three cell aggregatesin suspension then placed in 24-well plates in triplicate, at concentrations

of 5 x 10” cells/well. All 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-hum bromide-derived growth curves were compared with values gener-ated with a hemacytometer and trypan blue exclusion.

Northern Analysis. Polyadenylated RNA was isolated, and 10 �gRNA were separated by gel electrophoresis on 1 .5% agarose-formalde-hyde gels, which were alkali treated, transferred to nylon membranes

(Zeta-Probe; Bio-Rad, Hercules, CA), and hybridized as reported previ-

ously (11). cDNA probes were prepared as inserts of human TGF-f31

(ATCC 59955), human TGF-co (ATCC 59953), human PKC-p (ATCC

59289), human GAPDH (ATCC 57091 ; all obtained from American Type

Cell Growth & Differentiation 1155

Culture Collection, Rockville, MD), and hASH1 (a gift from Dr. DouglasBall, Johns Hopkins Department of Medicine, Division of Endocrinology)and were labeled by random priming with [a-�P]dCTP to a specrflc

activity of approximately 10� cpm/�g DNA(43). These blots were sequen-tially stripped and reprobed as per the manufacturer’s protocol (Bio-Rad).Images were quantitated using Phosphorlmager analysis by the Image-Quant program (Molecular Dynamics).

Flow Cytometric Analysis. Cells were prepared, and microfluorom-etry was assayed as described previously (13). The anti-N-CAM antibody(NCL-CD56) was from Novocastra Labs (Newcastle-upon-Tyne, UnitedKingdom); the anti-CD44 monoclonal antibody was derived from thehybridoma line H4C4 (44); and the fluoresceinylated goat antimouse an-thody used to detect the primary munne immunoglobulin was Obtainedfrom Boebringer Mannheim (Indianapolis, IN; Ref. 13).

AcknowledgmentsWe thank Drs. Jeremy Graff and Robert Casero for their critical review ofthe manuscript.

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