(CAN@ERRESEARCH58, 1631—1635,April15, 19981

drive the extension of pseudopodia in an invasive transformed epithelial cell variant.

Materials and Methods

Cells and CUltUre. MDCK II and MSV-MDCK cells were grown as

described previously (3, 4). BioCoat Matrigel invasion chambers (BectonDickinson) were used to select for the MSV-MDCK-INV subpopulation. TheMatrigel layer was rehydrated with DMEM for 2 h before 106 MSV-MDCK

cells were added to the upper chamber of the filter units. After 24 h, cells that

had passed through to the lower chamber were recuperated by trypsinization.

This procedure was repeated five times, and after cloning of the invasive

population, the MSV-MDCK-INV cell line was obtained.Antibodies and Reagents. Rhodamine-conjugated phalloidin was pur

chased from Molecular Probes (Eugene, OR). Monoclonal antibody to @-actinwas purchased from Sigma Chemical Co. (St. Louis, MO), and polyclonalantibody to ‘y-actinwas obtained from C. Bulinski (Columbia University, New

York, NY). Vitamin D-binding protein was purchased from Calbiochem (SanDiego, CA), and antibodies to vitamin D-binding protein were purchased fromDako (Carpinteria, CA). Secondary antibodies conjugated to either FITC orTexas Red were purchased from Jackson Immunoresearch Laboratories (WestGrove,PA).

Immunofluorescence Labeling. Immunofluorescence labeling of cells

plated sparsely on glass coverslips for 2 days was performed essentially asdescribed previously (4). Labeling of F-actin and G-actin was performed oncells fixed with 3% paraformaldehyde and permeabilized with 0.2% Tritonx-bOO.F-Actin was visualizedwith rhodamine-conjugatedphalboidin,andG-actin was visualized by the addition of vitamin D-binding protein to labelmonomeric actin, which was revealed by antibodies to vitamin D-bindingprotein (7, 8). (3- and ‘y-actindouble labeling was performed on cells fixed by

the addition of precooled (—80°C)methanol-acetone (80:20) directly to thecoverslips. All washings and incubations with both primary and secondaryantibodies were done with PBS containing 0.1 nmi Ca@ @,lmrvi Mg'@ @,and 1%BSA. Afterlabeling,the coverslipswere mountedin Airvol (Air ProductsandChemicals Inc., Allentown, PA) and viewed with a Zeiss Axioskop fluorescentmicroscope equipped with a 63X planapochromat objective. Confocal imageswere obtained with a Bio-Rad MRC-600 confocabmicroscope equipped witha Nikon 60X planapochromat objective.

Phagokinetic Track Motility Assay. Cell motility was quantified using aphagokinetic track assay as described previously (3). Cell migration on colloidal coated coverslips was visualized after 22 h with a 4X objective using

dark-field illumination. Images were acquired using a Princeton Microview1400 camera, and the area cleared of gold particles, over which the cell hadmoved (excluding the area ofthe cell), was quantified using a Northern Eclipseimage analysis system (Empix Imaging, Mississauga, Ontario, Canada). Theexperiment was performed at least three times, and equivalent results wereobtained.

Immunoblot. Cells cultured at approximately 70% confluency werescraped, lysed, and sonicated in lysis buffer consisting of PBS containing 1%SDS, 5 mMEDTA, andproteaseinhibitors.Proteincontentwas assayedusingthe BCA (bicinchoninic acid) protein assay (Pierce, Rocklord, IL), and 50 @.&gof protein were separated by SDS-PAGE and blotted onto nitrocellulose paper.The blots were blocked with 5% milk in PBS and incubated with primaryantibody and then with the appropriate secondary antibody conjugated tohorseradish peroxidase. The labeled bands were revealed by chemibuminescence and exposed to preflashed Kodak XRP-1 film. Band intensities within

1631

Advances in Brief

Increased @3-ActinExpression in an Invasive Moloney Sarcoma Virus-transformed

MDCK Cell Variant Concentrates to the Tips of Multiple Pseudopodia'

PhuongU. Le, Trung N. Nguyen,Patrick Drolet-Savoie,NicoleLeclerc,and Ivan R. Nabi2Dipartement de Pathologic et Biologic Cellulaire, Universitd de Montréal,Montréal,Québec,Canada H3T 1J4

Abstract

An Invasive variant of Maloney sarcoma virus-transformed MDCKcells (MSV.MDCK-INV), which was IsOlated by the repeated selection of

cells thatsuccessfully traversed a Matrigel-coated filter, exhibits increasedmotile ability and presents an elongated cell shape and numerous pseu

do@la. Although stress fibers are present in both MDCK and MSVMDCK ealls, MSV-MDCK-INV cells contain no stress fibers and exhibita denseconcentrationofactinatthetipsofpseudopodia.RelativetobothMDCK and MSV-MDCK cells, the MSV-MDCK-INV cells exhibit increased expression of I3.actin and redistribution of I3-actin to the tips ofpseudopodla. These actin concentrations are enriched In both F- andG-actln and, thus, represent dynamic regions ofactin cytoskeleton remodeling. The acquisition ofinvasive properties by epithelial transformants Is,therefore, associated with the Increased expression of I3-actin and itsconcentradon in actin-rich domains, which may drive pseudopodlal extension and facilitate tumor cell Invasion.

Introduction

Malignancy of tumorcells of epitheial origin is inversely associated with the expression of the cell adhesion molecule, E-cadhenn,identified as a tumor suppressor molecule (1). The acquisition ofmetastatic properties must, however, also require the expression ofmolecules that confer a motile and invasive phenotype. Transformation of the polarized epitheial MDCK cell line and loss of E-cadherinexpression is associated with the acquisition of invasive properties (2).Moloney sarcoma virus transformationof MDCK cells is also associated with increased expression of autocrine motility factor receptorand reorganization of the microtubule cytoskeleton (3, 4). A similarinverse relationship between E-cadherin and autocrine motility factorreceptor has also been observed in differentially malignant humanbladder carcinomas (5). The MSV-MDCK3 cell line is heterogeneous(3), and we have isolated an invasive MSV-MDCK cell variantbyselection of cells capable of invading through a Matrigel-coated filterunit. MSV-MDCK-INV cells exhibit multiple pseudopodia, the lossof actin stress fibers, and the formationof actin-nch domains at thetips of pseudopodia. These domains are enriched in (3-actin, andMSV-MDCK-INV cells express more j3-actinrelativeto MDCK andMSV-MDCK cells. @-Actin has previously been localized to dynamic

regions of motile cells (6), and our results implicate a role for (3-actinexpression in the formation of dynamic actin-rich domains, which

Received 12/16/97; accepted 3/3/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 These studies were supported by grants from the National Cancer Institute of Canada

(to I R. N.) with money raised by the Canadian Cancer Society and from the MedicalResearch Council of Canada (to N. L. and I. R. N.) and by salary support from the Fondade Ia Racherche en Santédu Québec(to N. L and I. R. N.).

2 To whom requests for reprints should be addressed, at Département de Pathologic et

Biologie Cellulaire, N-8l2, Universitéde Montréal,2900 Edouard Montpetit, Montréal,Québec,CanadaH3T 1J4.Phone:(514)343-6291;E-mail:nabii@ere.umontreal.ca.

3 The abbreviations used are: MSV-MDCK, Moloney sarcoma virus-transformed

MDCK;MSV-MDCK-INV,invasivevaiiantof MSV-MDCK;G- andF-actin,globularandfilamentousactin,respectively.

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@3-AC11NEXPRESSION IN AN INVASIVE MSV-MDCK VARIANT

the linear range of the densitometer were quantified by densitometry (Molecular Dynamics).

Results

The actin cytoskeleton of sparsely plated MDCK cells presentscortical actin fibers as well as stress fibers, which traverse the cell,whereas MSV-MDCK cells present a more disorganized actin cy

toskeleton with fewer stress fibers (Fig. 1, A and B). To isolate aninvasive MSV-MDCK cell variant, we plated MSV-MDCK cells inthe upper chamber of Matrigel invasion chambers and, after 24 h,collected by trypsinization those cells that had succeeded in passinginto the lower chamber. This procedure was performed three times.This invasive MSV-MDCK subpopulation presented numerous pseudopodia, no actin stress fibers, and an actin dense region at the tip of

Fig. 1. Isolation of MSV-MDCK-INV cells. F-Actin labeling with rhodamine phalloidin revealed that MDCK epithelial cells exhibit numerous cortical actin stress fibers (A), whereasMSV-MDCK cells exhibit a more disorganized actin cytoskeleton but still present a few actin stress fibers (B). To isolate invasive MSV-MDCK cells, we plated MSV-MDCK cellsin the upper chamber of Matrigel invasion chambers and, after 24 h, collected by trypsinization the cells that had passed to the lower chamber. This procedure was performed threetimes, and the selected cell population exhibited no actin stress fibers and multiple pseudopodia with dense actin labeling at their tips (C). After multiple passages, this phenotype waslost, and the cells resembled MSV-MDCK cells. Cloning of the invasive MSV-MDCK cell population generated a clone that resembled the selected cell population, exhibiting multiplepseudopodia densely labeled for actin: the MSV-MDCK-INV cell line (D). Phase contrast images of MSV-MDCK (E) and MSV-MDCK-INV (F) cells show the abundance ofpseudopodia in the MSV-MDCK-INV cell line. Cell motility was measured using a phagokinetic track cell motility assay, and the area that was displaced by the cells over a periodof 22 h was measured. G, the MSV-MDCK-INV clone was significantly more motile than MDCK and MSV-MDCK cells. Scale bar, 20 @a.i.

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(ä-ACTINEXPRESSION IN AN INVASIVE MSV-MDCK VARIANT

Fig. 2. ß-actinconcentrates at the tips of pseudopodia of MSV-MDCK-INV cells.MDCK (A and B), MSV-MDCK (C and D), and MSV-MDCK-INV (£and Fi cell lineswere immunofluorescemly labeled with polyclonal ami-y-actin (A. C. and £T)and monoclonal anti-ß-actin(B, D. and /O antibodies and visualized by confocal microscopy, ß-andy-actin are uniformly distributed throughout the cell in MDCK and MSV-MDCK cells. InMSV-MDCK-INV cells, ß-actinis visualized exclusively to the tips of pseudopodia.whereas y-actin isoform is localized to the tips of pseudopodia but remains distributed in

the cytoplasm. Scale bar. 20 ¡ÕM.

each pseudopod (Fig. 1C). After multiple passages of the cells, thephenotype was lost, and the cells resembled the parental MSV-MDCK

cell line. Repeated selection of invasive cells capable of passingthrough the invasion chamber did not stabilize the phenotype, and oneclone that presented the multiple pseudopod phenotype of the invasive

MSV-MDCK population (Fig. 1C) was selected and used for furtherstudy. In this cell line, subsequently referred to as MSV-MDCK-INV,

actin was concentrated at the tips of multiple pseudopodia (Fig. ID),as described for the selected cells, and this phenotype was stable overnumerous passages. Compared to the more spread morphology of theparental MSV-MDCK cells, MSV-MDCK-INV cells present an elon

gated morphology with numerous pseudopodia per cell (Fig. l, E andF). Essentially all of the MSV-MDCK-INV cells exhibit multiple

pseudopodia, demonstrating that the cell population is homogeneous(Fig. IF). Using a phagokinetic track cell motility assay, the selectedMSV-MDCK-INV cell line is consistently more motile than theparental MSV-MDCK cells, demonstrating that these invasive cells

express a distinct phenotype, including the formation of multiplepseudopodia and an enhanced motile capacity (Fig. 1C).

The two nonmuscle actin isoforms, ßand y, are expressed inMDCK and MSV-MDCK cells. The ß-actinisoform has been localized to the leading edge of motile fibroblasts (6). "y-Actin has been

shown to be distributed throughout the cytoplasm of various culturedcell lines (9, 10). To assess whether epithelial transformation and theacquisition of an invasive phenotype are associated with the alteredexpression of actin isoforms that have a defined role in cell motility,we determined the distribution of ß-and y-actin in MDCK, MSV-MDCK. and MSV-MDCK-INV cells (Fig. 2). Double immunofluo-rescent labeling with antibodies specific for ß-and y-actin revealedthat the distribution of ß-and y-actin was not dissimilar in thedifferentially motile MDCK and MSV-MDCK cells (Fig. 2). Both

isoforms were distributed throughout the cytoplasm as well as toperipheral cell regions, although the density of ß-actinlabeling wasslightly increased relative to y-actin in cortical regions of MDCK cellsand at the edges of lamellipodia in MSV-MDCK cells (Fig. 2. A-D).In contrast, in MSV-MDCK-INV cells, ß-actinwas localized exclusively to the tips of pseudopodia; although y-actin was also localized

to the tips of pseudopodia, it was also present throughout the cytoplasm (Fig. 2, £and F). By immunoblot, ß-actinlevels in MDCK andthe more motile MSV-MDCK cells were essentially equivalent butwere significantly increased in MSV-MDCK-INV cells (Fig. 3). Relative to y-actin levels, the ß-actinexpression of MSV-MDCK-INVcells increased 3-fold compared to either MSV-MDCK or MDCKcells. The enhanced invasive and motile capacities of the MSV-

go > >co e/)

ß-actin

Fig. 3. Increased expression of ß-actinin the invasive MSV-MDCK-INV cell line, ß-and y-actin expression levels were determined in MDCK, MSV-MDCK. and MSV-MDCK-INV cell lines by immunoblot. Fifty /ig of protein were loaded, and the blotsprobed with antibodies to either ß-or y-actin. as indicated. Compilation of the results ofthree independent experiments revealed that the ratio of the expression of ß-aclintoy-actin in MSV-MDCK cells was essentially equivalent to that of MDCK cells (1.05-±0.25-fold) but was significantly increased in MSV-MDCK-INV cells relative to MDCKcells (3.2- ±0.69-fold).

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13-ACTINEXPRESSION IN AN INVASIVE MSV-MDCK VARIANT

Fig. 4. F- and G-actin are present in the actinrich domains of MSV-MDCK-INV pseudopodia.The distributions of monomeric G-actin (A and C)and polymerized F-actin (B and D) were determinedin MSV-MDCK (A and B) and MSV-MDCK-INV(C and D) cells. G-Actin is found uniformly distributed throughout the cytoplasm of MSV-MDCKcells (A), but in MSV-MDCK-INV cells, it concentrates along with F-actin at the tips of pseudopodia(C and D), demonstratingthat theseregionsrepresent dynamic domains of actin remodeling. Scalebar, 20 pM.

MDCK-INV cell line are, therefore, associated with increased (3-actinexpression and its redistribution and concentration to the tips ofpseudopodia.

Nonfilamentous actin (G-actin) has been shown to concentrate inregions undergoing significant actin assembly activity (8, 11, 12).G-Actin can be labeled by vitamin D-binding protein, and its distribution can be revealed by labeling with antibodies to vitamin Dbinding protein (8). In MSV-MDCK cells, F-actin can be visualized inactin stress fibers, and G-actin is distributed uniformly throughout thecell cytoplasm (Fig. 4, A and B). In MSV-MDCK-INV cells, F-actinconcentrates along with G-actin at the tips of pseudopodia, identifyingthese (3-actin rich regions as dynamic domains of actin cytoskeletonremodeling (Fig. 4, C and D).

DIscussion

Dynamic assembly and disassembly of actin filaments result inlocalized remodeling of the actin cytoskeleton and protrusion ofcellular lamellipodia and pseudopodia implicated in cell motility (13).Although cellular transformation has long been associated with theloss of actin stress fibers and the diffuse distribution of cytoplasmicactin (14, 15), disruption of actin stress fibers is more specificallyassociated with increased motile and metastatic ability (16, 17). Thepresence of stress fibers in low-metastatic and their absence in highmetastatic K-l735 melanoma and UV-2237 fibrosarcoma variants(17) is consistent with our observation that the absence of stress fibers

is associated not with Moloney sarcoma virus transformation ofMDCK cells but with the acquisition of enhanced motile and invasive

properties by a subpopulation of MSV-MDCK cells. Pseudopod formation is an essential aspect of tumor cell motility and invasion, andthe presence of multiple pseudopodia on MSV-MDCK-INV cells ishighly consistent with the use of Matrigel invasion as a selectioncriterion for their isolation. In a similar fashion to the acm concentrations at the tips of MSV-MDCK-INV pseudopodia, F-actin concentrates in newly formed pseudopodia of motile neutrophils and infilopodia of neuronal growth cones (18, 19).

Multiple actin isoforms exist, and many nonmuscle cells express (3-and y-actin isofonns (20). (3-and y-actin isoforms differ by only fouramino acids near their NH2 terminus but exhibit different cellular

distributions. f3-Actin is enriched at the apical membrane, and ‘y-actinis enriched at the basolateral membrane of gastric parietal cells (21).(3-Actin and its mRNA are specifically distributed to peripheral ccllular regions and the leading edge of motile cells (6, 22, 23). (3-Actinhas recently been shown to concentrate in motile and dynamic regionsof neurons, such as growth cones and dendritic spines.4 The equivalent expression levels of (3-actin relative to y-actin in MDCK andMSV-MDCKcells demonstratesthat epithelialtransformationis notnecessarily related to altered (3-actin expression. Increased (3-actinexpression in MSV-MDCK-INV cells relative to parental MSVMDCK cells demonstrates that (3-actin expression levels may regulatethe enhanced pseudopodial extension and invasivity of MSV-MDCK

4 K. Micheva, A. Vallée, C. Beaulieu, and N. Leclerc. @-Actin is confined to structures

having high capacity of remodeling in developing and adult rat cerebellum, submitted forpublication.

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13-ACI1NEXPRESSIONIN AN INVASIVEMSV-MDCKVARIANT

INV cells. The increased expression of f3-actin and its distribution tothe tips of pseudopodiain MSV-MDCK-INV cells indicates that the13-actin concentration in these regions is significantly higher than inany cellular region of MDCK or MSV-MDCK cells. Transfection of13-actinintosmoothmusclecells inducedtheformationof actin-richprotrusions at the cell periphery (24) and modulation of the (3-actin:‘y-actinratio has been shown to influence myoblast morphology (25).

f3-Actin mRNA is targeted to the leading edge of motile fibroblasts,and serum-mediated signal transduction mechanisms induce the re

distribution of f3-actin mRNA to the leading edge of fibroblasts,implicating the peripheral distribution of (3-actin mRNA and its translation in enhanced cell motility (6, 10, 26, 27). It is conceivable thatthe stabilization of f3-actin mRNA at the tips of pseudopodia couldgenerate the localized concentrations of (3-actin seen in MSV-MDCKcells. Interestingly, expression of an acidic variant of (3-actin reducesthe metastaticability of Bl6 melanomacells and induces the formation of an increased number of stress fibers (28, 29). Although theseresults show that expression of a (3-actin variant and interference with

13-actin function can reduce tumor cell motility and metastasis, ourresults demonstrate, for the first time, that modulation of the expression and cellular distributionof wild-type (3-actinis directly associated with enhanced tumor cell motility and invasivity. Regulation of(3-actin expression and distribution might be important parametersdetermining pseudopodial protrusion and invasivity of tumor cells.

Such a concentrationof (3-actinmay be responsiblefor the formalion of highly dynamic actin domains, which drive pseudopodialextension. The dynamic exchange of actin between its monomeric orglobular and polymerized or filamentous form is an essential aspect ofcellular movement (13). Punctate structures that are rich in G-actinhave been located behind extending lamellipodia and may serve aspools of monomeric actin, which can be transformed rapidly intoF-actin, inducing leading edge extension (8). In contrast to the urnform distribution of G-actin in MSV-MDCK cells, in MSV-MDCKINV cells, dense concentrations of G-actin colocalized with F-actindensities at the tips of pseudopodia. These actin densities, whichrepresent the site of (3-actin concentration in MSV-MDCK-INV cells,are, therefore, the site of extensive actin filament assembly anddisassembly. Our results implicate a role for (3-actin expression in theformation of dynamic actin-rich domains that drive pseudopodialprotrusion in an invasive transformed epithelial cell variant.

Acknowledgments

We thankJeanLeveilléfor the photographicreproductionsandGinetteGuay for her excellent technical assistance.

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1998;58:1631-1635. Cancer Res   Phuong U. Le, Trung N. Nguyen, Patrick Drolet-Savoie, et al.   of Multiple PseudopodiaVirus-transformed MDCK Cell Variant Concentrates to the Tips

-Actin Expression in an Invasive Moloney SarcomaβIncreased

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