INTRODUCTION

Biliary glycoprotein (BGP), also known as CD66a due to itsexpression in granulocytes, is a cell surface glycoproteinmainly expressed on the lumenal surface of epithelial cells.BGP is a member of the CEA (carcinoembryonic antigen) genefamily, which in turn is a member of the immunoglobulinsuperfamily (Williams and Barclay, 1988). As part of ourongoing studies on the biology of BGP, we have investigatedits expression in the normal and malignant breast (Huang et al.,

1998). These studies were prompted by the observation thatBGP is expressed in a polarized, lumenal orientation in theliver (Wagener et al., 1983b), colon (Frangsmyr et al., 1995)and breast (Riethdorf et al., 1997), and is downregulated inpremalignant adenomas (Nollau et al., 1997) and colon tumors(Neumaier et al., 1993). Although we found that BGP isdownregulated in only 30% of breast cancers (Huang et al.,1998) compared to >90% in colon cancers (Neumaier et al.,1993), it is likely that BGP delivers a strong growth inhibitorysignal upon its expression in fully differentiated epithelial cells,

4193Journal of Cell Science 112, 4193-4205 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0702

Normal mammary epithelial cells express the cell surfaceprotein biliary glycoprotein (BGP or CD66a) in a polarizedmanner, suggesting that this protein may play a role in theformation of mammary acini. In order to test thishypothesis, we interrupted the expression of BGP in themammary epithelial line MCF10F when cultured in or onMatrigel, a source of extracellular matrix (ECM). Whenanalyzed by immunofluorescence confocal microscopy, theBGP staining is confined to the lumenal surface andcolocalizes with actin.

Sequential scanning electron microscopy demonstratesthat the MCF10F cells migrate to form clusters, followedby apoptotic cell death within the center, resulting in lumenformation. Transmission electron micrographs reveal thepresence of tight junctions and desmosomes between thecells, microvilli along the lumenal surface, and typicalapoptotic bodies within the lumen. When the MCF10F cellsare transfected with the BGP antisense gene and grown inMatrigel, they exhibit reduced acini formation (12% and20%) compared to untransfected cells (52%) or to cellstransfected with vector only (62%). Acini formation is alsosignificantly reduced when MCF10F cells grown inMatrigel are treated with anti-BGP antibody (18% at 100µg/ml), or recombinant soluble BGP (18% at 0.4 µM). Incontrast, the BGP-negative MCF7 breast tumor cell line,which does not form acini when grown in matrigel, exhibits>60% cell death with the occasional formation of acini,

when transfected with the BGP sense gene and grown inMatrigel. These results support the hypothesis that BGPplays a role in the normal differentiation program ofmammary epithelial cells, indicating that its expression isessential to the formation of the lumen. Furthermore, andas shown by others, the differentiation program dependson the presence of ECM. The lack of expression of BGP inthe MCF7 breast cancer cell line suggests that thedownregulation of BGP expression confers a growthadvantage to these cells in ECM. In addition, we found thatthe MCF10F cells could be separated into a BGP-positiveepithelial fraction (MCF10F-e), and a BGP-negativemyoepithelial fraction (MCF10F-m). When themyoepithelial cell-enriched fraction is grown on Matrigel,web-like structures are formed. These cells have a typicalspindle shape cell morphology and express keratin, α-smooth muscle actin and vimentin, markers of themyoepithelial cell phenotype. When MCF10F-m cells aretreated with IFNγ, they express CEA (carcinoembryonicantigen) but not BGP. Since breast carcinomas, especiallyin situ carcinomas, express CEA, this finding maysuggest a heretofore unappreciated relationship betweenmyoepithelial cells and breast cancer.

Key words: Biliary glycoprotein, CD66a, Mammary epithelial cell,Myoepithelial cell, Matrigel, Extracellular matrix, Morphogenesis

SUMMARY

Essential role of biliary glycoprotein (CD66a) in morphogenesis of the human

mammary epithelial cell line MCF10F

Jie Huang1, John D. Hardy2, Yanyu Sun3 and John E. Shively4,*1Graduate School of the City of Hope and Beckman Research Institute, Duarte, CA 91010, USA2Electron Microscopy Unit, Biology Division, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA3Division of Anatomic Pathology, City of Hope National Medical Center, Duarte CA 91010, USA4Division of Immunology, Beckman Research Institute of the City of Hope, Duarte CA 91010, USA*Author for correspondence (e-mail: jshively@coh.org)

Accepted 28 September; published on WWW 17 November 1999

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and that many cancers of epithelial origin overcome thisgrowth inhibition by downregulation of the BGP gene. Thisproperty of BGP is further indicated by its tumor growthinhibitory activity when the BGP gene is transfected andexpressed in prostate (Kleinerman et al., 1995), bladder(Kleinerman et al., 1996) or breast cancer cells (Luo et al.,1997). The fact that breast tumors show a lower incidence ofdownregulation of the BGP gene may relate to the changingrole of BGP during morphogenesis. In this respect, wehypothesize that BGP may play a role in the formation of thelumen. In order to test this hypothesis, we investigated BGPexpression in a mammary epithelial cell model ofmorphogenesis.

Normal mammary glands have a polarized orientation ofepithelial cells, with their secretory surfaces facing a centrallumen and the basal surfaces surrounded by a layer ofmyoepithelial cells. When these cells are separately isolatedand cultured together in Matrigel, a source of extracellularmatrix (ECM), they form a reconstituted alveolar structure(Gomm et al., 1997). When the epithelial cells are culturedseparately in Matrigel or collagen gel, respectively, they mayform either spheroids (Gomm et al., 1997) or branchingstructures (Foster et al., 1983).

In vitro models have shown that mammary epithelial cellsundergo differentiation to acini and tubule structures whengrown on Matrigel (Barcellos-Hoff et al., 1989; Bergstraesserand Weitzman, 1993; Gomm et al., 1997; Petersen et al., 1992).The spontaneously immortalized human breast cell lineMCF10 (Soule et al., 1990) mimics the behaviour of primarymammary epithelial cells when grown on Matrigel and hasbeen used as a model for the study of alveolar (Howlett et al.,1995; Petersen et al., 1992) and branching morphogenesis(Stahl et al., 1997). This cell line has been further divided intotwo lines designated MCF10A for attached and MCF10F forfloating (Soule et al., 1990). In the original report (Soule et al.,1990) both cell lines exhibited the polarized phenotype ofepithelial cells while neither cell line was observed to containmyoepithelial cells. In subsequent studies, MCF10A cells wereshown to favor acini structures when mixed with liquidMatrigel and grown in thick Matrigel culture (Howlett et al.,1995), but predominantly formed tubules when seeded on thetop of thin Matrigel cultures at a concentration of 2.5×104

cells/cm2 (Stahl et al., 1997). Tubule formation was found todepend on the seeding density, with the cells forming acini at1.25×104 cells/cm2 or below. Tubule formation (branchingmorphogenesis) was found to depend on the expression ofα6β4 integrin on these cells and the presence of laminin-5 inMatrigel (Stahl et al., 1997). Treatment of these cells withantibodies to laminin or α6β4 integrin blocked the formationof tubules on thin Matrigel. Similarly, treatment of the HMT-3522 breast epithelial cell line with blocking antibodies toα6β4 integrin prevented acini formation in thick Matrigel(Weaver et al., 1997).

In other studies, a variety of growth factors, includinghepatocyte growth factor (Brinkmann et al., 1995; Soriano etal., 1995), epidermal growth factor (Gomm et al., 1997),neuregulin/heregulin (Yang et al., 1995), keratinocyte growthfactor (Hirai et al., 1998) and fibroblast growth factor (Liand Shipley, 1991) were found to promote branchingmorphogenesis. In addition, cell surface glycoproteins onstromal cells or the epithelial cells such as epimorphin (Hirai

et al., 1998) play a role in morphogenesis of the mammarygland.

In this study, we have chosen the MCF10F cell line ratherthan MCF10A because MCF10F cells produce a higher levelof acini when grown in Matrigel (approx. 50%) compared toMCF10A cells (<20%). We show here that MCF10F cells canbe divided into epithelial and myoepithelial sublines, and thatwhen the epithelial cells are cultured on Matrigel they formacinar structures. Furthermore, interruption of BGP expressionwith anti-BGP antibody or a BGP antisense gene or treatmentwith recombinant soluble BGP significantly reduces theformation of acini.

MATERIALS AND METHODS

Cell culture and treatmentsMCF10F, MCF10A and MCF7 cell lines were obtained from ATCC.MCF10F and MCF10A cells were grown in mammary epithelial cellgrowth medium (MEGM) (Clonetics) with 10 ng/ml hEGF, 5 µg/mlinsulin, 0.5 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/mlamphotericin-B and 52 µg/ml bovine pituitary extract. MCF10F cellswere separated into two sublines, MCF10F-e (strongly adherent) andMCF10F-m (weakly adherent). MCF10F-e cells were grown inmammary epithelial cell growth medium, and MCF10F-m cells weregrown in DMEM-F12 (Gibco-BRL) with 5 µg/ml insulin, 5 µg/mltransferrin, 20 ng/ml epidermal growth factor, 5×10−8 Mdexamethasone and 5% FBS. MCF7 cells were maintained in MEMwith Earle’s salts and 10% FBS (Irvine Scientific). Matrigel culture:2.5×105 cells were plated either on a thin layer (0.5 mm) or in a thicklayer (1 mm) of Matrigel (Collaborative Biomedical Products) in 2-well chamber slides. To harvest cells, the gels were incubated withdispase (0.2 ml of 50 U/ml per cm2; Collaborative BiomedicalProducts) at 37°C for 2 hours. Dispase was inactivated by dilutionwith medium. Cytokine treatment: cells cultured as above were treatedwith either 500 U/ml of IFNγ or 50 ng/ml of TNF-α for the timesindicated.

MCF10F-e cells (2.5×105) were treated with anti-BGP mAb T84.1or control mAb T84.66 (anti-CEA) at concentrations of 10, 50 and100 µg/ml at room temperature for 15 minutes prior to plating inMatrigel. The same concentrations of antibodies were maintained inthe culture medium over the length of the experiment. A syntheticpeptide (BGP-N1B) containing residues 5-18 of the N-domain of BGP(Barnett et al., 1989) was mixed with MCF10F-e cells (2.5×105) at 1and 10 µM and cultured as above. A cDNA fragment containing theextracellular domains of BGP (N, A1, B1 and A2 domains) wascloned into a glutamine synthetase expression vector (details to bepublished elsewhere) and the construct was expressed in NS0 cells.Recombinant soluble BGP was purified from culture mediumsupernatant using T84.1 mAb affinity chromatography and used at 0.4µM to treat MCF10F cells (2.5×105) as described above.

Cell transfectionsA 1.6 kb full-length cDNA of BGP (BGPa) was cloned into the SalIand HindIII restriction sites of an expression vector pHβ-actin(Gunning et al., 1987) in either the sense or antisense orientation,designated as BGPa/pHβ and BGPAS/pHβ, respectively. The pHβvector contains a neomycin-resistant gene and a β-actin promoter.

MCF10F cells8×105 cells were plated in each well of 6-well plates and transfectedwith 1.5 µg of plasmid DNA constructs BGPa/pHβ, BGPAS/pHβ orpHβ-actin vector using Lipofectin (Life Technologies, Ltd), accordingto the manufacturer’s instructions. Cells were grown in DMEM-F12(Gibco-BRL) with 5 µg/ml insulin (Sigma), 5 µg/ml transferrin(Sigma), 20 ng/ml epidermal growth factor (Sigma), 5×10−8 M

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dexamethasone (Sigma) and selected with 0.4-0.5 mg/ml of geneticin(Life Technologies) for 4 weeks. The G418-resistant colonies wereexpanded and analyzed by FACS. Two of the stably transfected lines(AS6 and AS9) were grown in Matrigel and analyzed byimmunohistochemistry. Colonies were scored for the presence (acinusformation) or absence of lumen and compared by the chi-squared test.

MCF7 cellsThe cells were grown in 100 mm dishes until they reached 80%confluence, then the cells were transfected with 4 µg of plasmid DNAconstruct BGPa/pHβ or pHβ-actin vector, respectively, using thelipofectin method. G418-resistant cells were grown in Matrigel andscored for acinus formation as described above.

ImmunohistochemistryIn monolayer culture, cells were fixed with 10% formalin for 10minutes, followed by cold methanol for 5 minutes, and chilled acetoneat −20°C for 1 minute. In Matrigel culture, cells were first fixed with10% formalin for 10 minutes, embedded in 3% agarose, then treatedwith 10% formalin overnight, followed by 70% ethanol. Paraffin-embedded sections were prepared and used for Hematoxylin andEosin staining and immunohistochemistry. Monoclonal antibodiesagainst α-smooth muscle actin (1:10,000; Sigma), vimentin (1:200;Dako), pankeratin (1:100; Ventana) and cytokeratin-14 (1:20;Neomark) were used. Monoclonal antibody 4D1C2 (1 µg/ml) wasused to detect BGP (Drzeneik et al., 1991). Immunohistochemicalstaining was performed using the standard avidin-biotin-peroxidasetechnique. In brief, sections were blocked in 5% normal horse serumfor 20 minutes, incubated with primary antibodies at roomtemperature for overnight, and further incubated with biotinylatedhorse anti-mouse secondary antibody (1:400; Vector) for 1 hour. Afterwashing with PBS, the sections were incubated with avidin-biotin-peroxidase complex (1:200; Vector) for 1 hour and with 0.05% 3,3′-diaminobenzidine/0.03% H2O2 for 7 minutes, washed andcounterstained with Hematoxylin.

Electron microscopyFor scanning electron microscopy, the cells were grown on thinMatrigel on coverslips for 2-4 days, fixed for 3 hours with 1%glutaraldehyde in cacodylate buffer, washed 3×, and dehydrated bytreatment with graded ethanol (2× at 2 minutes each in 60%, 70%,80%, 95% and 100%). The cells were dried at the critical point undercarbon dioxide, mounted on aluminum stubs with colloidal silverpaste and sputter-coated with Pt/Pd (40/60) for 160 seconds. Scanningelectron microscopy was performed on a Philips SEM 505. Fortransmission electron microscopy, cells were fixed as above, washed3× and post-fixed with 1% osmium tetroxide for 1 hour at 4°C, washed3×, scraped from the coverslips and placed in microcentrifuge tubesfor graded ethanol rinses as above. The samples were treated withpropylene oxide, embedded in Eponate with araldite, and polymerizedfor 72 hours at 70°C. Sections (50 nm) were cut on an LKB UltrotomeIII, placed on 300 mesh, uncoated copper grids, and stained with 5%uranyl acetate for 10 minutes, followed by 1 minute in Sato’s leadstain. Transmission electron microscopy was performed on a PhilipsCM 10 TEM.

Western blotsMembrane extracts (30-50 µg total protein) prepared with 1% NP40and protease inhibitors were boiled in SDS-sample buffer before beingapplied to 8% reducing-SDS gels. After electrotransfer tonitrocellulose membranes (Bio-Rad) and blocking in TBS (100 mMTris-HCl, pH 7.5, 150 mM NaCl) buffer containing 5% nonfat drymilk overnight, the blots were incubated with mAb T84.1 (Wageneret al., 1983a) at a final concentration of 1 µg/ml for 1 hour, followedby incubation with goat anti-mouse IgG-peroxidase (1:3000;Boehringer Mannheim) for 1 hour. Detection was carried out usingthe chemiluminescence substrate and enhancer kit (Pierce).

RT-PCRTotal RNA was extracted from tissue culture cells using ToTALLYRNA Kit (Ambion). 1 µg of total RNA was used for synthesis of first-strand cDNA. Reverse transcription reaction was carried out at 42°Cfor 30 minutes as described previously (Huang et al., 1998), using 1µM BGP antisense primer (5′-GCTGGGCTTCAAAGTTCAGGGT-3′). In the PCR reaction, a final concentration of 1 µM of each BGPprimers (sense primer: 5′-CTGCAACAGGACCACATGCAAG-3′;antisense primer: 5′-GCTGGGCTTCAAAGTTCAGGGT-3′) wasused. PCR was carried out with denaturation at 94°C for 2 minutesfollowed by 30 cycles at 94°C for 1 minute, 55°C for 2 minutes, and72°C for 3 minutes. RNA from HT-29 cells was used as a positivecontrol (Huang et al., 1998). PCR products were separated byelectrophoresis on a 2% agarose gel and transferred to a nylonmembrane (Qiagen). Following prehybridization with 100 µg/mlsalmon sperm DNA in 5× SSC, 20 mM sodium phosphate, pH 7.0,7% SDS, 10× Denhardt’s solution, 10% dextran sulfate for 5 hours at50°C, the blot was hybridized with [γ-33P]ATP labeled BGP probe(5′-GAAAATGGCCTCTCACCTGG-3′) at 50°C, overnight.Autoradiography was performed using BioMax MR films.

Ribonuclease protection assayA 190-bp fragment of the BGP cDNA (1435-1625) was subclonedinto Bluescript at the HindIII/BamHI sites. BGP antisense riboprobewas synthesized and labeled with 50 µCi of [α-32P]dUTP usingMAXIscript T7/T3 Kit (Ambion). Samples (10 µg) of total RNA werecoprecipitated with the BGP probe and a 316-bp [α-35S]dUTP-labeledGAPDH probe (as internal control), and hybridized in hybridizationsolution containing 80% formamide at 42°C, overnight. Afterdigestion with RNases A and T1, the protected RNA hybrids wereprecipitated, dissolved in denaturing loading buffer, and separatedby electrophoresis on 7 M urea, 5% polyacrylamide gels.Autoradiography was performed at −70°C using BioMax MS filmswith intensifying screen. The intensities of bands were quantitatedusing a Bio-Rad scanning densitometer.

FACS analysisFor the detection of cell surface expression of BGP and cell sorting,MCF10F cells were first incubated with mAb T84.1 (1 µg/ml) at 4°Cfor 1 hour, washed with PBS, and then incubated with fluoresceinisothiocyanate-conjugated secondary antibody (goat anti-mouse IgG,1:250; Boehringer Mannheim) at 4°C for 1 hour. Cells were sorted onthe MoFlo Optical Bench (Cytomation). After sorting, T84.1 positivecells and T84.1 negative cells were cultured separately, both on plasticand in Matrigel, as described above.

Confocal microscopyThe cells were grown on Matrigel, fixed and permeabilized with 10%formaldehyde/0.1% Triton X-100 at room temperature for 10 minutesand stained with both FITC-conjugated phalloidin (200 ng/ml; Sigma)and mAb T84.1 (1 µg/ml) for 1 hour followed by Texas Red-conjugated goat anti-mouse IgG (1:100; Molecular Probes) for 1 hour.A Zeiss Model 310 confocal microscope was used to acquire data.

RESULTS

Characterization of MCF10F cell phenotypesIn order to study BGP expression in an in vitro model system,we chose MCF10F, a mammary epithelial cell line, which inpreliminary experiments produced high yields of acini whencultured in Matrigel. However, we also noted that this cell linecontained at least two phenotypes, prompting the followingphenotypic characterization. When MCF10F cells were grownin serum-free medium on plastic, the majority of cells exhibited

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a cobblestone morphology with occasional spindle-shapedcells. When the loosely attached cells were repetitivelypassaged on plastic with removal of tightly adhering cells,cultures containing >95% spindle-shaped cells were obtained.In contrast, repetitive passaging of the tightly attached cells onplastic and removal of the loosely attached cells led to >95%epithelial cells with a typical cobblestone morphology.Immunostaining of the spindle-shaped cells (MCF10F-m) waspositive for keratin, α-smooth muscle actin and vimentin (Fig.1A-C), confirming the myoepithelial phenotype of these cells.Immunostaining of the cobblestone-like cells (MCF10F-e) waspositive for keratin only (Fig. 1D-F), confirming their epithelialphenotype. When stained for BGP using a BGP-specificantibody, the MCF10F-e cells were weakly positive and themyoepithelial cells were negative (data not shown). When theMCF10F-e cells were grown in thick Matrigel they formedacini (approx. 50% of the colonies formed acini after 14 days)but no tubule structures, staining positively for BGP on thelumenal side (Fig. 2A,B). When grown on thin Matrigel, theMCF10F-m cells formed thin web-likestructures (Fig. 2C). In contrast, when grownon thin Matrigel the MCF10F-e cells formeddonut-shaped acini structures. When theacini structures were examined by confocalmicroscopy and stained for BGP and actin,they showed colocalization of BGP and actinon the lumenal surface (Fig. 2D).

Electron microscopyThe formation of acini by MCF10F-e cellsgrown on thin Matrigel was further studiedby electron microscopy. This approachpermitted the direct examination ofmorphological changes that the cellsundergo while developing acini. Scanningelectron micrographs of the MCF10F-e cellsgrown on thin Matrigel revealed smallcolonies (<8 cells per colony) with fewisolated cells after 1-2 days of culture (Fig.3A). After 2-4 days of culture, the coloniesincreased in size (>12 cells) and began toform central lumens (Fig. 3B,C). Closeinspection of the developing lumensrevealed bright, condensed bodies withnumerous blebs. The consistent finding ofbright, condensed bodies on the tops of thecolonies and within the developing lumensuggested that the lumen was formed by celldeath within the central region of the colony.In addition, the mature acini exhibited cellflattening with a polarized orientationaround the lumen. Cells at the base of thecolonies were strongly attached to the ECMand showed either evidence of pulling matrixfibrils up along the sides of the colonies (Fig.3B,C) or the extension of cellular processesto the ECM. Based on a close examinationof the fibrils (under TEM), we conclude thatthe fibrils originate from the ECM and notfrom the cell. A similar conclusion wasderived by Barcellos-Hoff et al. (1989), who

performed EM studies on acini formation by freshly isolatedmurine mammary epithelial cells grown on Matrigel.

To further confirm the cellular morphology, representativecolonies were embedded, sectioned and analyzed bytransmission electron microscopy (Fig. 4). The cell surfacesfacing the lumen contained microvilli, while the intercellularboundaries exhibited tight junctions and desmosomes, acharacteristic finding for mammary epithelial cells grown onMatrigel (Barcellos-Hoff et al., 1989). The lumen containednumerous small vesicles (20-50 nm), which were not presentelsewhere and probably represent vesicle formation by thedifferentiated cells. Close examination of the lumen revealedcellular bodies with well defined membranes, compactedchromatin, and condensed cytoplasm with well preservedorganelles (vacuoles, Golgi and mitochondria). In particular, asection through the nucleus of one of the bodies (Fig. 4B)reveals several areas of condensed chromatin and an irregularnuclear boundary. These cellular bodies correspond toapoptotic bodies described in detail by other investigators using

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Fig. 1. Characterization of MCF10F cell phenotypes. MCF10F-m cells (A-C) orMCF10F-e cells (D-F) were grown on plastic and immunostained with anti-pan-keratin(A,D), anti-α-smooth muscle actin (B,E) or anti-vimentin (C,F) antibodies. The spindle-shaped morphology of the MCF10F-m cells and their strong staining with all threeantibodies confirms their myoepithelial cell phenotype, while the MCF10F-e cells havean epithelial cell phenotype.

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electron microscopy as a definitive tool for the identificationof apoptosis (Tomei and Cope, 1991; Wyllie et al., 1980).

Upregulation of BGP mRNA and protein expressionOnce stable phenotypes were established for MCF10F-m andMCF10F-e cells, it was necessary to determine the effect ofMatrigel on BGP expression at the mRNA and protein levels.When mRNA levels were analyzed by RNase protectionassays, BGP mRNA increased by over five- to tenfold whencultured for 6 days on Matrigel (Fig. 5A). However, when theprotein levels of BGP were determined by western blotanalysis (Fig. 6A), only a modest increase (20-50%) in thelevel was observed before and after culture of MCF10F cellson Matrigel. While the mechanism of BGP mRNA inductionby Matrigel is unknown, previous studies on colonic epithelialcells have shown that BGP mRNA is strongly induced byTNFα and IFNγ (Chen et al., 1996; Takahashi et al., 1993).Therefore, we tested the effect of these cytokines on MCF10Fcells. When the cells grown on plastic were treated with 50ng/ml of TNFα or 500 U/ml of IFNγ, BGP expressionincreased over twofold when analyzed by RNase protection(Fig. 5B) or western blots (Fig. 6B). When the cells weregrown in Matrigel with the addition of 500 U/ml of IFNγ or50 ng/ml of TNFα, no change in rate of growth or morphologywas detected (data not shown), and a less dramatic increase inBGP protein expression was observed (Fig. 6A). For purposesof comparison with the non-tumorigenic MCF10F cell line, the

breast carcinoma cell line MCF7 was also analyzed for BGPexpression under the same conditions. MCF7 cells were foundnot to express BGP at either the mRNA (Fig. 5A) or the proteinlevel (Fig. 6D). However, these cells do express CEA, whichwas detected as a 180 kDa band on western blots with theT84.1 antibody (Fig. 6D), and confirmed by reaction with CEAspecific antibody T84.66 (data not shown). Similarly,MCF10F-m cells did not produce detectable amounts of BGP,but after treatment with IFNγ, CEA expression was induced(Fig. 6C). The CEA production of cytokine-treated MCF10F-m cells was confirmed by RT-PCR (data not shown).

Fig. 2. Immunostaining and confocal analysis of MCF10F-e cellsgrown in thick Matrigel or on thin Matrigel. (A) Hematoxylin andEosin staining of MCF10F-e cells grown in thick Matrigel for 14days. (B) Immunostaining of MCF10F-e cells grown in thickMatrigel with BGP-specific mAb 4D1C2. (C) Phase-contrast imageof MCF10F-m cells grown on thin Matrigel. (D) Confocalmicroscopic analysis of MCF10F-e cells grown on thin Matrigel.MCF10F-e cells were grown on thin Matrigel for 7 days, fixed andstained with mAb T84.1, and detected with Texas Red-conjugatedgoat anti-mouse IgG (right) or FITC-conjugated phalloidin (left).

Fig. 3. Scanning electron micrographs of MCF10F-e cells grown onthin Matrigel. (A) 2 days, (B,C) 4 days. (A) Side view of an earlycolony exhibiting a piled-up cell appearance with fibrillarconnections of the bottom cells to the Matrigel. (B) Top view of alater stage colony exhibiting the formation of bright, condensed cellbodies at the top of the colony. (C) A mature colony exhibiting aflattened morphology with a central lumen containing bright,condensed cell bodies.

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Since BGP mRNA can exist in at least fourisoforms, it was also necessary to perform anisoform-specific RT-PCR analysis (Takahashi et al.,1993) on mRNA from MCF10F-e cells before andafter growth in Matrigel. The analysis is based ontwo primers common to the four major isoforms ofBGP (Fig. 5C). This analysis (Fig. 5D) revealed thatonly three of the four BGP isoforms are produced,mimicking the situation in normal breast glands(Huang et al., 1998). No change in isoformexpression was found upon growth in Matrigel.

Morphology of BGP sorted cellsSince it was possible that the expression levels ofBGP played a role in lumen formation, we decidedto sort MCF10F cells into BGP high and lowpopulations in the expectation that their initial BGPlevels would influence their morphologies whencultured in Matrigel. Cell sorting analysis ofMCF10F cells revealed that 60-70% of the cells wereBGP positive. When the cells were sorted into BGPhigh (>90% positive) and BGP low (<10% positive)populations, passaged on plastic for 2 weeks, andreanalyzed, the BGP low population was 30%positive (Fig. 7A) and the BGP high population was80% positive (Fig. 7D). When grown on thinMatrigel, the BGP low population gave rise to eitheracini (Fig. 7B) or tubule structures (Fig. 7C), somewith a surrounding layer of myoepithelial cells. Incontrast, the BGP high population gave rise toexclusively acini-forming colonies (Fig. 7E). Whenthe BGP high cells were grown in thick Matrigel andstained for BGP, intense staining was observed at thelumenal surfaces of the acini (Fig. 7F). When theBGP low cells were grown on thin Matrigel andstained for BGP, intense staining was observed at thelumenal surfaces of the acinar and tubular structures(data not shown). Thus, we found that BGP lowsorted cells still retain the ability to express BGP, andwhen grown in or on Matrigel, can form glandularstructures, often including myoepithelial cells. Theseresults can be explained by assuming that while theBGP high cells contained few or no myoepithelialcells, the BGP low cells contained significantnumbers of myoepithelial cells. We also conclude

that either the presence of myoepithelial cells in the mixed cellcultures or the lower amounts of BGP induced tubule formation,a result usually seen with MCF10A cells.

Interruption of BGP expression and cell adhesion inMCF10F cellsIn order to directly probe the role of BGP in morphogenesis,we attempted to interrupt its expression via an antisense gene,and block its homotypic inter-cellular effects with anti-BGPantibody, synthetic BGP peptide and recombinant soluble BGP.MCF10F cells were transfected with the BGP sense andantisense genes and a vector control, and permanent lines wereestablished by selection by G418 resistance. A line establishedfrom the vector control transfected cells showed 70% BGP-

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Fig. 4. Transmission electron micrographs of MCF10F-e cells grown on thinMatrigel for 4 days. (A) A section of a polarized surface showing lumen (lu),microvilli (mi), vesicles (ve), desmosomes (de) and an apoptotic body (a).(B) Section of an apoptotic body (a) within the lumen exhibiting dark bands ofcondensed chromatin, convoluted nucleus, and condensed cytoplasm withretention of the cell membrane and organelles.

Table 1. Acinus formation for MCF10F cells transfectedwith the BGP antisense and sense gene

Cells Acini/colonies % acini P

Untransfected 52/100 52 −Vector 62/99 62 >0.05Antisense AS6 31/156 20 <0.001Antisense AS9 14/117 12 <0.001Sense S3 113/198 57 >0.05

Cells (2.5×105/well) were mixed with Matrigel, cultured for 14 days, fixed,embedded and stained with anti-BGP antibody 4D1C2. Two lines wereestablished from transfection of MCF10F cells with the BGP antisense gene(AS6 and AS9), one line from the BGP sense gene (S3) and one line fromtransfection with vector only (vector).

P values were calculated by the χ2 method.

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positive cells (Fig. 8A) compared to 60% for untransfectedcontrols (not shown). The slight increase in BGP-positive cellswas not significant. Two lines were established from the BGPantisense transfected cells, AS9 and AS6, showing 8% and30% positive staining for BGP, respectively (Fig. 8B, and not

shown). A line established from the BGP sense transfectedcells showed 90% positive staining for BGP (Fig. 8C). Whenthese lines were grown in thick Matrigel and scored for aciniformation by immunohistochemical staining, the antisensetransfected lines showed a significant reduction in acini

Fig. 5. BGP mRNA expression in MCF10F and MCF7 cells analyzed by RNase protection assay (A,B) and RT-PCR (C,D). (A) Total RNA wasisolated from MCF10F and MCF7 cells grown either on plastic (− Matrigel) or on Matrigel (+ Matrigel) for 6 days and probed for GAPDH andBGP. Colon carcinoma cell line HT-29 was used as a positive control. (B) MCF10F or MCF7 cells were treated with 500 U/ml of IFNγ or 50ng/ml of TNF-α for 6-24 hours, total RNA was isolated and probed for BGP and GAPDH. (C) Schema for PCR amplification of BGPa-disoforms and the expected size of the PCR products. (D) Total RNA was isolated from MCF10F and MCF7 cells grown either on plastic (− Matrigel) or on Matrigel (+ Matrigel) for 6 days and analyzed by RT-PCR. Colon carcinoma cell line HT-29 was used as a positive control,and water (none) as a negative control.

Fig. 6. Regulation of BGP proteinexpression in MCF10F and MCF7 cellsas analyzed by western blots. MCF10Fand MCF7 cells grown on thin Matrigel(A) or on plastic (B-D) were treated with500 U/ml of IFNγ or 50 ng/ml of TNF-αfor 1-4 days, as indicated. Membranepreparations (30-50 µg total protein)from these cells were separated by 8%reducing-SDS gels and immunoblottedwith mAb T84.1. In A, BGP-transfectedMC38 cells were used as a positivecontrol. Samples can be compared onlywithin each group (A-D), representingsamples transferred and developed fromseparate gels.

BGP Isoforms

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formation compared to the untransfected, vector transfected,and BGP sense transfected lines (Table 1). In the case of theuntransfected control, 52% of the colonies exhibited aciniformation compared to 62% for the vector transfected line(P>0.05). In the case of the antisense transfected lines AS6 andAS9, 20% and 12% positive acini formation were scored,respectively (P<0.001). As a further control, the sensetransfected line S3 scored 57% positive for acini (P>0.05).Thus, the inhibition of acini formation correlated with thelower expression levels of BGP in the antisense transfectedlines.

Immunostaining for BGP showed high levels of BGPexpressed in a polarized manner for the cells transfected withvector only (Fig. 9A), while the two lines obtained fromtransfection with the antisense BGP gene showed reducedstaining for BGP and reduced acini formation (Fig. 9B,C).These results further confirm that transfection of MCF10F cellswith the BGP antisense gene has lowered the expression levelsof BGP versus controls (untransfected and vector transfectedcells) and has otherwise not affected the ability of the cells togrow in Matrigel. A small amount of positive staining for BGPwas observed for both the AS6 and AS9 lines, accounting forthe reduced presence but not absence of acini. With this vectorsystem, we were unable to obtain cell lines completely lackingBGP expression; therefore, we did not expect 100% inhibitionof acini formation.

Treatment of MCF10F-e cells grown in thick Matrigel withincreasing concentrations of either anti-BGP antibody T84.1 orwith a single concentration of recombinant soluble BGP alsoresulted in a significant reduction of acini formation (Table 2).In this series of experiments, each treatment group included aseparate untreated control group. In the case of the anti-BGPantibody (T84.1)-treated cells, an antibody control (T84.66)was also included. While the control antibody T84.66 (an anti-CEA antibody) had no effect at three dose levels (10, 50 and100 µg/ml), the T84.1 anti-BGP antibody was able to reduce

J. Huang and others

Fig. 7. FACS analysis and morphology of BGP sorted cells.(A) FACS analysis of the BGP low population on plasticusing mAb T84.1, 2 weeks after cell sorting. The cells weretreated with T84.1 (1:1000) and FITC-conjugated anti-mouse IgG (1:500). The bar shows the cut-off for the cellsanalyzed with no primary antibody with the percentage ofpositive cells shown to the right of the bar. (B,C) Phase-contrast micrograph of the BGP low population grown onthin Matrigel. (D) FACS analysis of the BGP highpopulation on plastic using mAb T84.1, 2 weeks after cellsorting. The bar shows the cut-off for the cells analyzed withno primary antibody with the percentage of positive cellsshown to the right of the bar. (E) Phase-contrast micrographof the BGP high population grown on thin Matrigel.(F) Immunostaining of the BGP high cells grown in thickMatrigel with mAb 4D1C2.

Fig. 8. FACS analysis of MCF10F (A-C) and MCF7 (D-F) cellstransfected with BGP genes. The cells were treated with T84.1(1:1000) and FITC-conjugated anti-mouse IgG (1:500). The barshows the cut-off for the cells analyzed with no primary antibodywith the percentage of positive cells shown to the right of the bar.MCF10F: (A) vector only, (B) BGP antisense gene, line AS9,(C) BGP sense gene. MCF7: (D) untransfected control, (E) vectoronly, (F) BGP sense gene, line S6.

4201BGP expression in MCF10F cells

acini formation from 43% (control) to 18% (P<0.001) at thehighest concentration (100 µg/ml). The percentage reductionwas also singificant at the two lower dose levels with the lowestdose level bordering on significance (P<0.05). Thus, antibodyinhibited acini formation in a BGP-specific and dose-dependent manner.

It is important to note here that antibody T84.1 recognizesan epitope on the N-terminal domain of BGP, and that thisdomain has been previously shown to mediate BGP’s cell-celladhesion effects (Cheung et al., 1993; Obrink, 1997). Withthis in mind, we decided to test if either a synthetic peptideor a recombinant soluble form of BGP could inhibit aciniformation of MCF10F cells grown in Matrigel. When thesynthetic peptide N1B was tested at two levels (1 µM and 10µM) a slight, but statistically borderline, reduction in aciniformation was observed (Table 2). When recombinant solubleBGP was tested at 0.4 µM, a morepotent effect was observed (18% positiveacini formation versus 43% for thecontrol, P<0.001). Although insufficientquantities of recombinant soluble BGPwere available for a dose-response study,we can conclude that interruption ofBGP interactions at the cell surface byeither anti-BGP antibody or a solubleform of BGP resulted in reduced aciniformation.

Transfection of MCF7 cells with theBGP geneSince these studies provided strongevidence that expression of BGP isessential to lumen formation in the BGP-positive MCF10F model system, andbreast tumor lines lack BGP expression,we attempted to restore a more normalphenotype to MCF7 cells by transfectionwith the BGP gene. MCF7 cells expressthe oncofetal protein CEA, but not BGP(Figs 5, 6) and thus react with mAb T84.1(70% positive, Fig. 8D). A lineestablished from MCF7 cells transfectedwith vector only showed reducedpositivity for CEA (50% positive, Fig.8E). While the reduction in CEApositivity borders on being significant, themechanism of reduction is unknown, andmay be due to the G418 selectionconditions. A line (S1) established fromMCF7 cells transfected with the senseBGP gene showed 95% positivity forT84.1 (Fig. 8F) suggesting that BGP wasexpressed in addition to CEA. BGPexpression was confirmed by westernblot (data not shown). When twolines (S1 and S6) were grown inMatrigel and the colonies analyzed byimmunohistochemistry using the BGP-specific mAb 4D1C2, they showedsignificant BGP expression and cell death,and occasional acini formation, compared

to the untransfected and vector transfected lines whichshowed no BGP expression, no cell death and no aciniformation (Table 3). The untransfected cells (not shown) orvector transfected controls formed large spherical colonieswith no central lumen (Fig. 9D). In contrast, the BGP-transfected line exhibited significant cell death (approx. 60%of the colonies, Fig. 9E) with an occasional surviving colonyexhibiting lumen formation (Fig. 9F). The central dead cellsshowed intense staining for BGP (Fig. 9F) compared to anormal mouse IgG control (Fig. 9G). Cells within thesurviving colonies exhibited a polarized expression of BGParound the central lumen with dead cells within the lumenstaining intensely for BGP. These results demonstrate thatwhile BGP expression in MCF7/BGP cells grown on plasticis not growth inhibitory, it is growth inhibitory for MCF/BGPcells grown in Matrigel.

Fig. 9. Immunohistochemical analysis of MCF10F (A-C) and MCF7 (D-G) BGP transfectedcells grown in thick Matrigel. The colonies were stained with 4D1C2. MCF10F: (A) vectoronly, (B) antisense line AS6, (C) antisense line AS9. MCF7: (D) vector only, (E) coloniesundergoing cell death, (F) example of a surviving colony, (G) example of surviving colonystained with normal mouse serum (control).

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DISCUSSION

MCF10F cell phenotypesIn order to test our hypothesis that the cell-cell adhesionmolecule BGP played a role in lumen formation, we selectedMCF10F cells grown in or on Matrigel as a model system. Incontrast to the first report describing the isolation andcharacterization of MCF10A and MCF10F cell lines (Soule etal., 1990), we found that at least one of these lines (MCF10F),contained myoepithelial cells (Fig. 1). It is not clear from ourstudies whether these cells were always present ascontaminating cells, or whether they arose spontaneouslyduring routine cell culture. Due to their differential attachmentto plastic, it was possible to greatly enrich cultures in cellsbelonging to either the epithelial (MCF10F-e) or myoepithelial(MCF10F-m) phenotype. When cultured on Matrigel, theMCF10F-e cells produced acini structures while the MCF10F-m cells produced thin web-like structures. In order to maintainand expand the MCF10F-m population, it was necessary togrow these cells in serum-containing medium. In spite of theirexposure to serum, there was no effect on their morphologywhen grown on Matrigel. We are not aware of any otherpublications describing the presence of myoepithelial cells inMCF10 cell lines; however, most groups use the MCF10A linewhich was originally described as ‘tightly attached’. Since onlythe MCF10F-e cells expressed BGP and formed acini whengrown in or on Matrigel, we used these selected cells to furtherdetermine the role of BGP in lumen formation.

The potential role of apoptosis in lumen formationOnce we established that MCF10F-e cells formed exclusively

acini structures when grown in or on Matrigel and that BGPexpression was exclusively lumenal (Fig. 2), we proceeded toexamine the process in detail by SEM studies (Fig. 3). Whenseeded on Matrigel, the cells were uniformly dispersed assingle cells, but after 1-2 days migrated to form colonies of 4-8 cells, leaving large spaces between them. By 4 days, the cellsexhibited a flattened morphology, and the colonies formed acentral lumen with bright, condensed bodies observed withinthe lumen. We interpreted these results as follows: (1) the cellsmigrated and adhered to each other and to the Matrigel to forma stable colony, (2) the colonies increased in size by celldivision reaching a critical mass that (3) triggered lumenformation by cell death (bright, condensed bodies) of thecentral cells. Oblique views by scanning EM showed that thecolonies stuck up above the Matrigel surface, but were coveredwith fibrils maintaining contact with the ECM. The polarizedphenotype of the MCF10F-e cells was also confirmed bytransmission EM. The polarized surface of these cells hadfinger-like projections (small microvilli) with the production oflarge amounts of 20-50 nm vesicles. The cells formedintercellular tight junctions, including desmosomes, a furthercharacteristic of differentiated mammary epithelial cells(Barcellos-Hoff et al., 1989). A close examination of thecondensed bodies within the lumen revealed all thecharacteristics of apoptosis: the chromatin was condensed atthe edges of the nuclear membrane, which was highlyinvaginated, and the cytoplasm was condensed but stillcontained intact organelles and a plasma membrane. These hallmarks of apoptosis have been described for other cell systems(Tomei and Cope, 1991; Wyllie et al., 1980), and represent adefinitive proof of apoptotic bodies by TEM. In addition, andas described later, the cells within the lumen have not beensubjected to stress (lack of nutrients or oxygen) and thusappeared to have been programmed to die, rather thanundergoing necrosis. Finally, we note that while necrotic cellsare easily recognized by TEM, exhibiting dissolved nuclearand plasma membranes, and disintegrating cytoplasmicorganelles (Wyllie et al., 1980), none were observed in ourelectron micrographs. These results support the conclusion thatthe MCF10F-e cells have a differentiated phenotype typical ofpolarized mammary epithelial cells and that the central lumenis formed by apoptosis of the cells in the center of the colony.

Since BGP expression was limited to the lumenal surface in

J. Huang and others

Table 2. Acinus formation of MCF10F cells treated with antibodies, peptide or soluble BGPTreatment Concentration Acini/colonies % acini P

Control-a − 109/208 52.4 −Control-b − 61/124 49.2 −Control-c − 55/127 43.3 −mAb T84.66, a 10 µg/ml 74/136 54.4 >0.05mAb T84.66, b 50 µg/ml 102/203 50.2 >0.05mAb T84.66, c 100 µg/ml 29/85 34.1 >0.05mAb T84.1, a 10 µg/ml 80/194 41.2 <0.05mAb T84.1, b 50 µg/ml 65/209 31.1 <0.001mAb T84.1, c 100 µg/ml 18/100 18.0 <0.001Peptide N1B, a 1 µM 88/215 40.9 <0.05Peptide N1B, a 10 µM 88/204 43.1 >0.05Sol-BGP, c 0.4 µM 30/161 18.6 <0.001

Cells were analyzed as described in Table 1 in the presence of increasing concentrations of antibody (T84.66 or T84.1), peptide N1B, or a single concentrationof recombinant soluble BGP. Three sets of experiments were performed, each with its own control group, designated a, b and c, and indicated with theappropriate letter.

P values were calculated between the treatment group and appropriate control group.

Table 3. Colony and acini formation for MCF7 cellstransfected with the BGP gene

Cells Live/total % live Acini/total % acini

Untransfected 100/100 100 0/100 0Vector 100/100 100 0/100 0Sense S1 35/90 39 1/90 1Sense S6 30/98 31 2/98 2

Cells were analyzed as described in Table 1, and reported as either thenumber of live colonies/total colonies or the number of acini/total colonies.

Two lines containing the BGP sense gene were analyzed.

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the mature acini and the lumen formed by death of the cellswithin the center of the colony, it was tempting to speculatethat BGP was involved in the delivery of the death signal. Sincewe know that BGP expression by itself is not fatal, its actionon adjacent cells in the as-yet unformed lumen must require anadditional signal(s). The additional signal may depend on theloss of contact of the central cells to extracellular matrix(ECM), leading to a phenomenon refered to as anoikis (Frischand Ruoslahti, 1997). Anoikis is a popular theory used todistinguish tumorigenic from non-tumorigenic epithelial cells.It is postulated that normal epithelial cells die when they losecontact with ECM while tumor cells (due to some geneexpression change) continue to live. In our model system,MCF10F-e cells require both loss of contact with ECM andexpression of BGP to apoptose (form a central lumen), becausethe cells transfected with the BGP antisense gene form colonieswith no central lumen. Thus, the cells in the center of thesecolonies do not die even though they have lost contact with theECM. This fact demonstrates that central cells do not die dueto lack of nutrients or oxygen, but rather due to an inherentdifferentiation program. In support of this mechanism, theMCF7/BGP cells exhibit pronounced cell death during colonyformation in Matrigel, but grow well on plastic. In this case,the apoptotic signal is stronger for MCF7/BGP cells than forMCF10F-e cells, perhaps due to lower expression of cellsurface integrins (which interact with ECM) on MCF7 cells(Noel et al., 1993). Based on our studies, we propose thatanoikis may be a more complicated phenomenon than just theloss of cell contact with ECM.

The lack of expression of BGP in all breast tumor cell linesthat we have examined suggested that breast tumor cells mayescape apoptosis by downregulation of the BGP gene, asituation already found for colon cancer (Neumaier et al.,1993). However, we have found that BGP was onlydownregulated in 30% of breast cancers (Huang et al., 1998).This finding can be reconciled by postulating that the apoptoticsignal transduction mechanism was obviated in the other 70%of breast tumors. Nonetheless, it is intriguing that BGP generegulation is fundamentally different between colon and breastcancers.

Role of BGP in cell-cell adhesionMembers of the CEA gene family, including BGP, have beenpostulated to mediate homotypic cell adhesion (Benchimol etal., 1989). In particular rat BGP was originally cloned as anecto-ATPase by Lin and coworkers (Lin and Guidotti, 1989;Lin et al., 1991) and identified as the cell adhesion moleculeCell-Cell Adhesion Molecule-1 (CCAM1 or CCAM105) byObrink and coworkers (Aurivillius et al., 1990; Edlund et al.,1993; Hansson et al., 1989; Ocklind and Obrink, 1982; Odinet al., 1988). Transfection of the BGP gene into a variety ofnon-adhesive cell lines results in transient cell-cell adhesion,supporting this view (Oikawa et al., 1992; Rojas et al., 1990;Turbide et al., 1991). However, immunohistochemical stainingof BGP on colon (Frangsmyr et al., 1995), prostate (Hsieh andLin, 1994), breast (Huang et al., 1998; Riethdorf et al., 1997)and bile canalicular ducts (Svenberg et al., 1979) reveals adistinct polarized orientation of BGP, while Hansson et al.(1989) have observed BGP expression at both the lumenal andbasolateral surface of intestinal epithelial cells. To rationalizethe apparent discrepancy between the postulated role of BGP

in cell-cell adhesion versus its observed lumenal location, wepropose here that BGP-BGP intercellular interactions may bea transient phenomenon, not leading to permanent cell-cellcontacts, but rather guiding the cells towards formation of acinior tubules with a central lumen. Thus, when cells sense BGP-BGP intercellular contacts, they know that they have not yetachieved the formation of a polarized surface or lumen andcontinue the polarization program, but once the cells no longersense BGP-BGP intercellular contacts and BGP has migratedto an exclusively lumenal orientation they know that they haveachieved their polarized state and cease the polarizationprogram. In this scheme, BGP would be a dynamic cell-celladhesion molecule, in which its function as a cell adhesionmolecule is temporal and changes during the differentiationprogram.

A mechanism for how BGP may mediate transient celladhesion has been proposed by Obrink (1997). Since BGPmolecules may form inter- or intra-cellular dimers, they mayswitch between the two states to cause first adhesion (transdimers) and then disaggregation (cis dimers). If the BGPcytoplasmic domain confers the signal to switch between thetwo states, then its deletion would not be expected to preventcell adhesion. However, its deletion may affect the ability ofthe cell to continue the polarization program. Interactions ofthe BGP cytoplasmic domain with calmodulin (Edlund et al.,1996; Edlund and Obrink, 1993; Edlund et al., 1998) and thecytoskeleton (see Fig. 2D) could be key determinants of thisprogram. The expression of different isoforms (long and shortcytoplasmic forms) of BGP would give the cells the potentialto modulate the polarization program, with only the long formcapable of delivering a negative growth signal via its ITIM(Beauchemin et al., 1997; Huber et al., 1999). We predict thatthe ITIM of BGP must be fully phosphorylated (inhibitory)only in the completely polarized cells where further celldivision would be unnecessary or even disrupt the acinistructure.

Effect of cytokines on BGP expressionAs we have previously described for colon carcinoma celllines (Chen et al., 1996; Takahashi et al., 1993), BGP isstrongly induced by inflammatory cytokines such as IFN-γand TNF-α. In this study we found that both cytokines werecapable of inducing BGP in MCF10F-e, but not in BGPnegative MCF10F-m cells. However, CEA was induced byIFN-γ in the MCF10F-m cells. This intriguing finding mayprovide a clue to the high incidence of CEA production inbreast cancers, especially in in situ ductal carcinomas. Whilevery few breast carcinomas are believed to arise frommyoepithelial cells, we note here that we found bothepithelial and myoepithelial cells in the MCF10F cell line,suggesting that the two types of cells coexist and may undersome circumstances interconvert. It is also intriguing that theMCF7 breast carcinoma cell line produces CEA, but not BGP,even when treated with IFN-γ and TNF-α. Thus, thedownregulation of BGP in breast cancer epithelial cells is apermanent event and cannot be reversed by cytokines, anotherwise strong inducer of BGP. In this respect theregulatory phenotype of MCF7 cells resembles amyoepithelial rather than an epithelial one. Therefore, furtherstudies aimed at deciphering the regulatory changes in theBGP gene during tumorigenesis are warranted.

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ConclusionWe conclude that the MCF10F cell line is a valuable source ofboth mammary epithelial and myoepithelial cells, and in thecase of the MCF10F-e cells, is an excellent model for the studyof acini formation when grown in or on Matrigel. In addition,and under appropriate conditions, the two types of cellscan collaborate to form gland-like structures resemblingmyoepithelial-surrounded acini and tubules in the normalmammary gland. BGP is an excellent marker for cellpolarization in this system and is specific for epithelial cellexpression. BGP appears to follow a programmedreorganization of the cell surface proteins during lumenformation. We hypothesize here that the transient cell-celladhesion properties of BGP may assist in this process bysensing the position of the cell surface relative to other cellsduring the reorganization process. Finally, as demonstrated byinterruption of BGP expression by a BGP antisense gene, anti-BGP antibody, or recombinant soluble BGP, we showed thatBGP plays an essential role in lumen formation, most likely bypromoting apoptosis of the cells in the center of the colony thatlack contact with ECM.

Facilities for electron microscopy were supported by NIH grant S10RR01462 and NSF grant BNS 8415920. J.H. was partially supportedby a graduate fellowship from the American Cancer Society. Theauthors gratefully acknowledge the expert advice on electronmicroscopy by Dr Marcia Miller (Biology Dept, City of Hope).

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