Vol. 1, 407-420, September 1990 Cell Growth & Differentiation 407

Transforming Growth Factor-a Expression Is Enhanced inHuman Mammary Epithelial Cells Transformed by anActivated c-Ha-ras Protooncogene but not by thec-neu Protooncogene, and Overexpression of theTransforming Growth Factor-a ComplementaryDNA Leads to Transformation

Fortunato Ciardiello, Mary Lou McGeady, Nancy Kim,Fulvio Basolo, Nancy Hynes, Beatrice C. Langton,Hiroshi Yokozaki, Toshiaki Saeki, James W. [lliott,Hideo Masui, John Mendelsohn, Herbert Soule,Jose Russo, and David S. Salomon’

Laboratory of Tumor Immunology and Biology, National CancerInstitute, NIH, Bethesda, Maryland 20892 [F. C., M. L. M., N. K., H. Y.,1. 5., D. S. S.]; Department of Pathology, Michigan Cancer Foundation,Detroit, Michigan 48201 [F. B., I. W. E., H. S., I. R.J; Friedrich MiescherInstitut, P.O. Box 2543, Ch-4002 Basel, Switzerland [N. H.]; Departmentof Immunology, Triton Biosciences, Inc., Alameda, California 94501

[B. C. L.]; and Medical Receptor Biology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [H. M., J. M.]

AbstractMCF-1OA cells are a spontaneously immortalized nor-mal human mammary epithelial cell line. MCF-1OAcells were transfected with two expression vector plas-mids containing either a human point-mutated c-Ha-rasprotooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCE-1OA cells grow as colonies in softagar, exhibit a 3- to 4-fold increase in their growth ratein serum-free medium, and show a reduced mitogenicresponse to exogenous epidermal growth factor ([GE)or transforming growth factor-a (TGFa) as compared toMCF-1OA cells. c-Ha-ras-transfected MCF-1OA cells ex-press a 4- to 8-fold increase in TGFa mRNA levels andsecrete 4- to 6-fold more TGFa protein as compared toMCF-1OA cells. Addition of either an anti-TGFa neu-tralizing monoclonal antibody or an anti-[GE receptorblocking monoclonal antibody to the Ha-ras-trans-formed MCE-1OA cells produces a 50 to 80% inhibitionof colony formation of these cells in soft agar. c-neu-transfected MCF-1OA cells grow in soft agar and ex-hibit an increase in their growth rate in serum-freemedium at a level comparable to that observed in Ha-ras-transformed MCE-1OA cells. Addition of an anti-c-erb8-2 monoclonal antibody inhibits the anchorage-in-dependent growth of these cells in soft agar. However,c-neu-transformed MCF-1OA cells show no increase inTGFa secretion and no change in their responsivenessto exogenous [GE or TGFa. A recombinant retroviralvector containing the human TGFa gene was also intro-duced into MCF-1OA cells. TGFa-infeded MCF-1OAcells secrete 1 5- to 20-fold more TGFa protein than

Received 4/12/90.

1 To whom requests for reprints should be addressed, at LTIB, National

Cancer Institute, NIH, Bldg. 10, Room 5B39, Bethesda, MD 20892.

MCE-1OA cells, form colonies in soft agar, exhibit anenhanced growth rate in serum-free medium, and showa decreased mitogenic response to exogenous [GF orTGFa at a level equivalent to Ha-ras-transformed MCF-1OA cells. Growth of TGFa-infected MCF-1OA cells insoft agar is completely inhibited by anti-TGFa neutral-izing or anti-[GF receptor blocking monoclonal anti-bodies. These results suggest that TGFa is an intermedi-ary in the transformation of human mammary epithe-hal cells by an activated c-Ha-ras gene, but not by thec-neu gene, and demonstrate that overexpression ofthis growth factor is able to transform immortalizedhuman mammary epithelial cells which also express asufficient complement of functional [GF receptors.

Introduction

Cenetic alterations or changes in the expression of spe-cific cellular protooncogenes or tumor suppressor geneshave been detected in different human malignancies (1).Several protooncogenes, such as c-Ha-ras, c-erbB, c-erbB-2, int-2, c-myc, and the recessive tumor suppressorRB-i gene, have been implicated in the pathogenesis ofhuman breast cancer (2). Changes in these genes may beimportant for the control of cellular proliferation, theinitiation of transformation, and the progression to a

metastatic phenotype. In this respect, the introductionof single or multiple activated protooncogenes, such asc-Ha-ras, c-myc, or c-neu (the rat analogue of the humanc-erbB-2 gene), into primary or immortalized mousemammary epithelial cells can lead to in vitro transforma-tion and to in vivo tumonigenicity (3-7). Overexpressionof c-Ha-ras-specific mRNA and elevated levels of p2i�protein occur in 60 to 70% of human primary breasttumors and may be correlated with other prognosticmarkers, such as axillary lymph node involvement and amore advanced tumor stage (8-10). In addition, amplifi-cation of the c-erbB-2 protooncogene and/or overexpres-sion of p185�’8�2 protein have been detected in i5 to30% of primary human breast tumors and have beenshown to be important prognostic factors for a subset ofhuman breast cancers (11-13).

A structural and functional relationship exists betweenprotooncogenes and growth factors since certain pro-tooncogenes code for proteins that are growth factors,growth factor receptors, or effectors in the intracellularsignal transduction pathway for various growth factors(14). In addition, activation of protooncogenes may alsoindirectly lead to an increase in the production of endog-enous growth factors and/or growth factor receptors orto a change in the cellular response to growth factors or

408 Transformation of Human Mammary Epithelial Cells and TGF

growth inhibitors (i4, 15). In fact, rodent and human

breast cancer cells secrete several growth factors thatcould function in an autocnine and/or paracnine fashionto control cell growth (16-18).

Transforming growth factor a is a growth factor pro-

duced by several different primary tumors and tumor celllines that has been circumstantially implicated in regulat-ing the growth of cancer cells via an autocnine mechanism(19). TCFa2 is initially synthesized as a transmembraneglycosylated precursor that is cleaved to a 50-amino acidpeptide, exhibits partial amino acid sequence homology

to epidermal growth factor, and binds to the ECF recep-ton (20). Both the cell-associated precursor and the se-creted TCFa peptide are biologically active (2i, 22).TGFct is able to cooperate with other growth factors,such as transforming growth factor-a, platelet-derivedgrowth factor, and basic fibroblast growth factor, to re-versibly induce a transformed phenotype in nontrans-formed cells (23, 24). TCFa may be important in regulat-ing the growth of mammary epithelial cells since [CF isa potent mitogen for these cells and since [CF on TGFacan stimulate the lobuloalveolar development of themouse mammary gland in vitro and in vivo (25). TCFamRNA and protein have been detected in rodent andhuman mammary tumors and in several human breastcancer cell lines (26-32). More recently, expression ofthis growth factor has been found in normal rodent andhuman mammary epithelial cells in vitro and in vivo (33-37). TCFa is also an estrogen-inducible protein in rat andhuman breast cancer cells, suggesting that part of thegrowth-promoting effects of estrogen may be mediatedthrough this growth factor (28-30).

MCF-1OA cells are a spontaneously immortalized hu-man mammary epithelial cell line that was originallyestablished from an outgrowth of an s.c. mastectomy ofa woman with fibrocystic disease by maintenance in lowcalcium medium (38). MCF-1OA cells are near diploidand express epithelial-specific keratins and the mam-mary-specific milk fat globule antigen (38, 39). MCF-1OAcells are not transformed since they do not grow in softagar and are not tumonigenic in immunosuppressed mice.MCF-1OA cells offer distinct advantages over other pre-viously characterized normal human mammary epithelialcell lines since they are spontaneously immortalized andthey can be efficiently transfected by the calcium phos-phate precipitation method. MCF-1OA cells can be trans-formed after transfection with an expression vector plas-mid containing the human point-mutated c-Ha-ras pro-tooncogene under the transcriptional control of theMoloney sarcoma virus LTR and an internal 5V40 pro-moter (40).�

To ascertain whether TCFa may function as an inter-mediary in ras transformation of human mammary epi-thelial cells, Ha-ras-transformed MCF-1 OA cells were ana-lyzed for their mitogenic response to exogenous ECF or

2 The abbreviations used are: TGFa, transforming growth factor-a;

EGF, epidermal growth factor; MSV, Moloney sarcoma virus; LTR, longterminal repeat; MT-i, metallothionein-1, Cl, clone; kb, kilobase(s); CM,conditioned medium; RIA, radioimmunoassay; RRA, radioreceptor assay;

cDNA, complementary DNA; poly(A)�, polyadenylated; bp, base pair(s).3 F. Basolo, J. Elliott, L. Tait, X. Chen, T. M. Maloney, I. H. Russo, R.

Pauley, S. Momiki, J. Caamano, A. Klein-Szanto, M. Koszalka, and J.

Russo. Transformation of a human breast epithelial cell line by C-Ha-rasoncogene, submitted for publication.

TCFa, for the level of expression and secretion of en-dogenous TCFa, and for their growth in the presence ofanti-TCFa neutralizing and anti-[CF receptor blockingmonoclonal antibodies. In addition, to determinewhether overexpression of the c-neu protooncogene cantransform human mammary epithelial cells and, if so,whether TCFa is a growth factor involved in the trans-formation process induced by this oncogene, MCF-1OAcells were transfected with an expression vector plasmidcontaining the rat c-neu protooncogene under the tran-scniptional control of the MSV LTR. Finally, MCF-1OAcells were infected with an amphotropic retroviral vectorcontaining the human TCFa gene under the control ofthe mouse metallothionein-i promoter to ascertainwhether elevated and constitutive expression of thisgrowth factor is capable of transforming human mam-mary epithelial cells through an autocnine pathway.

Results

Transformation of MCF-1OA Cells by ras Leads to [n-hanced [xpression of TGFa. It has previously been dem-onstrated that MCF-1OA cells transformed by an activatedc-Ha-ras protooncogene, MCF-1OA Ha-ras clone 1 cells,can form colonies in soft agar and exhibit a 50% reduc-tion in their doubling time in serum-containing medium

as compared to the parental MCF-1OA cells (40). Ha-ras-transformed MCF-iOA cells show a significant reductionin the expression of cytokeratins and milk fat globuleantigens. In addition, these in vitro transformed cells formundifferentiated invasive carcinomas when injected intoirradiated nude mice after 3 to 5 weeks (4#{216})#{149}3From thesecells we have subsequently subcloned an additional cellline, designated MCF-iOA Ha-ras clone 2 cells. BothMCF-1OA Ha-ras Cl 1 and Cl 2 cells expressed approxi-mately a 5- to 10-fold increase in the levels of a 1 .4-kbc-Ha-ras mRNA transcript as compared to MCF-1OA cellsor to MCF-1OA cells transfected with the same plasmidcontaining only the neomycin resistance gene, MCF-1OAneo cells (Fig. 1A). In addition, there was a correspondingincrease in the levels of p2V� protein detected in theseclones by Western blotting analysis (Fig. 10).

The increase in p2lras protein expression in the twoMCF-1OA Ha-ras clones is functionally relevant sincesome of their biological properties are significantly al-tered as compared to the parental MCF-1OA cells. Twobiological characteristics of transformed cells in vitro aretheir ability to exhibit anchorage-independent growth insemisolid medium and to actively grow under serum-restricted conditions. MCF-1OA Ha-ras Cl 1 and Cl 2 grewas colonies in soft agar at a cloning efficiency of approx-imately 4 to 8% (Table 1). In contrast, MCF-iOA or MCF-1OA neo cells were unable to form colonies under com-parable anchorage-independent growth conditions. Inaddition, MCF-1OA neo cells formed duct-like structuresthat superficially resemble the normal architecture of themammary gland in vivo when they are grown in type Icollagen gels (Fig. 2A). However, MCF-1OA Ha-ras cellsformed disorganized outgrowths under the same growthconditions (Fig. 20). The two Ha-ras-transformed MCF-1OA clones also exhibited a 3-fold increase in their an-chorage-dependent growth rate in serum-free mediumthat lacks [CF as compared to MCF-1OA or MCF-1OAneo cells (Fig. 3, A and B). In this respect, MCF-iOA andMCF-1OA neo cells have an obligatory requirement forexogenous [CF to grow in either serum-containing or

Table 1 Biological properties of MCF-iOA Ha-ras, MCF-JOA, c-neu, and

MCF-1OA TGFa cells

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410 Transformation of Human Mammary Epithelial Cells and TGFa

1 OR cells into their CM during a 48-h period (Table 1).These levels were 4- to 6-fold higher than the TGFa basallevels detected in the CM from MCF-1OA or MCF-1OAfl�() cells. In addition, the amount of TCFa that is able tocompete with ‘2�l-ECF for binding to the EGF receptorin an EGF/TGFs RRA was equivalent to the amount ofactivity that could be measured in the TGFs RIA, sug-gesting that the majority of immunoreactive TCFa pro-duced l)y these cells is also biologically active. The in-crease in TGFe prot(’in secreted in the CM from theMCF-1OA Ha-ras clones is paralleled by an increase inthe level of TCFa mRNA expression in these cells. In thisrespect, a 4- to 8-fold increase in the levels of expressionof a 4.8-kb niRNA transcript could be detected by North-em blot analysis in the two MCF-1OA Ha-ras clones ascompared to MCF-1OA or MCF-1OA neo cells (Fig. 1B).This increase is specific for TGFa mRNA since no changesin the relative levels of �3-actin mRNA expression wereobserved in these c:ells (Fig. 1C).

We have previously demonstrated that the decreasedgrowth response to exogenous EGF or TGFe observed ina fl1OUSC mammary epithelial cell line, NOC-8, followingtransformation with a point-niutated c-Ha-ras protoon-cogene � also be due to a loss of or a reduction in thenumber of high affinity EGF receptors on these cells (41).To determine whether a similar phenomenon may occurin the MCF-1OA cells after ras transformation, we meas-ured the specific binding of different concentrations of21-EGF to these cells. Scatchard analysis of the EGF

binding isotherms shows that there was a single high

affinity class of EGF receptor sites with a comparabledissociation constant of 0.8 nrvi on all four of the MCF-1OA cell lines. MCF-1OA and MCF-1OA neo cells pos-sessed approximately 2.4 x lOs EGF binding sites/cell,whereas the two MCF-1OA Ha-ras clones had only a 15to 2O% reduction in the number of the EGF binding sitesas compared to the parental cells.

It is conceivable that TCFa in the MCF-1OA Ha-rascells may be functioning as an external autocrine growthfactor that interacts with the EGF receptors on the cellsurface and may therefore mediate some of the biologicaleffects of MS. To address this issue, the growth of MCF-1OA Ha-ras Cl 1 and Cl 2 cells in soft agar was assessedin the presence of different concentrations of a mousemonoclonal anti-TCFa neutralizing antibody, TAb 1 , thathas been generated against the 50-amino acid native,reconibinant human TCFs peptide, or of TAb 2, a mousemonoclonal anti-TCFs nonneutralizing antibody (42, 43).In addition, the ability of these cells to grow in soft agarwas evaluated in the presence of a mouse monoclonalanti-human ECF receptor blocking antibody, 528, thatinhibits the binding of 2�l-ECF to EGF receptors on A431human epidermoid carcinoma cells and on MDA-MB-468 human breast carcinoma cells, two high expressingECF receptor cancer cell lines, and that blocks the invitro and in vivo growth of these cells (44-46). As illus-trated in Fig. 4A (MCF-1OA Ha-ras CI 1) and 4B (MCF-1OA Ha-ras Cl 2), the TAb 1 anti-TCFs neutralizing anti-

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Transformation of MCF-1OA Cells by neu Fails toInduce an Increase in TGFa Expression. Amplificationand/or overexpression of the c-erbB-2 protooncogenehas been detected in 15 to 30% of primary human breastcancers and has been associated with a more aggressivedisease in a subset of these tumors (11-13). Therefore,to determine whether overexpression of this protoon-cogene could transform immortalized human mammaryepithelial cells and to ascertain whether TGFt may be agrowth factor that is involved in the transformation proc-

1� ess induced by this gene, we have transfected into MCF-r 1OA cells an expression vector plasmid containing the c-

neu cDNA under the transcriptional control of the MSVLTR and the neo gene (47). Following mass transfection

______________________________ _I_ of MCF-iOA cells and selection in C418-containing me-_____________________________ - dium for 3 weeks, 13 C418-resistant individual colonies

were cloned and expanded into cell lines. To ascertainwhether the transfected c-rieu cDNA was being tran-scribed in the G418-resistant clones, p�ly(��e RNA iso-lated from eight different clones was analyzed by North-em blotting after hybridization with the 32P-Iabeled nick-translated c-neu cDNA insert. As illustrated in Fig. 5A, all

of the MCF-1OA c-neu clones, but not the MCF-1OA neocells, expressed two c-neu-specific mRNA species of

approximately 9.5 kb and 8.5 kb that are consistent withthe expected full length genomic fusion c-neu cDNA-neo transcripts (47). In addition, a third 4.8-kb c-neu-specific mRNA species that is derived from the portionof the plasmid containing the c-neu cDNA could be

detected in the MCF-1OA c-neu clones. Equivalentamounts of RNA were loaded on the gel since the levelof expression of a 2.4-kb fl-actin mRNA was similaramong the MCF-1OA neo cells and the MCF-1OA c-neuclones (Fig. SB). In addition, Western blot analysisshowed a 5- to 10-fold increase in the levels of expression

of pi85�B�2 protein in several MCF-1OA c-neu clonesthat express high levels of the c-flea mRNA, as comparedto MCF-iOA neo cells (Fig. SC). To ascertain the cellulardistribution of the p185erbB2 protein in the c-neu-trans-fected cells, cytospin preparations of MCF-1OA neo andMCF-1OA c-neu CI 9 cells were assessed for immunoper-oxidase localization with the 21N anti-erbB-2 rabbit pol-yclonal antiserum that was used for the Western blot

analysis (48). There was weak but detectable pi8s�rbB2reactivity in the cytoplasm of the MCF-1OA neo cells,whereas in most of the MCF-1OA c-neu CI 9 cells a veryintense membrane-associated staining was observed

(data not shown).To determine whether overexpression of pi85erbB2

protein in the MCF-1OA c-neu clones is functionallysignificant for the acquisition of a transformed pheno-type, the ability of these cells to grow in anchorage-independent conditions was tested. As illustrated in Ta-ble 1, eight MCF-1OA c-neu clones have acquired theability to form colonies in soft agar. Moreover, five flea-transfected clones (MCF-iOA c-neu Cl 1, 2, 3, 8, and 9)were able to grow at a cloning efficiency comparable tothat observed in ras-transformed MCF-1OA cells. In ad-dition, these cells also formed disorganized outgrowthswhen grown in type I collagen gels (Fig. 2, B and C). Totest whether overexpression of pigserbB2 is responsiblefor the ability of neu-transfected MCF-1OA cells to growin anchorage-independent conditions, MCF-1OA c-neu

1 2 3 4 5

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body produced a dose-dependent inhibition of colonyformation in soft agar of both MCF-1OA Ha-ras clones. Amaximum inhibition of approximately 50% was observedbetween50and 100�ig/mlofTAb 1. Incontrast, 100�g/ml of TAb 2 nonneutralizing anti-TCFa antibody had noeffect upon the anchorage-independent growth of thesecells. Addition of the 528 blocking anti-ECF receptorantibody determined an even stronger dose-dependentinhibition of colony formation with a maximum of 75 to

Cell Growth & Differentiation 411

- I_ ;�: i� � � � �L& L& 9 U � 14: �rV t4:��(’) � � Lj.�O)� �,‘ oo�2�o�c�0

Fig. 3. Anchorage-dependent growth of MCF-1OA cells under serum-free medium conditions. A, 2 X 10� cells/well were seeded in 1 2 multiwellcluster dishes. Twenty-four h later, the cells were switched to PC-iserum-free medium and grown for 4 days in the presence (D) or in theabsence (0) of TGFa, 10 ng/mI, before being counted. Values represent

the average [± SD (bars)] of two different experiments, each performedin quadruplicate. B, 10� cells/well were seeded in 12 multiwell clusterdishes. Twenty-four h later, the cells were switched to PC-i serum-freemedium and counted every day for 5 days. #{149},MCF-1OA neo; �, MCF-1OA Ha-ras CI 1; 0, MCF-1OA TGFa Cl 4; 0, MCF-1OA c-neu Cl 9. Valuesrepresent the average of two different experiments in triplicate. SD was

less than 10%.

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Fig. 4. Effects of anti-humanneutralizing anti-TGFa (TAb 1) andof anti-human blocking anti-EGFreceptor (528) monoclonal anti-

bodies on the anchorage-inde-

pendent growth of MCF-1OA Ha-ras CI 1 (A) and CI 2 (B) cells andof MCF-1OA TGFa Cl 3 (C) and Cl

4 (0) cells. 2 x 10� cells wereplated in soft agar as described in“Materials and Methods” andtreated with different concentra-

tions of TAb 1, 528, or a nonneu-tralizing anti-TGFa antibody, TAb

2. Data are expressed as the per-centage of cell growth as colonies

greater than 50 �im in the presenceof the indicated antibody as com-pared to untreated controls. The

growth for untreated controls was:MCF-1OA Ha-ras CI 1, 892 cob-nies/dish; MCF-1OA Ha-ras Cl 2,

1580 colonies/dish; MCF-1OA

TGFa Cl 3, 1 1 79 colonies/dish;MCF-1OA TGFa Cl 4, 1338 cob-nies/dish. Values are the average(± SD) of quadruplicate determi-

nations.

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412 Transformation of Human Mammary Epithelial Cells and TGFa

Cl 1, 2, 8, and 9 cells were plated in soft agar in thepresence of TAb 250, a mouse monoclonal antibodyraised against the extracellular domain of the humanp185��2 protein (49). TAb 250 has been shown tospecifically inhibit the in vitro and in vivo growth ofseveral human breast cancer cell lines that overexpresspl85��2 (50). There is a specific dose-dependent inhi-bition of soft agar colony formation with a 70 to 90%maximum inhibitionbetween 50 and 100 zg/ml TAb 250in all of the clones tested (Fig. 6). This effect is specificfor the anti-erbB-2 monoclonal antibody since equivalentconcentrations ofanti-TCFct TAb 1 and anti-[CF receptor528 antibodies had no influence on the soft agar growthof these MCF-1OA c-neu clones (data not shown). Fur-thermore, as shown in Fig. 3B, neu-transformed MCF-1OA cells exhibited an increased growth rate in anchor-age-dependent serum-free conditions in absence of [CF

at a level that is comparable to the growth rate observedunder similar conditions in MCF-1OA Ha-ras cells. How-ever, in contrast to ras-transformed MCF-1OA cells andsimilarly to parental MCF-1OA cells, MCF-1OA c-neu cellsexhibited a 4- to S-fold increase in their anchorage-dependent growth response to exogenous ECF or TCFa(Fig. 3A). To assess whether neu-transfected MCF-1OAcells produce TCFa, CM from MCF-1OA c-neu Cl 1, 2, 3,5, 8, 9, 1 1 , and 14 cells was collected, concentrated, andanalyzed for the presence of immunoreactive TCFa. Allof the MCF-1OA c-neu clones secreted into their CMTCFcs protein at a level comparable to that found inMCF-1OA and MCF-1OA neo cells (Table 1).

Since it has previously been shown that in ras-trans-formed MCF-1OA cells there is a reduction in the expres-sion of epithelial keratins and of mammary epithelial cell-specific antigens, such as milk fat globule antigen (40),

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414 Transformation of Human Mammary Epithelial Cells and TGFa

Fig. 6. Effects of anti-human

blocking anti-erbB-2 (TAb 250)monoclonal antibody on the an-chorage-independent growth of

MCF-1OA c-neu Cl 1 (A), Cl 2 (8),CI 8 (C), and CI 9 (0) cells. 2 x i0�cells were plated in soft agar as

described in “Materials and Meth-ods� and treated with differentconcentrations of TAb 250 or TAb

2. Data are expressed as the per-centage of cell growth as coloniesgreater than 50 �m in the presence

of the indicated antibody as com-pared to untreated controls. Thegrowth for untreated controls was:MCF-1OA c-neu CI 1, 1208 cob-nies/dish; MCF-1OA c-neu CI 2,1089 colonies/dish; MCF-1OA c-neu CI 8, 900 colonies/dish; MCF-1OA c-neu CI 9, 951 colonies/dish.Values are the average (± SD) of

quadruplicate determinations.

protein. As shown in Table 1, all of the MCF-1OA TGFaclones secreted between 100 and 300 ng/i08 cells duringa 48-h conditioning period. There was a general corre-lation between the levels of immunoreactive TCFa asdetected by RIA and the levels of bioactive TCFa asdetermined by RRA. In addition, a 30 to 35% reductionin the total number of ECF receptor sites per cell and nochange in the [CF receptor Kd was observed in MCF-1OA TCFa cells as compared to parental MCF-iOA cells(data not shown).

Furthermore, as shown in Table 1, eight MCF-1OATCFa clones that were secreting more than 250 ng/108cells/48 h of TGFa were also able to grow as colonies insoft agar with a cloning efficiency comparable to that

observed in MCF-iOA Ha-ras and MCF-1OA c-neu clonesand formed disorganized outgrowths in type I collagengels. In addition, like ras- and neu-transformed MCF-iOAcells, MCF-1OA TCFct cells exhibited an enhanced growthrate in serum-free medium in the absence of exogenousECF (Fig. 3B). Moreover, MCF-1OA TCFa Cl 3 and Cl 4cells exhibited a 2-fold increase in their growth in serum-free medium in the presence of exogenous [CF or TCFafor 4 days that is comparable to the magnitude of themitogenic response to MCF-1OA Ha-ras cells to thesegrowth factors and significantly less than the 4- to 5-foldincrease found in the parental MCF-iOA cells or in MCF-iOA c-neu cells (Fig. 3A).

To determine whether the elevated levels of TCFa

Fig. 7. Northern blot analysisfor TGF0 mRNA in MCF-1OATGFcs clones. Total cellular RNA

)i0 pg/lane) was fractionated on

a i.2% denaturing agarose gel,transferred to nylon membrane,

and hybridized to the 32P-la-beled TGFa cDNA (A) insert and#{216}-actinprobe (B).

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416 Transformation of Human Mammary Epithelial Cells and TGFa

result in a rapid and specific increase in the productionand secretion of endogenous TCFa that is concomitantwith the acquisition of some transformation-associatedproperties in these cells, such as anchorage-independentgrowth in soft agar and anchorage-dependent growth inlow serum or in serum-free growth factor-depleted me-dium (41). In addition, the magnitude of increase in TCFaproduction is directly proportional to the levels of Ha-rasmRNA and of p2V� protein in ras-transformed NOC-8mouse mammary epithelial cells (56). These results sug-gest that TCFa may be functioning as one importantintermediary in the transformation process that is in-duced by an activated ras gene in rodent mammaryepithelial cells.

Although overexpression of c-Ha-ras mRNA and p2irasprotein has been detected in approximately 60 to 70%of primary human breast carcinomas (8-10), the role andthe potential interactions between ras and TCFa in thetransformation of human mammary epithelial cells havenot yet been clearly defined because only a few non-transformed human mammary epithelial cell lines havebeen described (57). MCF-iOA cells offer a unique sys-tem to study the mechanisms of transformation by var-ious activated cellular protooncogenes. These cells werederived following the in vitro spontaneous immortaliza-tion of a population of normal human mammary epithe-hal cells from an s.c. mastectomy maintained in lowcalcium medium, and they can be efficiently transfectedby the calcium phosphate precipitation method (38, 40).The present study is the first to demonstrate that trans-formation of a human mammary epithelial cell line by anactivated c-Ha-ras protooncogene can induce an in-crease in TCFa mRNA expression and TCFa proteinsecretion. This increase in TCFa production may partlyaccount for the enhanced growth rate of these cells inserum-free medium and for their ability to grow in softagar. The elevated levels of secreted TCFa may alsocontribute to their reduced mitogenic response to ex-ogenous [CF and TCFa. These results suggest that TCFamay function as a secreted autocrine growth factor inras-transformed MCF-iOA cells. In fact, either an anti-TCFa neutralizing antibody, TAb 1 , or an anti-ECF recep-tor blocking monoclonal antibody, 528, was able to sig-nificantly inhibit the growth of these cells in soft agar.We have previously demonstrated that TAb 1 can inhibitthe anchorage-independent growth of NOC-8 mousemammary epithelial cells transformed by overexpressionofthe human TCFa gene (43). Likewise, the 528 antibodyhas previously been shown to completely block thebinding of “tI-ECF to MCF-7 human breast cancer cellsand to inhibit the mitogenic effect of TCFa on these cells(45). In addition, this antibody can also inhibit the in vivogrowth of xenografts established from MDA-MB-468cells, a human breast cancer cell line that expressesapproximately 2 x i06 [CF receptor sites/cell (46). Theinability of either TAb i or 528 antibodies to completelyblock the soft agar growth of MCF-1OA Ha-ras cellssuggests that other endogenous growth factors may alsobe involved in mediating the effects of an activated rasgene in these cells. In fact, in ras-transformed NOC-8mouse mammary epithelial cells there is an increase inthe production of TCFa and of at least two other growthfactors, transforming growth factor-fl and insulin-likegrowth factor I (41).

The results described in the present study relating to

ras transformation and TCFct production in MCF-1OAcells are in contrast with those recently obtained in the184A1N4 human mammary epithelial cell line (35).184A1 N4 cells are significantly different from MCF-1OAcells since they were derived as an immortal cell linefollowing treatment of a normal human mammary epi-thelial outgrowth with benzo(a)pyrene, a chemical car-cinogen (57). Moreover, unlike MCF-1OA cells, a v-Ha-ras oncogene is insufficient by itself to transform184A1 N4 cells since the presence of an additional gene,such as SV4O large T antigen or v-mos oncogene, isrequired to complete this process (58). No significantchange in the levels of TCFa production could be de-tected in v-Ha-ras-infected 184A1 N4 cells or in 184A1 N4cells transformed by a combination of v-Ha-ras andSV4OT oncogenes as compared to parental 184A1 N4cells (35).

Overexpression of a point-mutated rat c-neu gene inNOC-8 and HC-i i mouse mammary epithelial cells iscapable of transforming these cells in vitro and in vivo(56).� However, overexpression of the c-neu protoonco-gene in HC-1 1 cells fails to induce a tumorigenic phe-notype.4 Amplification of the c-erbB-2 protooncogenehas been detected in a subset of primary human breasttumors, and activation of this gene apparently occurs byoverexpression and not by point mutation (1 1). There-fore, it might be possible to transform a population ofnormal human mammary epithelial cells by overexpress-ing the c-erbB-2 gene when it is placed under the tran-scriptional control of a strong retroviral promoter, as hasbeen demonstrated in mouse NIH-3T3 cells (59). Theresults of this study are the first to demonstrate thatoverexpression of the c-neu protooncogene in a normalhuman mammary epithelial cell line can lead to in vitrotransformation. Similar to ras-transformed MCF-i OA cells,MCF-1OA c-neu cells are able to clone in soft agar, toform disorganized outgrowths in type I collagen gels, andto exhibit a reduction in the expression of specific cyto-keratins and of milk fat globule antigens. In addition, bothras- and neu-transformed MCF-1OA cells exhibit an en-hanced anchorage-dependent growth rate in serum-freemedium as compared to parental MCF-1OA cells. How-ever, whereas MCF-1OA Ha-ras cells show an attenuatedmitogenic response to exogenous ECF or TCFa, MCF-iOA c-neu cells exhibit no change in their sensitivity tothese growth factors. This may relate to the fact thatMCF-1OA c-neu cells do not significantly differ from MCF-1OA cells with respect to the production of endogenousTCFa. A similar inability to find an increase in TCFaexpression was also demonstrated in NOC-8 and HC-1 1mouse mammary epithelial cells after transformation witha point-mutated c-neu protooncogene (56).�

These results suggest that neu may utilize a different

transformation pathway than an activated ras gene, atleast with respect to the involvement of TCFa as apotential autocrine growth factor. This possibility is sup-ported by the observations that either the neutralizinganti-TCFa TAb-i monoclonal antibody or the blockinganti-[CF receptor 528 monoclonal antibody was unable

4 N. E. Hynes, D. Taverna, I. M. Harwerth, F. Ciardiello, D. S. Salomon,T. Yamamoto, and B. Groner. Epidermal growth factor receptor, but notc-erbB-2 activation, prevents lactogenic hormone induction of the �-casein gene in mouse mammary epithelial cells. Mol. Cell. Biol., in press,1990.

Cell Growth & Differentiation 417

to appreciably affect the anchorage-independent growthof MCF-1OA c-neu cells. In contrast, the blocking anti-c-erbB-2 TAb 250 monoclonal antibody that was originallygenerated against the extracellular domain of the humanpi85��’ protein is able to significantly inhibit the an-

chorage-independent growth of MCF-iOA c-flea cells

that are expressing this protein on their surface. Although

MCF-i0A cells that are overexpressing the c-neu pro-

tooncogene exhibit anchorage-independent growth insoft agar, they are unable to form tumors. These results

suggest either that a sufficient level of c-neu expression

has not been achieved in these cells to induce a fully

malignant phenotype in vivo or that additional genetic

changes such as deletion or inactivation of a tumor

suppressor gene or cooperation with a second activatedprotooncogene are necessary for this progression to oc-cur. In fact, high levels of expression of the ECF receptorcan synergistically enhance the transformation of mouseNIH-3T3 cells that are overexpressing the rat c-neu pro-tooncogene (60). The inability of an overexpressed c-fleaprotooncogene to lead to a fully malignant phenotype invivo agrees with the results obtained with the normal andpoint-mutated c-erbB-2 gene in transgenic mice (61).Although mammary adenocarcinomas were found intransgenic lines harboring point-mutated c-neu or c-erbB-2 genes, no histological abnormalities or mammary tu-mors developed in transgenic mice possessing the normalc-erbB-2 gene under the control of a mouse mammarytumor virus LTR (5, 6, 61).

If TCFa is functioning as one important intermediaryin ras transformation of MCF-1OA human mammary epi-thelial cells, then the constitutive overexpression of thisgrowth factor may by itself be sufficient to transformthese cells. For example, the enhanced expression of therat or human TCFa genes can lead to the full transfor-mation of Rat-i fibroblasts but can only partially trans-form normal rat kidney cells or mouse NIH-3T3 fibro-blasts, suggesting that a threshold level of [CF receptorexpression might also have to be reached before trans-formation can occur (52, 62, 63). This is supported bythe observation that the inappropriate overexpression ofthe human [CF receptor in NIH-3T3 cells leads to theirtransformation only in the presence of exogenouslyadded ligand or in cells in which a TCFa expressionvector has also been introduced (64, 65). We have re-cently demonstrated that clones of NOC-8 mouse mam-mary epithelial cells that were derived after either trans-fection or infection with human TCFa cDNA expressionvectors and that secreted elevated amounts of biolog-ically active TCFa were capable of anchorage-independ-ent growth in soft agar and were tumorigenic in nudemice (43, 52). The present results extend these observa-tions and are the first to demonstrate that an immortal-ized population of human mammary epithelial cells canbe similarly transformed in vitro through an autocrinepathway involving TCFa. The levels of TCFa that werefound in the CM from the majority ofthe MCF-1OA TCFaclones were comparable to the amounts of TCFa pro-duced by the mouse mammary epithelial NOC-8 TCFatransfectants (43). Moreover, a major fraction of theimmunoreactive TCFa secreted by these clones wasbiologically active as assessed in a competitive ECF/TCFaRRA, suggesting that there is correct processing of theprotein in these cells. The MCF-1OA TCFa cells alsoexhibited several properties that were common to the

ras-transformed MCF-1OA cells. First, MCF-1OA TCFacells showed a selective growth advantage in serum-freemedium devoid of exogenous [CF. Second, MCF-iOATCFot cells grew at a rate comparable to ras-transformedMCF-10 cells in serum-free medium. Third, the TGFa-expressing MCF-1OA cells showed a diminished mito-genic response to exogenous [CF or TCFa. Finally, sev-eral MCF-1OA TCFa clones exhibited a cloning efficiencyin soft agar that was comparable to that observed in ras-transformed MCF-1OA cells. In fact, the ability of theMCF-1OA TCFa cells to form colonies in soft agar maybe related to some sort of threshold level of secretedTCFa protein since clones secreting less than 200 ng/108 cells of biologically active TCFa over a 48-h condi-tioning period failed to grow in soft agar. This is appar-ently not as crucial for MCF-1OA Ha-ras cells since theyare secreting levels of TCFa below this limit, suggestingthat the ras transformants may depend upon additionalendogenous growth factors for their proliferation in softagar. Nevertheless, TCFa is the major factor in controllingthe growth of the MCF-1OA TCFa cells in vitro sinceeither the anti-TCFa TAb 1 antibody or the anti-ECFreceptor 528 antibody can completely block their growthin soft agar. These results strongly support the conceptthat TCFa is functioning as a bona fide autocrine growthfactor in these cells by binding to ECF receptors on thecell surface. Analogous to MCF-1OA c-flea cells, MCF-1OA TCFa cells are not tumorigenic in vivo, suggestingthat additional genetic changes may be necessary to elicita fully malignant phenotype in these cells.

Collectively, the results of this study demonstrate thatras but not nea transformation in vitro of an immortalizedpopulation of human mammary epithelial cells can leadto an increase in the expression of TCFa that may beinvolved in regulating the growth of these cells. Further-more, transformation in vitro can also be accomplishedby overexpression of TCFa through an autocrine-de-pendent pathway in MCF-1OA cells that also express asufficient complement of functional [CF receptors. Inthis respect, mouse NOC-8 and HC-1 1 and human MCF-1OA mammary epithelial cells express between iO� and2 X i0� ECF receptor sites/cell (41).� Therefore, highlevels of expression of this growth factor as well as itscognate receptor are equally important in initiating and!or maintaining cellular transformation (52, 65). The ex-perimental findings of this study may also be of someclinical significance for the pathogenesis of human breastcancer since TCFa is expressed in a majority of primaryhuman breast tumors and since TCFa can be found inthe metastatic pleural effusions obtained from breastcancer patients (26, 32). In addition, EGF receptors havebeen detected in approximately 40 to 50% of primaryhuman breast tumors where coexpression of [CF recep-tor and TCFa mRNA has been demonstrated in a subsetof primary and metastatic breast cancers (31 , 66). Finally,there is a significant association between high levels of[CF receptor expression in primary human breast carci-nomas and poor prognostic factors such as an absenceof estrogen and progesterone receptors, axillary lymphnode involvement, and an increased rate of tumor cellproliferation (13, 66, 67).

Materials and Methods

Transfection, Infection, and Establishment of Cell Lines.MCF-1OA cells were grown in a 1:1 Dulbecco’s modified

418 Transformation of Human Mammary Epithelial Cells and TGFa

1. Furth, M., and Greaves, M. (eds.), Cancer Cells, Vol. 7. Cold Spring

Harbor, NY: Cold Spring Harbor Laboratory, 1989.

Eagle’s medium and Ham’s F12 mixture (v/v) supple-mented with 5% horse serum-20 m�i 4-(2-hydroxyethyl)-i -piperazineethanesulfonic acid, pH 7.4-4 mivi gluta-mine-i 00 units/mI penicillin-100 �zg/ml streptomycin(CIBCO, Crand Island, NY)-iOO ng/ml cholera toxin (ListBiological Laboratories, Campbell, CA)-10 ng/ml [CF-b�zg/ml insulin (Collaborative Research, Bedford, MA)-0.5�zg/mI hydrocortisone (Sigma Chemical Co, St. Louis,MO) in a humidified atmosphere of 95% air and 5% CO2at 37#{176}C.MCF-1OA Ha-ras cells and MCF-iOA cells trans-fected with a plasmid containing the activated c-Ha-rasprotooncogene cloned from the human T24 bladdercarcinoma cell line under the transcriptional control ofthe MSV LTR and an internal SV4O promoter, and theneomycin-conferring resistance gene, neo (40, 68). MCF-1OA neo cells are MCF-iOA cells transfected with thesame plasmid containing only the neo gene. MCF-iOAc-nea cells were generated by transfection of MCF-1OAcells with an expression vector plasmid, pDOL-VLJ8,containing the full length normal rat c-flea cDNA underthe transcriptional control of the MSV LTR and the neogene (47). Transfection by the calcium phosphate precip-itation method was performed as previously described

(43). Following 21 days of culture in selective mediumcontaining 400 pg/mI geneticin (C4i8, CIBCO), 13 mdi-vidual C418-resistant MCF-1OA c-neu clones were iso-lated and expanded into cell lines. MCF-iOA TCFa cellswere produced by infection of MCF-1OA cells with anamphotropic recombinant retrovirus, 1522, containingthe full length human TCFa cDNA under the transcrip-tional control of an internal mouse MT-i promoter andthe neo gene, as previously described (52). After C4b8selection, 15 individual MCF-iOA TCFa clones wereisolated and expanded in cell lines.

Monolayer Growth. 2 x 10� cells/well were seeded in12 multiwell cluster dishes (Costar, Cambridge, MA) inserum-containing medium. Twenty-four h later, the cellswere washed twice and incubated in PC-i serum-freemedium (Ventrex, Portland, ME) in the absence or thepresence of 10 ng/mI human recombinant ECF (Collab-orative Research) or iO ng/ml human synthetic TCFa(Bachem, Torrance, CA). After 4 days of treatment, thecells were trypsmnized and counted with a model ZBICoulter Counter (Coulter Electronics, Hialeah, FL). Todetermine the basal growth in absence of exogenous[CF or TCFe, i0� cells were seeded in 12 multiwellcluster dishes in serum-containing medium and after 24h were switched to PC-i serum-free medium. Each dayfor 5 days, the cells were trypsmnized and counted.

Soft Agar Growth 2 x i0� cells were seeded in 1 mlof 0.3% Difco Noble agar (Difco, Detroit, Ml) supple-mented with Dulbecco’s modified Eagle’s medium andHam’s F12 containing 5% horse serum. This suspensionwas layered over 1 ml of 0.8% agar medium base layerin 35-mm dishes (Costar). After 16 days, the cells werestained with nitro blue tetrazolium and colonies largerthan 50 �sm were counted with an Artek 880 colonycounter (Artek Systems, Farmingdale, NY).

Effects of Anti-TGEa, Anti-EGF Receptor, and Anti c-erb8-2 Antibodies on Anchorage-independent Growth.2 x i0� cells were seeded in soft agar, as describedabove, in the absence or presence of different concen-trations ofthe following antibodies: TAb 1 , an anti-humanTCFa neutralizing mouse monoclonal IgC1 antibody (42,43); TAb 2, an anti-human TCFr nonneutralizing mousemonoclonal IgC2a antibody (43); 528, an anti-human [CF

receptor blocking mouse monoclonal lgC2a antibody

(44); or TAb 250, an anti-human c-erbB-2 blocking mousemonoclonal lgCb antibody that has been generatedagainst the extracellular domain of the plg5erbB.2 protein(49).

Preparation of CM, RIA for TGFa, and RRA for TGFa/[GE. CM was collected over a 48-h period from cellsgrown in PC-i serum-free medium and concentratedusing a Ci8 Sep-pak column (Waters Instruments, Roch-ester, MN), as previously described (41). Determinationof TCFa protein in the CM by RIA and by RRA was

performed as previously described (41).

RNA Isolation and Northern Blot Analysis. Total cel-lular RNA was isolated by lysis of the cells in guanidmne

thiocyanate and centrifugation over a cesium chloridecushion (43). PoIy(A�” RNA was obtained by absorptionto and elution from an oligo (dt) cellulose column (41).Equivalent amounts of RNA were electrophoresedthrough a denaturing 1 .2% agarose-2.2 M formaldehydegel. Ethidium bromide staining of the gels showed that

each lane contained an equivalent amount of RNA. Thegels were then transferred to Biotrans nylon membranes(ICN Biomedicals, Costa Mesa, CA) and hybridized tothe appropriate 32P-labeled nick-translated cDNAprobes: a 406-bp EcoRl-Apal restriction fragment derivedfrom a human TCFa cDNA clone, pTCF-Ci (41); a 6.6-kb BamHl fragment of the human c-Ha-ras gene (41); a420-bp BamHl fragment derived from the rat c-flea gene(47); or a 770-bp human �3-actin cDNA probe (Oncor,Caithersburg, MD).

Western Blot Analysis. Protein Iysates were separatedby sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, transferred to nitrocellulose, and reacted witheither Yi3-259 rat monoclonal anti-p2V� antibody (8) orwith 2i N rabbit polyclonal anti�pb85er�)B2 antiserum (48),as previously described (8).

Immunocytochemistry. Cells fixed in 4% formaldehydewere incubated with the followingantibodies: 2iN rabbit

polyclonal anti-pi8S��2 antiserum (48); AE1/AE3, a poolof anti-human epithelial keratin mouse monoclonal an-tibodies (Hybritech, San Diego, CA); MC5 (kindly pro-vided by Dr. R. Ceriani, J. Muir Aging and Cancer Re-search Institute, Walnut Creek, CA) and MFA-breast (IRE-Celltarg, Belgium), two mouse monoclonal antibodiesagainst milk fat globule membrane antigens (51). Anti-body-specific reactivity was detected by immunoperox-idase staining using a commercially available avidin-bio-tin complex kit (Vectastain ABC kit; Vector Laboratories,Burlingame, CA).

Growth in Collagen Gels. Type 1 collagen (CollagenCorp., Palo Alto, CA) gels containing cells were preparedas previously described (69). Cell growth was monitoredafter 3 to 4 weeks.

Acknowledgments

The authors thank Dr. Robert Weinberg, Whitehead Institute for Biomed-

ical Research (Cambridge, MA) for generously providing the pDOL-VLJ8

expression vector plasmid containing the c-neu cDNA and Dr. WilliamGullick, Imperial Cancer Research Fund (London, England) for generouslyproviding the 21N anti-p18Y’�’ rabbit antiserum. Part of this researchwas supported by a grant from the NCI (CA38921) to J. Russo.

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Cell Growth & Differentiation 419

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