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ICANCERRESEARCH56. I 155-I 163. March I. 19961 Inhibitory Effects of Activin on the Growth and of Primary and Transformed Mammary Epithelial Cells'

Qiu Yan Liu, Birunthi Niranjan, Peter Gomes, Jennifer J. Gomm, Derek Davies, R. Charles Coombes, and Lakjaya Buluwela2

Departments of Medical Oncology (Q. Y. L, P. G.. J. J. G., R. C. C., L B.J and Biochemistry (Q. Y. L. L B.J. Charing Cross and Westminster Medical School, Fuiham Palace Road. London W6 8RF; Division of Cell Biology and Experimental Pathology. Institute of Research, 15 Cotswald Rood, Sutton. Surrey SM2 SNG (B. NJ; and FACS Analysis Laboratory. imperial Cancer Research Fund, Lincoln ‘sInnFields. London WC2A 3PX (D. DI, United Kingdom

ABSTRACT logical activities of activin. Indeed, two types of activin receptors have aLready been identified in the mouse (28) and several forms in Activin Is a member of the transforming growth factor fi superfamily, Xenopus (29, 30). The sequences of the Act-RI! (3 1), the TGF-@ type which is known to have activities Involved In regulating differentiation II receptor (32), the TGF-f3 type I receptor (33), and various activin and development. By using reverse transcrlption.PCR analysis on immu noafflnity.purlfied human breast cells, we have found that activin IJa and receptor-like (34) have been described. The comparison of these activin type II receptor are expressed by myoepithelial cells, whereas no sequences shows that they belong to a newly defined family of expression was detected In other breast cell types. In examining 15 breast membrane-bound, ligand-activated - kinases (35). cell lines, we have found only four (HBL-100, MCF1O-A,PMC-42, and BT Much is known about TGF-f3 expression in the breast and its effect 20) to be positive for activin @3amRNA, whereas all expressed the activin on the development of tubules from end buds (4, 5, 36). However, type II receptor. Furthermore, we have found activin A to be a potent currently, there is very little information as regards the expression of growth inhibitor of MCF-7 cells (at 2 ng/ml), where it causes an arrest in other members of the TGF-@3family in the breast, their role in breast G1. Activin A does not appear to have an effect on the cell cycle of primary morphogenesis, and their expression in breast cell lines. In this study, myoepithellal or luminal cells. However, we demonstrate that activin is an we have studied the cell-specific expression of the activin @a inhibitor of tubule formation by human mammary in vitro. subunit and Act-RIl gene in highly enriched populations of normal These are the first observations of activin and in the normal human breast and in human breast cell lines and suggest a role for human mammary epithelial (luminal and myoepithelial) and activin mammary cell growth and morphogenesis. cells (37), as well as in established breast cell lines. Our results show that in the normal human breast, activin @aandAct-RI! gene expres sion is restricted to the myoepithelial cells. In breast cell INTRODUCTION lines, however, activin expression was found in only 4 of the 15 cell lines examined, whereas all cell lines expressed the Act-RI! gene. We The TGF3-13s are powerful mediators of proliferation and differen demonstrate that activin inhibits the cell cycle of the breast carcinoma tiation in a variety of cell types (1). TGF-(3 is expressed in the breast cell line MCF-7 but not primary human breast epithelial cells. Fur (2, 3), where it regulates the growth and development of end buds into thermore, we have found that activin can inhibit morphogenic pro ducts (4, 5). TGF-@3has also been shown to inhibit the growth of cesses leading to tubule formation by normal breast organoids in vitro. breast carcinoma cell lines in vitro (6). It is now clear that the Our data suggest that, like TGF-@3,activin may be involved in pro “classical―TGF-f3sare part of a much larger supergene family (7), cesses that underlie mammary gland morphogenesis and breast car which includes the Müllerianduct-inhibiting substance, bone mor cinoma cell growth. phogenic , the Vg-l-related gene product, and the Drosophila complex, together with the activins and inhibins (8). MATERIALS AND METHODS were first described as gonadal that stimulate/inhibit pituitary follicle-stimulating production Purification of Normal Human Breast Cell Populations (9—11).In vivo activin and inhibin subunits are expressed in gonadal Normal human and epithelial cells were sorted and purified from tissues, where they are known to regulate gonadal hormone produc organoids derived from reduction mammoplasties by an immunoaffinity tech tion (12). Activin and inhibin are also expressed in a wide variety of mque (37). Briefly, myoepithelial cells were isolated by adhesion to magnetic tissues, where their function remains poorly characterized (13, 14). particles (Dynal dynabeads) coated with a monoclonal to the Specifically, activin has been shown to play a critical role in meso CALLA, while luminal epithelial cells were isolated with similar beads coated derm induction during early vertebrate development (15—19).Activin with a monoclonal antibody to the EMA. Stromal cells were prepared by is also likely to be involved in later developmental and differentiation depleting the original mixed cell population sequentially for CALLA and then events, as indicated by gene knockout studies (20). Furthermore, in for EMA-expressing cells. Primary human luminal cells were grown in luminal vitro, activin has been shown to influence neuronal cell survival (21, cell media composed of Ham's F12 supplemented with 10% FCS, 5 @xg/ml 22), as well as cell proliferation (23—26)and differentiation (27) of a , 5 @.tg/mlhydrocortisone,100 ng/ml cholera toxin, and 10 ng/ml EGF. variety of cell types. Because of these complex functions, it has been Primary human myoepithelial cells were grown in myoepithelial cell media composed of RPM! 1640 supplemented with 10% FCS, 5 @Wmlinsulin,5 proposed that multiple receptors may be involved in mediating bin @.tg/mlhydrocortisone, and 100 ng/ml cholera toxin. Primary human mammary fibroblasts were used for RNA extractions immediately after cell separation. Received 6/20/95; accepted 1/3/96. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage Cell Culture charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C.Section1734solelyto indicatethisfact. Human normal and carcinoma breast cell lines HBLIOO,MCF7, T-47D, @ This work was supported by grants from the Medical Research Council (United Kingdom), the Special Trustees of Chasing Cross and Westminster Medical School, and BT-20, BT-474, MDA-MB-l57, MDA-MB-23l, MDA-MB-36l, MDA-MB theCancerResearchCampaign. 415, MDA-MB-453, ZR-iS-i, SK-BR-3, PMC-42, CAL-5l, and MCFIOA 2 To whom requests for reprints should be addressed. were obtained from American Type Culture Collection. MCF-lOA was grown 3 The abbreviations used are: TGF, transforming growth factor; Act-RI!, activin type as described by Soule et a!. (38). All other cell lines were maintained in RPMI 11 receptor; CALLA, common acute lymphoblastic leukemia antigen; EMA, epithelial membrane antigen; RT-PCR, reverse transcription-PCR; BrdUrd, bromodeoxyundine; 1640 or DMEM supplemented with 10% FCS and grown at 37°Cin5% or HGF/SF, hepatocyte growth factor/scatter factor; Cdk, cyclin-dependent kinase. 10% CO2 atmosphere. 1155

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FACS Analysis solution with 40 @.d7.5%sodium hydrogen carbonate and 200 @.dmedium containing 15—20 organoids. The gels were then poured into 24-well tissue Exponentially growing cells were pulsed with 10 @MBrdUrdfor 2 h, culture dishes already containing 500 @tlof collagen gel base/well, allowed to harvested by trypsinization, washed in ice-cold PBS, and fixed in ice-cold 70% set, covered with media, and left overnight. The following day, human recom ethanol. The fixed cells were then washed twice with PBS and resuspended in binant HGF/SF (a kind gift from Dr. E. Gherardi, Imperial Cancer Research 2 M HC1 for 30 mm with mixing at intervals. The cells were then washed three Fund, Cambridge, England) was added to the media at 50 ng/ml and left for times in PBS-T (PBS plus 0.1% BSA-0.2% Tween 20, pH 7.4) and incubated 5-7 days with daily feeding with HGF-SF. Activin A was used at 2 ng/ml, and with neat anti-BrdUrd antibody (Scm-Lab) for 20 rain. After two washes with the treated cultures were refed daily. The media used was composed of RPM! PBS-I, the cells were then incubatedwith FITC-conjugatedrabbitantimouse 1640 supplemented with 5% FCS, 5 @gImiinsulin,5 pg/mi hydrocortisone, F(ab')2 fragments (DAKO) for 20 mm. The cells were then washed twice in and 100 ng/ml cholera toxin. The morphological effects of activin were PBS and treatedwith RNase (1 mg/mI)for 15 mm, followed with propidium assessed by simultaneous addition of HGF/SF and activin A. iodide (10 @@g/ml)for 10 mm, and analyzed using a Becton Dickinson FAC

5@pIus All incubations were carried out at room temperature. Extraction of RNA from Cell Lines and Dynabead-sorted Human Cell Growth Assays Breast Cells

Recombinant human activin A (Activin @ahomodimer) isolated from the Cells lines were harvested by treatment with trypsinlEDTA and pelleted by conditioned medium of transfected Chinese hamster cells was a gift from centrifugation. These were washed twice in PBS, and total RNA was prepared Dr J. C. Smith (Medical Research Council, National Institute of Medical by a NP4Odetergent lysis method (40). Ethanol-precipitated RNA was recov Research, London, England). Activin A in 4 mr@iHCIwas diluted with culture ered by centrifugation, washed once with 70% ethanol, dried under vacuum, medium and filter sterilized using a 0.2 p.m filter. and finally resuspended in autoclaved deionized water to a final concentration Growth Curves. MCF-7 cells were seeded at a concentration of 1 x l0@ of 1 mg/mi. RNA integrity was checked by electrophoresis on 1.5% agarose cells per 125 flask in RPM! 1640 supplemented with 5% FCS in the presence mini-gels. RNA from freshly sorted cells was prepared while still attached to or absence of 2 ng/ml activin A. The growth of MCF-7 cells was determined beads using the NP4Odetergent lysis method. In this case, RNA quality was by counting cells in trypsinized suspensions at specific time intervals. Each found to be the same as that prepared from cultured cells. point was performed in triplicate. Measurement of DNA Synthesis. DNA synthesis in MCF-7 cells was Synthesis of cDNA for PCR measured by [3H]thymidine incorporation. For this procedure, cells were placed in 24-well tissue culture plates at a density of 5 X 10―cells/wellin cDNA was synthesized from total RNA, essentially as described by cultured medium supplemented with 10% FCS. After 24 h, the medium was Luqmani et al. (41). Briefly, 2 @gtotalRNA were reverse transcribed in a changed for 1 ml offresh medium containing 5% FCS and human recombinant volume of 20 ,.d containing 50 mMIris (pH 8.3), 40 mMKCI, 6 mMMgCl2, activin A at concentrations between 1 and 10 ng/ml. After 30 h, 0.5 @.tCi 10mt@DU, 200 @LMeachofdATP, dCTP, dGTP, and dTFP (Pharmacia), 200 [3H]thymidine (30 Cilmmol; Amersham) was added to each well, and the cells ng pd(N)6 random primers (Pharmacia), and 5 units of AMV reverse tran were allowed to grow for an additional 17 h. Medium was removed, and the scriptase (Super RI grade; HI Biotechnology). The reaction mixture was cells were lysed in situ with 1% SDS. Cell lysates were precipitated onto incubated at 37°Cfor 60 mm, heated to 95°Cfor 5 mm, and stored at —20°C. Whatman 3M filter discs with 15% trichloroacetic acid and then washed with 5% trichioroacetic acid and ethanol. Filters were dried and counted in loluene Amplification and Analysis of Gene Sequences by PCR Scintillator cocktail (Canberra Packard) in a Tri-Carb liquid scintillation ana lyzer. [3H]Thymidine incorporation at each activin A concentration was de PCR amplification was performed as described by Luqmani et aL (41). termined in triplicate. Reaction mixtures, without template, were made in bulk and aliquoted into reaction tubes in lOO-pi volumes. Each PCR reaction contained 2 @dDNA Immunohistochemical of Breast Sections template. Negative controls, consisting of reaction mix without cDNA tern plate, were also carried out with each amplification. All reactions were over Anti-activin @3apolyclonal antibody raised in rabbit was a gift kindly laid with 100 @.dlight mineral oil (Sigma Chemical Co.). Amplification provided by Professor Nigel Holder (Kings College, London, England). Serial programs consisted of 35 cycles, each composed of a DNA denaturing step of sections of normal breast tissue (obtained using a Reichert-Jung 2800 Frigocut 95°Cfor 1 rain, a primer annealing step of 53°Cfor 1 mm, and a DNA E cryostatandkeptat —20°Cuntiluse) were airdriedandfixed in successive synthesis step of 72°Cfor 1 mm. A final synthesis step of 72°Cfor 5 mm was washes ofSO and 100%acetone for 5 mm, washed with PBS, and incubated for carried out. Amplification reactions were carried out on a Cornbi thennal 15 mm in (Avidin/ Blocking kit; Vector Laboratories), followed reactor (Hybaid). PCR products were analyzed on 2% NuSieve/1% HGT by washing in PBS. After an additional incubation for 15 mm in biotin composite agarose gels (FMC Bioproducts). PCR product identity was con (Avidin/Biotin Blocking kit; Vector Laboratories) and washing in PBS, the firmed by hybridization to cloned probes for CALLA, EMA, keratin 14,activin nonspecific binding sites were blocked by incubation in PBG buffer [10% goat 13a,Act-RH, and f3-actinsequences. serum (GIBCO-BRL), 5% bovine , fraction V, and BSA (Sigma Chemical Co.) in PBS] for 30 mm at room temperature. The slides were then incubated with 2.5 mg/nil polyclonal anti-activin (3aantibody overnight at 4°C. Oligonucleotide Primers After three washes in PBS, the slides were incubated with biotinylated anti Oligonucleotide primers were synthesized on a 380A DNA synthesizer rabbit IgO (Vectastain ABC kit) overnight at 4°Cand washed three times with (Applied Biosysterns) and purified by NAP-b gel filtration (Pharmacia). For PBS. The slides were then incubatedwith avidin/biotin/peroxidasereagent a given gene, PCR primer pairs were selected so that they were located in (Vectastain ABC kit), and the staining was visualized with 0.1 M diaminoben separate exons (42—46).The DNA sequences of PCR primers used were as zidine and 0.01% . Slides were counterstained in hematox follows: activin @a:primer1, 5'-TFAIGGAGCAGACCICGGAG-3', primer ylin and mounted with Histomount (National Diagnostics) and then photo 2, 5'-CCCITFAAGCCCAC'TTCCIC-3'; Act-LIR: primer 1, 5'-lTfGCCIG graphed on a Zeiss microscope. Parallel sections were stained simultaneously GAATGAAGCAIG-3', primer 2, 5'-AGAAGCCAGTFCCCAIAGGAC-3'; with a monoclonal antibody to cytokeratin 14 (Sigma Chemical Co.) by the CALLA:primer1, 5'-TFGIAAGCAGCCTCAGCC-3',primer2, 5'-TFGIC same protocol. CACC1TVFC'FCGG-3'; EMA: primer 1, 5'-GAGGAICCGCTCCACC'FCT Morphogenic Assays CAA-3', primer 2, 5'-CCAAGCTFCTGGGCACTGAACTFCTCTGGGTAG 3'; @3-:primer 1, 5'-AICAIG1TFGAGACCTTCAA-3', primer 2, 5'- Human mammary organoids were derived by progressive collagenase di ICCCAICACCAICTFCCA-3'; andcytokeratin14: primer1, 5'-CCCIAC gestion, followed by sedimentation and filtration to produce ductal and lobu rFCAAGACCATFGAGG-3', primer 2, 5'-GTCACGCAICTCGTFCAGAA loalveolar fragments (organoids; Refs. 37 and 39). Organoids were embedded 3'. The expected size for RT-PCR products from CALLA, activin @a,Act-RU, in collagen gels (rat tail type I; Becton Dickinson) by mixing 250 p3 collagen and f3-actinmRNAs were 460, 250, 487, 378, and 319 bp, respectively. Due to 1156

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A Gel Hybridization B Gel Hybridization 123 123 123 123

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Fig. 1. RT-PCR analysis of RNA prepared to C D immunoaffinity-purified human breast cells. The Gel Hybridization Gel Hybridization expression of CALLA, EMA, cytokeratin 14, ac tivin fin, Act-RI!, and @-actinwere determined by RT-PCR. Hybridization of RT-PCR products with 1 23 123 1 23 123 cloned probes for CALLA, EMA, activin (3a,Act lul, and @-actinare shown adjacent to the gel photographs of PCR products. Lanes 1—3ofeach panel correspond to CALLA-positive cells, EMA positive cells, and CALLA and EMA-negative cells, respectively. A, RT-PCR amplification of CALLA sequences; B, RT-PCR amplification of EMA sequences; C. RT-PCR amplification of activin 13asequences; D. RT-PCR amplification of Act-Ril sequences; E, RT-PCR amplification of @-actinsequences;F.RT-PCRamplificationof cytokeratin 14 sequences.

F Gel Hybridization Gel 1 23 123 M 1 23

alternative mRNA splicing, four EMA PCR products can be generated.4These tiveness of the purification of different breast cell types. Fig. 1A sizes were 254, 227, 218, and 191 bp. shows that the luminal and fibroblast cell populations were predom inantly devoid of CALLA sequences (contaminating myoepithelial RESULTS cells), while Fig. lB demonstrates that the myoepithelial cells are devoid of EMA sequences (contaminating luminal cells). Further ACtiVin @3aandAd-RH Gene Expression in Sorted Populations more, Fig. lF shows that cytokeratin 14 sequences can only be of Normal Human Breast Cells. Three different sorted populations amplified from cDNA made to myoepithelial cells. The differential of normal human breast cells from reduction mammoplasties were used in this study. These were populations of myoepithelial cells, amplification of CALLA and cytokeratin 14 sequences cannot be due luminal cells, and stromal cells. Total RNA isolated from each of to differences in the quality of the cDNA derived to the three popu these cell populations was reverse transcribed into cDNA and then lations because @-actinsequences amplify equally well in all cases used to amplify CALLA, EMA, cytokeratin 14, activin @a,Act-RI!, (Fig. 1E), and cytokeratin 14 is a marker of myoepithelial cells in the and f3-actin sequences. Fig. 1 shows the result of amplification of human breast (47). Our data show that this cell type is effectively these sequences from the sorted breast cell types. We have used PCR isolated in this purification step. Likewise, purification of EMA of CALLA, cytokeratin 14, and EMA sequences to assess the effec expressing, luminal cells also leads to a great enrichment of EMA sequences, as shown in Fig. lB; although in this case, some EMA

4 Q. Y. Liu et a!., manuscript in preparation. expression is seen in the CALLAIEMA-negative population (stromal 1157

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Fig. 3. Growth inhibition of MCF-7 by activin 100 A. A, dose-response relationship for the inhibition It) of DNA synthesis in MCF-7 cells by activin A 80 0 demonstrated by [3H]thymidine incorporation of MCF-7cellstreatedwithvaryingconcentrationsof >( activin A in the presence of 5% FCS. Each value 60 represents the mean of triplicate readings; bars, SE. 2 B. growth curve of activin A-treated MCF-7 cells. 40 E Replicate MCF-7 cultures were grown in RPM! E z 1640 supplemented with 5% FCS in the absence 5.' 20 4, (‘0')or presence(0)of activinA (2 ng/ml).The U medium was changed every second day. I 0) @ 0 2 4 6 8 10 12 0 4 6 8

Activln (ng/ml) Time (days)

cells). It is likely that this is due to EMA-expressing cells that failed reciprocal fall of cells in S phase starts 4—8h after treatment (Fig. 5, B to attach to beads. In summary, these PCR profiles suggest that we can and C). By 16 h, over 70% of the cells were arrested in [email protected] figure successfully enrich for luminal and myoepithelial cell populations. plateaus between 80 and 85% over the next 32 h. Similar experiments Hence, we carried out further experiments to look for activin f3a and with primary, purified luminal and myoepithelial cells showed that ac activin II receptor transcripts in the purified breast cell types. As tivin did not induce a G1 phase block in these cells types over a 48-h shown in Fig. 1, it was found that both activin @a(Fig. IC) and period of growth (Fig. 6) and did not affect cell growth over a period of Act-RI! (Fig. 1D) gene expression could be detected in myoepithelial 8 days, as judged by cell number (data not shown). (CALLA-expressing) cells. There appears to be little expression of Expression of Activin f3a and Act-RH in Human Breast Cell either activin @aorthe Act-RH in luminal and stromal mammary cell Lines. We have used PCR to investigate the expression of activin and populations. Additional PCR experiments failed to detect expression activin receptor in human breast cell lines. Two breast lines showing of the a subunit of inhibin in any of the cell populations derived above (data not shown). Using , we have further confirmed the tissue distribution of activin @ainnormal human breast. The results presented in Fig. 2 illustrate that activin @3aprotein expression is restricted to the myoepithelial cells, as confinned by coexpression of cytokeratin 14, a marker for human mammary myo epithelial cells (Fig. 2C). No activin staining was observed in either luminal or stromal cells. Effect of Activin A on the Growth of MCF-7 Cells. MCF-7 cells are known to be growth inhibited by TGF-@ (6, 48, 49). We have found that activin A was also growth inhibitory to MCF-7 cells and that there is a dose-dependent relationship between activin A concen tration and growth inhibition in these cells, as assessed by [3H]thy midine incorporation (Fig. 3A). Inhibition is detectable with 1 ng/ml of activin A, where growth was inhibited by 20% of control cultures. p The level of inhibition brought about by 5 and 10 ng/ml of activin A were similar and suggested that the maximum growth inhibition that B could be achieved was close to 60% of the control. Growth curves for MCF-7 cells in the presence of 2 ng/ml of activin A and untreated, control cultures are shown in Fig. 3B. From this it is seen that growth of MCF-7 cells is significantly inhibited by activin A over a period of 8 days. After 8 days, control cultures were confluent, containing approximately 4.2 X 106 cells, while activin A-treated cultures con mined 1.2 X 106 cells only. MCF-7 cells treated with activin A also showed a change in cell phenotype, typified by an increase in cell size and a dispersed colony morphology (Fig. 4). Effect of Activin A on the Cell Cycle of MCF-7 Cells and Primary Mammary Epithelial Cells. To define the mechanisms underlying the growth inhibition of MCF-7 by activin A, the cell cycle was examined. Fig. 5A shows that the growth arrest induced by activin A in these cells is achieved by a block in G1, which is accompanied by a reciprocal decrease in the number of cells in S phase. By Fig. 4. Morphology of MCF-7 cells grown in activin A. MCF 7 cells grown in RPMI 1640 supplemented with 5% FCS for 6 days, in the absence (A) or presence (B) of activin following the cell cycle in activin A-treated cultures at short time A (2 ng/ml). Note the increase in cell size and the marked dissociation of colonies in the intervals, it is seen that there is an accumulation of cells in G1. The activin-treated culture. 1159

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ACTIVIN AND HUMAN MAMMARY EPITHELIAL CELLS A. B. C.

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24 hrs. 48 hrs. 10 20 30 40 50 10 20 30 40 50 TIme, hrs. Time, hrs Fig.5. CellcycleanalysisofactivinA-treatedMCF7 cells.A,cellcycledistributionofMCF7 cellsafteractivinAtreatment.MCF7 cellsgrownin theabsence(0) or presence (U) of activin A (2 ng/ml) for 24 and 48 h, respectively, were pulsed with BrdUrd prior to FACS analysis to determine the proportion of the cells in each phase of the cell cycle. Activin A-treated cells display an accumulation in G1accompanied by a fail in S phase, while G2remains unchanged. B, a time course ofthe G1distribution ofMCF 7 cells grown in the absence (R) or presence ( •)of activin A (2 ng/ml). C. a time course of the S-phase distribution of MCF 7 cells grown in the absence (U) or presence ( •)of activin A (2 ng/ml). The proportion of cellsin G@andS phasewasestimatedbyFACSanalysisaftera 2-hpulsewithBrdUrd.Thesedataarerepresentativeofthreeseparateexperiments.

some normal characteristics (HBL-lOO and MCF-1OA) and 13 breast the presence of activin A, however, results in the inhibition of HGF/ carcinoma cell lines were examined. Table 1 shows that activin SF-induced tubule formation (Fig. 7D). Control, untreated, organoids receptor sequences were detected in all of the cell lines used. In (Fig. 7A) or organoids treated with activin A alone do not exhibit contrast, activin expression was detected in only four cell lines, i.e., tubule formation (Fig. 7C). HBL-l00, MCF-1OA, PMC-42, T474, and BT-20. Since our studies have shown that activin A expression is restricted to myoepitheial DISCUSSION cells in normal breast cells, we looked for CALLA expression, by PCR, in all of the above cell lines used. CALLA gene expression was Activin A and Act-RU Expression in the Human Breast. Estab detected in HBL-lOO, MCF-1OA, PMC-42, T474, and MDA-MB-453 lished breast cell lines generally show varying and incomplete luminal only (Table 1). Hence, in the breast cell lines examined, coexpression and myoepithelial characteristics. Therefore, such lines are of limited of activin, activin receptor, and CALLA were found in three cell lines, use in studies of assignment of gene expression to the specific cell i.e.,HBL-l00, MCF-1OA,andPMC-42. types that exist within the breast. Furthermore, until recently, it has Effects of Activin A on in Vitro Tubule Formation by Human been difficult to obtain highly pure primary cultures of luminal and Mammary Organoids. Human mammary organoids, when embed myoepithelial cell types in quantities required for such studies. Fur ded in collagen gels, respond to HGF/SF by forming extensive thermore, since both luminal and myoepithelial cells undergo major branching tubules (Fig. 7B). Treatment of organoids with HGF/SF in phenotypic changes in culture, including their irreversible conversion

MYOEPITHELIALCELLS LUMINALCELLS Gi Gi Gi Gi

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Fig. 6. Cell cycle analysis of activin A-treated primary human mammary luminal and myoepithelial cells. Freshly sorted luminal cells were grown in LC media in the absence (0) or presence(U)ofactivinA(2ng/ml)for24and48h,respectively.Similarly,freshlysortedmyoepithelialcellsweregrowninMCmediaintheabsence(0) orpresence(U)ofactivin A (2 ng/ml) for 24 and 48 h, respectively. The proportion of cells in each phase of the cell cycle was estimated by FACS analysis after a 4-h pulse with BrdUrd. 1160

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ACFIVIN AND HUMAN MAMMARY EPITHELIAL CELLS

Table1linesThe Acrivinf3a.Act-RI!.andCALLAgeneexpressioninhumanbreastcell finity purified cells for investigating the expression of activin f3a and expression of activin g3aand CALLA was determined by reverse PCR. Products the type II activin receptor in the human breast. Since it has been were analyzed by electrophoresis on a composite agarose gel (2% NuSieve agarose/l% HOT agarose), and fragment sizes were determined by co-electrophoresis of a digest of shown that activin expression in extra gonadal tissues is low (13), we 4@X-l74 DNA with the restriction enzyme HaeIII.Gene have used RT-PCR in our study. Using RNA made to sorted human SequenceActivin breast cell types, we have found that both the activin and the Act-RI! are expressed predominantly by myoepithelial cells. We have further @a Act-Rh CALLA bp)NonmalignantCell lines (250 bp) (487 bp) (460 confirmed this observation by immunohistochemical staining of nor mal breast tissue sections, where activin appears to be localized in and +MCF-lOAHBL-lOO + + around the myoepithelial cells. Interestingly, the distribution of ac +CarcinomaPMC42 + + tivin expression in the human breast resembles that of TGF-f33 in the mouse mammary gland (3). Our observations suggest that activin may +BT-20 + + —BT-474 + + have an autocrine mode of action on myoepithelial cells. In this +MDA-MB-453 — + context, activin is also expressed in an autocrine manner by +MDA-MB-157 - + -MDA-MB-231 - + parenchymal cells, where it acts as a negative regulator of DNA -MDA-MB-36l - + synthesis (26). In the case of breast epithelial cells, however, our data -MDA-MB-4l5 - + suggest that activin does not inhibit cell growth but may be involved -MCF7 - + -CAL-5l - + in regulating tubular morphogenesis (see below). Since in this study -SK-BR-3 — + we have only looked at the distribution of Act-IIR gene expression in —T-47D — + the human breast, our results do not preclude a paracnne mode of -ZR-iS-I - + — + — action for activin through other activin receptors and activin-like receptors (7, 35). Activin A and Act-RI! Expression and Activity in Breast Cell to mesenchymal and squamous epithelial like cells, respectively (50— Lines. We have observed expression of the activin receptor in all of 52), it is important to be able to reproducibly obtain large quantities the breast cell lines examined. These include 13 human breast carci of highly purified breast cell types. The recent ability to highly purify noma lines, the SV4O transformed line HBL-100 (53), and the spon myoepithelial and luminal cells by virtue of their expression of the taneously immortalized line MCF-lOA (38). The ubiquitous expres cell surface antigens CALLA and EMA, respectively, has provided an sion of the Act-RI! gene in breast cell lines, as opposed to the efficient way of obtaining the numbers of cells required for gene restricted expression in vivo, is interesting and may represent activa expression studies (37, 39). In this report, we have used immunoaf tion during breast cancer development. This possibility is supported

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Fig. 7. Morphogenic effects of activin A on human mammary organoids. A, control mammary organoids embedded in collagen gels and grown in the absence of HGF/SF and activin A. B, formation of branching tubules by mammary organoids embedded in collagen gels in response to HGF/SF. C, control mammary organoids embedded in collagen gels and grown in the presence of activin A. D. inhibition of HGF/SF induces branching tubes as a result of activin A treatment. 1161

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by recent PCR studies, which we have carried out on microdissected ACKNOWLEDGMENTS breast tumor material (54). Our preliminary results have shown that We thank Drs. Jim Smith and Ermanno Gherardi for generously providing Act-RI! gene expression can be detected in invasive carcinoma cells activin A and HGF/SF, respectively, and Drs. I. Kamalati and R. Brooks for (data not shown). Clearly, the role of Act-RH in breast tumor patho critical discussion of the work contained in this study. P. Gomes thanks the genesis requires further investigation. Royal Air Force for their continued support. In contrast to Act-RH, activin expression could only be detected in 4 of the 15 cell lines examined, demonstrating that, unlike TGF-j3, REFERENCES autocrine expression is not a feature of normal or transformed breast epithelial cells in culture (55). In the case of TGF-@, most of the I. Roberts, A. R., Flanders, K. C., Heine, U. I., Jakowlew, S., Komdaiah, P., Kim, S. J., and Spom, M. B. 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Qiu Yan Liu, Birunthi Niranjan, Peter Gomes, et al.

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