ICANCERRESEARCH56. I 155-I 163. March I. 19961 Inhibitory Effects of Activin on the Growth and Morphogenesis 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 Cancer 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 genes (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 serine-threonine 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 gene expression of the activin @a inhibitor of tubule formation by human mammary organoids in vitro. subunit and Act-RIl gene in highly enriched populations of normal These are the first observations of activin and activin receptor in the normal human breast and in human breast cell lines and suggest a role for human mammary epithelial (luminal and myoepithelial) and fibroblast 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 carcinoma 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 proteins, the Vg-l-related gene product, and the Drosophila decapentaplegic complex, together with the activins and inhibins (8). MATERIALS AND METHODS Activin and inhibin were first described as gonadal peptides that stimulate/inhibit pituitary follicle-stimulating hormone production Purification of Normal Human Breast Cell Populations (9—11).In vivo activin and inhibin subunits are expressed in gonadal Normal human fibroblasts 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 antibody 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 insulin, 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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1996 American Association for Cancer Research. ACfl@IN AND HUMAN MAMMARY EPITHELIAL CELLS 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 ovary 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).