InVitroCell.Dev.Biol.28A:716-724,November-December1992 © 1992TissueCultureAssociation 0883-8364/92 $01.50+O.00

C H A R A C T E R I Z A T I O N O F N O R M A L B R E A S T E P I T H E L I A L CELLS IN P R I M A R Y CULTURES: DIFFERENTIATION AND GROWTH FACTOR RECEPTORS STUDIES PHILIPPE BERTHON, GIANFRANCO PANCINO, PATRICIA DE CREMOUX, ALBERTO ROSETO, CHRISTIAN GESPACH, AND FABIEN CALVOI Laboratoire de Pharmacologic, Institut de Ggn&iqueMolgculaire, H~pital Saint Louis, F-75475 Paris Cedex 10 (P. B., P. de C., F. C.); Division d'Immunohistochimie Appliquge, Universitgde Technologic de Compidgne, F-60206 Compiegne Cedex (G. P., A. R.); and Inserm U.55, Unitg de recherches sur les Neuropeptides Digestifs et le Diabdte, HSpital Saint Antoine, F-75571 Paris Cedex 12 (C. G.) (Received 7 October 1991; accepted 31 March 1992)

SUMMARY The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures from breast tissue biopsies from 42 mammoplasty patients. Cells were grown for up to 7 mo. in low calcium medium. HMEC cultures displayed heterogeneous growth patterns, according to the average doubling time of 44 -4-6 h for 32 generations. Proliferation peaked at Day 30. HMEC maintained a normal karyotype and were organized in ductlike structures when cultured in collagen gel matrix. The cultures retained several phenotype traits of the epithelial lineage, including the expression of cytokeratins 18 and 19, specific mammary gland antigens, as shown by indirect HMEC immunostaining by the monoclonal antibodies DF3, EMA, 7B10, and 1BE12. Estrogen receptors were undetectable, whereas progesterone receptors were present at very low density. High-affinity cell surface receptors for epidermal growth factor (EGF) (Kd = 1.1 X 10 -1° M) were observed at a density of 50 000 to 100 000 sites per cell. Accordingly, [3H]thymidine incorporation in HMEC was optimally stimulated by EGF at concentrations of 10 -H to 10 -l° M. HMEC were also seen to possess functional VIP receptors linked to the adenylate cyclase system, as we previously observed in seven human breast cancer cell lines. These results show that long-term cultures of HMEC provide useful models for studying the growth and differentiation of the normal human mammary gland, and the role of growth factors and hormones in these functions. Key words: normal human mammary epithelial cells; cell culture; proliferation; differentiation. including normal mouse keratinocytes and neoplastic mammary cells, rat esophageal cells, human bronchial epithelial cells, and prostatic cells at different stages of neoplastic development (11,14,23,26,33,60). In recent years, several lines of evidence indicated that these culture conditions allow normal mammary epithelial cells to proliferate and maintain their differentiation characters. Using these culture conditions, we therefore characterized the different cell types arising from human mammary epithelial cells (HMEC) primary cultures according to different criteria, including a) cell proliferation in the presence or absence of EGF, estrogen, and progesterone; b) binding of these growth factors to their receptors; c) phenotypic traits related to the mammary epithelia, i.e. ultrastructural morphology and polarity, and the expression of the specific antigens and functional VIP receptors previously character ized in seven human breast cancer cell lines (17); and d) karyotype analysis and ability to grow in nude mice as test of breast tissue normality. This culture system may allow the study of the role of growth factors and hormones, alone or in combination, in the growth and differentiation of the breast epithelium (13).

INTRODUCTION Breast cancer is the main cause of death from cancer in Western countries. Most breast tumors arise from the junctions between acini and ducts. To understand the intrinsic and extrinsic factors, i.e. hormones and growth factors, involved in normal and pathologic growth of human breast epithelial cells, several in vitro reliable systems were developed. Cultures of normal human mammary epithelial cells were previously obtained using a medium supplemented with various factors, includingconditioned media, epidermal growth factor (EGF), dexamethasone, and insulin (32,59). In 1984, Hammond et al. (21) reported that bovine pituitary extracts promote clonal growth in serum-free medium containing growth factors and substrate adhesive factors. Most investigators used very similar media supplemented with normal calcium concentrations for shortand long-term cultures of normal breast tissue (2,45,46,51,65). McGrath (31) and Soule et at. (56,57) proposed lowering the calcium concentration to sustain continuous mammary cell growth in prolonged cultures. Low calcium medium is known to inhibit the growth of nontransformed mouse and human fibrobtasts (6,7) and to increase the proliferation and life span of various epithelial cells,

MATERIALSANDMETHODS Propagation of human mammary epithelial cells in long-term cultures. Normal breast cell cultures were grown according to the techniques de-

1 To whom correspondenceshould be addressed. 716

CHARACTERIZATION OF NORMAL HMEC scribed previously by Soule and McGrath (57). Tissue specimens from 42 women who underwent reduction mammoplasty for cosmetic reasons (mean age: 31 yr; range: 15 to 61 yr) were obtained in the operating room after the patients had given their informed consent, and were processed immediately. Tissues were mechanically dissociated with scissors and the tissue suspension was incubated at 37 ° C with constant shaking in medium containing 150 IU/ml hyaluronidase and 250 to 500 IU/ml collagenase, both from Sigma (St. Louis, MO) (59). Digestion was monitored under an inverted microscope. Released organoids were then separated by density gradient centrifugation using 1.077 g/ml lymphocyte separation medium. Cells were plated in medium A, made up of Dulbecco's modified Eagle's and Ham's F12 media (DMEM/F12, vol/vol) without calcium (IJB, France), containing 10 mM HEPES, 2 mM glutamine (IJB), 10 ttg/ml insulin (Sigma), 5 >( 10 -6 M cortisol (Sigma), 50 IU/ml penicillin, 50 #g/ml streptomycin, 100 ng/ml cholera toxin (Sigma), 2 ng/ml EGF (Paesel, West Germany), and 1.05 mM CaC12, supplemented with 5% horse serum (GIBCO, Grand Island, NY) Ca2+-chelated by Chelex 100 (Sigma) (8). Cells were cultured in a 5% CO2:95% air humidified incubator. After 7 to 10 days, medium A was replaced by the same medium containing 60 gM CaC12 instead of 1.05 mM (medium B). Cells were passaged by the transfer of either free-floating cells (FFC) or adherent cell layers removed by dispase grade II treatment (Boehringer Manheim, Germany). Living cells were evaluated by trypan blue exclusion. Tridimensional cultures in collagen gels were prepared as follows: 20 mg of rat tail collagen type 1 (Sigma) was dissolved in 10 ml 0.1 M acetic acid at 37" C for 30 min (66). This solution was diluted to a final concentration of 100 ~tg/ml, pH 7.4, by addition of culture medium B. Finally, 10 s FFC/ml were resuspended in this mixture and seeded in 35-mm petri dishes. lmmunostaining. Normal mammary epithelial cells were cultured in eight-well tissue culture chamber slides (Lab-Tek) and fixed before confluency at 4 ° C in methanol/acetone (2 vol/1 vol) for 10 min. After three washes in phosphate buffered sahne (PBS) containing 0.1% bovine serum albumin (BSA) they were processed for immunostaining or stored at - 8 0 ° C. HMEC-collagen gel cultures were tested on paraffin sections fixed in Bouin's fluid and counterstained using hematoxylin & eosin. Next, cells were preincubated with diluted normal horse serum to reduce nonspecific staining and incubated with various monoclonal antibodies (MAbs) for 1 h at room temperature. After three washes in PBS containing 0.1% BSA (pH 7.4), cells were incubated for another 30 min with biotinylated horse antimouse antibody and for 30 min with the avidin-biotin-peroxidase complex (Vectastain, Vector Lab., Burlingame, CA). After repeated washes, staining was revealed with 0.1% H202 and 27 ttg/ml 3-amino-9-ethylcarbazole in acetate buffet', pH 5.2. Slides were counterstained with Harris hematoxylin and mounted. Immunofluorescence studies were performed by treating the slides for 1 h with a rhodamine-labeled goat anti-mouse antibody at 6 #g/ml (Biosys, Compiegne, France). Antibodies. Three MAbs directed against cytokeratins were tested: a) anti-pan-cytokeratin MAb (KL1; Immunotech, France; final dilution 1:100) known to recognize mainly molecular weight (MW) 56 000 acidic cytokeratins but also others (64); b) anti-cytokeratin 19 MAb (Ks 19.1, Progen, final dilution 1:100); and c) anti-cytokeratin 18 MAb (M9, Monosan, final dilution 1:25)specific to the MW 40 000 and 45 000 acidic cytokeratin polypeptides observed in normal and tumoral breast tissue and in the breast adenocarcinoma cell line MCF-7 (35). The anti-vimentin MAb V3250 (final dilution 1:50) was from lmmunotech (19). The anti-actin MAb N.350 (final dilution 1:500) was from Amersham (28). The anti-a actin MAb 1A4 from smooth muscle (final dilution 1:400) was from Sigma (54). The anti o! tubulin MAb N.356 (final dilution 1:500) was from Amersham (5). MAb DF3, directed against human mammary epithelial mucin antigen (final dilution 1:200), was a generous gift from Otis, Paris, France (25). MAbs 7 B 10 and 1BE12, directed against the human mammary gland (final dilutions 1:100 and 1:200, respectively, i.e. 20 #g/ml) were produced in our laboratory (41,42). MAb E/29, directed against the epithehal membrane antigen (EMA, final dilution 1:50) was purchased from Dako (39). The anti-common acute lymphoblastic leukemia antigen (cALLA) MAb J5 (final dilution 1:50) was from Coulter (20), and the anti-pSz MAb (final dilution 1:25) was a gift from Dr. ]-F. Prud'homme (Inserm U135, Lekremlin-Bicetre, France) (47). For each assay, negative controls were run using an irrelevant MAb of the same Ig subclass. Cytogenetic analysis. Coleemide (0.2 #M) was added 4 to 12 h before harvesting the exponentially growing HMEC. Mammary cells were fixed

717

with a mixture of methanol/acetic acid (3 vol/1 vol). For banding, the chromosome preparations were treated with trypsin and stained with Giemsa (53). In each of four cuhures derived from different breast reduction patients, 40 metaphases were counted and at least 20 banded karyotypes were examined. Electron microscopy. Organoids obtained in tridimensional cultures in collagen gels were fixed in 3.7% paraformaldchyde for 1 h at romn temperature and rinsed 3 times in PBS. A similar procedure was applied to adherent cell monolayers. The fixed material was incubated with 1% osmium tetroxide for 1 h at 4 ° C, washed with distilled water, and embedded in Epon for 24 h at 60 ° C. Sections 1-#m thick prepared for light microscopy were stained with toluidine blue. Ultrathin sections were cut from selected areas on a Reiehert OMU4All mierotome. Grids were counterstained using standard methods and analyzed with a Philips EM 301 electron microscope. Hormone receptors. Estrogen and progesterone receptors (ESR and PGR) were determined using enzyme imnmnoassays (ESR-EIA and PGREIA). Cells were cultured for 48 h in charcoal dextran-stripped serum containing medium B, harvested using 2.5 mM EDTA in PBS, and counted. After three washes in PBS (pH 7.4), 2 X 106 cells were resuspended at 4 ° C in 500 #1 of freshly made extraction buffer (pH 7.6) containing, in raM, 8.8 NazHPO4, 1.1 KHzPO4, 3 sodium azide, 400 KCI, 10 NazMoO 4, 10% glycerol and 70 #g/ml dithiothreitol, and quick-frozen in liquid nitrogen. Cells were then thawed and homogenized using a Branson sonieator under nitrogen steam. The homogenate was centrifuged for 60 min at 105 000 Xg to yield the soluble eytoplasnfic and nuclear fractions that were used for ESR and PGR determination. Cell extracts were diluted with equal volumes of phosphate buffer (20 mM NazHPO4, 40 mM NaMoO4, 800 mM KCI, 5 mg/ml BSA, 100 mg/ml garamycin, and 2% charcoal dextran-treated serum) and incubated for 18 h at 0 ° to 4 ° C with nytex beads coated either with the anti-estrogen receptor antibody D547 or the anti-progesterone receptor antibody KD68 (Abbott, ESR-EIA and PGR-EIA kits). Beads were washed, incubated for 1 h at 37 ° C with peroxidase-conjugated anti-estrogen receptor antibody H222 or the anti-progesterone receptor antibody JZB39, and washed with distilled water. Beads were then treated for 30 min with the peroxidase substrate O-phenylenediamine and spectrophotometric measurements were performed at 492 ran. All experiments were done in duplicate. Epidermal growth factor binding studies. HMEC were plated for 24 h at 5 X 104 cells/well in 24 muhiwell dishes in medium B. Next, HMEC were cultured fro" 48 h in EGF-free medium B, supplemented with 5% charcoaldextran-treated horse serum, and for another 48 h in the presence or absence of 10 -9 M progesterone. EGF binding assays were performed on adherent cells after 1 h preincubation at 37 ° C with 1 ml DMEM/F12 medium supplemented with 0.1% BSA. This preincubation was repeated once, and HMEC were then incubated in triplicate at 4 ° C for 4 h, in 1 ml of the same medium containing 10 -t° M [12SI]EGF per well (mouse EGF from Amersham, specific activity: 100 ttCi/~g EGF), and concentrations of unlabeled EGF ranging from 10 - ' z M to 10 -a M (mouse EGF receptor grade; Paesel, West Germany). Cells were then washed in ice-cold medium. The supernatant was removed and cells were lysed for 30 rain at 37 ° C in 1 ml 0.1 N NaOH. Radioactivity was counted using an LKB automatic gamma counter (LKB Wallaeh, Finland). Effect of EGF on HMEC growth. Before the experiments, HMEC were cultured for 4 days in charcoal-dextran-treated horse serum without EGF. The medium was changed to serum-free medium B, supplemented with 0.025% BSA and increasing concentrations of EGF from 10 -12 to 5 X 10 -s M. After 20 h, 4/.tCi/ml [aH]thymidine (CEA, France) was added (1.6)< 10 -7 M) and HMEC were incubated for I h at 37 ° C. The cells were then washed twice for 10 min in three solutions: PBS (pH 7.4), ice-cold 5% (vol/vol) trichloroacetic acid, and cold ethanol. HMEC were allowed to dry, lysed with 1 ml 0.1 M NaOH at 37 °. C fur 30 min, and counted in a Beckman liquid scintillator.

Vasoaetive intestinal peptide (VtP) receptor binding and cAMP generation. Porcine VIP was purchased from Pr. V. Mutt (Department of Biochemistry II, Karolinska Institutet, Stockholm, Sweden). This VIP was used because its amino acid sequence is identical to that of human VIP. The homogeneous VIP monoiodinated on tyrosine 10 has a specific activity of 2000 Ci/mmol, and was found to retain" the full biological activity of the native neuropeptide on VIP receptors in the human colonic adenoeareinoma cell line HT-29 (30). Binding assays were performed on HMEC primary euhures at Day 30, as previously described (17). Briefly, 200 000 cells

718

BERTHON ET AL.

Fig. 1. Phase contrast microscopy of normal HMEC at 36 days of primary culture in medium B. X320.

were incubated for 30 min at 15 ° C in 500 #l of the binding assay buffer containing 35 mM Tris-HCl (pH 7.5), 1.2% BSA, 50 mM NaCI, 0.6 mg/ml bacitracin, 5 × 10 -11 M [12Sl]VIP, and concentrations of unlabeled VIP ranging from 10 - n to 10 7 M. Cells were then washed and centrifuged, and the pellet was counted for cell-associated [1251]VIPin a Packard autogamma counter. Generation of cAMP was measured in isolated HMEC prepared

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Doys of culture FIC. 2. Growth rate of normal HMEC cultured in medium B. About 3 × 105 normal cells issued from the FFC in primary culture were plated in 25-cm2 flasks. Medium was changed 1 day later and then every other day. Data are the means of four different cultures performed in duplicate. Monolayer reached confluency between Days 9 and 11. Inset, comparison of the growth rate of cells issued from FFC (solid circles) and of cells removed by dispase digestion (open circles).

from Day 30 primary cultures. Cells were incubated for 60 min at 15 ° C in 500 tti of 35 mM Tris-HC1 buffer (pH 7.5) containing 50 mM NaC1, 1 mM 3-isobutyl-l-methylxanthine (IBMX) as phosphodiesterase inhibitor, 2% BSA, and various concentrations of VIP or its structurally related peptides. These were porcine secretin, donated by Pr. E. Wiinsch (Max Plank Institut i'fir Biochemie, 8033 Martinsried, Germany), human peptide with N-terminal histidine and C-terminal methionine (PHM), human peptide with Nterminal histidine and C-terminal valine (PHV), and helospectin I purchased from Peninsula Laboratories (St. Helens, England), human pancreatic growth hormone-releasing factor (hpGRF) given by Drs. J. Rivier and W. Vale (Salk Institute, San Diego, CA), helodermin purchased from Novabiochem (Switzerland), and synthetic ovine hypothalamic PACAP-38 synthesized by solid-phase techniques and generously donated by Dr. A. Arimura, Tulane University, Belle Chasse, CA) (34). The reaction was stopped by adding 50 #1 of 11 N HC104. Cyclic AMP was determined using a radioimmunoassay (18). Soft agarose cloning experiments. About 4 × 104 FFC from two culture specimens were resuspended in low calcium medium B supplemented with 0.3% agarose and seeded on a layer of 0.5% agarose in petri dishes. The dishes were examined after 12 to 15 days of culture at 37 ° C. Tumorigenicity. For transplantation studies, FFC or adherent cells were used. About 5 X 106 cells were suspended in 100 #1 PBS and inoculated subcutaneously into six athymic nude mice (Nude/Swiss), 5 wk old. Tumorigenicity was activated in the mice by the slow release of 17/3estradiol after subcutaneous implantation of one 0.5-mg estradiol tablet per mouse (ICI). For periods of up to 3 mo. all animals were monitored weekly for the presence of a tumor. RESULTS

Growth Properties of Primary HMEC Cultures All 42 specimens obtained from reduction mammoplasty patients were successfully propagated in culture. Epithelial cell colonies ap-

CHARACTERIZATION OF NORMAL HMEC TABLE 1 CHARACTERIZATION OF SOME DETERMINANTS OF THE MAMMARY EPITHELIA USING IMMUNOFLUORESCENCE AND IMMUNOCYTOCHEMICAL STAINING ON HUMAN MAMMARY EPITHELIAL CELLS IN PRIMARY CULTURE" Antigen

MAb

Percent Cell +

Large Cells

Small Cells

Pan-cytokeratin Cytokeratin 18 Cytokeratin 19 Vimentin a-Actin smooth muscle Tubulin Mammary epithelial mucin Breast cancer associated gp Breast cancer associated gp EMA cALLA Protein pS2

KL1 M9 Ksl9-1 V-3250 1A4 N-356 DF3 1BE12 7B10 E/29 J5 /

100% >90% >80% 26% 0% 100% 77% 71% 50% 90% (500. Inset, phase contrast microscopy of FFC cultured in collagen gel ) helodermin > PHM, PHV > helospectin I >> hpGRF, secretin. Similar selectivity was observed for the VIP receptors characterized in human breast cancer cell lines, in brain, and in peripheral tissues (12,17,49). These results therefore suggest that VIP has a regulatory physiologic function in the normal human mammary gland (4). In conclusion, our HMEC in culture were characterized according to a set of specific human mammary epithelial determinants, they maintained an extended growth potential that was dependent on growth factors, and in athymic nude mice did not display any neoplastic potential. This implies that the intrinsic proliferation properties of our primary cultures can be used to study individual or combined effects of hormones and growth factors and therefore to induce neoplastic progression in normal human mammary epithelial cells after genomic insertion of functional oncogenes.

ACKNOWLEDGEMENTS

3.

4. 5. 6. 7.

8.

9. 10. 11.

12.

13. 14. 15.

16. 17.

18.

19. 20.

The authors thank Dr. Henri Magdelenat of the Curie Institute, Paris, France for his advice on steroid and EGF receptor assays. 21.

REFERENCES 1. Ances, I. G. Serum concentrations of epidermal growth factor in human pregnancy. Am. J. Obstet. Gynecol. 115:357-362; 1973. 2. Band, V.; Sager, R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that sup-

22. 23.

723

ports long-term growth of both cell types. Proc. Natl. Acad. Sci. USA 86:1249-1253: 1989. Bandyopaadhyay, G. K.; Imagawa, W.; Wallace, D. R., et al. Proliferative effects of insulin and epidermal growth factor on mouse mammary epithelial cells in primary culture. J. Biol. Chem. 263:75677573: 1988. Berthon, P.; Delbourg, V.; Taillemite, J-L., et al. Functional VIP receptors in normal, spontaneously immortalized and cancerous mammary epithelial cells. Regul. Peptides 26:141a; 1989. Blose, S. H.; Mehzer, D. I.; Feramisco, J. R. 10 nm filament induced to collapse in cell microinjection with antibodies against tubulin. J. Cell Biol. 95:229a; 1982. Boynton, A. L.; Whitfield, J. F.; lsaacs, R. J., et al. Control of 3T3 cell proliferation by calcium. In Vitro 10:12-17; 1974. Boynton, A. L.; Whitfield, J. F.; Isaacs, R. J., et al. The control of human WI-38 proliferation by extracellular calcium and its elimination by SV40 virus-induced proliferative transformation. J. Cell. Physiol. 92:241-248; 1977. Brennan, J. K.; Mansky. J.; Roberts, G., et al. Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation in vitro. In Vitro 11:354-360; 1975. Brown. C. F.; Teng, C. T.; Pentecost, B. T., et al. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells. Mol. Endocrinol. 3:1077-1083; 1989. Carpenter, G.; Cohen, S. Epidermal growth factor. Ann. Rev. Biochem. 48:193-216; 1979. Chaproniere, D. M.; McKeehan, W. L. Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. Cancer Res. 46:819-824; 1986. Chastre. E.; Emami, S.; Gespach, C. Expression of membrane receptors and (proto)oncogenes during the ontogenic development and neoplastic transformation of the intestinal mueosa. A review article. Life Sci. 44:1721-1742; 1989. Colomb, E.; Berthon, P.; Dussert, C., et al. Estradiol and EGF requirement for cell cycle progression of normal human mammary epithelial cells in culture. Int. J. Cancer 49:932-937; 1991. Cussenot, O.; Berthon, P.; Faille, A., et al. Immortalization of human adult normal prostatic epithelial cells by liposomes containing SV40. J. Urol. 143:881-886; 1991. Fitzpatrick, S. L.; Brightwell, J.; Wittliff, J. L., et at. Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen receptor and progesfin receptor levels. Cancer Res. 44:34483453; 1984. Fitzpatrick, S. L.; Lachance, M. P.; Schultz, G. S. Characterization of epidermal growth factor receptor and action on human breast cancer cells in culture. Cancer Res. 44:3442-3447; 1984. Gespach, C.; Bawab, W.; De Cremoux, P., et al. Pharmacology, molecular identification and functional characteristics of vasoactive intesfinal pepfide receptors in human breast cancer cells. Cancer Res. 48:5079-5083; 1988. Gespach, C.; Hui Bon Hoa, D.; Rosselin, G. Regulation by vasoactive intestinal peptide, histamine, somatostatin-14 and -28 of cyclic adenosine monophosphate levels in gastric glands isolated from the guinea pig fundus and antrum. Endocrinology 112:1597-1606; 1983. Gown, A. M.; Vogel, A. M. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies. J. Cell Biol. 95:414-424; 1982. Greaves, M. F.; Hariri, G.; Newman, R. A., et at. Selective expression of the common acute lymphoblastic leukemia (gpl00) antigen on immature lymphoid cells and their malignant counterpart. Blood 61:628-639; 1983. Hammond, S. L.; Ham, G. H.; Stampfer, M. R. Serum free growth of human mammary epithelial cells: a rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc. Natl. Acad. Sci. USA 81:5435-5439; 1984. Haslam, S. Z. Cell to cell interactions and normal mammary gland function. J. Dairy Sci. 71:2843-2854; 1988. Hennings, H.; Michael, D.; Cheng, C., et al. Calcium regulation of

724

24,

25. 26. 27.

28. 29. 30.

31. 32.

33. 34.

35. 36. 37. 38. 39. 40. 41. 42.

43. 44. 45.

BERTHON ET AL. growth and differentiation of mouse epidermal cells in culture. Cell 19:245-254; 1980. Imai, Y.; Leung, C. K.; Friesen, H. G., et at. Epidermal growth factor receptors and effect of epidermal growth factor on growth of human breast cancer ceils in long-term tissue culture. Cancer Res. 42:4394-4398; 1982. Kufe, D.; Inghirami, G.; Abe, M., et al. Differential reactivity of a novel monoclonal antibody (DF3) with human malignant versus benign breast tumors. Hybridoma 3:223-232; 1984. Lechner, J.; Haugen, A.; Autrup, H., et al. Clonal growth of epithelial cells from normal human bronchus. Cancer Res. 41:2294-2304; 1981. Le Meuth, V.; Farjaudon, N.; Bawab, W., et al. Characterization of binding sites for VIP-related peptides and activation of adenylate cyclase in developing pancreas. Am. J. Physiol. 260:G265-G274; 1991. Lin, J. J. Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc. Natl. Acad. Sci. USA 78:2335-2339; 1981. Malet, C.; Gompel, A.; Spritzer, P., et al. Tarnoxifen and hydroxytamoxifen isomers versus estradiol effects on normal human breast cells in culture. Cancer Res. 48:7193-7199; 1988. Marie, J-C.; Hui Bon Hoa, D.; Jackson, R., et al. The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22. Regul. Pept. 12:113-123; 1985. McGrath, C. M.; Soule, H. D. Calcium regulation of normal human mammary epithelial cell growth in culture. In Vitro 20:652-662; 1984. McGrath, C. M.; Soule, H. D. Renewal inhibition of human mammary cell growth in vitro: cortisol and the recruitment of cells to terminal differentiation. J. Cell. Physiol. 116:385-396; 1983. Medina, D.; Oborn, C. J. Growth of preneoplastic mammary epithelial cells in serum-free medium. Cancer Res. 40:3982-3987; 1980. Miyata, A.; Arimura, A.; Dahl, R. R., et at. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem. Biophys. Res. Commun. 164:567-574; 1989. Moll, R.; Achtstatter, T.; Becht, E., et al. Cytokeratins in normal and transitional epithelium. Am. J. Pathol. 132:123-144; 1988. Murphy, L. C.; Murphy, L. J.; Dubik, D., et at. Epidermal growth factor gene expression in human breast cancer cells: regulation of expression by progestins. Cancer Res. 48:4555--4560; 1988. Murphy, L. J.; Sutherland, R. L.; Stead, B., et at. Progestin regulation of epidermal growth factor receptor in human mammary carcinoma cells. Cancer Rcs. 46:728-734; 1986. Nunez, A. M.; Jakowlew, S.; Briand, J. P., et al. Characterization of the estrogen-induced pS 2 protein secreted by the human breast cancer cell line MCF-7. Endocrinology 121:1759-1765; 1987. Ormerod, M. G.; Steele, K.; Westwood, J. H., et al. Epithelial membrane antigen: partial purification, assay and property. Br. J. Cancer 48:533-541; 1983. Osborne, C. K.; Hamilton, B.; Nover, M. Receptor binding and procesS: ing of epidermal growth factor by human breast cancer cells. J. Clin. Endocrinol. Metab. 55:86-93; 1982. Pancino, G-F.; Charpin, C.; Calvo, F., et at. A novel monoclonal antibody (7B10) with differential reactivity between human mammary carcinoma and normal breast. Cancer Res. 47:4444-4452; 1987. Pancino, G-F.; Charpin, C.; Osinaga, E., et al. Characterization and distribution in human tissues of a glycoproteic antigen defined by monoclonal antibody 1 BE 12 raised against the human breast cancer cell line T47D. Cancer Res. 50:7333-7342; 1990. Pekonen, F.; Partanen, S.; Makinen, T., et al. Progestin regulation of epidermal growth factor in human mammary carcinoma cells. Cancer Res. 46:728-734; 1986. Petersen, O. W.; Hoyer, P. E.; Van Deurs, B. Frequency and distribution of receptor-positive cells in normal, nonlactating human breast tissue. Cancer Res. 47:5748-5751; 1987. Petersen, O. W.; Van Deurs, B. Distinction between vascular smooth muscle cells and myoepithelial cells in primary monolayer cultures

46. 47.

48.

49. 50. 51.

52. 53. 54. 55. 56. 57. 58.

59. 60. 61. 62.

63.

64. 65.

66. 67.

of human breast tissue. In Vitro Cell. Dev. Biol. 25:259-266; 1989. Petersen, O. W.; Van Deurs, B. Preservation of defined phenotypic traits in short-term cultured human breast carcinoma derived epithelial cells. Cancer Res. 47:856-866: 1987. Prud'homme, J-F,; Jolivet, A.; Pichon, M-F., et al. Monoclonal antibodies against native and denaturated forms of estrogen-induced breast cancer protein (BCEI:pS2) obtained by expression in Escherichia coll. Cancer Res. 50:2390-2396: 1990. Richards, J.; Imagawa, W.; Balakrishnan, A., et al. The lack of effect of phenol red or estradiol on the growth response of human, rat and mouse mammary cells in primary culture. Endocrinology 123:1335-1340; 1988. Rosselin, G. The receptors of the VIP family peptides (VIP, secretin, GRF, PHI, PHM, GIP, glucagon and oxytomodulin). Specificities and identity. Peptides 7:89-100; 1986. Rudland, P. S.; Barraclough, R. Stem cells in mammary gland differentiation and cancer. J. Cell Sci. Suppl. 10:95-114; 1988. Rudland, P. S.; Hugues, C. M.; Ferns, S. A., et al. Characterization of human mammary cell types in primary culture: immunofluorescent and immunocytochemical indicators of cellular heterogeneity. In Vitro Cell. Dev. Biol. 25:23-36; 1989. Russo, J.; Mills, M. J.; Moussatli, M. J., et al. Influence of breast development on the growth properties of primary cultures. In Vitro Cell. Dev. Biol. 25:643-649; 1989. Seabright, M. A. A rapid banding technique for human chromosomes. Lancet 2:971-972; 1971. Skalli, O.; Ropraz, P.; Trzeciak, A., et al. A monoclonal antibody against ,~-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103:2787-2796; 1986. Smith, J. A.; Winslow, D. P.; Rudland, P. S. Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial and stromal cell lines in culture. J. Cell. Physiol. 119:320-326; 1984. Soule, H. D.; Maloney, T. M.; Wolman, S. R., et at. Isolation and characterization of a spontaneously immortalized human breast cell line, MCF-10. Cancer Res. 50:6075-6085; 1990. Soule, H. D.; McGrath, C. M. A simplified method for passage and long-term growth of human mammary epithelial cells. In Vitro Cell. Dev. Biol. 22:6-12; 1986, Stampfer, M. R.; Bartley, J. C. Induction of transformation and continuous cell lines from normal human mammary epithelial cells exposed to benzo-a-pyrene. Proc. Natl. Acad. Sci. USA 82:2394-2398; 1985. Stampfer, M. R.; Hallowes, R. C.; Hackett, A. J. Growth of normal human mammary cells in culture. In Vitro 16:415-425; 1980. Stoner, G.; Babcock, M. Influence of growth factors on proliferation of normal and chemically transformed rat esophageal epithelial cells. Proc. Am. Assoc. Cancer Res. 23:42; 1982. Taketani, Y.; Oka, T. Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy. FEBS Lett. 152:256-260; 1983. Taylor-Papadimitriou, J.; Stampfer, M.; Bartek, J., et at. Keratin expression in human mammary epithehal cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J. Cell Sci. 94:403-413; 1989. Valverius, E. M.; Bates, S. E.; Stampfer, M. R., et at. Transforming growth factor a production and epidermal growth factor expression in normal and oncogene transformed human mammary epithelial cells. Mol. Endocrinol. 3:203-214; 1989. Viac, J.; Reano, A.; Brochier, J., et al. Reactivity pattern ofa monoclohal anti-keratin antibody (KL1). J. Invest. Dermatol. 81:351-354; 1983. Yang, J.; Richards, J.; Bowman, P., et at. Sustained growth and threedimensional organization of primary mammary tumor epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci. USA 7 6 : 3 4 0 1 3405; 1979. Yang, N-S.; Kube, D.; Park, C., et al. Growth of human mammary epithehal cells on collagen gel surfaces. Cancer Res. 4 1 : 4 0 9 3 4100; 1981. Zajchowski, D.; Band, V.; Pauzie, N., et al. Expression of growth factors and oncogenes in normal and tumor-derived human mammary epithehal cells. Cancer Res. 48:7041-7047; 1988.

Characterization of normal breast epithelial cells in primary cultures: differentiation and growth factor receptors studies.

The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures from breast tissue bi...
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