Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells

EDMOND GODFROED1*, MAURICE GEUSKENS2't, THIERRY DUPRESSOIR3, ISABELLE PARENT1 and CLAUDE SZPIRER1

1Laboratoire de Biologie du De'veloppement and 2Laboratoire de Cytologie et d'Embryologie Mol4cu.la.ires, Department de Bwlogie MoUculaire, University Libre de Bruxelles, Belgium 3Laboratoire d'Oncologie MoUculaire, U186 INSERM et CNRS URA 0156, Institut Pasteur, BP 245, F-59019 Lille cedex, France * Present address: Unite de Neurochimie, Faculty de M&iecine, University Catholique de Louvain, Clos Chapelle-aux-Champs, 30 bte 31, B-1200, Bruxelles, Belgium t Author for correspondence at Laboratoire de Cytologie et d'Embryologie mol6culaires, Department de Biologie Mol6culaire, University libre de Bruxelles, 67 rue des Chevaux, B-1640, Rhode-St-Genese, Belgium

Summary

Normal human mammary epithelial cells and estab- surface of normal mammary cells in primary culture. lished tumour cells of the same origin express three By ultrastructural immunocytochemistry, these cyto- to eight cytokeratins, which are distributed through- keratins were localized on blebs formed by the cell out the cytoplasm in the form of intermediate surface. Cytokeratins 8,18 and 19 were also detected filaments. The combined use of the iodogen and the in the culture medium of mammary carcinoma two-dimensional gel electrophoresis methods has cells. allowed us to demonstrate the presence of cytokera- tins 8, 18 and 19 on the outer surface of established human mammary carcinoma cells, in particular Key words: cytokeratins, mammary carcinoma cells, MCF-7, MCF-7 cells, while they were absent from the immunocytochemistry, electron microscopy, 2-D electrophoresis.

Introduction structures was demonstrated in many epithelial cultured cells or epithelial tissues (Franke et al. 19816; Franke et al. The cytoskeleton of eukaryotic cells is composed of three 1985; Geiger et al. 1983). On the other hand, a recent study important filamentous structures: the 8 run actin fila- on mouse mammary epithelial cells indicates that cyto- ments (microfilaments), the 23 run tubulin filaments keratins and other cytoskeletal proteins are noncovalently (microtubules) and the 7-11 run intermediate-sized fila- associated with lipids, especially with neutral lipids and ments. The latter structure is the most complex filamen- phospholipids, suggesting direct participation of the lipid tous system. In most epithelial cells, the constitutive bilayer in the association of the plasma membrane with proteins of intermediate filaments are a complex class of tonofilaments (Asch et al. 1990). In addition, studies on a proteins called cytokeratins. At least 20 different cyto- human cervical carcinoma cell line, ME 180, indicate that keratins have been identified in human epithelial cells some keratins penetrate the cell membrane to reach the (Moll et al. 1982; Franke et al. 1981a; Franke et al. 1983; extracellular environment (Vidrich et al. 1983; Zimmer- Moll et al. 1990). It has been shown that expression of man et al. 1982), while a shedding of proteolytically cytokeratins depends on the epithelium type as well as on processed keratins in the culture medium of MCF-7 the degree of cellular differentiation (Franke et al. 1981a; carcinoma epithelial cells has been reported by Chan et al. Kim et al. 1983; Kim et al. 1984a; Fuchs et al. 1981). In (1986). We demonstrate here that human mammary addition, carcinoma cells continue to express to a large carcinoma cells expose cytokeratins 8, 18 and 19 on their extent the cytokeratins of the corresponding normal cells outer surface, while normal mammary epithelial cells do (Franke et al. 1979; Wu and Rheinwald, 1981; Kim et al. not exhibit this property. Cytokeratins 8, 18 and 19 were 19846; Van Muijen et al. 1984; Van Muijen et al. 1986; Ben- also found in the culture medium of mammary carcinoma Zeev et al. 1986). cells. In both normal and carcinoma cells, the cytokeratin network exhibits an arborizing distribution throughout the cytoplasm, terminating at the plasma membrane Materials and methods (Henderson and Weber, 1981). The nature of the specific interactions between the cytokeratin filaments and the Mammary carcinoma cell lines cell membrane remains unclear. However, it is known that Cultures of established human mammary cell lines (MCF-7, the cytokeratin filaments are attached to desmosomes Evsa-T, HBH0O) were carried out at 37 °C in Dulbecco's modified (Franke et al. 1985); interconnection between these two Eagle's medium (DMEM) supplemented with 10% foetal calf Journal of Cell Science 99, 595-607 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 595 serum (FCS) (Gibco), kanamycin (10/jgml 1) and mycostatin of Oakley et al. (1980), with minor modifications. After electro- (lO/zgrnl"1). MCF-7, derived from a human pleural effusion of a phoresis, the gels were soaked in 10% acetic acid and 50% breast adenocarcinoma was isolated by Soule et al. (1973). Evsa-T methanol for 30 min, then in 1 % acetic acid and 5 % methanol for is a human breast carcinoma cell line established by Lippman et 2h, and finally, in 10% unbuffered glutaraldehyde for 30 min. al. (1977). HBL-100 is an epithelial cell line derived from human After the last step, the gels were extensively washed with breast milk of an apparently normal lactating woman (Gaffney et distilled water and placed in ammoniacal silver solution (0.075% al. 1979). HBL-100 cells are able to form colonies in soft agar. NaOH, 0.35% NH4OH, 0.9% AgNO3, w/v) for 15min. The Recently, Caron de Fromentel and co-workers (1985) demon- staining became visible when the gels were transferred in a fresh strated that HBL-100 harbours simian virus 40 (SV4O) genetic solution of 0.0025% citric acid and 0.02% formaldehyde. The information stably integrated into the cellular genome; this SV40 staining was stopped with 10 % acetic acid. SDS-polyacrylamide genetic information possesses transforming activity (Vanhamme gel electrophoresis (SDS-PAGE) was performed as described by and Szpirer, 1988). The MCF-7 carcinoma cell line and the HBL- Laemmli (1970). 100 non-tumoural cell line were obtained from the American Type Culture Collection. The Evsa-T carcinoma cell line was obtained Anti-cytokeratin antibodies from Dr G. Leclercq (Jules Bordet Institute, Brussels, Belgium). The following monoclonal antibodies were used: the anti- Clones of the MCF-7 and of the Evsa-T cell lines isolated in our cytokeratin KER 20-80 (Monosan) and PKK1 (Labsystems). laboratory were used during the experiments. These antibodies were used in the characterization of the cytokeratin expressed by human mammary carcinoma cell lines Normal mammary cell culture and normal mammary epithelial cells. The monoclonal antibody Normal human mammary epithelial cells (NHME cells) were 16-88, a human IgM, was received from Dr M. G. Hannah used as normal control cells. They were grown and maintained (Bionetics Research, Inc., 1330-A Piccard Drive, Rockville, according to the method of Soule et al. (1973) with some Maryland, USA) via Professor J. Urbain (Laboratoire de Physiolo- modifications. Fresh mammary gland pieces, surgically excised gie Animate, D6partement de Biologie Mol6culaire, Umversite during mammoplasties of healthy, premenopausal, multiparous Libre de Bruxelles, Belgium). The characterization of this human or nulliparous women, were obtained from Dr J. Pellerin (Service monoclonal antibody has been described by Haspel et al. (1985). de Chirurgie Plastique Reconstructrice, H6pital Regional Cen- The anti-cytokeratin 18 (clone CK2) and anti-cytokeratin pan tral, F 59000 Lille, France). Fat was carefully removed from the (clone Lu-5) antibodies were obtained from Boehringer Mann- tissue prior to manual slicing. Slices were then cut into 2-5 mm3 heim Biochemical and used to examine the organization of the pieces and further digested with collagenase (type III, Worth- cytokeratin network in normal and transformed mammary ington; 200 I.U. ml"1) and hyaluronidase (type II, Sigma; epithelial cells. The anti-cytokeratin (clone CAM 5.2) specific for lOOi.u.ml"1) in DMEM supplemented with 5% FCS, penicillin cytokeratins 8, 18 and 19 was obtained from Becton Dickinson (lOOi.u.ml"1), streptomycin (lOO^gml"1) and fungizone Immunocytometry Systems. The polyclonal anti-keratin (code (1.25^gml"1), at 37°C, overnight, with gentle shaking. The 1084) was obtained from Bio-Yeda. This antibody stains specifi- digested tissue was then filtered through a 60 fan nylon mesh cally networks of tonofilaments in epithelial cells in culture and (Hyland) and retained cell clumps were seeded on plastic culture in tissue sections. It does not react with mesenchymal, glial, dishes (9 mm diameter, Falcon) and fed with Joklik modified neuronal or muscle cells. Eagle's medium (JMEM) supplemented with Ca2+-depleted FCS, insulin UO^gml"1), cholera toxin (lOOngml"1), cortisol (0.5^M) Protein immunoblotting 1 and epidermal growth factor (20ngml~ ). Antibiotics and Proteins separated either by two-dimensional gel electrophoresis antifungics were added to that basal medium. Each NHME or by SDS— polyacrylamide gel electrophoresis (SDS-PAGE) were culture was assessed as epithelial by immunofluorescent staining transferred electrophoretically to nitrocellulose sheets for 60 min (Debus et al. 1982) of the cytokeratin 18 with a monoclonal at 4°C and 400 mA in 20 mM Tris-HCl buffer (pH 8.3) containing antibody (Boehringer Mannheim). 150 mM glycine and 20 % methanol (Towbin et al. 1979). Keratins were visualized by double antibody-peroxidase staining using Iodination of membrane surface proteins of human antibodies to keratins as primary antibody and peroxidase- mammary epithelial cells conjugated rabbit anti-mouse IgG as secondary antibody. When 16-88 antibody was used as primary antibody, the secondary Iodogen method. Cells were gently harvested with a rubber 7 antibody was a monoclonal mouse antibody against human IgM policeman and washed three times with PBS; 10 cells were 126 (Janssen Biochemica) and the third antibody was a peroxidase- radiolabelled for 5 min with 500 ^cytoskeletons as described by Oyamada and Mori (1985): they were briefly washed with cytoskeleton stabilization buffer (CSS) Gel electrophoresis (10mM pipes, lmM MgCl2, 2mM EGTA, 5% glycerol, 5% Two-dimensional gel electrophoresis was performed as described polyethylene glycol (PEG), pH6.9) and then permeabilized with by O'Farrell (1975). The gels were stained by the silver technique CSS buffer supplemented with 0.2 % Triton X-100 for 4 min and 596 E. Godfroid et al. washed again in CSS buffer. After two rinses in PBS, cells were foetal calf serum. Labelled cells and the corresponding con- fixed for 10 min with 3.5 % formaldehyde in PBS and incubated in ditioned culture medium were harvested. The culture medium O.lMglycine methanol/PBS(l/l, v/v) for 30 min. After this step, was dialysed for 48 h, at 4°C, in 50 mM ammonium hydrogen the cells were washed in ethanol/PBS for 30 min. The cells were carbonate, at pH7.5, and then lyophilized. Labelled cells and the incubated with anti-cytokeratin 18 from Boerhinger Mannheim corresponding lyophilized culture medium were solubilized in or anti-cytokeratins from human callus (Monosan) routinely used 200 ;/l lysis buffer, as described by O'Farrell (1975). at 1:20 dilution. After a 1 min rinse in PBS, coverslips were processed for protein A-immunogold staining and silver intensi- fication using the Bio-Rad kit adapted from the methods of Results Danscher and Norgaard (1983) and De Brabander et al. (1977). In the case of NHME cultures, the cells were fixed with methanol/ Characterization and spatial organization of cytokeratins acetic acid (1/1, v/v) and then permeabilized and washed in CSS expressed in human mammary epithelial cells buffer as described above. After one rinse in TBS buffer (20 mM In general, human mammary epithelial cells synthesize a Tris-HCl, 500 min NaCl, pH7.5) for 15 min, the cells were incubated with antibodies and processed for the immunoperoxi- subset of three to eight cytokeratins (cytokeratins 5, 7, 8, dase staining in the following sequence: (1) 3 % gelatin in TBS for 14, 15, 17, 18 and 19, according to the catalogue published lh at room temperature followed by washes with TTBS (TBS by Moll et al. 1982). We have analysed by two-dimensional supplemented with 0.05% Tween 20) (3x10 min). (2) Anti- gel electrophoresis and immunoblotting, the cytokeratin cytokeratin monoclonal antibody (Monosan, 1:20) diluted in expression in two human carcinoma cell lines (Evsa-T and TTBS/gelatin (TTBS supplemented with 1 % gelatin), followed by MCF-7), in the non-tumoral (but transformed) mammary washes with TTBS (3x20 mm). (3) Rabbit anti-mouse IgG cell line HBL-100, and in several primary cultures of antibodies conjugated with horseradish peroxidase diluted in normal human mammary epithelial cells (NHME cells) TTBS/gelatin for lh at room temperature, followed by washes isolated from fresh mammary gland pieces, surgically with TTBS (3x20min). (4) 0.1M Tris-HCl buffer, pH7.6, excised during mammoplasties of healthy women. Three containing 0.04% 3,3'-diaminobenzidine (DAB, Sigma) and 0.3% to five cytokeratins are expressed in the samples tested: H2O2for 10-15 min. After three rinses with TBS (3x15 min), and one wash with PBS, the cells were mounted with 50 % glycerin in cytokeratins 8, 18 and 19 are present in cells of carcinoma PBS. lines and in NHME cells; cytokeratins 7 and 17 are also detected in Evsa-T and in NHME cells. These results are Electron microscopy summarized in Table 1. It must be noted that keratin 19 For standard observations, cells were seeded in 4-well culture was weakly detected in HBL-100 cells, while, according to dishes (Nunc) and fixed in situ for 2 h, at room temperature, in 3 % Bartek et al. (1986), this keratin was not present in these glutaraldehyde in 0.1M cacodylate buffer, at pH7.2. After an cells. overnight wash in buffer, the cells were fixed again for 1 h in 2 % OsO^ in the same buffer. After ethanol dehydration, the cells were Using specific monoclonal antibodies (CAM 5.2, pKKl embedded in situ in Epon. Ultrathin sections, made parallel to the and Ker 20-80) to cytokeratins, we have examined by growth plane, were stained with uranyl acetate and lead citrate immunocytochemistry the cytokeratin network in these and examined in a AEI EM6B electron microscope, at 60 kV. For normal and mammary tumour cells. Cells of the HBL-100 immunocytochemistry of surface cytokeratins, cells were rinsed x line show a disorganized network of the cytokeratin in PBS, incubated with the primary antibody (16-88, 65/jgml~ filaments, often concentrated in a juxtanuclear position of PBS containing 0.01M NaN3) for lh at 37°C, washed three whereas the tumour-derived cell lines (Evsa-T and MCF- times in PBS and fixed for 5 min at 4°C in 0.05 % glutaraldehyde in PBS. After three washes in PBS and one in PBS containing 7) exhibit an arborizing distribution of the cytokeratin 0.1M glycine for 20 min, the cells were incubated with the filaments throughout the cytoplasm terminating at the secondary antibody, a murine monoclonal antibody against plasma membrane (Fig. 1). In the NHME cells, the human IgM (MH 15-01-M02 from Janssen Biochemica; SO^gml"1 cytokeratin filaments form a network throughout the of TTBS/gelatin) for 1 h at 37°C. After three washes in PBS, the cytoplasm (Fig. 1). cells were incubated for lh at 37"C in goat anti-mouse IgG In conclusion, the expression of cytokeratins (level of antibodies adsorbed to 15 nm gold particles (GAM IgG-Gl5 from expression, type of cytokeratins expressed) and the Amersham Life Sciences Products) diluted 1/100 in TTBS/ gelatin. This step was followed by three washes in PBS and distribution of the cytokeratin filaments throughout the fixation in 3% glutaraldehyde in 0.1M cacodylate buffer, at cytoplasm are similar in the carcinoma and normal pH7.2, for 30 min at room temperature. The cells were then postfixed in OSO4, dehydrated, embedded and sectioned as for the standard observations. When a mouse monoclonal antibody (Lu-5, 20/igml"1 or CAM 5.2, 25/igml"1) was used as first antibody, the Table 1. Cytokeratin expression in human mammary cells were treated directly, after three washes in PBS, with GAM carcinoma cell lines and the corresponding normal IgG-G15 as secondary antibody. For intracellular cytokeratins, epithelial cells cells were washed with PBS, treated with CSS buffer as for light Keratins' microscopic immunocytochemistry, then with the same solution containing 0.2% Triton X-100, and again with CSS buffer, each Cells 17 18 19 time for 5 min at 37°C. The cells were then fixed with 0.05% HBL 100 glutaraldehyde in PBS for 10 min, at 4°C, washed in PBS and MCP-7 glycine-containing PBS, as described above. The incubations with Evsa-T secondary and tertiary antibodies were also carried out as for NHMEt surface cytokeratin localization. —, Negative; +, positive; + + , strongly positive; + + + , very strongly 3B positive. [ S]methionine labelling of MCF-7 cells •Cytokeratin numbers are from the catalogue published by Moll and After seeding in Falcon dishes, MCF-7 cells were allowed to grow co-workers (1982). Cytokeratins were identified on two-dimensional gel to subconfluence and were then washed three times with large electrophoresis according to their electrophoretic properties. volumes of PBS. Cells were preincubated for 1 h in methionine- Immunoblots were performed from two-dimensional gel electrophoresis. free DMEM medium, and then incubated for 4, 8, 16 and 24 h in The monoclonal anti-cytokeratin antibodies used were KER 20-80, pKKl and CAM 5.2. methiomne-free medium with added [^SJmethionine (Amer- t Normal human mammary epithelial cells. sham; 250//Ci/106 cells) supplemented with 1% dialysed

Cell surface cytokeratins 597 Fig. 1. Cytokeratin network in HBL 100 (A), MCF-7 (B), Evsa-T (C) and NHME cells (D). Carcinoma cells were permeabilized with 0.2% Triton X-100 before incubation with the anti-cytokeratin 18 monoclonal antibody and then processed for immunogold staining (A,B,C). NHME cells were incubated with anti-cytokeratin 8, 18 and 19 antibody (Monosan) and processed for immunoperoxidase staining (D). X1250. human mammary epithelial cells, whereas HBL-100 cells (Fig. 2B). This result shows that the labelled proteins, and show a lower level of cytokeratin expression and a peculiar in particular the cytokeratins, were derived from live distribution of cytokeratin type intermediate filaments cells. In order to confirm these results and to minimize the (Fig. 1). handling of cells before iodination, undetached mammary carcinoma cells growing in monolayers, were incubated with iodobeads and Na I. Cytokeratins 8,18 and 19 were Iodination of intact cells labels cytokeratins also radiolabelled under these conditions. A picture The possibility that cytokeratins might be accessible on identical to Fig. 2A was obtained (data not shown). the outer side of the cell surface was investigated by To confirm further the finding that the labelled iodination of intact cells. We used the iodogen method, cytokeratins were exposed on the outer side of cell surface, which permits the labelling of proteins exposed at the iodinated MCF-7 cells were treated with 0.2 % trypsin for outer face of the plasma membrane. The protein extracts of 5 min, lysed and analysed by two-dimensional gel electro- the radiolabelled cells were then separated by two- phoresis; under these conditions, the labelling of all dimensional gel electrophoresis (Fig. 2A). The autoradio- cytokeratins disappeared while numerous labelled pep- graph in Fig. 2A shows that the three major cytokeratins tides of smaller molecular weight appeared (Fig. 2C). In (cytokeratins 8,18 and 19) expressed in the mammary cell addition, when MCF-7 cells were harvested by trypsin lines were radiolabelled; actin was not labelled. As a treatment before iodination (rather than using a rubber control experiment, protein extracts were prepared from policeman, or rather than being iodinated as adherent lysed MCF-7 cells and then labelled by the iodogen cultures), no labelling of cytokeratins could be detected. method: in this mixture, all cytokeratin variants (basic These last results exclude the possibility that iodination and acids) were labelled as well as actin and other affects intracellular proteins. polypeptides (Fig. 2D). Intact normal human mammary epithelial cells were To exclude the possibility that the labelled cytokeratins iodinated using the iodobead method. In this case, resulted from the intracytoplasmic labelling of lysed cells, cytokeratins were not radiolabelled (Fig. 2E). iodinated cells were replated and only the cells attached Taken together, these results strongly indicate that within lh (i.e. viable cells) were collected and then cytokeratins are exposed on the surface of human examined by two-dimensional gel electrophoresis. The mammary carcinoma cells and not on the surface of the cytokeratin labelling subsisted in these conditions corresponding normal cells. 598 E. Godfroid et al. ftl 6.1 5.7 5.2 i i i

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D O 6.1 5.7 5.2 6.1 5.7 5.2 UJ I I I I I _L 0 2

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Fig. 2. Two-dimensional gel electrophoresis of protein extracts of MCF-7 cells, which were radiolabelled according to the iodogen method. (A) Intact MCF-7 cells. (B) MCF-7 cells replated for 1 h. (C) MCF-7 cells treated with trypsin after iodination. (D) Lysed MCF-7 cells in dilute PBS buffer prior to the iodination procedure. (E) Intact NHME. All the autoradiographs were exposed for 24 h except those in D (4h). Arrowheads indicate the actin position. 8, 18 and 19 indicate the position of the basic and acidic variants of cytokeratins 8, 18 and 19, respectively. In C and E, the arrows indicate the theontical position of cytokeratins 8, 18 and 19.

Cell surface cytokeratins 599 B

8

•\ .19

Fig. 3. Immunonoblots of protein extracts of MCF-7 cells separated by two-dimensional gel electrophoresis. Keratins are visualized by double antibody-alkaline phosphatase staining method using CAM 5.2 (A) and 16-88 (B) antibodies as primary antibody. 8, 18 and 19 indicate the position of cytokeratins 8, 18 and 19, respectively.

Reactivity of external proteins with monoclonal NHME), although cytokeratins were efficiently immuno- antibodies to cytokeratins precipitated in each case (Western analysis of the In order to determine whether the cytokeratins detected immunoprecipitates; data not shown). on the surface of the cells by iodination were accessible to In the second procedure, the 16-88 monoclonal anti- antibody molecules, a panel of monoclonal antibodies body, but none of the monoclonal anti-cytokeratin anti- (KER 20-80, PKK1, CK2, Lu-5, CAM 5.2) and one bodies was found to react with labelled cytokeratins. polyclonal anti-keratin (code 1084) antibody prepared SDS-gel electrophoresis of the immmunoprecipitates against cytokeratins were screened for their capacity to from MCF-7 and Evsa-T (data not shown) mammary react with epitopes of external cytokeratins. In addition, carcinoma cells showed that five different radiolabelled we also used a monoclonal antibody prepared against polypeptides (molecular weight: 29, 40, 45, 52, 75(xlO3) colorectal carcinoma cells, called antibody 16-88 (Haspel had been precipitated (Fig. 4A, MCF-7 lane I). These et al. 1986). Before immunoprecitating the cell extracts, proteins were analysed by two-dimensional gel electro- 3 we checked the specificity of the monoclonal antibodies by phoresis (Fig. 4C). Three proteins (40, 45 and 52xlO Mr) the immunoblotting procedure (Fig. 3 and Table 1). As were detected by autoradiography and upon immunoblot- shown in Fig. 3A (and as expected), the CAM 5.2 antibody ting were recognized by specific monoclonal antibodies identifies three proteins in MCF-7 cells that have the against cytokeratins (CAM 5.2; data not shown). The 29 electrophoretic properties of cytokeratins 8, 18 and 19. 3 and 75xlO Mr proteins were not visible after two- Fig. 3B shows that the 16-88 antibody detects the same dimensional gel electrophoresis (Fig. 4C); these two three proteins as the anti-cytokeratin CAM 5.2 (although proteins thus have an isoelectric pH outside the pH range 16-88 reacts more weakly with the cytokeratin 8); it thus (4.5 to 6.5) used and therefore are not cytokeratins. This appears that the 16-88 antibody reacts with cytokeratins. observation also excludes the possibility that the 29 and As shown below (Fig. 7), the 16-88 antibody also reacts 75 x 103 proteins are heavy and light chains of immuno- with cytokeratins from normal (NHME) cells. globulins artefactually labelled (their isoelectric pH is 6.5 MCF-7, HBL-100 and NHME cells were radiolabelled by and 5.0, respectively). No labelled protein was detected in the iodogen method. The labelled cells were either directly the case of HBL-100 cells. The 16-88 monoclonal antibody solubilized in the lysis buffer and incubated with mono- did not detect any protein in the case of NHME cells clonal antibodies reacting with cytokeratins (the lysis (Fig. 4A, NHME lane I). buffer used does not dissociate most antigen-antibody These results show that one human monoclonal anti- complexes; see Materials and methods and Fig. 4) or were body known to react with cytokeratins (16-88, Fig. 3) is immediately incubated in the presence of these mono- able to react with three cytokeratins (cytokeratins 8, 18 clonal antibodies (prior to the lysis procedure). and 19) when intact mammary carcinoma cells are In the first procedure, labelled cytokeratins were incubated with this antibody. Therefore, at least a portion recognized by anti-cytokeratin antibodies and by the of these peptides must be present on the outer surface of 16—88 monoclonal antibody in the case of the carcinoma the carcinoma cells. In contrast, they are not present on cell line Evsa-T (data not shown) and MCF-7 (Fig. 4A, the cell surface of normal mammary epithelial cells MCF-7:lane L). Similar but much weaker labelling was (NHME cells), and cannot be detected on the surface of the observed in the case of the SV40-transformed cell line non-tumoural (but transformed) mammary cells from the HBL-100 (Fig. 4A, HBL-100:lane L), probably as the result HBL-100 line. This last result could be explained, as of the low level of cytokeratin expression in these cells (see above, by the low level of cytokeratin expression in these Table 1; and the work of Chang et al. 1982). No labelled cells, which would be below the limit of the sensitivity of protein was detected in the case of NHME cells (Fig. 4A, the method. In addition, two other peptides, which do not 600 E. Godfroid et al. NHME HBL 100 MCF-7 I L T H x10~3 -75

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Fig. 4. (A) Autoradiograph of a one-dimensional SDS-PAGE of 12B pi immunoprecipitated I-labelled 6.5 6.15.7 5.2 5.0 6.56.15.75.25.0 proteins from normal mammary i II ii i II II epithelial cells (NHME) and cells 3 from mammary cancer lines x10~ (HBI^lOO and MCF-7) with the 16—88 antibody. Cells were lysed with TDS buffer after iodination - 75 - performed on intact cells, and then incubated with the 16-88 antibody (L), or the iodination - 52 - and the immmunoprecipitating - 45 - lfl-u procedures were performed on - 40 - intact cells (I). (B and C) Autoradiographs of two- dimensional gel electrophoresis of MCF-7 cells either lysed before addition of the 16-88 antibody (B; see L, in A) or treated with the antibody before B lysis (C; see I, in A). possess the electrophoretic properties and the molecular associated with the surface blebs of Evsa-T cells, and weight of cytokeratins, were iodinated and co-precipitated weaker labelling with the surface of HBL-100 cells (data under these conditions; these two peptides either share not shown). When the incubation of MCF-7 cells with the antigenic determinants with cytokeratins or form a first antibody was omitted, no surface labelling (Fig. 5D), complex with cytokeratins present on the cell surface. or only a few occasional gold particles, was observed, while the omission of the first two antibodies completely abolished the labelling. Ultrastructural observations Permeabilized cells were also incubated with the 16-88 In order to determine the location of the cytokeratins on antibody. In this case, the gold labelling was associated the cell surface, an electron-microscopic study of cells with the tonofilaments throughout the cytoplasm of the treated with several monoclonal antibodies was carried cells (Fig. 6B), confirming the previous observation that out. this antibody reacts with cytokeratins. Mammary epithelial cell lines (MCF-7, Eusa-T and No surface labelling of MCF-7 cells was observed with HBL-100). Intact MCF-7 cells were incubated with the the monoclonal anti-cytokeratin pan Lu-5 or with the 16-88 antibody, with a mouse anti-human IgM secondary monoclonal anti-cytokeratin CAM 5.2, while a labelling of antibody, and finally with a gold-conjugated goat anti- the cytoplasmic tonofilaments was observed after cell mouse IgG antibody. Moderate labelling was observed at permeabilization (Fig. 6A: Lu-5). the surface of numerous bulbous expansions (called blebs) Primary cell cultures of normal human mammary projecting from the cell surface (Fig. 5A). At higher epithelium. Cells from three different donors were used. magnification, the gold particles appeared associated with The epithelial origin of these cells was confirmed by the a flocculent material (Fig. 5B, 5C). Similar labelling was presence of tonofilaments (Fig. 7B) and of occasional duct- Cell surface cytokeratins 601 •Jf

5A

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B

D

Fig. 5. (A,B,C) MCF-7 cell incubated with the 16-88 as first antibody, followed by immunogold localization of its binding sites. (A) Shows the density of the surface labelling, localized on blebs, at low magnification. The arrowheads point at the gold particles, n, nucleus; if, intermediate filaments or tonofibrils. X31.500. (B and C) Illustrate the association of the labelling with flocculent material at the surface of the blebs, x 90 000. Bar, 0.1 /an. (D) Shows the absence of labelling at the surface of the blebs when incubation with the first antibody is omitted before incubation with the mouse anti-human IgM antibody and the gold-labelled tertiary antibody, x50000. Bar, I/on.

602 E. Godfroid et al. Fig. 6. (A) MCF-7 cell permeabilized before incubation with anti-cytokeratin pan Lu-5, followed by immunogold localization of its binding sites. The cytoplasmic tonofilament bundles are labelled, x90 000. (B) MCF-7 cell permeabilized before incubation with the 16—88 antibody and immunogold localization of its binding sites. Gold particles are associated with cytoplasmic tonofibrils. X90000. Bars, 0.1 //m. like vacuoles in their cytoplasm, and of desmosomes at the culture medium: the cells were incubated for 4, 8, 16 intercellular junctions (not shown in the figures). Micro- and 24 h in methionine-free medium with added villi, but practically no blebs, were observed at their [35S]methionine, supplemented with dialysed foetal calf surface (Fig. 7A). serum. The concentrated radiolabelled MCF-7 culture After incubation of these cells with the 16-88 antibody, medium waa then analysed by two-dimensional gel no surface labelling was observed (Fig. 7A). The same electrophoresis followed by autoradiography. Radioactive result was obtained with the CAM 5.2 antibody. spots at the positions of cytokeratins were clearly When permeabilized cells were incubated with the identified under these conditions (no radioactive spot at 16-88 antibody, the gold labelling was associated with the the position of actin molecules was detected; see Fig. 8). cytoplasmic tonofilaments (Fig. 7B) more particularly at These labelled proteins were detected even after the the periphery of the cells. shorter incubation time (4h). Using the immunoblotting technique with monoclonal antibodies against cytokera- Presence of cytokeratins in the culture medium of MCF-7 tins, we showed that these radioactive proteins were cells indeed cytokeratins 8,18 and 19 (cytokeratin 8 itself being distributed into 7 isoelectric point variants). Chan and co-workers (1986) have reported the identifi- cation and characterization of a major group of polypep- tides released into the culture medium by MCF-7 cells. It Discussion was considered that these proteins were apparently breakdown products of intracellular keratin proteins. We Previous studies have reported that cytokeratins are have also studied the proteins released by MCF-7 cells into distributed throughout the cytoplasm, terminating at the Cell surface cytokeratins 603 7A B

Fig. 7. (A) Mammary epithelial cells from normal donor, in primary culture, incubated with the 16—88 antibody, followed by Lmmunogold localization of its binding sites. No labelling of the cell surface, x 30 000. Bar, 0.5/on. Inset: enlargement of the framed region: x 90 000. Bar, 0.1/an. (B) Mammary epithelial cell permeabil^zed before incubation with the 16-88 antibody and immunogold localization of its binding sites. The gold particles are associated with the cytoplasmic tonofilaments, more particularly at the cell periphery. X30000. Bar, 0.5/un. Inset: enlargement of the framed region: X90000. Bar, 0.1/un.

604 E. Godfroid et al. 6.1 5.7 5.2

H ALB Fig. 8. Two-dimensional gel X1CT" electrophoresis of [36S]methionine-labelled proteins from culture medium of MCF-7 52 cells. The labelling time was 4 h. 45 The fluorograph was exposed for 40 19 12 h. 8, basic and acidic variants of cytokeratin 8; 18, basic and acidic variants of cytokeratin 18; 19, cytokeratin 19. ALB indicates the position of foetal calf serum albumin. The arrowhead indicates the theoritical position of actin.

plasma membrane. Recent biochemical studies have cytoplasmic tonofibrils (i.e. cytokeratin filaments) present suggested that intermediate filaments might be anchored in tumour and normal mammary cells. to the submembrane cytoskeleton via specific protein- This antibody clearly recognized an epitope shared by aceous linkers (Georgatos et al. 1987). Other studies have intermediate filament proteins; it reacted with cytokera- shown that cytokeratin intermediate filaments are at- tins 8, 18 and 19 in immmunoblotting experiments tached to the plasma membrane via the dense cytoplasmic (Fig. 3B). The results of immunoprecipitating exper- plaques of desmosomes (Franke et al. 1985), and that iments confirmed the above observations: the 16-88 cytokeratins can easily associate with lipids (Asch et al. antibody permitted the immunoprecipitation of five 3 1990). different proteins (29, 40, 45, 52 and 75xlO Mr) from The combined use of the iodogen method, which labels intact cells and three different proteins (40, 45 and 3 proteins exposed on the cell surface, and the two- 52xlO Mr) from permeabilized cells. Among the five 3 dimensional gel electrophoresis method, has permitted us different proteins, three proteins {40, 45 and 52xlO Mr) to detect the presence of cytokeratins at the surface of cells were cytokeratins 8, 18 and 19. The other ones (29 and 3 from mammary carcinoma lines. The analysis of the 75xlO Mr) could not be identified. culture medium of MCF-7 cells also showed that cytokera- It has been recently reported that a loss of plasma tins 8, 18 and 19 are released into the culture medium, in membrane integrity, not detectable morphologically, in agreement with the recent results of Chan and co-workers human mammary epithelial cells cultivated in vitro, (1986). On the other hand, ultrastructural studies have under conditions of oxygen-glucose deprivation, could shown an association of cytokeratins with secretory permit anti-keratin antibodies to bind to the cytoskeleton granules (Bendayan, 1985) and mucin droplets (Worhol within live, unfixed cells (Dairkee et al. 1991). However, and Roth, 1985). such transmembrane access of antibodies to cytoplasmic We have studied by immunocytochemistry the distri- keratins during our experiments, when the cells were not hution of keratin proteins on the surface of different permeabilized or lysed, can be excluded for the following mammary epithelial cells (tumour and normal). The reasons, most of which have already been pointed out membrane of many transformed and neoplastic cells above: (1) our cells were not cultivated under conditions of develops numerous bulbous expansions called blebs. The nutrient deprivation. (2) Only one monoclonal antibody number and kind of surface processes are generally (16-88) recognizes an epitope on carcinoma mammary greater on cancer cells in culture than on normal cells epithelial cells, and such an epitope is not detectable with (Kessel and Shih, 1974) and can vary according to the the same antibody on the normal counterparts of these conditions under which the cells are grown (Vic et al. cells. (3) Trypsin treatment abolishes the iodination of 1982). We have shown that cytokeratins are localized on carcinoma cell surface cytokeratins. (4) These surface blebs, at the surface of mammary epithelial tumour cells. cytokeratins are localized on surface blebs of carcinoma Such external cytokeratins were not detected in the case of cells by immunogold cytochemistry, at the electron normal mammary cells. microscope level. By ultrastructural immunocytochemistry, only one of In conclusion, using different biochemical and immuno- the monoclonal antibodies to cytokeratins (16-88, of logical methods, we have shown that cytokeratins 8, 18 human origin) recognized an antigenic determinant and 19 can be expressed at the surface of mammary present at the surface of human carcinoma mammary carcinoma cells but not at the surface of normal mammary cells, but absent from the surface of normal epithelial epithelial cells. Cytokeratins are localized on the surface mammary cells. This antibody also reacted with the of plasma membrane expansions or blebs. The presence of

Cell surface cytokeratins 605 cytokeratin molecules in the culture medium of mammary Phenotype. (ed. A. L. Levine, G. F. Vande Woude, W. C. Topp and J. D. Watson), vol. 1, pp. 177-190. Cold Spring Harbor Laboratory Press, tumour cells supports the idea that cytokeratins are NY. metabolically released (Brabon et al. 1984), possibly FRANKS, W. W., SCHILLER, D. L., HATZFELD, M. AND WINTER, S. (1983). during cell division (Bjorklund and Bjorklund, 1983). Protein complexes of intermediate-sized filaments: Melting of A protein isolated from human placenta and several cytokeratin complexes in urea reveals different polypeptide separation carcinomas, tissue polypeptide antigen (TPA), has been characteristics. Proc. natn. Acad. Sci. U.S.A. 80, 7113-7117. FRANKE, W. W., SCHILLER, D. L., MOLL, R., WINTER, S , SCHMID, E., studied previously (Bjorklund and Bjorklund, 1983). From ENGELBRECHT, I., DENK, H., KREPLER, R. AND PLATZER, B. (1981a). partial sequence data and immunocytochemistry studies, Diversity of cytokeratins: Differentiation specific expression of Weber et al. (1984) demonstrated that TPA is related to cytokeratins polypeptides in epithelial cells and tissues. J. molec. Biol. cytokeratins 8, 18 and 19. Using cultures of synchronized 153, 933-959. human cancer HeLa cells, Bjorklund and Bjorklund (1983) FRANKS, W. W., SCHMID, E., GRUND, C, MULLER, H., ENGELBRECHT, I., MOLL, R., STADLER, J. AND JARASCH, E.-D. (19816). Antibodies to high demonstrated that TPA is released from these cells into molecular weight polypeptides of desmosomes: Specific localization of the culture medium. They have also shown that the a class of junctional proteins in cells and tissues. Differentiation 20, released TPA is included in numerous Talebs'. It is thus 217-241. possible that the cytokeratins 8, 18 and 19 that were FRANKE, W. W., SCHMID, E., WEBER, K. AND OSBORN, M. (1979). Hela cells contain intermediate-sized filaments of the prekeratin type. Expl detected in the culture medium of mammary carcinoma Cell. Res. 118, 95-109. cells (MCF-7) could be derived from glomerular expan- FUCHS, E., COPPOCK, S. M., GREEN, H. AND CLEVELAND, D. W. (1981). sions (blebs) released from the cell membrane. Two distinct classes of keratin genes and their evolutionary significance. Cell 27, 75-84 GAFFNEY, E V., PIGOTT, D. A. AND GRIMALDI, M. A. (1979) Human We thank Professors R. Tencer and J. Urbain, Drs M. Slaoui serum and the growth of human mammary cells. J. natn. Cancer Inst. and E. Pays for helpful discussions, M. Goldfinger for help and 63, 913-918. advice, and Drs M. Hannah and N. Pomato (Bionetics Research, GEIGBR, B., SCHMID, E. AND FRANKE, W. W. (1983). Spatial distribution Inc., 1330-A Piccard Drive, Rockville, Maryland) for the gift of the of proteins specific for desmosomes and adhaerens junctions in 16-88 IgM antibody. 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Cell surface cytokeratins 607