Proc. Nati. Acad. Sci. USAVol. 85, pp. 136-140, January 1988Cell Biology

HT-29 cells are an in vitro model for the generation of cell polarityin epithelia during embryonic differentiation

(intestinal cell differentiation/membrane markers/immunocytochemistry/adenocarcinoma)

ANDRE LE BivIC*t, MICHEL HIRNa, AND HUBERT REGGIO**Laboratoire de Diffdrenciation Cellulaire, Facultd des Sciences, and tCentre d'Immunologie de Marseille-Luminy, 70 Route Leon Lachamp, F-13288Marseille Cedex 9, France

Communicated by G. E. Palade, September 14, 1987

ABSTRACT A monoclonal antibody that recognizes amembrane glycoprotein specific for the apical membrane ofhuman colonic epithelial cells has been used to follow thedifferentiation and polarization of a cell line, HT-29, derivedfrom a human colon adenocarcinoma. When these cells formeda polarized epithelium, the antigen was concentrated at theapical plasma membrane. It was also found intracellularly invesicles and vacuoles. When HT-29 cells were undifferentiatedand unpolarized, the antigen was not expressed significantly atthe plasma membrane but was found concentrated in themembranes of intracellular vacuoles. Cells not yet organizedinto an epithelium may thus synthesize a membrane proteinspecific for their future apical membranes and store it intra-cellularly until the polarization process takes place. Interme-diary stages of differentiation were occasionally recognized.They are characterized by a small number of cells surroundingan intercellular lumen. These lumina displayed apical mem-brane features (the presence of the apical antigen, of somemicrovilli, and of junctional complexes), although the cellswere not fully differentiated. The differentiation process inHT-29 cells is apparently similar to that observed duringembryonic development ofthe intestine. Therefore, HT-29 cellsrepresent a useful model system to study epithelial differenti-ation in vitro.

Attempts have been made to study cell polarity as illustratedby the specific organization of various domains of the plasmamembrane in epithelia. Most studies have been performed onpolarized cell lines to elucidate the mechanism by which cellssort membrane proteins destined for each domain (for reviewsee refs. 1-3). In contrast, the generation of polarizedepithelia during embryonic differentiation is poorly under-stood. In the intestine, a polystratified epithelium is rapidlyremodeled into a monolayer covering longitudinal ridges ofconnective tissue (4-6). The polarization of the cells in thismonolayer has not been studied, because membrane markersexpressed at these early stages were unavailable. In thisrespect the cell line HT-29, originating from a human colonadenocarcinoma (7), seems to be a useful in vitro model, sincean early expressed apical antigen has been characterized (8,9) and since the degree of differentiation and polarization canbe modulated in vitro under specific culture conditions (10,11). In the presence of 25 mM glucose, the cells grow as anonpolarized multilayer; whereas, when carbohydrate isdepleted, they differentiate and acquire the morphologicalfeatures ofabsorptive epithelial cells or goblet cells organizedin a polarized monolayer. This cell line has some biochemicalfeatures similar to those of the embryonic colon, as exem-plified by the type and the molecular weight ofsome enzymes(11, 12).

In this paper we have used the monoclonal antibody (mAb)517 that recognizes a membrane glycoprotein (antigen 517)specific for the apical membrane of human colonic epithelialcells (8, 9) to follow the fate of apical membrane duringdifferentiation of HT-29 cells. When these cells were orga-nized in an epithelial monolayer, antigen 517 was concen-trated at the apical plasma membrane. When they were notso organized, the protein was not or was weakly expressed onthe plasma membrane but was concentrated in the membraneofintracellular vacuoles. Thus, even in undifferentiated cells,this protein is sorted from basolateral membrane proteins thatwere found uniformly distributed over the cell surface.

MATERIALS AND METHODSReagents. mAb 517 is an IgG1 produced by immunization

with HT-29-18 cells and characterized in human colon as anapical membrane glycoprotein (8, 9). Peroxidase-labeled Fabfragments of sheep anti-mouse and anti-rabbit IgG werepurchased from Pasteur Production (Marnes la Coquette).Molecular weight markers were from Bio-Rad. Phenylmeth-ylsulfonyl fluoride, papain, and endoglycosidase F were fromBoehringer Mannheim. Glutaraldehyde was from Ladd Re-search Industries (Burlington, VT). Diaminobenzidine hy-drochloride, gelatin, human transferrin, proteinase K, andsaponin were from Sigma. Culture medium and supplementswere from Flow Laboratories and from Eurobio (Paris).

Tissue Culture. HT-29-18, a multipotent clone of HT-29cells, was a gift from D. Louvard and C. Sahuquillo-Merino(Institut Pasteur, Paris). They were grown in Dulbecco'smodified Eagle's medium supplemented with 25 mM glucose,2 mM glutamine, human transferrin (10 .ug/ml), penicillin (50international units/ml), streptomycin (50 ug/ml), and 10%(vol/vol) fetal calf serum. The medium was changed everyday to prevent glucose exhaustion, which could initiatedifferentiation (10). Differentiated cells were grown in thesame conditions except glucose was replaced by 5 mMgalactose and each nonessential amino acid was added to afinal concentration of 0.1 mM. The cells were confluent 1week before use.

Immunofluorescence. Cells were fixed with 2% (vol/vol)formaldehyde in 0.1 M potassium phosphate buffer (pH 7.4)at 40C for 1 hr. Thin frozen sections (0.5 jum) were cutperpendicularly to the monolayer according to Tokuyasu (13)and were incubated with fluids from an antibody-producing Bhybridoma diluted 1:50 in isotonic phosphate-buffered salinecontaining 0.2% gelatin. Rhodamine-labeled sheep anti-mouse IgG was diluted 1:500 in the same mixture containingin addition 5% (vol/vol) rat serum, which significantlyreduced the background. Cultured cells grown on coverslips

Abbreviation: mAb, monoclonal antibody.tTo whom reprint requests should be addressed.

136

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Proc. Natl. Acad. Sci. USA 85 (1988) 137

were monitored for surface or internal labeling as describedby Wang et al. (14).

Electron Microscopy. Immunoperoxidase labeling was per-formed as described by Reggio et al. (15). For HT-29 cells,the best fixation was obtained with 2% (vol/vol) formalde-hyde and 0.01% glutaraldehyde in 0.1M potassium phosphate(pH 7.2). Controls included mAbs with different stainingpatterns or mAbs irrelevant to this cell type.Membrane Preparation. Membranes were purified from

tissue culture cells according to Gratecos et al. (16). The firstpellet was used as a total cellular membrane fraction. Mem-brane proteins were extracted with Triton X-114 according toBordier (17).Gel Electrophoresis and Immunoblotting. NaDodSO4/

PAGE was carried out as described by Laemmli (18), using5-15% linear polyacrylamide gradient slab gels. The proteinswere transferred from the gel to nitrocellulose (19), and theantigen was detected by peroxidase-conjugated antibodiesand visualized with diaminobenzidine (20). The reaction wasquantitated by scanning the filters on a Vernon photodensi-tometer.

Specific Hydrolysis. Papain cleavage was performed ac-cording to Louvard et al. (21). Proteinase K hydrolysis (2mg/ml) was carried out for 12 hr at 370C in 10 mM Tris-HCI,pH 7.4. Endoglycosidase F was used according to Elder andAlexander (22).

RESULTSCharacterization of the Antigen 517. In HT-29-18 cell

extracts, mAb 517 recognized a major band with an apparentmolecular mass of 170 kDa (Fig. 1, lanes b and d). Densi-

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FIG. 1. (A) Identification of the antigen recognized by the mAb517 in cultured cell lines. Total solubilized membrane proteins (100jug) were separated by NaDodSO4/PAGE and stained with Coomas-sie blue (lanes a and c) or by immunoperoxidase labeling of a replicawith the mAb 517 (lanes b and d). Total membrane extract fromundifferentiated HT-29-18 cells (lanes a and b) and from differenti-ated HT-29-18 (lanes c and d) are shown. (B) Enzymatic digestion ofthe antigen 517 in differentiated HT-29-18 cells. An extract of totalsolubilized membranes (100 jtg) was enzymatically digested andelectrophoresed on NaDodSO4/polyacrylamide gels, and a replicawas labeled with the mAb 517 by the immunoperoxidase technique(lanes e, f, g, and h). Lanes: e, total solubilized membrane extractincubated without enzyme; f, same sample as in lane e afterproteinase K treatment; g, total solubilized membrane extractincubated without enzyme; h, same sample as in lane g afterendoglycosidase F treatment. (C) Extraction of the antigen 517 fromdifferentiated cells with Triton X-114. Lanes: i, detergent phase; j,aqueous phase. Molecular weight (mw) markers: myosin, P-galac-tosidase, phosphorylase b, bovine serum albumin, and ovalbumin.

tometry indicated that differentiation induced a 5-fold in-crease in the amount ofantigen 517 (data not shown). A minorband was occasionally observed at 90 kDa. This was pre-sumably a degradation product, as shown by proteinase Kdigestion, which resulted in the accumulation at this positionof all material that could be revealed in immunoblots (Fig. 1,lane f). Antigen 517 was also sensitive to papain (data notshown). Since the immunologically cross-reactive antigenfrom human colon has been shown to be an N-glycosylatedmembrane protein (9), a similar characterization was per-formed for antigen 517 on membrane extracts from HT-29418cells. It was cleaved by endoglycosidase F (Fig. 1, lane g).Upon extraction with Triton X-114, the antigen 517 wasrecovered in the detergent phase (Fig. 1, lanes h and i). Thesedata indicate that the antigen obtained from cultured cellswas a membrane glycoprotein similar to the one observed inhuman colon (9).Immunofluorescence Localization. Antigen 517 was local-

ized in frozen thin sections (Fig. 2) or in whole cells afterpermeabilization with Triton X-100 (data not shown). Bothmethods gave consistent results, but frozen thin sectionsprovided more precise information. When HT-29-18 cellswere grown in glucose-containing medium, a multilayer wasformed. The plasma membrane of most of the cells was notlabeled or only faintly labeled, but the antigen 517 wasexpressed quite strongly in intracellular vesicles (Fig. 2 a andb). All cells were labeled, but the intensity of the reactiondisplayed some heterogeneity from cell to cell. In rareinstances, antigen 517 was concentrated at the plasma mem-brane of a few cells where it labeled the intercellular cyststhat formed occasionally within the multilayer (Fig. 2 c andd). Control experiments performed with a mAb that recog-nized a basolateral membrane glycoprotein in human colon(molecular mass, 38 kDa; ref. 8) produced complementarylabeling: the plasma membrane was labeled except in thecysts (Fig. 2 e andf). When these cells further differentiatedand became organized into an epithelium after culturing themin the presence of galactose, the mAb 517 intensely labeledthe apical but not the basolateral plasma membrane (Fig. 2 gand h). Internal vesicles were labeled as in the undifferenti-ated cells, and few intercellular cysts were still present andlabeled. Surface staining indicated that nearly all cells of themonolayer expressed the antigen 517 (data not shown).The time course of the formation of a polarized monolayer

in HT-29-18 cells plated in glucose-free medium was ofparticular interest. Under these conditions, polarization as anepithelial monolayer did not commence before confluencyand was found to take several days. At early stages numerouslarge intercellular cysts could be observed when other areaswere already forming a polarized monolayer (Fig. 2 i and j).Therefore, antigen 517 should prove to be a useful marker toinvestigate polarization in HT-29-18 cells in early differenti-ation stages, when intestinal enzymes such as alkaline phos-phatase normally found in adult colon are not yet expressed(6, 23).

Ultrastructural Studies. The labeling of undifferentiatedHT-29-18 cells with the mAb 517 displayed heterogeneityfrom cell to cell as observed by immunofluorescence; twophenotypes probably corresponding to different stages ofdifferentiation were observed.

(i) Undifferentiated cells not polarized as an epithelium.These cells represented the major fraction of the cell popu-lation that normally grew as a multilayer (90-95%). Actuallythis fraction was dependent on culture conditions. It de-creased when the cells were kept at confluency withoutchanging the medium daily, presumably because differenti-ation began as a result of glucose consumption. Serialsectioning indicated that most of the nonpolarized cells werenot in contact with the intercellular lumina described laterand were devoid ofjunctional complexes.

Cell Biology: Le Bivic et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 2. Immunofluorescent localization of the antigen 517 on frozen sections from HT-29-18 cells with the corresponding phase-contrastmicrographs. (a-d) Undifferentiated HT-29-18 cells. (e and f) Same cells with a mAb specific for the basolateral domain in colonic cells (8).(g and h) Differentiated HT-29-18 cells 1 week after confluency. (i and j) Differentiated HT-29 cells 2 days after confluency. Arrows designatethe membrane of the cysts. Arrowheads show discontinuous labeling of apical membranes during early stages of differentiation.

In these cells the antigen 517 was also detected intracellu-larly in vesicles and vacuoles (Fig. 3a). However, in rareinstances, weak labeling was seen on the plasma membrane.Thus, HT-29 cells were able to sort an apical membraneprotein before being organized as an epithelium. The labelingwas not due to endogenous peroxidase since it was notobserved with nonimmune serum or with antibodies havingother specificities (data not shown). The observation ofvacuoles was not due to cross-sectioning of invaginations ofthe plasma membrane; by serial sectioning, such vacuoleswere never found in connection with extracellular spaces.

(ii) Polarized andpartially differentiated cells. These wereobserved in rare instances where the cells formed intercel-lular cysts similar to lumina and occasionally displayedwell-formed microvilli on their surfaces. In this case, thelabeling was concentrated in the membrane of the cysts (Fig.3b). These were closed by structures morphologically similarto tight junctions that prevented the diffusion of the antigen517 within the membrane, since the adjacent plasma mem-brane was not labeled (Fig. 3b). Intracellular vacuoles,

labeled with mAb 517 were still observed, although theyappeared to be less numerous.

Fully differentiated and polarized HT-29 cells could belabeled on their apical surface without membrane permeabili-zation (Fig. 4). In this case the mAb 517 labeled the brushborder, indicating that the antigenic determinant recognizedwas oriented toward the outer part of the plasma membrane.In general, fully differentiated and polarized tissue culturecells were difficult to permeabilize with saponin, except whenfixed with formaldehyde alone. In this case, intracellularvesicles and vacuoles were also labeled, but the morphologywas poorly preserved (data not shown).

DISCUSSIONIn this paper we have used a monoclonal antibody specific forthe apical membrane of human colonic epithelial cells tofollow the differentiation and polarization of a cultured cellline (HT-29-18) originating from a human colon adenocarci-noma. In these cells the antigen recognized was a membraneglycoprotein with an apparent molecular mass of 170 kDa.

138 Cell Biology: Le Bivic et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 3. Immunoperoxidase localization of the antigen 517 in undifferentiated HT-29-18 cells. Reaction product was observed intracellularlyin vesicles and vacuoles (a). The plasma membrane was not labeled (arrowheads). When intercellular cysts were observed (<10%1 of the cells),the labeling was also concentrated on the membrane of the large intercellular lumina (Lu) (b); junctional complexes (JC) enclosed the lumina,and microvilli were often present. The adjacent plasma membrane was not labeled (arrowheads). Arrows indicate translucent vesicles oftenvisible in the vicinity of the lumina, which were not labeled (b). G, Golgi complex; N, nucleus; M, mitochondrion. (Unstained section.)

When the cells differentiated and organized in a continuousepithelium, the antigen was concentrated at the apical mem-brane.The localization of antigen 517 in nondifferentiated HT-

29-18 cells before their organization as a polarized epitheliumis of particular interest since the antigen was not significantlyexpressed at the plasma membrane but was concentrated inthe membrane of intracellular vacuoles. The nature and therole of such structures remains to be established, they mayrepresent a storage unit for apical proteins or they may bepart of the endosome system. These results indicate thatundifferentiated and nonpolarized HT-29 cells have a func-tional sorting system that prevents at least one apical proteinfrom accumulating in the plasma membrane in the absence ofapical domains and before the development oftightjunctions.When such apical domains were created by the formation ofintercellular lumina between a few cells, antigen 517 accu-mulated in that membrane. The presence of such a sortingsystem suggests that the plasma membrane of undifferenti-ated HT-29 cells is similar to a basolateral membrane. Asimilar hypothesis has been proposed (24) and may be ofgeneral interest concerning undifferentiated cells. Our ob-servations also indicate that an apical plasma membraneprotein may be synthesized and stored intracellularly beforethe formation ofan epithelium in which the cells are polarizedand have distinct apical and basolateral plasmalemmal do-mains. This seems also to be the case for sucrase-isomaltase,but the internal localization of the molecule was not inves-tigated (25). Upon differentiation, this enzyme was expressedat the cell surface but only by a fraction of HT-29 cells

(50-60%) (25). The nature of the signal necessary for polar-ization is still unknown. The formation of a junctionalcomplex between two cells may be the crucial event. Apicalmembrane proteins generally expressed in differentiated andundifferentiated cells, such as antigen 517, should prove to beuseful tools to elucidate the differentiation process.The role of the intercellular cysts is not yet clear. Similar

structures have been described (26) in MDCK cells, but inthat case, there was no apparent relation with development.In HT-29 cells, it is known that they are not in contact withthe external medium (27).Although the cysts involve a small fraction of the cell

population, antigen 517 allows their identification as an apicaldomain even when they do not display microvilli. In HT-29-18 cells, grown in glucose, it is clear that the intercellularcysts are not fully differentiated since alkaline phosphatase,which is a differentiation marker in colon (6), was not foundat their apical membrane (23). Actually, villin, a cytoskeletalprotein involved in the bundling of actin filaments in micro-villi (28) is expressed intracellularly as a diffuse set ofmolecules in undifferentiated HT-29-18 cells (29). Similarobservations were made during the development of thechicken embryo (30). We did not detect villin in the microvilliof the cysts by immunofluorescence or immunoperoxidaselabeling (unpublished results). Upon differentiation, bothproteins are integrated in the brush border of HT-29 cells andtheir expression is enhanced (23, 29, 31). Intercellular cystsmay represent initiation sites for the differentiation and forthe reorganization ofthe epithelium in a monolayer. Actually,the final step of the differentiation process in HT-29 cells

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Cell Biology: Le Bivic et A 139

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Proc. Natl. Acad. Sci. USA 85 (1988)

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FIG. 4. Immunoperoxidase localization of the antigen 517 in

differentiated HT-29-18. Cells grown in galactose formed a polarized

monolayer and displayed a well-developed brush border that was

strongly labeled by mAb 517. BB, brush border; BL, basolateral

membrane; N, nucleus; M, mitochondrion. (Unstained section.)

could occur by a mechanis 'M similar to that observed during

embryogenesis in the small intestine (5, 32) or the colon (6).

The similarity of the molecular mass of the antigen 517 in the

embryo and in these cells, as opposed to the adult colon (8),

supports the hypothesis of the embryonic character of HT-29

cells. Therefore, they may prove to be a useful in vitro model

system to study the events leading to the polarization of an

epithelium during embryogenesis.

We thank A. Berud for technical assistance; M. Berthoumieux for

photographic work; D. Louvard and C. Sahuquillo-Merino for the

gift of cell lines; E. Coudrier and C. Huet for helpful discussions; M.

Pinto for useful advice; L. Blair and L. Leserman for reading the

manuscript; and A. Zweibaum for the gift of intestinal biopsies. This

work was supported b the Centre National de la Recherche

Scientifique (UA 179), by the Institut National de la Sant6 et de la

Recherche Mmuicale (CRE 847 012), and by the Fondation pour la

RechercheMe dicaleFranHaise.

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