Epithelial Polarity, Villin Expression, and Enterocytic Differentiation of Cultured Human Colon Carcinoma Cells: a Survey of Twenty Cell Lines1

[CANCER RESEARCH 48, 1936-1942, April 1, 1988] Epithelial Polarity, Villin Expression, and Enterocytic Differentiation of Cultured Human Colon Carcinoma Cells: A Survey of Twenty Cell Lines1

Isabelle Chantret,2 Alain Barbat, Elisabeth Dussaulx, Michael G. Brattain, and Alain Zweibaum

UnitÃ©deRecherches sur le MÃ©tabolismeet la DiffÃ©renciationde Cellules en Culture, INSERM U178, 94807 Villejuif Cedex, France [I. C., A. B., E. D., A. Z.], and Bristol-Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 [ M. G. B.J

ABSTRACT binding protein known to be specifically associated with the cytoskeleton of the brush border microvilli (17), has been re Twenty human colon carcinoma cell lines were studied for their ability ported only in Caco-2 (18) and HT-29 cells (18-20). Surpris to develop some of the characteristics of the normal intestinal epithelium, ingly, villin was expressed in the latter cells even when undif- e.g., epithelial polarity, presence of the actin-binding protein villin, or the ferentiated, although at a lower level and with a different occurrence of an enterocytic differentiation either when cultured under cellular localization (18-20). That other colon cell lines should standard conditions, as for Caco-2 cells, or when grown in a glucose-free medium, as for IIT-29 cells. Except for the regular presence of villin, also express some of these differentiation features is suggested by the observations that (a) villin is present in a number of which can be considered a marker of the colonie origin of the cells, the colon tumors3 (18) and (b) an enterocytic differentiation is cell lines of this study could be classified into four types with regard to their differentiation characteristics. In type 1 (only one cell line, i.e., associated with some colon tumors (21). The purpose of the Caco-2) the cells undergo spontaneously an enterocytic differentiation present work was to screen 20 established human colon carci characterized by a polarization of the cell layer with the formation of noma cell lines, with Caco-2 and HT-29 being used as controls, domes and the presence of an apical brush borderthe membraneof which for the expression of cell polarity, the presence of villin, and is endowed with hydrolases such as sucrase-isomaltase, lactase, amino- their ability to develop an enterocytic differentiation, either peptidase N, dipeptidylpeptidase IV and alkaline phosphatase. In type 2 when grown under standard culture conditions, like Caco-2 (three cell lines: HT-29, HCT-EB, and HCT-GEO) the cells are undif- cells (13) or when adapted to grow in a glucose-free medium, ferentiated when grown in the presence of glucose but undergo an enter like HT-29 cells (14-16). ocytic differentiation when grown in the absence of glucose. In type 3 (eight cell lines: HCT-GLY, HCT-FET, HCT-FRI, HCT-CBS, HCT- Ã•LA, (oils, HRT-18, and SW-1116) the cells are organized into a MATERIALS AND METHODS polarized ntonolayer with the formation of domes but without any enter ocytic differentiation characteristics, whatever the culture conditions. In Cell Lines and Culture Conditions. The 20 cell lines used were type 4 (eight cell lines: HCT-116a, HCT-R, HCT-RCA, HCT-Moser, established lines developed from human colorectal adenocarcinoma. HCT-8R, SW-480, LS-174T, and Vaco-9P) the cells are organized into The cell lines HT-29 (22), SW-480 (23), SW-1116 (23), LS 174 T (24), a multilayer without any feature of epithelial polarity or enterocytic and Caco-2 (25) were obtained from Dr. J. Fogh (Sloan Kettering differentiation, whatever the culture conditions. Memorial Cancer Center, Rye, NY); HRT-18 (26) and HCT-8R (27) from Dr. W. A. F. Tompkins (University of Illinois, Urbana, IL); Co- 115 (28) from Dr. B. Sordat (Swiss Institute for Experimental Cancer INTRODUCTION Research, Epalinges/Lausanne, Switzerland); and Vaco 9P (29) from Dr. J. K. V. Willson (William S. Middleton Memorial Veterans Hos Cultured human colon carcinoma cell lines provide a useful pital, Madison, WI). The cell lines HCT-ALA, HCT-CBS, HCT-EB,, tool to understand how the neoplastic process interferes with HCT-FET, HCT-FRI, HCT-GEO, HCT-GLY, HCT-Moser, HCT-R, HCT-RCA, and HCT-116a were established as reported (30). The cells the regulation of cellular differentiation. Only a limited number were cultured in Dulbecco's modified Eagle's minimum essential me of colon cell lines have been characterized thus far with regard dium (Eurobio, Paris, France) supplemented with 10% inactivated to differentiation features associated with the absorptive cells (56Â°C,30 min) fetal bovine serum (Boehringer, Mannheim), except for of the colonie epithelium such as its ultrastructural morphology, Caco-2 cells which needed 20% serum and 1% nonessential amino its structural and functional polarity, or the expression of acids. The cell lines were seeded at 4 x IO4 cells/cm2 in either plastic specific proteins. Cell polarity, as assessed by the ability of the flasks (Corning Glass Works, Corning, NY), or 35-mm Petri dishes cells to form domes when grown on an impermeable support and cultured at 37Â°Cin an atmosphere of 90% air-10% CO2. For (1), the presence of tight junctions, or vectorial transport prop growth in glucose-free medium the cells were switched from the stand erties, has been reported in six cell lines only, namely T84 (2, ard culture medium to the same medium devoid of glucose supple 3); Caco-2 (4, 5); HCA-7 (6); clones of HT-29 cells such as HT- mented with 10% dialyzed inactivated fetal bovine serum and 2.5 m\i 29-19A (7, 8), HT-29-18C,, and HT-29-18N2 (9); HRT-18; and inosine as reported previously (16). The culture medium was changed Co-115 (10). The occurrence of an enterocytic differentiation daily regardless of the culture condition under investigation. Enzyme Assays. Enzyme activities were performed on the cell ho- with the concomitant presence of an apical brush border and of brush border-associated hydrolases specific for the small intes mogenates (H) and on membrane fractions prepared according to the same protocol as used for the preparation of brush border-enriched tine (11) and the fetal colon (12) has been reported in two cell fractions (P2) (31). Sucrase (EC 3.3.1.48) activity was measured accord lines only: Caco-2, in which it occurs spontaneously under ing to the method of Messer and Dahlqvist (32), dipeptidylpeptidase standard culture conditions (13); and HT-29 cells which are IV (EC 3.4.14.5) according to that of Nagatsu et al. (33) using 1.5 mM differentiated only when adapted to grow in a glucose-free glycyl-L-proline-4-nitroanilide as substrate, aminopeptidase N (EC medium (14-16). The presence of villin, a Ca2+-regulated actin- 3.4.11.2) according to the method of Maroux et al. (34) using L-alanine- /j-nitroanilide as substrate, alkaline phosphatase (EC 3.1.3.1) according Received 7/2/87; revised 10/19/87; accepted 12/29/87. to the method of Garen and Levinthal (35) withp-nitrophenylphosphate The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in as substrate, and lactase activity (EC 3.2.1.23) according to the method accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of Messer and Dalqvist (32), with lactose as substrate, in the presence 1Supported by the Association pour la Recherche sur le Cancer, the Fondation FranÃ§aisepour la Recherche MÃ©dicale,thÃ©Groupement des Entreprises Fran 3R. Moll, S. Robine, B. Dudouet, and D. Louvard. Villin: a cytoskeletal Ã§aisesdans la Lutte contre le Cancer, and NIH Grant CA34432. protein and an early differentiation marker that is expressed in a limited array of 2To whom requests for reprints should be addressed. human adenocarcinomas, submitted for publication. 1936

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. MARKERS OF COLON1C CELL LINES of 0.2 HIMp-chloromercuribenzoate, an inhibÃtorof acid /3-galactosidase In Group 3 (8 cell lines) neither domes nor cysts could be (36). Results are expressed as milliunits/mg protein. One unit is defined as the activity that hydrolyzes 1 ^mol of substrate per min at 37Â°C. observed at phase contrast microscopy. At transmission elec tron microscopy the cells which were examined formed a mul Proteins were assayed by the method of Lowry et al. (37). tilayer with no apical tight junctions or brush border as already Antibodies. Mouse monoclonal antibody HBB 3/775/42 specific for shown for some of the cell lines of this group such as SW-480 dipeptidylpeptidase IV from human small intestine (38) was obtained orHCT-8Rcells(10). from Dr. H. P. Hauri (Biocenter of the University of Basel, Basel, Switzerland). Polyclonal rabbit antibody against sucrase-isomaltase Expression of Villin. Villin, as detected in cellular extracts by "Western blot" analysis, was present in all the tested cell lines purified from Caco-2 cells was produced in rabbits (39). Rabbit polyclonal antibodies against porcine villin (18) were a gift from Dr. Daniel grown under standard culture conditions. However, apparent Louvard (Institut Pasteur, Paris, France). Anti-rabbit sheep antiglobu- quantitative differences of expression from one cell line to lins labeled with horseradish peroxidase or fluorescein isothiocyanate another were observed (Fig. 5). The degree of expression of were obtained from Institut Pasteur Production (Marnes-la-Coquette, villin was independent of both the polarization and the differ France). Anti-mouse swine antiglobulins labeled with fluorescein iso entiation of the cells (Table 1). thiocyanate were obtained from Nordic Immunological Laboratories Enterocytic Differentiation. Previous experience showed that (Tilburg, The Netherlands). the differentiation of Caco-2 and HT-29 cells was complete and Indirect Immunofluorescence. Indirect immunofluorescence assays optimum only late after confluency (13-16). Consequently, all were performed as reported previously (13, 15) on cells grown in plastic flasks. cell cultures in the present study were evaluated 30 days after Transmission Electron Microscopy. Transmission electron micros seeding. With the exception of Caco-2 cells none of the 19 copy was performed as reported previously (13, 15), on cells grown in other cell lines exhibited any sign of enterocytic differentiation Petri dishes. when grown under standard culture conditions, i.e., in the Immunoblotting of Villin. Cellular extracts for immunoblotting analy presence of glucose. This was demonstrated by: (a) the absence sis of villin were prepared as follows. Cells grown on Petri dishes were of immunofluorescent staining of the cell layers with antibodies rinsed three times with phosphate-buffered saline. To each dish were directed against brush border-associated hydrolases (see Fig. added 400 ^1 of 20 mM Tris buffer, pH 8.8 containing 2 mivi CaCl2, 8a); (b) the activities of brush border-associated hydrolases 2% Triton X-100, and a cocktail of protease inhibitors [phenylmethyl- which were tested in both the cell homogenates (H) and a sulfonyl fluoride (1 mM), antipain (1 ^g/ml), pepstatin A (I /Â¿g/ml), membrane-enriched fraction (P2) and were found to be either and benzamidine (15 j/g/ml)]. After 5 min at room temperature, the mixture was centrifuged (2 min at 1,000 x g) and yielded one pellet absent, as in the case of sucrase and laclase, or very low, as in (l*i) and a supernatant (S, ). S, was centrifuged 2 min at 10,000 x g and the case of dipeptidylpeptidase IV, aminopeptidase N, and yielded the second supernatant S2 which was stored at â€”70Â°C.Proteins alkaline phosphatase (Table 1); and (c) the low enrichment of S2 (100 or 200 ^g) were separated on 7.5% sodium dodecyl sulfate- factor of enzyme activities (?2/H) which was always much lower polyacrylamide gel electrophoresis in the buffer system of Laemmli than found in Caco-2 cells (Table 1) and was consistent with (40) and transferred to nitrocellulose sheets by the method of Burnette the absence of an apical brush border as disclosed by transmis (41) as modified by Coudrier et al. (42). Villin was identified with sion electron microscopy. antivillin antibodies and revealed with the peroxidase technique. In order to find out whether some of these cell lines would express an enterocytic differentiation when grown in glucose- free medium, 15 cell lines, including HT-29 cells used as a RESULTS control, were switched to inosine-supplemented glucose-free Epithelial Polarity. The different cell lines could be classified medium. These cell lines were passaged every 10 days and into three groups on the basis of their ability to form a polarized tested for their differentiation characteristics on day 30 of epithelium, as judged by phase contrast and transmission elec passage 5, a passage number which has been found to be tron microscopy (Table 1). Group 1 (10 cell lines) was charac optimum for the highest degree of differentiation of HT-29 terized by the formation of domes which occurred after the cells cells to be expressed in such glucose-free culture conditions had reached confluency. The size and number of domes varied (16). Only two cell lines (HCT-GEO and HCT-EB), in addition from one cell line to another. Their morphology at phase to the previously described HT-29 cells (14-16), exhibited signs contrast microscopy (Fig. 1) was the same as already reported of enterocytic differentiation when cultured in the absence of for other dome-forming cell lines. At transmission electron glucose. Under such culture conditions both cell lines formed a microscopy all dome-forming cell lines appeared as polarized polarized monolayer with the presence of an apical brush border monolayers with the presence of apical tight junctions (Fig. 2). (Figs. 6 and 7). The degree and type of enzymatic differentiation With the exception of Caco-2 cells (13) none of these cell lines of both cell lines were, however, different from those observed displayed an apical brush border; only irregular microvilli were in HT-29 cells (Table 2). HCT-EB cells exhibited an enzymatic present on the apical surface of the cell layer (Fig. 2, inset). differentiation restricted to the expression of dipeptidylpepti Group 2 (HT-29, HCT-GEO, and HCT-EB) was characterized dase IV (Fig. 8) and alkaline phosphatase (Table 2); no brush by the presence, at phase contrast microscopy, of intercellular border-associated hydrolases could be detected in differentiated cysts (Fig. 3), the total surface of which covered 50% of the cell HCT-GEO cells as disclosed by the absence or immunofluores layer of HCT-GEO and HCT-EB at late stationary phase. At cent reactivity of the cell layer with anti-hydrolase antibodies transmission electron microscopy these cysts were of two types, (not shown) and the absence of increase of enzyme activities concomitantly present in each cell line; some were lined with a (Table 2). regular and well organized brush border (Fig. 4a) whereas others were devoid of it (Fig. 4b). In both types of cysts the DISCUSSION cells were polarized with the presence of apical tight junctions towards the lumen of the cysts (Fig. 4). Except for the presence The results reported here show that some of the differentia of these cysts the general organization of the cell layer was that tion features normally associated with the structural or func of a nonpolarized multilayer with no brush border being present tional organization of the absorptive cells of the colonie epithe at its apical surface, i.e., towards the culture medium (Fig. 4b). lium are either totally, partially, or not at all preserved in 1937

Table 1 Polarization and differentiation characterization of 20 cultured human colon carcinoma cell lines grown under standard culture medium conditions (25 mm glucose) Electron microscopyCell activities"S17615658.900000000000000000000000000000000000000DPP-IV13512959.621874.17304.39424.7111079.714493.5749711605.5284.0451.311655.9393241154.8835036252.50448344.49434.81719111.252162.33.1AP20617998.72126.07334.75295.821051404823.48708.88405.05122.416619212416.5210577610.8382.71335183.600342717.45214.2APNenzyme ffCaco-2lines Polarization L+ Villin*25 +HT-29 Domes 1"10'10^+ +++2108.418

+HCT-GLY Cysts 000000000000+ +++110.62

+HCT-FET Domes +++31.52

+HCT-FRI Domes +++73.51

+HCT-CBS Domes +++11.03

+HCT-ALA Domes +++51.71

+HCT-GEO Domes +++32.52

+HCT-EB Cysts 00+ +++31.56

+HCT-116a Cysts 0000000000000000000000+ +++335.515

NDHCT-R ++81.910

NDHCT-RCA +++131.312

NDHCT-Moser +++181.512

NDHCT-8RCo-115 +82

++19

+HRT-18 Domes +151.70

+SW-480LS-174TSW-1116Domes +115

++91.81

+220

+Vaco-9P Domes +111

Cysts +BB' 00Specific +++100.9

" Enzyme activities were measured after 30 days in culture. Results are the mean of 5 different passages. SD (not shown) are less than 5%. * Results correspond to the intensity of the bands of immunoblotting (see Fig. 5), with lanes 1, 2 corresponding to +++, lanes 3, 4 to ++, and lanes 5, 6 to +. ' TJ, tight junctions; BB, brush border, L, lactase; S, sucrase; DPP-IV, dipeptidylpeptidase IV; APN, aminopeptidase N; AP, alkaline phosphatase; ND, not done. d Enzyme activity in the cell homogenates (H). ' Enzyme activity in the membrane-enriched fraction (I*,). ' Enrichment factor (ratio of activity P2/H).

1938

Fig. 1. Phase contrast micrograph showing the presence of domes in post-confluent cul tures of (a) Co-1 IS (focused on the monolayer) and (ft) HCT-GLY cells (focused on the top of the dome), x 70.

Fig. 2. Transmission electron microscopy of postconfluent (day 30) SW-1116 cells, x 3,8^0. Sections are perpendicular to the bot tom of the flask. Note the organization of the cells into a polarized monolayer with the pres ence of tight junctions (arrow and inset, x 18,200). Note the presence of irregular microvilli.

degree of differentiation (18). That villin is also regularly ex pressed in cultured cell lines derived from colon cancers further confirms the interest of this protein as a marker of the colonie origin of colon cancer cells (18-20). With regard to the morphological and functional differentia tion characteristics normally associated with the absorptive cells of the adult or fetal colon the cell lines of this study can be classified into four main types. Type 1: Cell Lines Which Express Spontaneously the Differ entiation Characteristics of Mature Enterocytes. Only one cell line has been found thus far to belong to this type, namely Caco-2. The differentiation of these cells is characterized by the concomitant association of three criteria: (a) the organization of the cells into a polarized monolayer; (/>)the presence of an apical brush border; and (c) the presence of brush border- associated hydrolases such as sucrase-isomaltase, lactase, aminopeptidase N, dipeptidylpeptidase IV, and alkaline phospha- Fig. 3. Phase contrast micrograph of the cell layer of HCT-EB cells (day 30) tase (13, 38). As reported previously this type of differentiation grown in a standard culture medium (i.e., in the presence of glucose). Note the is close to that found in the fetal colon (12, 21, 38, 43, 44). presence of very numerous intercellular cysts, x 100. Since the same type of differentiation has been observed in some colon cancers (21) it is most likely that other colon established colon cancer cell lines grown in culture. carcinoma cell lines, which have not been investigated yet, Villin appears to be the most regularly preserved colonie should express spontaneously the same type of differentiation. marker as it is present, although at different levels, in all the Type 2: Cell Lines the Differentiation of Which Can Be 20 cell lines of this study. Its expression is independent of both Modulated. Three cell lines belong to this type, namely, HT- the polarization and the differentiation of the cells. This obser 29, HCT-EB, and HCT-GEO. These cell lines are undifferen- vation is in good agreement with previous results which showed tiated when grown under standard culture conditions, i.e.. in that villin is regularly present in colon cancers, whatever their the presence of glucose. Their differentiation occurs only when 1939

Fig. 4. Transmission electron microscopy of (a) HCT-EB (x 3,000) and (Ã©)HCT-GEO (x 1,500) cells showing the two morphological types of intercellular cysts with the presence (a) or absence (b) of an ultraluminal brush border. Sections are perpendicular to the bot tom of the flask. Cells were examined after 30 days in culture in the presence of glucose.

kD 1 23456

116 -

92.5-

Fig. 5. Immunological detection of villin in cellular extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred onto nitro cellulose sheets. Cells were tested at day 30 of culture under standard conditions. Lane 1, Caco-2; Lane 2, Vaco-9P; Lane 3, HCT-116; Lane 4, SW-480; Lane 5, LS-174T; Lane 6, SW-1116. The same quantity of protein was loaded in each slot. Similar results were obtained with cell extracts from the other 14 cell lines (see Table 1). A/I, molecular weight in thousands. they are grown in the absence of glucose. Under such culture conditions they display a morphological enterocytic differentia tion characterized by the organization of the cells into a polar ized monolayer with the presence of an apical brush border. They differ, however, as to their degree of enzymatic differen tiation: HT-29 cells express the same hydrolases as Caco-2 Fig. 6. Transmission electron microscopy of postconfluent (day 30) HCT- cells, except for laclase; HCT-EB express only dipeptidylpep- GEO cells grown for S passages in inosine-supplemented glucose-free medium. tidase IV and alkaline phosphatase; whereas HCT-GEO differ Section is perpendicular to the bottom of the flask, x 3,300. Note the organization of the cells into a polarized monolayer with an apical brush border. Compare entiated cells express no enzymes at all. The particularities of with the same cells grown in the presence of glucose (Fig. 46). this type of cell raise at least two questions, (a) Why would these three cell lines only, but not others, undergo a modulatable with the neoplastic process may interfere with the overall reg differentiation? (b) Why would the absence of glucose allow the ulation of the differentiation of the cells (for review see Ref. differentiation of these cells? Thus far these questions remain 47): in Caco-2 cells an inhibition of the expression of sucrase- unanswered. Concerning the first question and with regard to isomaltase has been shown to be concomitant with an increase the analogy which can be established with fetal differentiation of glucose utilization in cells treated with forskolin (48) and it is of interest to note that HT-29, HCT-EB, and HCT-GEO monensin (49); whereas in HT-29 cells grown in the presence cells display, when undifferentiated, intercellular cysts which of glucose the absence of expression of sucrase-isomaltase has closely resemble the "secondary lumens" observed in the early been shown to be a consequence of an impairment of the post- stages of intestinal development (45, 46). The reason why the translational processing of the enzyme (39). absence of glucose in the culture medium permits the differen Type 3: Undifferentiated Polarized Cell Lines. These cell lines tiation of these three cell lines remains a challenging and are characterized by their ability to form domes and by the unanswered question. There are, however, some lines of evi organization of the cells into a polarized monolayer with apical dence that the alterations of glucose metabolism associated tight junctions, but without the presence of brush border micro- 1940

Fig. 7. Transmission electron microscopy of postconfluent (day 30) HCT-EB cells grown in the presence of glucose (a) or after 5 passages in inosine-supplemented glucose-free medium (b, c). Sections are perpendicular to the bottom of the flask. In a, the cells are undifferentiated and form a nonpolarized multilayer, x 1,000. In b, the cells are organized into a polarized monolayer with the presence of tight junctions and a well developed apical brush border, x 3,000. c. higher magnification of a detail of e, (x 20,000) showing a tight junction and regular brush border microvilli.

Table 2 Effect of glucose deprivation on the enzymatic differentiation ofHT-29, HCT GEO, and HCT EB cells Specific activity (milliunits/mg protein) Cells grown in the presence of 25 mM glucose" Cells grown for S passages in inosine supplemented glucose-free medium

IV50176963210APN12612741AP14748850S218NDNDNDNDLNDNDNDNDNDNDDPPIV10140153213182APN13201316AP105411014125 HT-29HCT

GEOHCTEBHPiHP2HP2SÂ»NDNDNDNDNDNDLNDNDNDNDNDNDDPP

* Cells were tested after 30 days in culture. 4 S, sucrase; L, laclase; DPP-IV, dipeptidylpeptidase IV; APN, aminopeptidase N; AP, alkaline phosphatase; ND, not detectable; H, homogenate; PÂ¡,membrane- enriched fraction.

Fig. 8. Immunofluorescence staining with monoclonal antibody against dipeptidylpepti dase IV of postconfluent (day 30) HCT-EB cells grown in the presence of glucose (left) or after 5 passages in inosine-supplemented glu cose-free medium (right). Note the absence of binding of the antibodies to the cell surface of glucose-fed cells (left) and the presence of an apical staining in glucose-deprived cells (right). x 380.

villi and without any significant expression of brush border- with no apparent personality represent less than one-half of the associated hydrolases. Whatever the culture conditions this type cell lines of this study. appears to be more frequent than should be expected from the These four types of cells already appear, because of the limited number of dome-forming colonie cell lines reported in differences in their organization, differentiation, and sensitivity to differentiation-permissive conditions, as potential tools for the literature (4-8, 10), as was found in 8 of the 20 cell lines of studying, at the cellular and molecular levels, the mechanisms this study. which underlie the regulation of the expression of a number of Type 4: Undifferentiated Unpolarized Cell Lines. These cell cellular constituents and functions associated with the intestinal lines grow as multilayers of unpolarized cells and do not display epithelium as well as the levels where the neoplastic process any differentiation features such as brush border microvilli or interferes with these mechanisms. Whether these different cell brush border-associated hydrolases. Interestingly these cell lines types may have any prognostic significance with regard to the 1941

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1988 American Association for Cancer Research. Epithelial Polarity, Villin Expression, and Enterocytic Differentiation of Cultured Human Colon Carcinoma Cells: A Survey of Twenty Cell Lines

Isabelle Chantret, Alain Barbat, Elisabeth Dussaulx, et al.

Cancer Res 1988;48:1936-1942.

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