[CANCER RESEARCH 48, 1936-1942, April 1, 1988]

Epithelial Polarity, Villin Expression, and Enterocytic Differentiation of CulturedHuman Colon Carcinoma Cells: A Survey of Twenty Cell Lines1

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

Unitéde Recherches sur le Métabolismeet la Différenciationde Cellules en Culture, INSERM U178, 94807 Villejuif Cedex, France [I. C., A. B., E. D., A. Z.], andBristol-Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 [ M. G. B.J

ABSTRACT

Twenty human colon carcinoma cell lines were studied for their abilityto develop some of the characteristics of the normal intestinal epithelium,e.g., epithelial polarity, presence of the actin-binding protein villin, or theoccurrence of an enterocytic differentiation either when cultured understandard conditions, as for Caco-2 cells, or when grown in a glucose-freemedium, as for IIT-29 cells. Except for the regular presence of villin,which can be considered a marker of the colonie origin of the cells, thecell lines of this study could be classified into four types with regard totheir differentiation characteristics. In type 1 (only one cell line, i.e.,Caco-2) the cells undergo spontaneously an enterocytic differentiationcharacterized by a polarization of the cell layer with the formation ofdomes and the presence of an apical brush borderthe membraneof whichis endowed with hydrolases such as sucrase-isomaltase, lactase, amino-peptidase N, dipeptidylpeptidase IV and alkaline phosphatase. In type 2(three cell lines: HT-29, HCT-EB, and HCT-GEO) the cells are undif-ferentiated when grown in the presence of glucose but undergo an enterocytic differentiation when grown in the absence of glucose. In type 3(eight cell lines: HCT-GLY, HCT-FET, HCT-FRI, HCT-CBS, HCT-ÕLA, (oils, HRT-18, and SW-1116) the cells are organized into apolarized ntonolayer with the formation of domes but without any enterocytic differentiation characteristics, whatever the culture conditions. Intype 4 (eight cell lines: HCT-116a, HCT-R, HCT-RCA, HCT-Moser,HCT-8R, SW-480, LS-174T, and Vaco-9P) the cells are organized intoa multilayer without any feature of epithelial polarity or enterocyticdifferentiation, whatever the culture conditions.

INTRODUCTION

Cultured human colon carcinoma cell lines provide a usefultool to understand how the neoplastic process interferes withthe regulation of cellular differentiation. Only a limited numberof colon cell lines have been characterized thus far with regardto differentiation features associated with the absorptive cellsof the colonie epithelium such as its ultrastructural morphology,its structural and functional polarity, or the expression ofspecific proteins. Cell polarity, as assessed by the ability of thecells to form domes when grown on an impermeable support(1), the presence of tight junctions, or vectorial transport properties, has been reported in six cell lines only, namely T84 (2,3); Caco-2 (4, 5); HCA-7 (6); clones of HT-29 cells such as HT-29-19A (7, 8), HT-29-18C,, and HT-29-18N2 (9); HRT-18; andCo-115 (10). The occurrence of an enterocytic differentiationwith the concomitant presence of an apical brush border and ofbrush border-associated hydrolases specific for the small intestine (11) and the fetal colon (12) has been reported in two celllines only: Caco-2, in which it occurs spontaneously understandard culture conditions (13); and HT-29 cells which aredifferentiated only when adapted to grow in a glucose-freemedium (14-16). The presence of villin, a Ca2+-regulated actin-

Received 7/2/87; revised 10/19/87; accepted 12/29/87.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by the Association pour la Recherche sur le Cancer, the FondationFrançaisepour la Recherche Médicale,théGroupement des Entreprises Françaisesdans la Lutte contre le Cancer, and NIH Grant CA34432.

2To whom requests for reprints should be addressed.

binding protein known to be specifically associated with thecytoskeleton of the brush border microvilli (17), has been reported only in Caco-2 (18) and HT-29 cells (18-20). Surprisingly, villin was expressed in the latter cells even when undif-ferentiated, although at a lower level and with a differentcellular localization (18-20). That other colon cell lines shouldalso express some of these differentiation features is suggestedby the observations that (a) villin is present in a number ofcolon tumors3 (18) and (b) an enterocytic differentiation is

associated with some colon tumors (21). The purpose of thepresent work was to screen 20 established human colon carcinoma cell lines, with Caco-2 and HT-29 being used as controls,for the expression of cell polarity, the presence of villin, andtheir ability to develop an enterocytic differentiation, eitherwhen grown under standard culture conditions, like Caco-2cells (13) or when adapted to grow in a glucose-free medium,like HT-29 cells (14-16).

MATERIALS AND METHODS

Cell Lines and Culture Conditions. The 20 cell lines used wereestablished lines developed from human colorectal adenocarcinoma.The cell lines HT-29 (22), SW-480 (23), SW-1116 (23), LS 174 T (24),and Caco-2 (25) were obtained from Dr. J. Fogh (Sloan KetteringMemorial 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 CancerResearch, Epalinges/Lausanne, Switzerland); and Vaco 9P (29) fromDr. J. K. V. Willson (William S. Middleton Memorial Veterans Hospital, Madison, WI). The cell lines HCT-ALA, HCT-CBS, HCT-EB,,HCT-FET, HCT-FRI, HCT-GEO, HCT-GLY, HCT-Moser, HCT-R,HCT-RCA, and HCT-116a were established as reported (30). The cellswere cultured in Dulbecco's modified Eagle's minimum essential me

dium (Eurobio, Paris, France) supplemented with 10% inactivated(56°C,30 min) fetal bovine serum (Boehringer, Mannheim), except for

Caco-2 cells which needed 20% serum and 1% nonessential aminoacids. The cell lines were seeded at 4 x IO4 cells/cm2 in either plasticflasks (Corning Glass Works, Corning, NY), or 35-mm Petri dishesand cultured at 37°Cin an atmosphere of 90% air-10% CO2. For

growth in glucose-free medium the cells were switched from the standard culture medium to the same medium devoid of glucose supplemented with 10% dialyzed inactivated fetal bovine serum and 2.5 m\iinosine as reported previously (16). The culture medium was changeddaily regardless of the culture condition under investigation.

Enzyme Assays. Enzyme activities were performed on the cell ho-mogenates (H) and on membrane fractions prepared according to thesame protocol as used for the preparation of brush border-enrichedfractions (P2) (31). Sucrase (EC 3.3.1.48) activity was measured according to the method of Messer and Dahlqvist (32), dipeptidylpeptidaseIV (EC 3.4.14.5) according to that of Nagatsu et al. (33) using 1.5 mMglycyl-L-proline-4-nitroanilide as substrate, aminopeptidase N (EC3.4.11.2) according to the method of Maroux et al. (34) using L-alanine-/j-nitroanilide as substrate, alkaline phosphatase (EC 3.1.3.1) accordingto the method of Garen and Levinthal (35) withp-nitrophenylphosphateas substrate, and lactase activity (EC 3.2.1.23) according to the methodof Messer and Dalqvist (32), with lactose as substrate, in the presence

3R. Moll, S. Robine, B. Dudouet, and D. Louvard. Villin: a cytoskeletal

protein and an early differentiation marker that is expressed in a limited array ofhuman adenocarcinomas, submitted for publication.

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of 0.2 HIMp-chloromercuribenzoate, an inhibítor of acid /3-galactosidase(36). Results are expressed as milliunits/mg protein. One unit is definedas the activity that hydrolyzes 1 ^mol of substrate per min at 37°C.

Proteins were assayed by the method of Lowry et al. (37).Antibodies. Mouse monoclonal antibody HBB 3/775/42 specific for

dipeptidylpeptidase IV from human small intestine (38) was obtainedfrom Dr. H. P. Hauri (Biocenter of the University of Basel, Basel,Switzerland). Polyclonal rabbit antibody against sucrase-isomaltasepurified from Caco-2 cells was produced in rabbits (39). Rabbit poly-clonal antibodies against porcine villin (18) were a gift from Dr. DanielLouvard (Institut Pasteur, Paris, France). Anti-rabbit sheep antiglobu-lins labeled with horseradish peroxidase or fluorescein isothiocyanatewere obtained from Institut Pasteur Production (Marnes-la-Coquette,France). Anti-mouse swine antiglobulins labeled with fluorescein isothiocyanate were obtained from Nordic Immunological Laboratories(Tilburg, The Netherlands).

Indirect Immunofluorescence. Indirect immunofluorescence assayswere performed as reported previously (13, 15) on cells grown in plasticflasks.

Transmission Electron Microscopy. Transmission electron microscopy was performed as reported previously (13, 15), on cells grown inPetri dishes.

Immunoblotting of Villin. Cellular extracts for immunoblotting analysis of villin were prepared as follows. Cells grown on Petri dishes wererinsed three times with phosphate-buffered saline. To each dish wereadded 400 ^1 of 20 mM Tris buffer, pH 8.8 containing 2 mivi CaCl2,2% Triton X-100, and a cocktail of protease inhibitors [phenylmethyl-sulfonyl fluoride (1 mM), antipain (1 ^g/ml), pepstatin A (I /¿g/ml),and benzamidine (15 j/g/ml)]. After 5 min at room temperature, themixture was centrifuged (2 min at 1,000 x g) and yielded one pellet(l*i) and a supernatant (S, ). S, was centrifuged 2 min at 10,000 x g andyielded the second supernatant S2 which was stored at —70°C.Proteins

of S2 (100 or 200 ^g) were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the buffer system of Laemmli(40) and transferred to nitrocellulose sheets by the method of Burnette(41) as modified by Coudrier et al. (42). Villin was identified withantivillin antibodies and revealed with the peroxidase technique.

RESULTS

Epithelial Polarity. The different cell lines could be classifiedinto three groups on the basis of their ability to form a polarizedepithelium, as judged by phase contrast and transmission electron microscopy (Table 1). Group 1 (10 cell lines) was characterized by the formation of domes which occurred after the cellshad reached confluency. The size and number of domes variedfrom one cell line to another. Their morphology at phasecontrast microscopy (Fig. 1) was the same as already reportedfor other dome-forming cell lines. At transmission electronmicroscopy all dome-forming cell lines appeared as polarizedmonolayers with the presence of apical tight junctions (Fig. 2).With the exception of Caco-2 cells (13) none of these cell linesdisplayed an apical brush border; only irregular microvilli werepresent on the apical surface of the cell layer (Fig. 2, inset).Group 2 (HT-29, HCT-GEO, and HCT-EB) was characterizedby the presence, at phase contrast microscopy, of intercellularcysts (Fig. 3), the total surface of which covered 50% of the celllayer of HCT-GEO and HCT-EB at late stationary phase. Attransmission electron microscopy these cysts were of two types,concomitantly present in each cell line; some were lined with aregular and well organized brush border (Fig. 4a) whereasothers were devoid of it (Fig. 4b). In both types of cysts thecells were polarized with the presence of apical tight junctionstowards the lumen of the cysts (Fig. 4). Except for the presenceof these cysts the general organization of the cell layer was thatof a nonpolarized multilayer with no brush border being presentat its apical surface, i.e., towards the culture medium (Fig. 4b).

In Group 3 (8 cell lines) neither domes nor cysts could beobserved at phase contrast microscopy. At transmission electron microscopy the cells which were examined formed a multilayer with no apical tight junctions or brush border as alreadyshown for some of the cell lines of this group such as SW-480orHCT-8Rcells(10).

Expression of Villin. Villin, as detected in cellular extracts by"Western blot" analysis, was present in all the tested cell lines

grown under standard culture conditions. However, apparentquantitative differences of expression from one cell line toanother were observed (Fig. 5). The degree of expression ofvillin was independent of both the polarization and the differentiation of the cells (Table 1).

Enterocytic Differentiation. Previous experience showed thatthe differentiation of Caco-2 and HT-29 cells was complete andoptimum only late after confluency (13-16). Consequently, allcell cultures in the present study were evaluated 30 days afterseeding. With the exception of Caco-2 cells none of the 19other cell lines exhibited any sign of enterocytic differentiationwhen grown under standard culture conditions, i.e., in thepresence of glucose. This was demonstrated by: (a) the absenceof immunofluorescent staining of the cell layers with antibodiesdirected against brush border-associated hydrolases (see Fig.8a); (b) the activities of brush border-associated hydrolaseswhich were tested in both the cell homogenates (H) and amembrane-enriched fraction (P2) and were found to be either

absent, as in the case of sucrase and laclase, or very low, as inthe case of dipeptidylpeptidase IV, aminopeptidase N, andalkaline phosphatase (Table 1); and (c) the low enrichmentfactor of enzyme activities (?2/H) which was always much lowerthan found in Caco-2 cells (Table 1) and was consistent withthe absence of an apical brush border as disclosed by transmission electron microscopy.

In order to find out whether some of these cell lines wouldexpress an enterocytic differentiation when grown in glucose-free medium, 15 cell lines, including HT-29 cells used as acontrol, were switched to inosine-supplemented glucose-freemedium. These cell lines were passaged every 10 days andtested for their differentiation characteristics on day 30 ofpassage 5, a passage number which has been found to beoptimum for the highest degree of differentiation of HT-29cells to be expressed in such glucose-free culture conditions(16). Only two cell lines (HCT-GEO and HCT-EB), in additionto the previously described HT-29 cells (14-16), exhibited signsof enterocytic differentiation when cultured in the absence ofglucose. Under such culture conditions both cell lines formed apolarized monolayer with the presence of an apical brush border(Figs. 6 and 7). The degree and type of enzymatic differentiationof both cell lines were, however, different from those observedin HT-29 cells (Table 2). HCT-EB cells exhibited an enzymaticdifferentiation restricted to the expression of dipeptidylpeptidase IV (Fig. 8) and alkaline phosphatase (Table 2); no brushborder-associated hydrolases could be detected in differentiatedHCT-GEO cells as disclosed by the absence or immunofluorescent reactivity of the cell layer with anti-hydrolase antibodies(not shown) and the absence of increase of enzyme activities(Table 2).

DISCUSSION

The results reported here show that some of the differentiation features normally associated with the structural or functional organization of the absorptive cells of the colonie epithelium are either totally, partially, or not at all preserved in

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Table 1 Polarization and differentiation characterization of 20 cultured human colon carcinoma cell lines grown under standard culture medium conditions(25 mm glucose)

ElectronmicroscopyCell

lines PolarizationffCaco-2

Domes+HT-29

Cysts+HCT-GLY

Domes+HCT-FET

Domes+HCT-FRI

Domes+HCT-CBS

Domes+HCT-ALA

Domes+HCT-GEO

Cysts+HCT-EB

Cysts+HCT-116a

NDHCT-R

NDHCT-RCA

NDHCT-Moser

NDHCT-8RCo-115

Domes+HRT-18

Domes+SW-480LS-174TSW-1116

Domes+Vaco-9P

Cysts +BB'

L+

1"10'10^+

000000000000+

00+

0000000000000000000000+

00Specific

enzymeactivities"S17615658.900000000000000000000000000000000000000DPP-IV13512959.621874.17304.39424.7111079.714493.5749711605.5284.0451.311655.9393241154.8835036252.50448344.49434.81719111.252162.33.1AP20617998.72126.07334.75295.821051404823.48708.88405.05122.416619212416.5210577610.8382.71335183.600342717.45214.2APN

Villin*25

+++2108.418

+++110.62

+++31.52

+++73.51

+++11.03

+++51.71

+++32.52

+++31.56

+++335.515

++81.910

+++131.312

+++181.512

+82

++19

+151.70

+115

++91.81

+220

+111

+++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).

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

Fig. 2. Transmission electron microscopyof postconfluent (day 30) SW-1116 cells, x3,8^0. Sections are perpendicular to the bottom of the flask. Note the organization of thecells into a polarized monolayer with the presence of tight junctions (arrow and inset, x18,200). Note the presence of irregular micro-villi.

Fig. 3. Phase contrast micrograph of the cell layer of HCT-EB cells (day 30)grown in a standard culture medium (i.e., in the presence of glucose). Note thepresence of very numerous intercellular cysts, x 100.

established colon cancer cell lines grown in culture.Villin appears to be the most regularly preserved colonie

marker as it is present, although at different levels, in all the20 cell lines of this study. Its expression is independent of boththe polarization and the differentiation of the cells. This observation is in good agreement with previous results which showedthat villin is regularly present in colon cancers, whatever their

degree of differentiation (18). That villin is also regularly expressed in cultured cell lines derived from colon cancers furtherconfirms the interest of this protein as a marker of the colonieorigin of colon cancer cells (18-20).

With regard to the morphological and functional differentiation characteristics normally associated with the absorptive cellsof the adult or fetal colon the cell lines of this study can beclassified into four main types.

Type 1: Cell Lines Which Express Spontaneously the Differentiation Characteristics of Mature Enterocytes. Only one cellline has been found thus far to belong to this type, namelyCaco-2. The differentiation of these cells is characterized by theconcomitant association of three criteria: (a) the organizationof the cells into a polarized monolayer; (/>)the presence of anapical brush border; and (c) the presence of brush border-associated hydrolases such as sucrase-isomaltase, lactase, ami-nopeptidase N, dipeptidylpeptidase IV, and alkaline phospha-tase (13, 38). As reported previously this type of differentiationis close to that found in the fetal colon (12, 21, 38, 43, 44).Since the same type of differentiation has been observed insome colon cancers (21) it is most likely that other coloncarcinoma cell lines, which have not been investigated yet,should express spontaneously the same type of differentiation.

Type 2: Cell Lines the Differentiation of Which Can BeModulated. Three cell lines belong to this type, namely, HT-29, HCT-EB, and HCT-GEO. These cell lines are undifferen-tiated when grown under standard culture conditions, i.e.. inthe presence of glucose. Their differentiation occurs only when

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Fig. 4. Transmission electron microscopyof (a) HCT-EB (x 3,000) and (é)HCT-GEO(x 1,500) cells showing the two morphologicaltypes of intercellular cysts with the presence(a) or absence (b) of an ultraluminal brushborder. Sections are perpendicular to the bottom of the flask. Cells were examined after 30days in culture in the presence of glucose.

kD 1 23456

116 -

92.5-

Fig. 5. Immunological detection of villin in cellular extracts analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred onto nitrocellulose 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 eachslot. 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 cultureconditions they display a morphological enterocytic differentiation characterized by the organization of the cells into a polarized monolayer with the presence of an apical brush border.They differ, however, as to their degree of enzymatic differentiation: HT-29 cells express the same hydrolases as Caco-2cells, except for laclase; HCT-EB express only dipeptidylpep-tidase IV and alkaline phosphatase; whereas HCT-GEO differentiated cells express no enzymes at all. The particularities ofthis type of cell raise at least two questions, (a) Why wouldthese three cell lines only, but not others, undergo a modulatabledifferentiation? (b) Why would the absence of glucose allow thedifferentiation of these cells? Thus far these questions remainunanswered. Concerning the first question and with regard tothe analogy which can be established with fetal differentiationit is of interest to note that HT-29, HCT-EB, and HCT-GEOcells display, when undifferentiated, intercellular cysts whichclosely resemble the "secondary lumens" observed in the early

stages of intestinal development (45, 46). The reason why theabsence of glucose in the culture medium permits the differentiation of these three cell lines remains a challenging andunanswered question. There are, however, some lines of evidence that the alterations of glucose metabolism associated

Fig. 6. Transmission electron microscopy of postconfluent (day 30) HCT-GEO cells grown for S passages in inosine-supplemented glucose-free medium.Section is perpendicular to the bottom of the flask, x 3,300. Note the organizationof the cells into a polarized monolayer with an apical brush border. Comparewith the same cells grown in the presence of glucose (Fig. 46).

with the neoplastic process may interfere with the overall regulation of the differentiation of the cells (for review see Ref.47): in Caco-2 cells an inhibition of the expression of sucrase-isomaltase has been shown to be concomitant with an increaseof glucose utilization in cells treated with forskolin (48) andmonensin (49); whereas in HT-29 cells grown in the presenceof glucose the absence of expression of sucrase-isomaltase hasbeen shown to be a consequence of an impairment of the post-translational processing of the enzyme (39).

Type 3: Undifferentiated Polarized Cell Lines. These cell linesare characterized by their ability to form domes and by theorganization of the cells into a polarized monolayer with apicaltight junctions, but without the presence of brush border micro-

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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-supplementedglucose-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. Inb, 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 magnificationof 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 supplementedglucose-free medium

HT-29HCT

GEOHCTEBHPiHP2HP2S»NDNDNDNDNDNDLNDNDNDNDNDNDDPP

IV50176963210APN12612741AP14748850S218NDNDNDNDLNDNDNDNDNDNDDPPIV10140153213182APN13201316AP105411014125

* 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; P¡,membrane-

enriched fraction.

Fig. 8. Immunofluorescence staining withmonoclonal antibody against dipeptidylpeptidase IV of postconfluent (day 30) HCT-EBcells grown in the presence of glucose (left) orafter 5 passages in inosine-supplemented glucose-free medium (right). Note the absence ofbinding of the antibodies to the cell surface ofglucose-fed cells (left) and the presence of anapical staining in glucose-deprived cells (right).x 380.

villi and without any significant expression of brush border-

associated hydrolases. Whatever the culture conditions this typeappears to be more frequent than should be expected from thelimited number of dome-forming colonie cell lines reported inthe literature (4-8, 10), as was found in 8 of the 20 cell lines of

this study.Type 4: Undifferentiated Unpolarized Cell Lines. These cell

lines grow as multilayers of unpolarized cells and do not displayany differentiation features such as brush border microvilli orbrush border-associated hydrolases. Interestingly these cell lines

with no apparent personality represent less than one-half of thecell lines of this study.

These four types of cells already appear, because of thedifferences in their organization, differentiation, and sensitivityto differentiation-permissive conditions, as potential tools forstudying, at the cellular and molecular levels, the mechanismswhich underlie the regulation of the expression of a number ofcellular constituents and functions associated with the intestinalepithelium as well as the levels where the neoplastic processinterferes with these mechanisms. Whether these different celltypes may have any prognostic significance with regard to the

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original tumor is still an unanswered question and remains anopen field of investigation.

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1988;48:1936-1942. Cancer Res   Isabelle Chantret, Alain Barbat, Elisabeth Dussaulx, et al.   Survey of Twenty Cell LinesDifferentiation of Cultured Human Colon Carcinoma Cells: A Epithelial Polarity, Villin Expression, and Enterocytic

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