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1991;51:1270-1277. Cancer Res Max Reynier, Hélène Sari, Manola d'Anglebermes, et al. Enterocytic-differentiated HT29 Human Colonic CellsDifferences in Lipid Characteristics of Undifferentiated and

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(CANCER RESEARCH 51. 1270-1277, February 15. 1991]

Differences in Lipid Characteristics of Undifferentiated and Enterocytic-differentiated HT29 Human Colonie Cells1

Max Reynier,2 HÃ©lÃ¨neSari, Manola d'Anglebermes, Edouard Ah Kye, and Lyda Pasero

Institut de Chimie Biologique, CNRS-U&4 202, UniversitÃ©d'Aix-Marseille I, 3 place Victor Hugo, I3331 Marseille cedex 3, France

ABSTRACT

In this paper we compared several lipid characteristics of the homog-enate and the corresponding plasma membrane in Undifferentiated anddifferentiated HT29 human colon cancer cells, using normal humancolonie cells as a reference. Electron microscopy showed that HT29 cellswere morphologically Undifferentiated when cultured in the presence ofeither glucose or inosine without glucose at early confluency. On thecontrary, HT29 cells cultured at late confluency in a glucose-free mediumcontaining inosine or grown in nude mice exhibited an enterocytic differentiation with the presence of tight junctions and an apical brush border.The cell homogenate and the plasma membrane were prepared from eachcell type. The study of specific marker enzymes showed the same degreeof purity in all plasma membranes, with a highly marked increase ofbrush border-associated hydrolases (A'-aminopeptidase and alkaline

phosphatase) only in the organelles isolated from differentiated HT29and colonie cells. Respective similar increases in the amount of freecholesterol and phospholipid and in the free cholesterol:phospholipidmolar ratio were found in the plasma membrane as compared with thehomogenate in all HT29 cell types. This ratio, due to an increasedphospholipid content in both homogenate and plasma membrane, waslowered in colonie cells. No differences in the phospholipid profile werefound between the homogenates of all cell types and the plasma membrane of Undifferentiated HT29 cells, with the exception of a decrease ofcardiolipin in this organelle. On the contrary, the plasma membranephospholipid composition was different from that of the correspondinghomogenate in differentiated HT29 and colonie cells. The most strikingchanges were a highly increased sphingomyelin amount and concomitantdecreases in phosphatidylethanolamine, phosphatidylserine, and cardiolipin. Moreover, differences in the percentage of phosphatidylcholineplus sphingomyelin as well as in phosphatidylcholine:sphingomyelin,phosphatidylethanolamine, and/or phosphatidylcholine molar ratios werealso found. The monounsaturated:polyunsaturated fatty acid ratio inphosphatidylethanolamine was similar in differentiated HT29 and coloniecells and lower than in Undifferentiated HT29 cells. A decrease in thislatter ratio in phosphatidylcholine was also observed in colonie cells andHT29 cells grown in nude mice. These changes were essentially due toopposite variations in the percentage of palmitoleic acid and those oflinoleic and/or arachidonic acids in both phospholipids. Thus, these dataindicate that Undifferentiated HT29 cells were characterized by theabsence of a specific phospholipid composition in their plasma membrane,which is suggested to be related to altered phospholipid sorting. Theplasma membrane phospholipid profile reversed essentially to the normalpattern when HT29 cells recovered the ability to differentiate.

INTRODUCTION

Various attempts have been performed to determine thepossible changes in phospholipid patterns associated with malignancy (1-5) and/or differentiation (6-9) and to correlatethese variations with the organization and functioning of cellular membranes (10, 11). However, the relevance of these data

Received 6/14/90: accepted 11/27/90.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.

1This work was supported in part by grants from FNCLCC and CNRS andwas performed in CNRS-URA 202 as directed by Dr. J. Marvaldi.

! To whom requests for reprints should be addressed.

is still uncertain, and no general trend is discernible concerningphospholipid modifications. Whether restoration of ultrastructural characteristics during the differentiation of tumor cells isaccompanied by the regression of biological and biochemicalfeatures, typical of a malignant phenotype, is a question stillunanswered (6, 12, 13).

The human colon adenocarcinoma cell line HT29 appears tobe a valuable experimental model to determine the respectiveinfluence of the malignant transformation and differentiationin the expression of cellular constituents and functions of theintestinal epithelium, since these cells are available under eitheran Undifferentiated or a differentiated state (14-18). The differentiation process of this cell line is reversible and mimics thenormal intestinal ontogenesis and epithelial crypt-to-villus migration. However, these cells are malignant and of colonieorigin, and the differentiated HT29 cells exhibit the closestanalogies to human fetal colon (16, 19, 20). In spite of theselimitations, recent reports are indicative of the potential interestin this experimental model (21-27) to determine the respectiveinfluence of neoplasia and differentiation in the expression ofmorphological and structural events.

Lipids are involved in a broad spectrum of cellular functions(10, 11). In particular, changes in the total cholesterol:phospholipid molar ratio as well as in the relative percentageand degree of fatty acid saturation of phospholipids have beenfound to influence a number of important functions in the ratintestinal antipodal plasma membranes (28-30).

In this study, we compare the phospholipid composition ofthe whole cell homogenate and the corresponding plasma membrane fraction of Undifferentiated and differentiated HT29 cellswith human normal mature colonie cells used as a reference.The most striking change was that the specific phospholipidprofile in the plasma membrane of normal colonie cells did notoccur in Undifferentiated HT29 cells but was recovered indifferentiated cells, suggesting that the phospholipid polarizeddelivery to the cell surface is severely altered in relation to theneoplastic process.

MATERIALS AND METHODS

Cultured Cells, Tumors in Nude Mice, and Normal Adult ColonieCells. The established cell line HT29 (HT29-Glc), which was derivedfrom a human colon adenocarcinoma (31), was cultured in Dulbecco'smodified Eagle's medium (Gibco-Brl, Cergy Pontoise, France) containing 25 HIMglucose and supplemented with 10% (v/v) PCS' (Seralab-

Biosys, CompiÃ¨gne, France) (15, 18). These cells were adapted in onestep to grow in the same medium devoid of glucose and pyruvate(Eurobio, Paris, France), supplemented with 2.5 mM inosine (SigmaChimie, La VerpilliÃ¨re, France) and 10% dialyzed FCS. Under theseconditions, no significant cell loss was observed and the resulting HT29cells (HT29-Ino) exhibited a confluency-dependent enterocytic differentiation as previously reported (17, 18, 22). Both cell types wereroutinely subcultured by trypsinization (0.25% trypsin plus 0.53 mM

3The abbreviations used are: PCS, fetal calf serum; DMA, dimethylacetals;PBS, phosphate-buffered saline: TLC. thin-layer chromatography.

1270

LIPID CHANGES DURING DIFFERENTIATION OF TUMOR CELLS

EDTA in PBS) every week. The cells were seeded at 10 x 10" cells/150-cm2 plastic flask and maintained at 37Â°Cin a humidified atmos

phere of 95% air and 5% CO2. The culture medium was renewed daily.The experiments were performed at days 24 to 28 after seeding forHT29-G1C cells. The HT29-Ino cells (passages 7 to 12) were used atearly (18 to 20 days in culture) or late (30 to 32 days in culture)confluency and will be referred to as early confluent HT29-Ino cellsand late confluent HT29-Ino cells, respectively. Cell layers were rinsed3 times with PBS, pH 7.2, and immediately fixed in situ for electronmicroscopy or mechanically scraped for biochemical analyses. HT29-N cells were obtained as previously reported (14, 16, 32) from tumorsdeveloped in athymic nu/nu mice (Cseal-Cnrs, OrlÃ©ans,France) afters.c. injection of 10 x 10' HT29-Glc cells into animals fed under

conventional sterile conditions. After 30 to 40 days of growth, solidtumors were excised immediately after decapitation, the connectivetissue was discarded, and the tumors were minced with surgical scissors.Normal adult colonie cells were scraped, using a glass microscopecoverslip from fresh surgical colon fragments extensively prewashedwith 0.24 M NaCl. The dislodged cells were epithelial cells as found bycytological studies and as reported previously (33).

Transmission Electron Microscopy. Cell layers in the culture flasksor tumor samples were fixed for l h with 2.5% glutaraldehyde in 0.1 Mphosphate buffer, pH 7.4, and then with 1% osmium tetroxide in 0.2M phosphate buffer. After extensive washing with the previous buffer,the fixed samples were dehydrated in graded alcohols and embedded inEpon. Thin sections were cut with a LKB 2088 ultratome, contrastedwith uranyl acetate and lead citrate, and examined with a Jeol 100 Celectron microscope.

Preparation of Whole Cell Homogenate and Plasma Membrane Fraction. Plasma membrane fractions were isolated essentially according toWarren (34) and Perdue (35). All procedures were carried out at 4Â°C.

Cells were washed 3 times with 0.16 M NaCl and twice with 0.05 MTris-HCl, pH 7.4, by resuspension and centrifugation. The cell pelletswere suspended at a concentration of 1 x IO7 cells/ml in 0.005 M

MgCl2:0.05 M Tris-HCl, pH 7.4, allowed to stand for 10 min, anddisrupted by 20 gentle strokes in a Dounce homogeneizer. This disruption step was repeated once more for human normal colonie cells. Thewhole-cell homogenates were sedimented at 105,000 x g for 1 h, andthe resulting pellets were homogeneized in 20% sucrose (w/w). Fourml of this particulate suspension were layered onto a 30-ml continuousdensity gradient of sucrose (20 to 45%, w/w) with 2 ml of 50% sucrosesolution on the bottom. After 15 h of centrifugation at 25,000 rpm ina SW27 Beckman rotor, plasma membranes were found at a band ofdensity 1.08 to 1.15 on the basis of enzymatic criteria. This layer washarvested, diluted with 0.16 M NaCl, and sedimented at 105,000 x gfor 45 min. The amount of protein recovered in this plasma membranefraction represented about 2.5% of the cell homogenate protein, whichis usual for plasma membrane preparation from various cell lines (36,37).

Enzymatic Assays. Ouabain-sensitive, (Na+,K+)-dependent ATPase

(EC 3.6.1.3) activity was measured according to the method of Gratecoset al. (38). 5'-Nucleotidase (EC 3.1.3.5) activity was determined by

liberation of inorganic phosphate (39). A'-Aminopeptidase (EC 3.4.11.2)and alkaline phosphatase (EC 3.1.3.1) activities were measured spectro-photometrically using appropriate substrates (40, 41). Rolenone-insen-sitive NADPH-cytochrome c reducÃase(EC 1.6.99.3) was detected bythe reduction of oxidized cytochrome c at 550 nm (42). Measurementofcytochromecoxidase(EC 1.9.3.1) was done according to the methodof Cooperstein and Lazarow (43). Protein content was determined bythe method of Lowry et al. (44) using bovine serum albumin as astandard. The enzyme activities are expressed as milliunits per mg ofprotein. One unit is defined as the activity that utilizes 1 ^mol ofsubstrate per min under experimental conditions. Alkaline phosphatase,7V-aminopeplidase, and 5'-nucleotidase were utilized as apical membrane markers; (Na+,K+)-ATPase for the basolateral membrane; andcytochrome c oxidase and NADPH-cytochrome c reducÃasefor themitochondria and the endoplasmic reticulum, respectively.

Extraclion and Analyses of Lipids. Lipids from cell homogenates andplasma membrane fractions were extracted as previously described (5)

by 2-h stirring in 20 volumes of chloroform:melhanol (2:1, v/v) al roomtemperature according to the technique of Folch et al. (45). Afterpartition with 0.2 volume of 0.85% KCI, the lower organic phase wasevaporated to dryness, resuspended in a small volume of chloro-form:melhanol (2:1, v/v), and stored al â€”¿�20Â°Cunder nitrogen. Free

cholesterol was estimated using an enzymatic melhod utilizing cholesterol oxidase, catalase, and acetylacelone (Boehringer-Mannheim, Mey-lan, France). Phosphorus in phospholipid was eslimated by the methodof Ames (39).

Phospholipids (150 nmol) were fractionated by two-dimensionalTLC on 20-x 20-cm precoated silica gel H plates (Merck, Nogent-sur-Marne, France) using chloroform:acetone:methanol:acelic acid:waler(50:20:10:15:5,v/v) in ihe firsl dimension and chloroform:-methanohpetrol etheracetic acid:boric acid (40:20:30:10:1.8, v/v/v/v/w) in ihe second dimension. Phospholipids were delected by sprayingwith a specific reagent (46) and idenlified by comparison wilh referenceslandards. After Ihe first migration, plates were sprayed with 0.005 MHgCU in 0.1 M acetic acid and dried under vacuum for l h forplasmalogen determinalion. Individual spols were scraped off andanalyzed for phosphorus coment as described above.

Fally acid analyses of phosphalidylcholine and phosphalidylelha-nolamine isolated from preparative TLC of cell homogenate phospho-lipids (750 nmol) were performed as described earlier (5). Fatly acidmelhyl eslers and DMA of both isolated phospholipids were preparedby boron Irifluoride Iransmelhylalion and analyzed isolhermally al185Â°Cby gas-liquid chromalography wilh a Varian 1440 gas chromal-

ograph equipped with a hydrogen flame ioni/ai ion detector in a columnof 7% butanediolsuccinale on Gas-Chrom Q, 100/200 meshes (Gerdel,Suresnes, France) wilh a nitrogen flow rate of 28 ml/min. Peaks wereidentified by comparison wilh ihe relenlion lime of reference slandardsand were quaniiiated by integralion of iheir areas wilh a Varian CDSIII calculator.

RESULTS

Ultrastructural Characterization of HT29 Cells Grown in Culture and in Nude Mice Tumors. HT29-Glc cells at late con-fluency displayed disorganized multilayers of undifferentiatedcells (Fig. IA). These cells exhibited cytoplasmic projectionsrandomly dispersed at the cell periphery and interacted bynumerous desmosomes but lacking tight junctions. Early confluent HT29-Ino cells formed essentially a monolayer with

some multilayered regions of unpolarized cells (Fig. ÃŒB).Onlyspare, irregular microvilli were present on the surface layer withfew tight junctions. At late confluency, HT29-Ino cells wereorganized into a typical polarized monolayer with well-developed junctional complexes and characteristic apical brush border microvilli (Fig. 1C). HT29-N cells showed the presence ofintercellular cysts with a regular and well-organized brush border in the majority of them (Fig. \D).

Enzymatic Characterization of the Homogenates and PlasmaMembrane Fractions from Differentiated and UndifferentiatedCells. The specific activity of several marker enzymes wasdetermined in both the whole-cell homogenate and the plasmamembrane fraction prepared from each cell type. The resultsare presented in Table 1. For each plasma membrane marker,the respective specific activities were nearly similar in all homogenates. These values were markedly increased in the corresponding plasma membrane fractions except for both brushborder-associated hydrolases in the morphologically undifferentiated cells. In HT29-G1C and early confluent HT29-Ino cells,the activities of the A'-aminopeptidase and alkaline phosphatasewere, respectively, either only moderately or not significantlyenhanced in plasma membranes compared with homogenates.On the other hand, plasma membrane fractions were depleted

1271


, -bb

C DFig. 1. Ultrastructural characterization of HT29 cells grown in vitro and in nude mice. A, HT29 cells grown in the presence of 25 HIM glucose (28 days after

seeding): vertical section showing a multilayer of undifferentiated cells with desmosomes (d) and cytoplasmic processes (cp) (X 3,600). B, early confluent HT29 cellsgrown in the absence of glucose and in the presence of 2.5 mM inosine (19 days after seeding): vertical section showing a monolayer of predifferentiated cells withirregular and sparse microvilli (m) and tight junctions ((/') (x 6,000). C, HT29 tumors in nude mice (36 days after injection): cross-section showing the presence of a

regular brush border (bb) lining the luminal membrane of cells organized around intercellular crypts (x 20,000). D, late confluent HT29 cells grown in the presenceof 2.5 TOMinosine and absence of glucose (30 days after seeding): vertical section showing a monolayer of polarized and differentiated cells with apical brush bordermicrovilli (bb), desmosomes (</), and tight junctions (tj) (x 5,000). Inset, cross-section of brush border microvilli. Note, as in C, the two leaflets of the plasmamembrane with the outer leaflet coated with filamentous material and the core of microfilaments which extend into the cytoplasm (x 20,000).

Table 1 Distribution of marker enzymes in whole-cell homogenates and plasma membrane fractionsPreparation of whole-cell homogenates and plasma membrane fractions as well as the enzymatic assays was described in "Materials and Methods."

Cells

HT29-GIC HT29-Ino HT29-N Normal colonie

EarlyconfluentMarker

enzymes(Na\K*)-ATPase

5'-NucleotidasejV-AminopeptidaseAlkaline phosphataseCytochrome c oxidaseNADPH cytochrome c

reducÃaseWhole-cell

homogenate36.4

53.85.30.66.24.814.6Â°

13.01.90.21.21.4Plasma

membrane189.2

Â±33.1247.8 Â±45.1

18.2 Â±2.61.8 Â±1.12.0 Â±1.30.7 Â±0.2Whole-cell

homogenate55.2*

44.7 Â±14.86.1 Â±1.91.0 + 0.3

11.8*3.2Â»Plasma

membrane152.3*

181.2 + 22.415.3 + 2.42.1 Â±1.3

2.9*1.2Â»Late

confluentWhole-cell

homogenate46.0

Â±12.1NDC

6.0 Â±1.41.3 Â±0.2

10.3 + 2.14.9 + 2.7Plasma

membrane134.7

Â±22.1ND

39.6 Â±2.67.0 + 2.12.1 Â±1.51.5 + 0.7Whole-cell

homogenate50.9

+ 9.932.6 Â±0.8

7.1 Â±1.01.3 + 0.2

16.2 Â±3.91.8+ 1.1Plasma

membrane140.2

Â±40.5158.9 Â±6.6108.9 Â±37.3

5.1 Â±2.44.9 + 2.12.4 Â±0.3Whole-cell

homogenate43.5

Â±12.430.2 Â±6.611.9 Â±3.5

1.6 Â±0.415.6 + 2.45.2 Â±0.9Plasma

membrane181.4

Â±28.9128.9 Â±19.267.2 + 25.1

6.4+ 1.20.5 Â±0.32.3 Â±1.1

Â°Mean Â±SD of 3 to 5 separate independent experiments, milliunits/mg of protein. One unit is defined as the activity that utilizes 1 Â»jmolof substrate/min under

the experimental conditions.* Values obtained in duplicate from one experiment.c ND, not determined.

of cytochrome c oxidase and NADPH-cytochrome c reducÃase,and the contamination by mitochondria and endoplasmic retic-ulum was calculated to be less than 5% (data not shown).

Comparative Free Cholesterol and Phospholipid Contents ofCell Homogenates and Plasma Membrane Fractions. As shownin Fig. 2, the free cholesterohphospholipid ratio was higher in

1272


I ] Plasma membrani

Cell homogenate

T

a b c d eFree cholesterol

3 b c dPhospholipid

a b c d eFree cholesterol/phospriolipid

Fig. 2. Free cholesterol and phospholipid contents of whole-cell homogenatesand plasma membrane fractions. Free cholesterol and total lipid phosphorus wereestimated as described in "Materials and Methods." From these values, the freecholesterol:phospholipid ratios were calculated, a, HT29-Glc; Â¿>,early confluentHT29-Ino; c, late confluent HT29-Ino; d, HT29-N; e, normal colonie cells.Columns, mean; bars, SD (n = 3 to 5). *, Significantly different from respectivevalues found in e with P < 0.05 (determined by Cochran's / test).

the plasma membrane fraction than in the cell homogenate foreach cell type. Moreover, this ratio in both cell homogenatesand plasma membrane fractions was, respectively, similar in allundifferentiated and enterocytic-differentiated HT29 cells andwas enhanced as compared with that of normal colonie cells.This increase was due to the reduced amount of phospholipidin all types of HT29 cells, whereas the free cholesterol contentwas similar in these and normal colonie cells.

Phospholipid Composition of the Cell Homogenates andPlasma Membrane Fractions. Except for cardiolipin which waslowered in all plasma membrane fractions, comparison of thephospholipid composition between the cell homogenate and thecorresponding plasma membrane fraction was allowed to distinguish two groups among the studied cells according to thedifferentiation state (Table 2). In both HT29-Glc and earlyconfluent HT29-Ino cells, the percentage distribution of phosphorus among the major phospholipid classes was similar inthe cell homogenate and plasma membrane fraction. On thecontrary, the plasma membrane phospholipid composition wasdifferent from that of the cell homogenate in late confluentHT29-Ino, HT29-N, and normal colonie cells. The most striking changes were the approximately 3-fold increase in theamount of sphingomyelin and a concomitant slight decrease ofphosphatidylethanolamine in the plasma membrane fraction in

comparison to the respective cell homogenate. Moreover, therewas a 2-fold decrease in the proportion of phosphatidylserinein the plasma membrane fraction of HT29-N and normal

colonie cells, whereas such a change was not found in lateconfluent HT29-Ino cells. Table 2 also shows that the percentage of the two choline-containing phospholipids, which represented about 50% of the total phospholipid in all cell homogenates, was unchanged in the plasma membrane fractions of bothundifferentiated cells but was clearly enhanced in those ofdifferentiated cells. Similarly, the phosphatidylcholine:sphingo-myelin and phosphatidylcholine:phosphatidylethanolamine ratios in the plasma membrane fractions were, respectively,greatly decreased and moderately increased in the three differentiated cell types, while the phosphatidylcholine:phospha-tidylserine ratio was only enhanced in plasma membrane fractions of HT29-N and normal colonie cells.

Unsaturated Fatty Acid Composition of Phosphatidylcholineand Phosphatidylethanolamine. The unsaturated fatty acid patterns of phosphatidylethanolamine and phosphatidylcholinepurified from cell homogenates are reported in Tables 3 and 4,respectively, and the ratio of monounsaturated to polyunsatu-rated fatty acids in both phospholipids is shown in Fig. 3. Whencompared with normal colonie cells, both undifferentiatedHT29 cells were similarly characterized by a high proportionof palmitoleic acid in both phospholipids associated with lowpercentages of either linoleic, linolenic, and arachidonic acidsin phosphatidylethanolamine or linoleic and arachidonic acidsin phosphatidylcholine. Consequently, the monounsaturated:polyunsaturated fatty acid ratios in both undifferentiated HT29cells were similarly increased 2- to 4-fold in phosphatidylethanolamine and 6- to 8-fold in phosphatidylcholine as comparedwith those found in normal colonie cells. In both differentiatedHT29 cells, the monounsaturatedrpolyunsaturated fatty acidratio of phosphatidylethanolamine was similarly decreased ascompared with that found in undifferentiated cell types and hadthe same value as in normal colonie cells. This reversion to thenormal was due to a partial decrease in the palmitoleic acidproportion coupled with an increase only in the relative percentage of either arachidonic acid in late confluent HT29-Inocells or both linoleic and linolenic acids in HT29-N cells. Theunsaturated fatty acid pattern of phosphatidylcholine exhibitedsimilar changes in HT29-N cells but was unmodified in lateconfluent HT29-Ino cells as compared with undifferentiated

Table 2 Phospholipid composition of cell homogenates and plasma membrane fractionsPhospholipids in the organic phase of Folch's extracts from cell homogenates and plasma membrane fractions were analyzed by two-dimensional TLC and were

quantitated by estimation of the inorganic phosphate.

Cells

HT29-G1C HT29-lno HT29-N Normal colonie

Early confluent Late confluent

PhospholipidsWhole-cell

homogenatePlasma

membraneWhole-cell

homogenatePlasma

membraneWhole-cell

homogenatePlasma

membraneWhole-cell

homogenatePlasma

membraneWhole-cell

homogenatePlasma

membranePC"SMPEPIPSCLPC+SMPC/SMPC/PEPC/PS48.5Â±2.8*5.2

Â±1.622.8Â±4.36.7Â±1.29.5Â±1.55.7Â±2.053.7Â±3.59.3Â±1.72.1

Â±0.15.1Â±1.146.7

Â±1.16.4Â±0.622.3Â±1.28.3Â±1.012.3Â±2.11.6+0.7'53.1

Â±1.27.3Â±0.72.1

Â±0.13.8Â±0.749.5

Â±1.03.31.226.8

3.99.60.76.60.74.2

1.252.82.315.0

0.61.80.27.5

0.947.0

Â±0.45.40.827.4

0.58.91.07.81.11.8

0.8'52.4

1.28.7Â±1.4'1.7

Â±0.06.0Â±0.95

1.5 Â±2.3 5 1.9 Â±0.9 43.2 Â±0.9 44.3 Â±0.4 46.3 Â±0.3 5 1.3 Â±0.83.2Â±0.7 15.7+1.0' 3.9+1.8 14.6 Â±0.6' 5.3 + 0.6 14.8 +0.4'27.9Â±1.6 20.1 + 0.9' 34.8 Â±2.6 25.3Â±1.3C 27.2 Â±2.419.7+1.9'6.2+1.4

7.6 Â±0.8 8.0 + 0.8 7.9 + 0.7 7.6 + 0.8 8.2 Â±0.47.2+ 0.6 5.5+1.3 6.0 + 0.7 3.2 Â±1.2' 6.3+1.1 2.9 Â±0.4'3.8+ 0.9 0.0' 2.8+1.0 0.8 Â±0.3' 6.7 Â±0.50.0'54.7Â±2.4 67.6+1.2' 47.1 + 2.8 58.9+1.1' 51.6+1.1 66.1 Â±1.8'16.1Â±0.4 3.3 Â±0.2' 11.1 Â±0.5 3.0 + 0.3' 8.7+1.1 3.5 Â±0.2'1.8Â±0.2 2.6 Â±0.1' 1.2 Â±0.1 1.8 + 0.1' 1.7 Â±0.2 2.6 Â±0.3'7.1Â±0.7 9.4+1.6 7.2 Â±1.0 13.8+1.4' 7.3 Â±1.3 17.7+1.4'

" PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine + plasmalogens; PI, phosphatidylinositol: PS, phosphatidylserine; CL, cardiolipin.* Mean Â±SD (n = 3 to 5), expressed as molar percentage of total phospholipid. From these values, the mean Â±SD of the sum of the percentages of PC + SM and

the ratios of the percentage of PC to that of SM, PE, or PS were calculated.' P < 0.05 for values between plasma membrane fraction and cell homogenate of the same cell type (determined by Cochran's t test).

1273


Table 3 Unsaturated fatly acid and DMA composition ofphosphatidylethanolamine

Fatty acid methylesters and DMA of phosphatidylethanolamine purified fromcell homogenates were prepared by boron trifluoride transmethylation and analyzed by gas-liquid chromatography as described in "Materials and Methods."

Cells

HT29-Ino

Fattyacids16:1

u)718:1o)918:2^)618:3

o)320:3u)620:40.620:5w3DMAHT29-G1C17.1

Â±2.6"39.3

Â±4.33.3Â±1.20.3

Â±0.12.4Â±0.37.9Â±1.83.8

Â±0.16.0Â±2.7Early

confluent21.4

Â±0.732.21.21.26.42.37.62.60.3Â«0.20.81.21.3Late

confluent6.2Â±2.0*26.6

Â±3.51.4Â±0.30.7

Â±0.32.7Â±1.912.9

Â±0.3*3.3

Â±1.68.0Â±0.9HT29-N8.2

Â±0.7*24.118.91.61.28.42.09.61.81.9*0.3*0.20.70.41.5Normalcolonie3.1

Â±0.5*29.4

Â±2.612.4Â±2.8*2.9

Â±0.5*2.2

Â±0.314.9Â±3.0*1.6

Â±0.115.6Â±1.1*

Â°Mean Â±SD of 3 to 5 determinations, expressed as weight percentage of total

fatty acids.* P < 0.05 compared with values in HT29-Glc cells (determined by Cochran's

t test).

Table 4 Unsaturated fatty acid composition ofphosphatidylcholinePhosphatidylcholine purified after TLC of cell homogenate phospholipids was

treated by boron trifluoride and analyzed by gas-liquid chromatography.

Cells

HT29-Ino

Fattyacids16:1

o)718:10,918:2o)6I8:3o>320:3

0)620:40)620:5

U3HT29-G1C21.340.81.70.51.30.91.3"4.60.30.20.60.30.60.4Early

confluent30.8

Â±1.927.5Â±2.32.3Â±1.3r0.7

Â±0.40.7Â±0.30.3Â±0.2Late

confluent22.4

Â±1.530.0Â±3.03.2Â±0.20.4Â±0.10.6

Â±0.1â€”HT29-N11.

9Â±0.8*25.415.80.20.51.70.40.3*0.10.30.2*0.2

+ 0.1Normal

colonie5.6Â±0.4*38.3

Â±3.820.1Â±1.9*â€”0.8

Â±0.22.3Â±0.3*0.3

Â±0.1Â°Mean Â±SD of 3 to 5 determinations, expressed as weight percentage of total

fatty acids.* Ps 0.05 compared with values in HT29-Glc cells (determined by Cochran's

t test.

PE

Js/PolyunsaturatedfatHnounsaturated

I1IiIii_I_l

njÃ•Ã•

Fig. 3. Monounsaturated:polyunsaturated fatty acid ratios in phosphatidylcho-line and phosphatidylethanolamine. a to c. as in Fig. 2. *, Significantly differentfrom respective values found in a with P < 0.01 for phosphatidylcholine and P <0.05 for phosphatidylethanolamine (determined by Cochran's t test). Columns,mean: bars, SD (n = 3 to 5).

cells. In addition, DMA that originate from plasmalogens werepresent only in phosphatidylethanolamine with about a 2-foldincreased level in normal colonie cells. The differences in fattyacid composition of both phosphatidylethanolamine and phosphatidylcholine found between the early confluent and the lateconfluent HT29-Ino cells were not due to a depletion of essential fatty acids in the culture medium, since the level of these

components was unchanged after 3 days of cell growth withoutmedium change (data not shown).

DISCUSSION

This investigation revealed marked differences in the phos-pholipid pattern between undifferentiated and differentiatedHT29 human colonie adenocarcinoma cells. The phospholipidcomposition of the plasma membrane in undifferentiated cellsresembled that of the whole cell homogenate. On the contrary,significant differences in the phospholipid composition betweenthe cell homogenate and the plasma membrane found in normalmature colonie cells occurred in differentiated HT29 cells.Moreover, changes in the monounsaturated:polyunsaturatedfatty acid ratio in phospholipids were also found to be relatedto the differentiation state of this cell line.

As previously reported (14-18), the present ultrastructuraldata showed that HT29 cells cultured either in the presence ofglucose (15, 18) or at early confluency in a glucose-free mediumcontaining inosine (17, 18) were essentially undifferentiated.On the contrary, HT29 cells grown in nude mice (14, 16, 32)or cultured at late confluency in the presence of inosine withoutglucose (17, 18) displayed the presence of regular apical micro-villi and tight junctions.

In order to determine the possible relationship between thephospholipid pattern and differentiation in the HT29 cell system, the plasma membrane fractions were prepared from different HT29 cell types and human normal colonie cells. Exceptfor the brush border-associated hydrolases, the distribution ofmarker enzymes always showed respective similar enhancements of the plasma membrane enzymes and the same decreaseof intracellular enzymes in all plasma membrane fractions ascompared with the corresponding cell homogenates. Thus, itcan be considered that the degree of purity of the plasmamembrane prepared from all cell types was nearly the samewith a minor contamination with intracellular organelles.

In contrast with these data, the specific activities of the brushborder-associated hydrolases in the plasma membrane were

similarly highly increased as compared with those of the cellhomogenate in both morphologically differentiated HT29 andnormal colonie cells. This marked enhancement of alkalinephosphatase and aminopeptidase activities was not found inthe plasma membrane isolated from undifferentiated HT29cells. This distribution of brush border-associated hydrolasesrelated to the differentiation state of HT29 cells is consistentwith previous studies (17, 22) as well as with ultrastructuraldata reported above.

The lipid composition of these purified membranes and theircorresponding cell homogenates was investigated in undifferentiated and differentiated HT29 cells, and in normal coloniecells.

Changes in the total or free cholesterol:phospholipid ratio ofcell homogenate, plasma membrane, and various intracellularmembranes occur often in relationship with cellular transformation or differentiation. In this study, however, the amountsof free cholesterol and total phospholipid, and consequently thefree cholesterol:phospholipid molar ratio in both cell homogenate and plasma membrane fraction, did not differ betweenundifferentiated and differentiated HT29 cells. A pronouncedincrease of phospholipid content in both cell homogenate andplasma membrane that resulted in a decrease in the free choles-terol:phospholipid ratio was found in normal colonie cells.Recent results indicated that the levels of free cholesterol and

1274


total phospholipid were, respectively, slightly or greatly increased in human colonie tumors as compared with normalintestinal mucosa (47). The reasons for this discrepancy are atthis time unknown.

In normal colonie cells, comparative phospholipid analysesof the cell homogenate and the plasma membrane showed ahigh increase in sphingomyelin content and a moderate increasein phosphatidylcholine amount, associated with slight decreasesin phosphatidylethanolamine and phosphatidylserine contentsand the absence of cardiolipin in the plasma membrane. Thesedifferences, notably the enrichment of sphingomyelin, the absence of cardiolipin, and the increase of free cholesterol, are inline with the general trend of lipid localization in normaleukaryotic cells (48). These results were also consistent withsimilarities in the distinct phospholipid composition of eachantipodal plasma membrane observed in the rat colonocyte andenterocyte (28, 29, 49, 50), the brush border domain having aspecially high sphingomyelin amount and the basolateral domain being enriched to a lesser extent in phosphatidylcholine.

The differential phospholipid pattern between the cell homogenate and the plasma membrane found in normal coloniecells was also seen in differentiated HT29 cells, with the exception of certain variations which were less acute or abolishedaccording to the differentiated cell type. It seems likely thatthese slight differences in the plasma membrane phospholipidcomposition between differentiated HT29 and normal coloniecells were, at least in part, dependent on the differentiationstate of HT29 cells (16, 19, 20).

On the contrary, in undifferentiated HT29 cells, the plasmamembrane-phospholipid composition was similar to that of cellhomogenate. This organelle had only a moderate increase ofthe sphingomyelin content and a decrease of cardiolipin. Thisequalization of the phospholipid pattern between the plasmamembrane and the cell homogenate has already been reportedin some experimental tumors, notably various rat hepatomasas compared with normal liver (2, 51, 52). This loss of specificphospholipid composition in the plasma membrane was notdue to cross-contamination and intermembrane phospholipidredistribution during subcellular fractionation or related to cellgrowth, but it was dependent on the malignancy of the tumor(52).

Moreover, the phospholipid composition of all cell homoge-nates was similar and, therefore, the biosynthesis of thesecomponents was possibly unchanged in different cell types. Onthe contrary, the phospholipid composition of the plasma membrane was a function of the differentiation state, suggesting thatthe polarized distribution of the phospholipids between theexoplasmic and cytoplasmic leaflets of the antipodal domainsin epithelial cells (53) was probably altered in undifferentiatedHT29 cells, but essentially restored in their enterocytic-differ-

entiated counterparts. It was recently reported that the relativerate of translocation, and not the synthetic step, was the mostimportant factor controlling the phospholipid segregation between apical and basolateral membranes in rat renal proximaltubule cells (54). These probable altered sorting and targetingof phospholipids in undifferentiated HT29 cells are also consistent with the abnormal expression and distribution of othercell surface molecules recently observed in these cells whencompared with their differentiated counterparts (21, 23-27). In

particular, as already indicated above in this study, the levels ofboth alkaline phosphatase and /V-aminopeptidase were not in

creased in the undifferentiated plasma membrane.The differences in acyl group composition of total or individ

ual phospholipids in relation to cell transformation or differentiation have often been reported. Tumor cells were usuallycharacterized by a lower proportion of polyunsaturated fattyacids and a higher percentage of monounsaturated fatty acidsthan the corresponding normal cells (1,5, 55). In agreementwith this general tendency, the monounsaturatedrpoly-unsaturated fatty acid ratio in both phosphatidylcholine andphosphatidylethanolamine had the highest value in undifferentiated tumoral HT29 cells but the lowest in normal coloniecells. These changes were essentially the result of an oppositevariation in the palmitoleic acid proportion and those of lino-

leic, arachidonic, and/or linolenic acids in both phospholipids.The differentiation of HT29 cells leads to a partial reversion ofthe fatty acid pattern characteristic of normal colonie cells.Except for phosphatidylcholine in one differentiated HT29 celltype, the monounsaturated:polyunsaturated fatty acid ratioshad the same values. However, this restoration was related tolower changes in the proportion of the fatty acids than thedifferences found between undifferentiated HT29 cells and normal colonie cells. In particular, there was only a decrease in theamount of either arachidonic acid or linoleic and linolenic fattyacids, depending on the differentiated HT29 cell type. Thereason for the absence of fatty acid reversion in phosphatidylcholine in differentiated HT29-N cells is unknown but mightbe due, at least in part, to the low and quite similar proportionof arachidonic acid in this phospholipid in all HT29 cell typesas compared with that in phosphatidylethanolamine. These dataare consistent with changes in fatty acyl profile observed duringin vitro differentiation of various cultured cells (8, 56-59),although controversial data were reported in Friend erythroleu-kemia cells (7, 57). The fatty acyl changes found in this studydid not appear to be due to variations in essential fatty aciduptake related to different nutritional conditions. They mightbe due rather to alterations in the activities of enzymes responsible for the transformation of linoleic and linolenic precursorsin other polyunsaturated fatty acids (55, 60). The decreasedcontents of arachidonic acid and both linoleic and linolenicprecursors found in undifferentiated HT29 cells might also berelated to an increased formation of leukotrienes B4, sulfido-peptide leukotrienes, and prostaglandin E2 as observed in human colonie adenocarcinomas when compared with autologousnormal colonie mucosa (47).

In summary, the results reported here, for the first time toour knowledge, show that the absence of a characteristic phospholipid plasma membrane composition observed in undifferentiated HT29 cells reversed essentially to a normal pattern inenterocytic-differentiated counterparts. Moreover, it seems rea

sonable to suggest that these significant phospholipid changesmight be important in different cellular properties related tomalignancy or differentiation in the HT29 experimental modeland, in turn, in the human gut. In particular, the phosphatidyl-choline:phosphatidylethanolamine ratio is known to be a determinant in cell-cell interaction, cell-substrate adhesion, endocy-

tosis, receptor binding, and metastatic potential (1,61,62). Thelevel of polyunsaturated fatty acids affects lectin-induced agglu-tinability, receptor interactions, cytolysis, proliferation, tumor-igenicity, and metastatic potential (1, 5, 11, 63, 64). Recentlysphingomyelin and more specifically its breakdown products,which are protein kinase C inhibitors and may function assecond messengers, appear to be active in signal transduction,receptor activities, phorbol ester-induced responses, action oftumor promoters, and growth factors (65).

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ACKNOWLEDGMENTS

We are obliged to Dr. A. Zweibaum and Dr. G. Trugnan for the giftof HT29-Ino cells and Dr. S. Champion for critically reading themanuscript. We are very grateful to F. <Cannellini and R. Soirat forthe cell culture, M. Roccabianca for the electron microscopy, and J. J.Roccabianca for the photographs.

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