(CANCER RESEARCH 51. 1270-1277, February 15. 1991]
Differences in Lipid Characteristics of Undifferentiated
differentiated HT29 Human Colonie Cells1
and Enterocytic-
Max Reynier,2 Hélène
Sari, Manola d'Anglebermes, Edouard Ah Kye, and Lyda Pasero
Institut de Chimie Biologique, CNRS-U&4 202, Universitéd'Aix-Marseille I, 3 place Victor Hugo, I3331 Marseille cedex 3, France
ABSTRACT
In this paper we compared several lipid characteristics of the homogenate and the corresponding plasma membrane in Undifferentiated and
differentiated HT29 human colon cancer cells, using normal human
colonie cells as a reference. Electron microscopy showed that HT29 cells
were morphologically Undifferentiated when cultured in the presence of
either glucose or inosine without glucose at early confluency. On the
contrary, HT29 cells cultured at late confluency in a glucose-free medium
containing inosine or grown in nude mice exhibited an enterocytic differ
entiation with the presence of tight junctions and an apical brush border.
The cell homogenate and the plasma membrane were prepared from each
cell type. The study of specific marker enzymes showed the same degree
of purity in all plasma membranes, with a highly marked increase of
brush border-associated hydrolases (A'-aminopeptidase and alkaline
phosphatase) only in the organelles isolated from differentiated HT29
and colonie cells. Respective similar increases in the amount of free
cholesterol and phospholipid and in the free cholesterol:phospholipid
molar ratio were found in the plasma membrane as compared with the
homogenate in all HT29 cell types. This ratio, due to an increased
phospholipid content in both homogenate and plasma membrane, was
lowered in colonie cells. No differences in the phospholipid profile were
found between the homogenates of all cell types and the plasma mem
brane of Undifferentiated HT29 cells, with the exception of a decrease of
cardiolipin in this organelle. On the contrary, the plasma membrane
phospholipid composition was different from that of the corresponding
homogenate in differentiated HT29 and colonie cells. The most striking
changes were a highly increased sphingomyelin amount and concomitant
decreases in phosphatidylethanolamine, phosphatidylserine, and cardi
olipin. Moreover, differences in the percentage of phosphatidylcholine
plus sphingomyelin as well as in phosphatidylcholine:sphingomyelin,
phosphatidylethanolamine, and/or phosphatidylcholine molar ratios were
also found. The monounsaturated:polyunsaturated fatty acid ratio in
phosphatidylethanolamine was similar in differentiated HT29 and colonie
cells and lower than in Undifferentiated HT29 cells. A decrease in this
latter ratio in phosphatidylcholine was also observed in colonie cells and
HT29 cells grown in nude mice. These changes were essentially due to
opposite variations in the percentage of palmitoleic acid and those of
linoleic and/or arachidonic acids in both phospholipids. Thus, these data
indicate that Undifferentiated HT29 cells were characterized by the
absence of a specific phospholipid composition in their plasma membrane,
which is suggested to be related to altered phospholipid sorting. The
plasma membrane phospholipid profile reversed essentially to the normal
pattern when HT29 cells recovered the ability to differentiate.
INTRODUCTION
Various attempts have been performed to determine the
possible changes in phospholipid patterns associated with ma
lignancy (1-5) and/or differentiation (6-9) and to correlate
these variations with the organization and functioning of cell
ular 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 in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from FNCLCC and CNRS and
was 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 concerning
phospholipid modifications. Whether restoration of ultrastruc
tural characteristics during the differentiation of tumor cells is
accompanied by the regression of biological and biochemical
features, typical of a malignant phenotype, is a question still
unanswered (6, 12, 13).
The human colon adenocarcinoma cell line HT29 appears to
be a valuable experimental model to determine the respective
influence of the malignant transformation and differentiation
in the expression of cellular constituents and functions of the
intestinal epithelium, since these cells are available under either
an Undifferentiated or a differentiated state (14-18). The differ
entiation process of this cell line is reversible and mimics the
normal intestinal ontogenesis and epithelial crypt-to-villus mi
gration. However, these cells are malignant and of colonie
origin, and the differentiated HT29 cells exhibit the closest
analogies to human fetal colon (16, 19, 20). In spite of these
limitations, recent reports are indicative of the potential interest
in this experimental model (21-27) to determine the respective
influence of neoplasia and differentiation in the expression of
morphological 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 percentage
and degree of fatty acid saturation of phospholipids have been
found to influence a number of important functions in the rat
intestinal antipodal plasma membranes (28-30).
In this study, we compare the phospholipid composition of
the whole cell homogenate and the corresponding plasma mem
brane fraction of Undifferentiated and differentiated HT29 cells
with human normal mature colonie cells used as a reference.
The most striking change was that the specific phospholipid
profile in the plasma membrane of normal colonie cells did not
occur in Undifferentiated HT29 cells but was recovered in
differentiated cells, suggesting that the phospholipid polarized
delivery to the cell surface is severely altered in relation to the
neoplastic process.
MATERIALS AND METHODS
Cultured Cells, Tumors in Nude Mice, and Normal Adult Colonie
Cells. The established cell line HT29 (HT29-Glc), which was derived
from a human colon adenocarcinoma (31), was cultured in Dulbecco's
modified Eagle's medium (Gibco-Brl, Cergy Pontoise, France) contain
ing 25 HIMglucose and supplemented with 10% (v/v) PCS' (SeralabBiosys, Compiègne, France) (15, 18). These cells were adapted in one
step to grow in the same medium devoid of glucose and pyruvate
(Eurobio, Paris, France), supplemented with 2.5 mM inosine (Sigma
Chimie, La Verpillière, France) and 10% dialyzed FCS. Under these
conditions, no significant cell loss was observed and the resulting HT29
cells (HT29-Ino) exhibited a confluency-dependent enterocytic differ
entiation as previously reported (17, 18, 22). Both cell types were
routinely 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.
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LIPID CHANGES DURING DIFFERENTIATION
EDTA in PBS) every week. The cells were seeded at 10 x 10" cells/
150-cm2 plastic flask and maintained at 37°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 for
HT29-G1C cells. The HT29-Ino cells (passages 7 to 12) were used at
early (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 cells
and late confluent HT29-Ino cells, respectively. Cell layers were rinsed
3 times with PBS, pH 7.2, and immediately fixed in situ for electron
microscopy or mechanically scraped for biochemical analyses. HT29N cells were obtained as previously reported (14, 16, 32) from tumors
developed in athymic nu/nu mice (Cseal-Cnrs, Orléans,France) after
s.c. injection of 10 x 10' HT29-Glc cells into animals fed under
conventional sterile conditions. After 30 to 40 days of growth, solid
tumors were excised immediately after decapitation, the connective
tissue was discarded, and the tumors were minced with surgical scissors.
Normal adult colonie cells were scraped, using a glass microscope
coverslip from fresh surgical colon fragments extensively prewashed
with 0.24 M NaCl. The dislodged cells were epithelial cells as found by
cytological studies and as reported previously (33).
Transmission Electron Microscopy. Cell layers in the culture flasks
or tumor samples were fixed for l h with 2.5% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4, and then with 1% osmium tetroxide in 0.2
M phosphate buffer. After extensive washing with the previous buffer,
the fixed samples were dehydrated in graded alcohols and embedded in
Epon. Thin sections were cut with a LKB 2088 ultratome, contrasted
with uranyl acetate and lead citrate, and examined with a Jeol 100 C
electron microscope.
Preparation of Whole Cell Homogenate and Plasma Membrane Frac
tion. Plasma membrane fractions were isolated essentially according to
Warren (34) and Perdue (35). All procedures were carried out at 4°C.
Cells were washed 3 times with 0.16 M NaCl and twice with 0.05 M
Tris-HCl, pH 7.4, by resuspension and centrifugation. The cell pellets
were 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, and
disrupted by 20 gentle strokes in a Dounce homogeneizer. This disrup
tion step was repeated once more for human normal colonie cells. The
whole-cell homogenates were sedimented at 105,000 x g for 1 h, and
the resulting pellets were homogeneized in 20% sucrose (w/w). Four
ml of this particulate suspension were layered onto a 30-ml continuous
density gradient of sucrose (20 to 45%, w/w) with 2 ml of 50% sucrose
solution on the bottom. After 15 h of centrifugation at 25,000 rpm in
a SW27 Beckman rotor, plasma membranes were found at a band of
density 1.08 to 1.15 on the basis of enzymatic criteria. This layer was
harvested, diluted with 0.16 M NaCl, and sedimented at 105,000 x g
for 45 min. The amount of protein recovered in this plasma membrane
fraction represented about 2.5% of the cell homogenate protein, which
is usual for plasma membrane preparation from various cell lines (36,
37).
Enzymatic Assays. Ouabain-sensitive, (Na+,K+)-dependent ATPase
OF TUMOR CELLS
by 2-h stirring in 20 volumes of chloroform:melhanol (2:1, v/v) al room
temperature according to the technique of Folch et al. (45). After
partition with 0.2 volume of 0.85% KCI, the lower organic phase was
evaporated to dryness, resuspended in a small volume of chloroform:melhanol (2:1, v/v), and stored al —¿20°C
under nitrogen. Free
cholesterol was estimated using an enzymatic melhod utilizing choles
terol oxidase, catalase, and acetylacelone (Boehringer-Mannheim, Meylan, France). Phosphorus in phospholipid was eslimated by the method
of Ames (39).
Phospholipids (150 nmol) were fractionated by two-dimensional
TLC on 20-x 20-cm precoated silica gel H plates (Merck, Nogent-surMarne, 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 spraying
with a specific reagent (46) and idenlified by comparison wilh reference
slandards. After Ihe first migration, plates were sprayed with 0.005 M
HgCU in 0.1 M acetic acid and dried under vacuum for l h for
plasmalogen determinalion. Individual spols were scraped off and
analyzed for phosphorus coment as described above.
Fally acid analyses of phosphalidylcholine and phosphalidylelhanolamine isolated from preparative TLC of cell homogenate phospholipids (750 nmol) were performed as described earlier (5). Fatly acid
melhyl eslers and DMA of both isolated phospholipids were prepared
by boron Irifluoride Iransmelhylalion and analyzed isolhermally al
185°Cby gas-liquid chromalography wilh a Varian 1440 gas chromalograph equipped with a hydrogen flame ioni/ai ion detector in a column
of 7% butanediolsuccinale on Gas-Chrom Q, 100/200 meshes (Gerdel,
Suresnes, France) wilh a nitrogen flow rate of 28 ml/min. Peaks were
identified by comparison wilh ihe relenlion lime of reference slandards
and were quaniiiated by integralion of iheir areas wilh a Varian CDS
III calculator.
RESULTS
Ultrastructural Characterization of HT29 Cells Grown in Cul
ture and in Nude Mice Tumors. HT29-Glc cells at late confluency displayed disorganized multilayers of undifferentiated
cells (Fig. IA). These cells exhibited cytoplasmic projections
randomly dispersed at the cell periphery and interacted by
numerous desmosomes but lacking tight junctions. Early con
fluent HT29-Ino cells formed essentially a monolayer with
some multilayered regions of unpolarized cells (Fig. ÃŒB).
Only
spare, irregular microvilli were present on the surface layer with
few tight junctions. At late confluency, HT29-Ino cells were
organized into a typical polarized monolayer with well-devel
oped junctional complexes and characteristic apical brush bor
der microvilli (Fig. 1C). HT29-N cells showed the presence of
(EC 3.6.1.3) activity was measured according to the method of Gratecos
intercellular cysts with a regular and well-organized brush bor
et al. (38). 5'-Nucleotidase (EC 3.1.3.5) activity was determined by
der in the majority of them (Fig. \D).
liberation of inorganic phosphate (39). A'-Aminopeptidase (EC 3.4.11.2)
Enzymatic Characterization of the Homogenates and Plasma
and alkaline phosphatase (EC 3.1.3.1) activities were measured spectrophotometrically using appropriate substrates (40, 41). Rolenone-insenMembrane Fractions from Differentiated and Undifferentiated
sitive NADPH-cytochrome c reducÃ-ase(EC 1.6.99.3) was detected by Cells. The specific activity of several marker enzymes was
determined in both the whole-cell homogenate and the plasma
the reduction of oxidized cytochrome c at 550 nm (42). Measurement
ofcytochromecoxidase(EC
1.9.3.1) was done according to the method
membrane fraction prepared from each cell type. The results
of Cooperstein and Lazarow (43). Protein content was determined by are presented in Table 1. For each plasma membrane marker,
the method of Lowry et al. (44) using bovine serum albumin as a
the respective specific activities were nearly similar in all ho
standard. The enzyme activities are expressed as milliunits per mg of
mogenates. These values were markedly increased in the cor
protein. One unit is defined as the activity that utilizes 1 ^mol of
responding plasma membrane fractions except for both brush
substrate per min under experimental conditions. Alkaline phosphatase,
border-associated hydrolases in the morphologically undiffer
7V-aminopeplidase, and 5'-nucleotidase were utilized as apical mem
entiated cells. In HT29-G1C and early confluent HT29-Ino cells,
brane markers; (Na+,K+)-ATPase for the basolateral membrane; and
the activities of the A'-aminopeptidase and alkaline phosphatase
cytochrome c oxidase and NADPH-cytochrome c reducÃ-asefor the
were,
respectively, either only moderately or not significantly
mitochondria and the endoplasmic reticulum, respectively.
enhanced in plasma membranes compared with homogenates.
Extraclion and Analyses of Lipids. Lipids from cell homogenates and
plasma membrane fractions were extracted as previously described (5) On the other hand, plasma membrane fractions were depleted
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LIPID CHANGES DURING DIFFERENTIATION
,
OF TUMOR CELLS
-bb
D
C
Fig. 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 cells
grown 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 with
irregular 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 presence
of 2.5 TOMinosine and absence of glucose (30 days after seeding): vertical section showing a monolayer of polarized and differentiated cells with apical brush border
microvilli (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 plasma
membrane 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 fractions
Preparation of whole-cell homogenates and plasma membrane fractions as well as the enzymatic assays was described in "Materials and Methods."
Cells
HT29-GIC
confluentMarker
enzymes(Na\K*)-ATPase
HT29-Ino
Early
HT29-N
Normal colonie
confluentWhole-cell
homogenate36.4membrane189.2homogenate55.2*
membrane152.3*
homogenate46.0
membrane134.7homogenate50.9membrane140.2homogenate43.5
membrane181.4
+ 9.9
±33.1
±12.1
±22.1
±40.5
±12.4
±28.9
NDC
53.8
13.0 247.8 ±45.1 44.7 ±14.8 181.2 + 22.4
ND
32.6 ±0.8
158.9 ±6.6 30.2 ±6.6 128.9 ±19.2
15.3 + 2.4
67.2 + 25.1
5.3 1.9
18.2 ±2.6
6.1 ±1.9
6.0 ±1.4
39.6 ±2.6
7.1 ±1.0
108.9 ±37.3 11.9 ±3.5
1.0 + 0.3
7.0 + 2.1
1.3 + 0.2
6.4+ 1.2
0.6 0.2
1.8 ±1.1
2.1 ±1.3
1.3 ±0.2
5.1 ±2.4
1.6 ±0.4
11.8*
2.9*
10.3 + 2.1
4.9 + 2.1 15.6 + 2.4
6.2 1.2
2.0 ±1.3
2.1 ±1.5 16.2 ±3.9
0.5 ±0.3
3.2»Plasma
1.2»Late
4.9
+
2.7Plasma
1.5
+
0.7Whole-cell
1.8+
1.1Plasma
4.814.6°
1.4Plasma0.7 ±0.2Whole-cell
2.4 ±0.3Whole-cell
5.2 ±0.9Plasma2.3 ±1.1
5'-Nucleotidase
jV-Aminopeptidase
Alkaline phosphatase
Cytochrome c oxidase
NADPH cytochrome c
reducÃ-aseWhole-cell
°Mean ±SD of 3 to 5 separate independent experiments, milliunits/mg of protein. One unit is defined as the activity that utilizes 1 »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 reducÃ-ase,
and the contamination by mitochondria and endoplasmic reticulum was calculated to be less than 5% (data not shown).
Comparative Free Cholesterol and Phospholipid Contents of
Cell Homogenates and Plasma Membrane Fractions. As shown
in Fig. 2, the free cholesterohphospholipid
ratio was higher in
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LIPID CHANGES DURING DIFFERENTIATION
I
] Plasma membrani
Cell homogenate
T
a b c d e
Free cholesterol
3
b c d
Phospholipid
a b c d e
Free cholesterol/phospriolipid
Fig. 2. Free cholesterol and phospholipid contents of whole-cell homogenates
and plasma membrane fractions. Free cholesterol and total lipid phosphorus were
estimated as described in "Materials and Methods." From these values, the free
cholesterol:phospholipid ratios were calculated, a, HT29-Glc; ¿>,
early confluent
HT29-Ino; c, late confluent HT29-Ino; d, HT29-N; e, normal colonie cells.
Columns, mean; bars, SD (n = 3 to 5). *, Significantly different from respective
values found in e with P < 0.05 (determined by Cochran's / test).
the plasma membrane fraction than in the cell homogenate for
each cell type. Moreover, this ratio in both cell homogenates
and plasma membrane fractions was, respectively, similar in all
undifferentiated and enterocytic-differentiated HT29 cells and
was enhanced as compared with that of normal colonie cells.
This increase was due to the reduced amount of phospholipid
in all types of HT29 cells, whereas the free cholesterol content
was similar in these and normal colonie cells.
Phospholipid Composition of the Cell Homogenates and
Plasma Membrane Fractions. Except for cardiolipin which was
lowered in all plasma membrane fractions, comparison of the
phospholipid composition between the cell homogenate and the
corresponding plasma membrane fraction was allowed to dis
tinguish two groups among the studied cells according to the
differentiation state (Table 2). In both HT29-Glc and early
confluent HT29-Ino cells, the percentage distribution of phos
phorus among the major phospholipid classes was similar in
the cell homogenate and plasma membrane fraction. On the
contrary, the plasma membrane phospholipid composition was
different from that of the cell homogenate in late confluent
HT29-Ino, HT29-N, and normal colonie cells. The most strik
ing changes were the approximately 3-fold increase in the
amount of sphingomyelin and a concomitant slight decrease of
phosphatidylethanolamine
in the plasma membrane fraction in
OF TUMOR CELLS
comparison to the respective cell homogenate. Moreover, there
was a 2-fold decrease in the proportion of phosphatidylserine
in the plasma membrane fraction of HT29-N and normal
colonie cells, whereas such a change was not found in late
confluent HT29-Ino cells. Table 2 also shows that the percent
age of the two choline-containing phospholipids, which repre
sented about 50% of the total phospholipid in all cell homoge
nates, was unchanged in the plasma membrane fractions of both
undifferentiated cells but was clearly enhanced in those of
differentiated cells. Similarly, the phosphatidylcholine:sphingomyelin and phosphatidylcholine:phosphatidylethanolamine
ra
tios in the plasma membrane fractions were, respectively,
greatly decreased and moderately increased in the three differ
entiated cell types, while the phosphatidylcholine:phosphatidylserine ratio was only enhanced in plasma membrane frac
tions of HT29-N and normal colonie cells.
Unsaturated Fatty Acid Composition of Phosphatidylcholine
and Phosphatidylethanolamine. The unsaturated fatty acid pat
terns of phosphatidylethanolamine
and phosphatidylcholine
purified from cell homogenates are reported in Tables 3 and 4,
respectively, and the ratio of monounsaturated to polyunsaturated fatty acids in both phospholipids is shown in Fig. 3. When
compared with normal colonie cells, both undifferentiated
HT29 cells were similarly characterized by a high proportion
of palmitoleic acid in both phospholipids associated with low
percentages of either linoleic, linolenic, and arachidonic acids
in phosphatidylethanolamine
or linoleic and arachidonic acids
in phosphatidylcholine. Consequently, the monounsaturated:
polyunsaturated fatty acid ratios in both undifferentiated HT29
cells were similarly increased 2- to 4-fold in phosphatidyletha
nolamine and 6- to 8-fold in phosphatidylcholine as compared
with those found in normal colonie cells. In both differentiated
HT29 cells, the monounsaturatedrpolyunsaturated
fatty acid
ratio of phosphatidylethanolamine
was similarly decreased as
compared with that found in undifferentiated cell types and had
the same value as in normal colonie cells. This reversion to the
normal was due to a partial decrease in the palmitoleic acid
proportion coupled with an increase only in the relative per
centage of either arachidonic acid in late confluent HT29-Ino
cells or both linoleic and linolenic acids in HT29-N cells. The
unsaturated fatty acid pattern of phosphatidylcholine exhibited
similar changes in HT29-N cells but was unmodified in late
confluent HT29-Ino cells as compared with undifferentiated
Table 2 Phospholipid composition of cell homogenates and plasma membrane fractions
Phospholipids 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
Early confluent
HT29-N
Normal colonie
Late confluent
Whole-cell
Whole-cell
Whole-cell
Whole-cell
Whole-cell
Plasma
Plasma
Plasma
Plasma
Plasma
Phospholipids
membrane
membrane
homogenate
membrane
homogenate
homogenate
homogenate
membrane
homogenate
membrane
2.8*5.2
±
PC"SMPEPIPSCLPC+SMPC/SMPC/PEPC/PS48.5
1.16.4
±
1.03.3
±
0.45.4
±
0.83.2
1.5 ±2.3
5 1.9 ±0.9
43.2 ±0.9
44.3 ±0.4
46.3 ±0.3
5 1.3 ±
0.4'27.9
±0.7
15.7+1.0'
3.9+1.8
14.6 ±0.6'
5.3 + 0.6
14.8 +
1.622.8
±
0.622.3
±
0.827.4
1.226.8
19.7+1.9'6.2+1.4
±1.6
20.1 + 0.9'
34.8 ±2.6
25.3±1.3C 27.2 ±2.4
4.36.7
±
0.58.9
1.28.3
±
3.99.6
0.47.2
7.6 ±0.8
8.0 + 0.8
7.9 + 0.7
7.6 + 0.8
8.2 ±
1.29.5
±
1.012.3
±
0.76.6
1.07.8
0.4'3.8
+ 0.6
5.5+1.3
6.0 + 0.7
3.2 ±1.2'
6.3+1.1
2.9 ±
1.55.7
±
2.11.6
±
0.74.2
1.11.8
0.7'53.1
+
0.8'52.4
0.0'54.7
+ 0.9
0.0'
2.8+1.0
0.8 ±0.3'
6.7 ±0.5
2.053.7
±
1.252.8
1.8'16.1
±2.4 67.6+1.2'
47.1 + 2.8
58.9+1.1'
51.6+1.1
66.1 ±
1.28.7
3.59.3
±
1.27.3
±
2.315.0
1.4'1.7
±
0.2'1.8
±0.4
3.3 ±0.2'
11.1 ±0.5
3.0 + 0.3'
8.7+1.1
3.5 ±
1.72.1
±
0.72.1
±
0.61.8
0.3'7.1
±0.2
2.6 ±0.1'
1.2 ±0.1
1.8 + 0.1'
1.7 ±0.2
2.6 ±
±0.15.1
±0.13.8
0.27.5
±0.06.0
±0.7
9.4+1.6
7.2 ±1.0
13.8+1.4'
7.3 ±1.3
17.7+1.4'
±1.146.7
±0.749.5
0.947.0
±0.95
" PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine + plasmalogens; PI, phosphatidylinositol: PS, phosphatidylserine; CL, cardiolipin.
* Mean ±SD (n = 3 to 5), expressed as molar percentage of total phospholipid. From these values, the mean ±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).
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LIPID CHANGES DURING DIFFERENTIATION
Table 3 Unsaturated fatly acid and DMA composition of
phosphatidylethanolamine
Fatty acid methylesters and DMA of phosphatidylethanolamine purified from
cell homogenates were prepared by boron trifluoride transmethylation and ana
lyzed by gas-liquid chromatography as described in "Materials and Methods."
Cells
OF TUMOR CELLS
components was unchanged after 3 days of cell growth without
medium change (data not shown).
DISCUSSION
This investigation revealed marked differences in the phospholipid pattern between undifferentiated and differentiated
Fatty
acids16:1
confluent21.4 confluent6.2
colonie3.1
HT29 human colonie adenocarcinoma cells. The phospholipid
2.6"39.3
±
2.0*26.6
±
0.7*24.118.91.61.28.42.09.61.81.9*0.3*0.20.70.41.5Normal
±
±0.5*29.4
composition of the plasma membrane in undifferentiated cells
u)718:1
±0.732.21.21.26.42.37.62.60.3«0.20.81.21.3Late
o)918:2^)618:3
4.33.3
±
3.51.4
±
2.612.4
±
resembled that of the whole cell homogenate. On the contrary,
±2.8*2.9
1.20.3
±
±0.30.7
0.5*2.2
±
significant differences in the phospholipid composition between
0.32.7
±
o)320:3
±0.12.4
u)620:4
0.37.9
±
1.912.9
±
0.314.9
±
the cell homogenate and the plasma membrane found in normal
±0.3*3.3
3.0*1.6
±
0.620:5
1.83.8
±
mature colonie cells occurred in differentiated HT29 cells.
w3DMAHT29-G1C17.1
±0.16.0
1.68.0
±
±0.115.6
±1.1*
Moreover, changes in the monounsaturated:polyunsaturated
±2.7Early
±0.9HT29-N8.2
°Mean ±SD of 3 to 5 determinations, expressed as weight percentage of total
fatty acid ratio in phospholipids were also found to be related
to the differentiation state of this cell line.
fatty acids.
* P < 0.05 compared with values in HT29-Glc cells (determined by Cochran's
As previously reported (14-18), the present ultrastructural
t test).
data showed that HT29 cells cultured either in the presence of
glucose (15, 18) or at early confluency in a glucose-free medium
containing inosine (17, 18) were essentially undifferentiated.
Table 4 Unsaturated fatty acid composition ofphosphatidylcholine
Phosphatidylcholine purified after TLC of cell homogenate phospholipids was
On the contrary, HT29 cells grown in nude mice (14, 16, 32)
treated by boron trifluoride and analyzed by gas-liquid chromatography.
or cultured at late confluency in the presence of inosine without
Cells
glucose (17, 18) displayed the presence of regular apical microHT29-Ino
villi and tight junctions.
In order to determine the possible relationship between the
phospholipid pattern and differentiation in the HT29 cell sys
Fatty
acids16:1
confluent30.8 confluent22.4
colonie5.6
±0.8*25.415.80.20.51.70.40.3*0.10.30.2*0.2
9
0.4*38.3
±
tem, the plasma membrane fractions were prepared from dif
1.530.0
±
o)718:1
1.927.5
±
0,918:2
3.03.2
±
3.820.1
±
2.32.3
±
ferent HT29 cell types and human normal colonie cells. Except
1.9*—0.8
±
o)6I8:3o>320:3
1.3r0.7
±
0.20.4
±
for the brush border-associated hydrolases, the distribution of
0.10.6
±
marker enzymes always showed respective similar enhance
0.40.7
±
0.22.3
±
0)620:4
±
0.30.3
±
±0.1—HT29-N11. 0.3*0.3
0)620:5
ments of the plasma membrane enzymes and the same decrease
+
0.1Normal
0.4Early
±0.2Late
±0.1
U3HT29-G1C21.340.81.70.51.30.91.3"4.60.30.20.60.30.6
of
intracellular enzymes in all plasma membrane fractions as
°Mean ±SD of 3 to 5 determinations, expressed as weight percentage of total
compared
with the corresponding cell homogenates. Thus, it
fatty acids.
* Ps 0.05 compared with values in HT29-Glc cells (determined by Cochran's
can be considered that the degree of purity of the plasma
t test.
membrane prepared from all cell types was nearly the same
with a minor contamination with intracellular organelles.
In contrast with these data, the specific activities of the brush
border-associated hydrolases in the plasma membrane were
PE
similarly highly increased as compared with those of the cell
fatHnounsaturated
Js/Polyunsaturated
njÕÕ
homogenate in both morphologically differentiated HT29 and
normal colonie cells. This marked enhancement of alkaline
phosphatase and aminopeptidase activities was not found in
the plasma membrane isolated from undifferentiated HT29
cells. This distribution of brush border-associated hydrolases
related to the differentiation state of HT29 cells is consistent
with previous studies (17, 22) as well as with ultrastructural
I1IiIii_I_l
data reported above.
The lipid composition of these purified membranes and their
corresponding cell homogenates was investigated in undiffer
entiated and differentiated HT29 cells, and in normal colonie
cells.
Fig. 3. Monounsaturated:polyunsaturated
fatty acid ratios in phosphatidylchoChanges in the total or free cholesterol:phospholipid ratio of
line and phosphatidylethanolamine. a to c. as in Fig. 2. *, Significantly different
cell
homogenate, plasma membrane, and various intracellular
from respective values found in a with P < 0.01 for phosphatidylcholine and P <
0.05 for phosphatidylethanolamine (determined by Cochran's t test). Columns,
membranes occur often in relationship with cellular transfor
mean: bars, SD (n = 3 to 5).
mation or differentiation. In this study, however, the amounts
of free cholesterol and total phospholipid, and consequently the
cells. In addition, DMA that originate from plasmalogens were free cholesterol:phospholipid molar ratio in both cell homoge
present only in phosphatidylethanolamine
with about a 2-fold nate and plasma membrane fraction, did not differ between
increased level in normal colonie cells. The differences in fatty
undifferentiated and differentiated HT29 cells. A pronounced
acid composition of both phosphatidylethanolamine
and phos
increase of phospholipid content in both cell homogenate and
phatidylcholine found between the early confluent and the late plasma membrane that resulted in a decrease in the free cholesconfluent HT29-Ino cells were not due to a depletion of essen
terol:phospholipid ratio was found in normal colonie cells.
tial fatty acids in the culture medium, since the level of these
Recent results indicated that the levels of free cholesterol and
HT29-Ino
1274
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LIPID CHANGES DURING DIFFERENTIATION
total phospholipid were, respectively, slightly or greatly in
creased in human colonie tumors as compared with normal
intestinal mucosa (47). The reasons for this discrepancy are at
this time unknown.
In normal colonie cells, comparative phospholipid analyses
of the cell homogenate and the plasma membrane showed a
high increase in sphingomyelin content and a moderate increase
in phosphatidylcholine amount, associated with slight decreases
in phosphatidylethanolamine
and phosphatidylserine contents
and the absence of cardiolipin in the plasma membrane. These
differences, notably the enrichment of sphingomyelin, the ab
sence of cardiolipin, and the increase of free cholesterol, are in
line with the general trend of lipid localization in normal
eukaryotic cells (48). These results were also consistent with
similarities in the distinct phospholipid composition of each
antipodal plasma membrane observed in the rat colonocyte and
enterocyte (28, 29, 49, 50), the brush border domain having a
specially high sphingomyelin amount and the basolateral do
main being enriched to a lesser extent in phosphatidylcholine.
The differential phospholipid pattern between the cell ho
mogenate and the plasma membrane found in normal colonie
cells was also seen in differentiated HT29 cells, with the excep
tion of certain variations which were less acute or abolished
according to the differentiated cell type. It seems likely that
these slight differences in the plasma membrane phospholipid
composition between differentiated HT29 and normal colonie
cells were, at least in part, dependent on the differentiation
state of HT29 cells (16, 19, 20).
On the contrary, in undifferentiated HT29 cells, the plasma
membrane-phospholipid composition was similar to that of cell
homogenate. This organelle had only a moderate increase of
the sphingomyelin content and a decrease of cardiolipin. This
equalization of the phospholipid pattern between the plasma
membrane and the cell homogenate has already been reported
in some experimental tumors, notably various rat hepatomas
as compared with normal liver (2, 51, 52). This loss of specific
phospholipid composition in the plasma membrane was not
due to cross-contamination and intermembrane phospholipid
redistribution during subcellular fractionation or related to cell
growth, but it was dependent on the malignancy of the tumor
(52).
Moreover, the phospholipid composition of all cell homogenates was similar and, therefore, the biosynthesis of these
components was possibly unchanged in different cell types. On
the contrary, the phospholipid composition of the plasma mem
brane was a function of the differentiation state, suggesting that
the polarized distribution of the phospholipids between the
exoplasmic and cytoplasmic leaflets of the antipodal domains
in epithelial cells (53) was probably altered in undifferentiated
HT29 cells, but essentially restored in their enterocytic-differentiated counterparts. It was recently reported that the relative
rate of translocation, and not the synthetic step, was the most
important factor controlling the phospholipid segregation be
tween apical and basolateral membranes in rat renal proximal
tubule cells (54). These probable altered sorting and targeting
of phospholipids in undifferentiated HT29 cells are also con
sistent with the abnormal expression and distribution of other
cell surface molecules recently observed in these cells when
compared with their differentiated counterparts (21, 23-27). In
particular, as already indicated above in this study, the levels of
both alkaline phosphatase and /V-aminopeptidase were not in
creased in the undifferentiated plasma membrane.
The differences in acyl group composition of total or individ
OF TUMOR CELLS
ual phospholipids in relation to cell transformation or differ
entiation have often been reported. Tumor cells were usually
characterized by a lower proportion of polyunsaturated fatty
acids and a higher percentage of monounsaturated fatty acids
than the corresponding normal cells (1,5, 55). In agreement
with this general tendency, the monounsaturatedrpolyunsaturated fatty acid ratio in both phosphatidylcholine and
phosphatidylethanolamine
had the highest value in undiffer
entiated tumoral HT29 cells but the lowest in normal colonie
cells. These changes were essentially the result of an opposite
variation in the palmitoleic acid proportion and those of linoleic, arachidonic, and/or linolenic acids in both phospholipids.
The differentiation of HT29 cells leads to a partial reversion of
the fatty acid pattern characteristic of normal colonie cells.
Except for phosphatidylcholine in one differentiated HT29 cell
type, the monounsaturated:polyunsaturated
fatty acid ratios
had the same values. However, this restoration was related to
lower changes in the proportion of the fatty acids than the
differences found between undifferentiated HT29 cells and nor
mal colonie cells. In particular, there was only a decrease in the
amount of either arachidonic acid or linoleic and linolenic fatty
acids, depending on the differentiated HT29 cell type. The
reason for the absence of fatty acid reversion in phosphatidyl
choline in differentiated HT29-N cells is unknown but might
be due, at least in part, to the low and quite similar proportion
of arachidonic acid in this phospholipid in all HT29 cell types
as compared with that in phosphatidylethanolamine.
These data
are consistent with changes in fatty acyl profile observed during
in vitro differentiation of various cultured cells (8, 56-59),
although controversial data were reported in Friend erythroleukemia cells (7, 57). The fatty acyl changes found in this study
did not appear to be due to variations in essential fatty acid
uptake related to different nutritional conditions. They might
be due rather to alterations in the activities of enzymes respon
sible for the transformation of linoleic and linolenic precursors
in other polyunsaturated fatty acids (55, 60). The decreased
contents of arachidonic acid and both linoleic and linolenic
precursors found in undifferentiated HT29 cells might also be
related to an increased formation of leukotrienes B4, sulfidopeptide leukotrienes, and prostaglandin E2 as observed in hu
man colonie adenocarcinomas when compared with autologous
normal colonie mucosa (47).
In summary, the results reported here, for the first time to
our knowledge, show that the absence of a characteristic phos
pholipid plasma membrane composition observed in undiffer
entiated HT29 cells reversed essentially to a normal pattern in
enterocytic-differentiated counterparts. Moreover, it seems rea
sonable to suggest that these significant phospholipid changes
might be important in different cellular properties related to
malignancy or differentiation in the HT29 experimental model
and, in turn, in the human gut. In particular, the phosphatidylcholine:phosphatidylethanolamine
ratio is known to be a deter
minant in cell-cell interaction, cell-substrate adhesion, endocytosis, receptor binding, and metastatic potential (1,61,62). The
level of polyunsaturated fatty acids affects lectin-induced agglutinability, receptor interactions, cytolysis, proliferation, tumorigenicity, and metastatic potential (1, 5, 11, 63, 64). Recently
sphingomyelin and more specifically its breakdown products,
which are protein kinase C inhibitors and may function as
second messengers, appear to be active in signal transduction,
receptor activities, phorbol ester-induced responses, action of
tumor promoters, and growth factors (65).
1275
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LIPID CHANGES DURING DIFFERENTIATION
ACKNOWLEDGMENTS
We are obliged to Dr. A. Zweibaum and Dr. G. Trugnan for the gift
of HT29-Ino cells and Dr. S. Champion for critically reading the
manuscript. We are very grateful to F. <Cannellini and R. Soirat for
the cell culture, M. Roccabianca for the electron microscopy, and J. J.
Roccabianca for the photographs.
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Differences in Lipid Characteristics of Undifferentiated and
Enterocytic-differentiated HT29 Human Colonic Cells
Max Reynier, Hélène Sari, Manola d'Anglebermes, et al.
Cancer Res 1991;51:1270-1277.
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