Download Differential expression of the human mucin genes MUC1 to MUC5 in

Journal of Cell Science 106, 771-783 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
771
Differential expression of the human mucin genes MUC1 to MUC5 in
relation to growth and differentiation of different mucus-secreting HT-29
cell subpopulations
Thécla Lesuffleur1,*, Nicole Porchet2, Jean-Pierre Aubert2, Dallas Swallow3, James R. Gum4, Young S.
Kim4, Francisco X. Real5 and Alain Zweibaum1
1INSERM U178, 16 avenue Paul-Vaillant Couturier, 94807
2INSERM U16, Lille, France
3MRC Human Biochemical Genetics Unit, London, UK
4Gastroenterology Research Laboratory, VA Hospital, San
5Institut Municipal d’Investigacio Mèdica, Barcelona, Spain
Villejuif Cedex, France
Francisco, CA, USA
*Author for correspondence at Unité de Recherches sur la Différenciation Cellulaire Intestinale
SUMMARY
Mucin expression was analysed, in relation to cell
growth, in parental HT-29 cells and in two populations
of mucus-secreting HT-29 cells selected by adaptation
to methotrexate (HT29-MTX) or 5-fluorouracil (HT29FU). These two populations express mature mucins that
differ in their immunoreactivity to antibodies against
gastric (HT29-MTX) or colonic mucins (HT29-FU). In
the parental population, at late confluency, only very
few cells produce mucins or the MUC1 glycoprotein, this
being consistent with the low level of expression of the
mRNAs corresponding to the MUC1 to MUC5C mucin
genes. In the HT29-MTX and HT29-FU populations, the
appearance of mucus droplets, as shown by histochemistry and immunofluorescence, starts a few days after
confluency, progressively involving a greater proportion
of cells and reaching a steady state at late confluency.
The MUC1 glycoprotein appears earlier, already being
detectable in preconfluent cells. Its distribution is
restricted to the apical surface of the cells and is dis-
tinct from that of the mucus droplets. In both populations the growth-related levels of MUC1 mRNA are concordant with the apparent levels of expression of the
MUC1 glycoprotein. The levels of MUC2, MUC3, MUC4
and MUC5C mRNAs differ from one population to
another and, within each population, according to the
stage of the culture. The highest levels of MUC2 and
MUC4 mRNAs are found in the HT29-FU cells, whereas
the highest levels of MUC3 and MUC5C are found in
the HT29-MTX cells, suggesting that the differences
observed in the mature mucins expressed by either population may be related to which MUC genes are
expressed. In both populations significant or even high
levels of MUC mRNAs are already present in early cultures, i.e. at a stage when the mature mucins are not yet
detectable, suggesting that mucin maturation is a later
event.
INTRODUCTION
apomucins. MUC1 (Gendler et al., 1987; Lan et al., 1990)
encodes a transmembrane mucin-like glycoprotein
expressed in many epithelial cell types. MUC2 and MUC3
cDNA clones have been isolated from a human small intestine cDNA library (Gum et al., 1989, 1990, 1992) and from
tracheobronchial libraries (Gerard et al., 1990; Jany et al.,
1991). MUC4 and a family of other partial cDNAs included
under the name of MUC5 have been isolated from a tracheobronchial library (Crepin et al., 1990; Nguyen Van
Cong et al., 1990; Porchet et al., 1991; Aubert et al., 1991;
Dufosse et al., 1993). The only full-length cDNA sequence
currently available is that of MUC1 (Gendler et al., 1990;
Lan et al., 1990), although most of the MUC2 cDNA has
been sequenced (Gum et al., 1992). Mucin-encoding genes
It has long been recognized that intestinal mucins are highly
heterogeneous in their glycosylation, since this is readily
detectable by a variety of biochemical and immunological
techniques (Wesley et al., 1985; Podolsky, 1985a,b; Podolsky et al., 1986). In contrast, less is known about the synthesis and regulation of expression of apomucins or their
structure and heterogeneity. Recent advances in two areas,
namely the cloning of cDNAs encoding apomucins and the
isolation of permanent lines of cultured mucus-secreting
cell populations, have facilitated the analysis of this problem.
It is now known that several distinct genes encode the
Key words: mucins, MUC genes, HT-29
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T. Lesuffleur and others
seem to be characterized by a high level of polymorphism
due to the presence of variable numbers of tandem repeat
sequences (Swallow et al., 1987; Gendler et al., 1988; Griffiths et al., 1990; Fox et al., 1992; Nguyen Van Cong et
al., 1990; Porchet et al., 1991; Toribara et al., 1991; Gross
et al., 1992). The identification of several distinct mucinencoding genes raises a number of general questions regarding the biology of mucus-secreting cells. (1) How many
apomucin genes can be expressed in one tissue? (2) Are
multiple mucin genes expressed by a single cell? (3) What
is the relationship between morphological differentiation
and mucin mRNA and protein synthesis? (4) what is the
relationship between apomucin structure and glycosylation?
The mucus-secreting subpopulations isolated from the
HT-29 cell line should help in answering these questions.
This cell line is of particular interest with regard to mucins.
Indeed, from the original population, which contains a small
proportion (<0.5%) of goblet cells (Augeron and Laboisse,
1984; Lesuffleur et al., 1990, 1991c), several groups have
isolated, through selective pressure, clones or subpopulations of mucus-secreting cells (Augeron and Laboisse,
1984; Huet et al., 1987; Hafez et al., 1990; Kreusel et al.,
1991; Devine et al., 1991). There is some evidence that the
mucins secreted by HT-29 cells are heterogeneous as to the
oligosaccharide epitopes that they express. Mucins secreted
by the clones isolated from galactose-adapted cells (Huet
et al., 1987) were found to share epitopes with normal
colonic mucins (Phillips et al., 1988), whereas those
secreted by the clones isolated from sodium butyrate-treated
cells (Augeron and Laboisse, 1984) are characterized by
sialylated oligosaccharide chains of gastric type (Capon et
al., 1992). Accordingly, the small proportion of goblet cells
present in the parental line were shown to share antigenic
properties with either colonic or gastric mucins as disclosed
by their immunoreactivity to polyclonal antibodies raised
against crude colonic or gastric mucins (Lesuffleur et al.,
1990).
We have recently isolated, through selective pressure
with methotrexate (MTX) (Lesuffleur et al., 1990, 1991a;
Dahyia et al., 1992) or 5-fluorouracil (FU) (Lesuffleur et
al., 1991c), two distinct subpopulations of mucus-secreting
HT-29 cells that maintain their ability to differentiate under
normal culture conditions. These two populations differ as
to the morphology of the cells and to the type of immunoreactivity of their mucins. Cells adapted to 10−6 M MTX form
a homogeneous monolayer of polarized goblet cells (Fig.
1a) that exhibit a discrete apical brush border endowed with
intestinal hydrolases and secrete mucins of gastric
immunoreactivity (Lesuffleur et al., 1990, 1991a), two
characteristics that are reminiscent of the differentiation of
the human fetal colonic epithelium (Zweibaum et al., 1991;
Bara et al., 1986). Cells adapted to 10−5 M FU form a mixed
population involving a majority (80%) of polarized domeforming cells, devoid of a brush border, and a minority
(20%) of mucus-secreting cells that, in contrast to MTXadapted cells, are organized into multilayered foci of nonpolarized goblet cells that accumulate mucins within the
intercellular spaces (Fig. 1b); these mucins being of colonic
immunoreactivity (Lesuffleur et al., 1991c). These differentiation characteristics, like those reported by the other
groups, have mainly been analysed in late post-confluent
cultures and nothing is known of the time-course of the
goblet cell differentiation process in relation to cell growth,
a relationship that has been well documented in enterocytelike differentiated cultured cells like Caco-2 cells or other
HT-29 cell populations (Zweibaum et al., 1991).
The aims of this study were to analyse the time course
of differentiation of mucus-secreting MTX and FU cells, to
examine the expression of the mucin gene mRNAs in relation to the growth and to the phenotype of these two populations compared with the parental line, and to determine
whether the differences in phenotype observed between
MTX and FU cells are associated with differences in the
mucin genes that are expressed in these cells.
MATERIALS AND METHODS
Cell culture
The parental HT-29 cell line (Fogh and Trempe, 1975), obtained
from late Dr Jorgen Fogh (Memorial Sloan Kettering Cancer
Center, Rye, NY), was established from a blood group A patient
(J. Fogh, personal communication). The cell line was used
between passages 169 and 182 and is referred to as HT-29. The
subpopulations obtained by adaptation to 10−5 M 5-fluorouracil
(Lesuffleur et al., 1991c) and 10−6 M methotrexate (Lesuffleur et
al., 1990) are referred to as HT29-FU and HT29-MTX, respectively. Drug-adapted cells were used after several weekly passages
(4 to 20) in the absence of drug. They were routinely grown in
25 or 75 cm 2 plastic T-flasks (Corning Glassworks, Corning, NY)
in Dulbecco’s modified Eagles’s minimum essential medium supplemented with 10% heat-inactivated (56°C, 30 minutes) fetal
bovine serum (Boehringer Mannheim Biochemicals, Mannheim,
Germany) at 37°C in a 10% CO2/90% air atmosphere. For maintenance or experimental purposes all cells were seeded at 2×104
cells per cm2. The medium was changed daily.
Antibodies and lectins
Rabbit polyclonal antibodies against human gastric (L56) and
colonic (L53) mucins were prepared as reported (Lesuffleur et al.,
1990); a pool of mAbs against mucin M1 epitopes that specifically react with gastric mucins (Bara et al., 1986) was obtained
from Dr J. Bara (INSERM U55, Paris); mAb ZE4 was raised
against normal human small intestinal mucosa and was found to
react with colonic and small intestinal mucins, but not with gastric mucins (D. Swallow, unpublished results); mAbs BC1, BC2
and BC3 (Xing et al., 1989), and LICR LON M8 (McIlhinney et
al., 1985), which recognize the tandem repeat sequence of the
MUC1 gene product were obtained from Dr McKenzie (University of Melbourne, Australia) and The Ludwig Institute for Cancer
Research, Sutton, UK, respectively. Dipeptidylpeptidase-IV (DPPIV) was detected using mAb HBB 3/775/42 (Hauri et al., 1985)
raised against the human small intestinal enzyme (obtained from
Dr H. P. Hauri, Biocenter, Basel, Switzerland) and a rabbit
immune serum raised against the porcine kidney enzyme (Darmoul et al., 1991) (obtained from Dr G. Trugnan, INSERM U239,
Paris). Polyclonal rabbit antibodies against porcine villin (Robine
et al., 1985) were obtained from Dr D. Louvard (Institut Pasteur,
Paris). The following monoclonal antibodies were used for studying the saccharide epitopes associated with mucins: mAbs against
AB blood group antigens from the Second International Workshop and Symposium on Monoclonal Antibodies against Human
Red Blood Cells and Related Antigens (Oriol et al., 1990),
obtained from Dr R. Oriol (INSERM U178, Villejuif); 7LE against
Lea antigen (Bara et al., 1987), 2-25LE against Leb (Bara et al.,
1986), and 12-4LE against Ley (Bara et al., 1988), obtained from
Mucin gene expression in HT-29 cells
773
Dr J. Bara (INSERM U55, Paris); Cu-1 against Tn antigen (Takahashi et al., 1988) and B72.3 against sialyl-Tn (Nuti et al., 1982),
obtained from Dr K. O. Lloyd (Memorial Sloan Kettering Center,
New York, NY); G14 against αGalNAc (Dippold et al., 1987),
obtained from Dr W.G. Dippold (Johannes Gutenberg Universität,
Mainz, Germany); K21 against precursor type 1 (Rettig et al.,
1985), obtained from Dr W. J. Rettig (Memorial Sloan Kettering
Center, New York, NY); H15 against sialyl-Lea, obtained from Dr
J. Sakamoto (Aichi Cancer Center, Nagoya, Japan). Ulex
europaeus, Maackia amurensis and Sambucus nigra lectins
labelled with rhodamine were from EY laboratories (San Mateo,
CA, USA).
tion buffer containing 50% formamide, 5× SSC, 1× Denhardt’s
solution, 0.1% SDS, 20 mM sodium phosphate (pH 6.5), and 250
mg/ml denatured salmon sperm DNA. Filters were then hybridized
with the 32P-labeled probe for 20 hours at 42°C in prehybridization buffer containing 10% dextran sulphate (Thomas, 1980).
Blots were washed twice with 2× SSC, 0.1% SDS at room temperature, once with 0.1× SSC, 0.1% SDS at 55°C, and once, using
the same buffer, at 65°C for 15 minutes. Blots were then processed
for autoradiography. To normalize for RNA, filters were dehybridized and rehybridized with the actin cDNA probe.
Immunofluorescence and histochemistry
RESULTS
Indirect immunofluorescence was performed on unpermeabilized
cell layers and on cryostat sections of cell layer rolls as previously reported (Lesuffleur et al., 1990, 1991a,b). Cell layers and
sections were fixed (15 minutes, room temperature) in either
paraformaldehyde (3.5% in PBS Ca2+,Mg2+-free) or ethanol. Fluorescein-coupled sheep anti-rabbit and anti-mouse globulins were
from Institut Pasteur Production (Marnes la Coquette, France).
Rhodamine-coupled sheep anti-rabbit and anti-mouse globulins
were from Boehringer Mannheim. Periodic acid/Schiff and Alcian
blue staining (pH 2.5) of mucins was done on cryostat sections
of cell layers fixed in ethanol following standard procedures.
cDNA probes
MUC1 was detected using PUM24P (Yonezawa et al., 1991),
MUC2 with SMUC41 (Gum et al., 1989), MUC3 with SIB124
(Gum et al., 1990), MUC4 with JER64 (Porchet et al., 1991) and
MUC5C with JER58 (Crepin et al., 1990; Aubert et al., 1991).
Actin was detected with cDNA probe pA2 (Cleveland et al., 1986).
Northern blot analysis
Total RNA was extracted using guanidium isothiocyanate and centrifugation through a CsCl gradient (Chirgwin et al., 1979).
Poly(A)+ RNA was purified by chromatography on oligo(dT)-cellulose (Davis et al., 1986). Samples (4 mg) of mRNAs were fractionated by electrophoresis in 1% agarose gels after denaturation
in 1mM glyoxal (Thomas, 1980). Fractionated samples were transferred to reinforced nitrocellulose (Schleicher and Schuell, Dassel,
Germany) in the presence of 2 M ammonium acetate, 0.02 M
NaOH. Filters were incubated overnight at 42°C in prehybridiza-
Characteristics of mucin expression in relation to
growth and differentiation phenotype
The morphological differentiation of the cells, as well as
the secretion of mucins when it occurs, is a growth-related
phenomenon, starting after the cells have reached confluency, i.e. under our culture conditions, between 7 and 9
days, depending on the cell populations
In the parental HT-29 cells, goblet cells are detectable
only after confluency, which is complete at day 7, and will
never exceed 0.2% of the cells, even at late confluency, as
already reported (Lesuffleur et al., 1990). Their mucins
react either with anti-gastric or anti-colonic antibodies (Fig.
2). A few cells also express MUC1 (Fig. 2).
HT29-MTX cells reach confluency on day 8. A gel
becomes visible on the surface of the cell layer after 14
days of culture. This mucous gel becomes more conspicuous in the following days and continues to be present during
all the complete period of culture (up to 60 days). This
growth-related process is best visualized by indirect
immunofluorescence of the mucus present on the cell surface at different times in culture (Fig. 3), as well as by histological staining of cryostat sections of the cell layer,
which shows that the proportion of cells that express mucus
increases progressively with the age of the culture, reaching 100% after 21 days and remaining stable thereafter (Fig.
4). The pattern of indirect immunofluorescence staining
Fig. 1. Transmission electron microscopy of postconfluent cultures (day 28) of (a) HT29-MTX cells (bar, 6.25 mm) and (b) HT29-FU
cells (bar, 7.7 mm). Sections are perpendicular to the bottom of the flask (arrows).
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T. Lesuffleur and others
Fig. 2. Indirect immunofluorescence staining of paraformaldehyde-fixed cryostat sections of late post-confluent cultures (day 28) of
parental HT-29 cells with (a) polyclonal antiserum L-53 against colonic mucins; (b) polyclonal antiserum L-56 against gastric mucins;
(c) mAb BC2 against MUC1 peptide. The same results as shown in a and b were obtained with mAb ZE4 and mAb against M1 antigen,
respectively (not shown). Note that very few cells express immunoreactive material. Bar, 100 µm.
Fig. 3. Detection by indirect immunofluorescence, with the polyclonal antiserum L-56, of mucus secreted on the cell surface of cultures of
HT29-MTX cells in relation to cell growth; a, b, c, d, e and f correspond to days 7, 10, 12, 14, 21 and 28, respectively. Assays were
performed on unpermeabilized cultures fixed with ethanol. In order not to remove the mucous gel, the medium was aspirated gently from
the flask, which was kept horizontal, and ethanol was added without prior rinsing of the cell layer. The same results were obtained with
mAbs against M1 antigens. The small mucin granules present in all the pictures do not correspond to intracellular mucins but to small
clusters of mucins present on the cell layer surface as demonstrated by scanning electron microscopy or by their disappearance after
extensive rinsing of the cell layer prior to fixation (not shown). Bar, 100 µm.
Mucin gene expression in HT-29 cells
with anti-gastric mucin antibodies exactly corresponds to
the Alcian blue staining. These mucins appear to carry
terminal oligosaccharides with the structure NeuAcα2-
775
3Galβ-, as indicated by double immunofluorescence labeling with Maackia amurensis lectin (Fig. 5 and Table 1). A
high proportion of the mucin droplets also express termi-
Fig. 4. Alcian blue staining of ethanol-fixed cryostat sections of the cell layer of HT29-MTX cells in relation to cell growth. The numbers
indicate the day after seeding. Unlike the preparation shown in Fig. 3, the cell layer was rinsed with PBS prior to harvesting and freezing,
so that the mucus stained corresponds to the intracellular mucus, as in Fig. 1a. The same pattern of mucus expression was observed by
PAS staining or indirect immunofluorescence with mAbs against M1 antigens or polyclonal antiserum L-56 (not shown). Bar, 100 µm.
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T. Lesuffleur and others
Fig. 5. Double immunofluorescence labeling of an ethanol-fixed frozen cryostat section of a late post-confluent culture (day 28) of HT29MTX cells with (a) polyclonal antibodies L-56 and fluorescein-labelled globulins; and (b) Maackia amurensis labelled with rhodamine.
Bar, 40 µm.
nal αGalNAc and sialyl-Le a (Table 1). They do not express
blood group A, H, Lea and Le b antigens (Table 1). At late
confluency (after 21 days) a small proportion of mucin
droplets (<2%) also reacts with anti-colonic antibodies (not
shown). In contrast to the mucous secretions, MUC1 is
already expressed in 50% of the cell population in preconfluent cultures, with this proportion increasing to the entire
cell layer after 14 days and remaining stable thereafter (Fig.
6). MUC1 is not colocalized with the mucous droplets, but
is associated with the apical brush border, as substantiated
by its colocalization with DPP-IV or villin (Fig. 7).
The same growth-related expression of mucus secretion
is also observed in HT29-FU cells,which reach confluency
on day 9. In this population, however, the proportion of
mucus-secreting cells never exceeds 20%, even in late postconfluent cultures (Fig. 8). These mucins react with polyclonal and monoclonal antibodies against colonic mucins
(L-53 and ZE4) and appear to express unfucosylated linear
A antigen (Fig. 9) as deduced from the analysis of their
immunoreactivity to the panel of anti-blood group A antigens tested, as well as NeuAcα2-3Gal, as indicated by their
reactivity to Maackia amurensis (Table 1). A proportion of
these mucins also express Lea (Fig. 9), Tn, sialyl-Tn,
αGalNAc and sialyl-Lea (Table 1). As with HT29-MTX
Table 1. Immunoreactivity of mucins from HT29-M and HT29-FU cells to antibodies and lectins that recognise the
ABH and Lewis blood group structures
Approximate % of labelled
mucin droplets
mAbs and lectins
1, 2, 5, 6, 12, 14, 16, 19, 20*
13*
9*
48, 49, 50*
26, 28*
7Le
2.25Le
12.LE
Cu-1
B72-3
G14
K21
H15
Ulex europaeus
Maackia amurensis
Sambucca nigra
Specificity
ALey,
HT29-M
HT-29-FU
0
100
0
0
0
0
0
0
5
10
80
0
60
0
100
0
100
100
0
0
0
50
0
0
50
50
100
0
50
0
100
0
ALeb,
A type1, 2, 3, 4, 5, 6,
unfucosylated linear A, Forssman
A type2, 6, ALey, unfucosylated linear A
A type 3, 4, 5
A type 2, ALey, B
B
Le a
Le b
Le y
Tn
Sialyl-Tn
Terminal αGalNAc
Precursor type 1
Sialyl Le a
H
NeuAcα2-3Galβ-R
NeuAcα2-6Galβ-R
The results were obtained by immunofluorescence double-labelling assays with anti-gastric and anti-colonic mucin antibodies.
*The numbers correspond to the mAbs listed by Oriol et al. (1990).
Mucin gene expression in HT-29 cells
777
Fig. 6. Immunofluorescence detection of MUC1 peptide with mAb BC2 in paraformaldehyde-fixed cryostat sections of cultures of HT29MTX cells in relation to cell growth; a, b and c correspond to days 7, 14 and 21, respectively, and are from the same culture as shown in
Fig. 3. Note that MUC1 is already expressed at day 7, i.e. when no mucins can be detected by Alcian blue staining. Bar, 100 µm.
Fig. 7. Phase-contrast microscopy and double immunofluorescence staining of paraformaldehyde-fixed cryostat sections of late postconfluent cultures (day 21) of HT29-MTX cells; (a), (b) and (c), the same section labelled with: (b) the polyclonal antiserum L-56 against
gastric mucins; and (c) the mAb BC2 against MUC1 peptide; (d), (e) and (f), same section labelled with: (e) the polyclonal antiserum
against DPP-IV; and (f) the mAb BC2. Note the difference in distribution of MUC1 and mucins and the apical colocalization of MUC1
and DPP-IV. The same results as in (c) and (f) were obtained with the mAbs BC1, BC3 and LICR LON M8 (not shown). The same
results as in (e) were obtained with anti-villin antibodies (not shown). Bar, 40 µm.
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T. Lesuffleur and others
Fig. 8. Alcian blue staining of ethanol-fixed cryostat sections of HT29-FU cells in relation to cell growth. The numbers indicate the days
after seeding. The same pattern of mucin expression was observed with PAS staining or indirect immunofluorescence with the mAb ZE4
or the polyclonal antiserum L-53 against colonic mucins (not shown). Bar, 100 µm.
cells, MUC1 is already expressed at the apical surface of a
high proportion of cells in preconfluent cultures (day 7, not
shown), i.e. when mucous secretions are not yet detectable
by histological or immunological techniques. The proportion of cells expressing MUC1 increases during the days
following and is stable after day 14 (Fig. 9). Expression of
MUC1, as demonstrated by double-immunofluorescence
staining (not shown) is restricted to these polarized cells,
organized into a monolayer, which form the majority of the
HT29-FU population.
Differential and growth-related expression of
mucin genes mRNAs
Days 7, 14 and 21 were chosen for a comparative analysis
of the expression of the apomucin mRNAs in the three cell
populations, since these represent stages at which no, little
or extensive, mucus is produced by the cultures of MTX
and FU cells. As shown in Fig. 10, the presence and the
levels of expression of the MUC mRNAs vary with the time
in culture and the phenotype of the cells.
Two transcripts of 6.5 kb and 4 kb of MUC1, presumably corresponding to the two MUC1 alleles, are expressed
in all three populations. They are already expressed at a
high level in early cultures (day 7) in all populations. Except
for HT-29 cells, which show a decrease of these transcripts
with time, their levels remain high in the two differentiated
populations.
MUC2 mRNA is expressed at a low level in the parental
HT-29 cells and its level decreases with time. In the HT29FU cells it is expressed at much higher levels, but also
decreases with time. In the HT29-MTX cells the level of
expression is low in early cultures but increases with time.
The highest levels of MUC3 mRNA are found in the
MTX cells. In these cells the transcript is already detectable
at day 7; it greatly increases at day 14, remaining stable
thereafter. It is poorly expressed in HT-29 and not
detectable in the FU cells.
MUC4 mRNA is the most highly expressed in the HT29FU cells, with its level of expression decreasing from day
7 to day 21. It is poorly expressed in HT-29 and HT29MTX cells.
The highest levels of MUC5C mRNA are found in the
HT29-MTX cells, with the level increasing dramatically
between 7 and 14 days of culture. A similar increase in
expression with time was not detected in the other cell populations where the expression remains low.
Because of the stable expression of mucins in the late
post-confluent cultures of HT29-MTX cells and the
expression of some mucins of colonic phenotype at very
late confluency, poly(A)+ RNAs prepared from cultures of
Mucin gene expression in HT-29 cells
779
Fig. 9. Immunofluorescence detection of mucins and the MUC1 peptide in cryostat sections of post-confluent cultures (day 21) of HT29FU cells. In (a) and (b), ethanol-fixed sections were double-labelled with: (a) the polyclonal antiserum L-53 against colonic mucins; and
(b) the mAb 021 against blood group A antigen (see Table 1), showing that all the mucin droplets express both types of immunoreactive
material. In (c) and (d), ethanol-fixed sections were double-labelled with: (c) the polyclonal antiserum L-53; and (d) the mAb 7LE against
Lea antigen showing that only half of the mucin droplets express Lea. In (e) paraformaldehyde-fixed sections labelled with mAb BC2,
showing an apical expression of MUC1. Bar, 100 µm.
HT29-MTX cells harvested over a 60-day period were
hybridized with MUC2, MUC3 and MUC5C cDNAs. As
shown in Fig. 11, the levels of MUC3 and MUC5C mRNAs
are stable from day 21 onwards. Interestingly, the level of
MUC2 mRNA increases in late cultures.
DISCUSSION
Although a number of mucus-secreting HT-29 clones or
sublines have been isolated and used as in vitro models for
the analysis of functions associated with goblet cells, little
is known about the kinetics of differentiation of these cells
or which genes encode the mucins they secrete. Most
studies so far have been restricted to the ultrastructural or
histochemical analysis of cells at late confluency (Augeron
and Laboisse, 1984; Huet et al., 1987; Hafez et al., 1990;
Lesuffleur et al., 1990, 1991a,c; Kreusel et al., 1991), or to
the characterization of the oligosaccharide structures associated with their mucins (Phillips et al., 1988; Capon et al.,
1992). The differences in the oligosaccharide structures
observed by Phillips et al. and Capon et al. would suggest,
notwithstanding the different approaches used (immuno-
logical or biochemical) that the clones or subpopulations
analysed are different from one laboratory to another. This
is not surprising in view of the heterogeneity of the parental
cell line. Even less is known about which MUC genes are
expressed in these cells. Ogata et al. (1992) did not detect
any MUC1 to MUC4 mRNA in Northern blots of total RNA
from parental HT-29 cells. In a study on preconfluent cultures Dahyia et al. (1992) observed, by slot blot analysis of
total RNA, an increased expression of MUC1, but not of
MUC2 and MUC3, in MTX cells in comparison with the
parental HT-29 cells. The MUC1, but not MUC2 glycoprotein, was detected, by Western blot analysis, in the
mucins extracted from the gel secreted by a population of
mucus-secreting cells derived from sodium butyrate-treated
HT-29 cells (Devine et al., 1991).
The results reported here describe the growth-related
appearance of mucins, the corresponding time-course of
expression of the mucin gene mRNAs and the relationship
between the type of mucins secreted by the two different
populations of mucus-secreting HT-29 cells analysed and
the type of mucin genes that they express.
The appearance of mucus in both MTX and FU cells, as
disclosed by histological and immunohistological methods,
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T. Lesuffleur and others
MUC1
-actin
MUC2
MUC3
MUC4
MUC5C
-actin
Fig. 10. Northern blot hybridization of MUC1, MUC2, MUC3,
MUC4 and MUC5C cDNAs with poly(A)+ RNAs from cultures of
the parental HT-29 (P), HT29-FU (F) and HT29-MTX (M) cells
in relation to cell growth; (a), (b) and (c) correspond to days 7, 14
and 21, respectively. The results presented for MUC2, MUC3,
MUC4 and MUC5C represent successive hybridisation of a single
filter. The MUC1 results shown correspond to another filter.
Similar results were obtained from three different Northern blots
corresponding to experiments with cells at different passages.
Fig. 11. Growth-related expression of MUC2, MUC3 and MUC5C
mRNAs in long-term cultures of HT29-MTX cells; cDNAs were
hybridized with poly(A)+ RNAs using the same filter for all
hybridizations; a, b, c, d, e, f and g correspond to days 7, 14, 21,
28, 40, 50 and 60, respectively.
appears to be a growth-related process that starts at confluency and progressively involves an increasing proportion
of cells. This process differs in its time-course with each
of the mucins examined, the epithelial mucin MUC1 being
detected much earlier than the mucous droplets. It is also
associated with quantitative and qualitative differences in
MUC gene expression.
In HT29-MTX and FU cells the MUC1 protein is
expressed on the apical surface of the cells, like in other
systems of polarized cells (Ormerod et al., 1981; Corcoran
and Walker, 1990), and its localization is distinct from that
of the mucous droplets as disclosed by immunofluorescence. In contrast to the late appearance of mucous droplets
in these cells, the MUC1 protein is already present in preconfluent cultures. This high expression of the MUC1 protein is consistent with the observation that the levels of
MUC1 mRNA are high and almost constant during cell
growth in the two populations. In the parental line, however, unlike in the differentiated populations, the level of
MUC1 mRNA is high in early cultures, but decreases with
the time in culture. What mechanism is responsible for the
decrease in the steady-state level of MUC1 mRNA in these
cells remains to be elucidated.
The expression of MUC2, MUC3, MUC4 and MUC5C
mRNAs differs from one population to another and, within
each population, according to the stage of the culture.
MUC2 and MUC4 are expressed at the highest levels in
FU cells, the cells that express mucins of colonic
Mucin gene expression in HT-29 cells
immunoreactivity, whereas the highest levels of MUC3 and
MUC5C are found in the MTX cells, the cells that express
mucins of gastric immunoreactivity. It has been postulated
that the type of mucin peptide may determine the type of
carbohydrate present in the final mature mucin (Gum et al.,
1989). Although the epitopes recognized by the polyclonal
and monoclonal antibodies against gastric and colonic
mucins used in this study have not been identified, it is
clear that mucins from MTX and FU cells carry different
oligosaccharide structures. There is indirect evidence that
the oligosaccharide species associated with the mucins from
MTX cells are the same as those that have been characterized on the mucins produced by clone 16E (Capon et al.,
1992): they react with the lectin from Maackia amurensis,
which binds to the major oligosaccharide species found in
Cl.16E mucins, and we have previously shown that mucins
from Cl.16E immunoreact with the polyclonal antibodies
against gastric mucins (L-56) used in this study (Lesuffleur
et al., 1991b). Although further compositional studies are
required to determine precisely which oligosaccharide
structures are associated with the mucins from each population, it is clear that MUC2 and MUC4 are expressed concomitantly with the mucins of colonic-like phenotype (from
FU cells), while MUC3 and MUC5C are expressed concomitantly with the mucins of gastric-like phenotype (from
MTX cells). These results are interesting in view of recent
studies using in situ hybridization (Audié et al., 1993) and
apomucin-specific antibodies (Gambus et al., 1993; Ho et
al., 1993; and unpublished observations) which indicate that
high levels of MUC2 are expressed in the colon, but not in
the stomach, and the reciprocal pattern is observed with
MUC5C.
It is noteworthy that the expression of the MUC mRNAs
occurs earlier than the production of mucus (detected histologically) in the differentiated cells. This discrepancy
would suggest that the process of mucin biosynthesis in
these cells involves a growth-related time-lag between the
activation of the mucin genes, which are responsible for the
biosynthesis of the mucin peptides, and the onset of their
glycosylation. It would therefore be of interest to detect the
newly synthesized apomucins in early cultures. However,
using the monoclonal antibody LDQ10 (Gambus et al.,
1993) against MUC2 peptide, or polyclonal antibody M3P
against MUC3 repeat peptide (Gum et al., 1990) we were
unable to detect either of these these peptide antigens by
immunofluorescence, in 7-day-old cultures of FU and MTX
cells, respectively (unpublished results). This will be further investigated by electron microscopy and immunogold
labeling, since Egea et al. (1993) have successfully demonstrated MUC2 in the RER in post-confluent MTX cells by
this method, even though it is undetectable by immunofluorescence.
It is also possible and likely that other mucin genes are
expressed in these cells, such as the human mucin gene
MUC6, which has been identified recently (Toribara et al.,
1993), or other unidentified mucin genes.
Thus, notwithstanding the fact that they are malignant,
and may show abnormal apomucin synthesis and glycosylation, these two different populations of mucus-secreting
cells isolated from the HT-29 cell line appear to be relevant in vitro models for studying the sequence of regula-
781
tory events that are associated with the onset and maintenance of the biosynthesis of mucins, and the relationship
between the type of mucin gene expressed and the type of
glycosylation of the mature mucin.
This work was supported in part by Association pour la
Recherche sur le Cancer, Fondation pour la Recherche Médicale
Française, INSERM-CSIC Cooperation Agreement, grant SAL900853 from the Plan Nacional de I+D (Ministerio de Educacion,
Madrid), the Veterans Affairs Medical Research Service, and
NATO grant 0789/88. Part of this work has been presented at the
2nd International Workshop on Carcinoma-Associated Mucins
(Cambridge, August 1992).
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(Received 27 May 1993 - Accepted 27 July 1993)