[CANCER RESEARCH 47. 1426-1433. March 1. 1987]
A and H Blood Group Antigens as Markers of Sucrase-Isomaltase from the
Enterocyte-like Differentiated Human Colon Carcinoma Cell Lines HT-29
and Caco-21
Isabelle Chantret,2 Guillemette Chevalier, Elisabeth Dussaulx, and Alain Zweibaum
L'ailé
de Recherches sur le Métabolismeet la Différenciationde Cellules en Culture, INSERM U178, BâtimentINSERM, 16 avenue Paul Vaillant Couturier, 94807
Villejuif Cedex, France
ABSTRACT
The purpose of this work was to investigate whether sucrase-isomaltase from enterocyte-like differentiated human colon carcinoma cell lines
carries blood group antigens of the ABH system. Six cultured lines of
blood group A (HT-29, SW-480, Co-115) or O phenotype (Caco-2, HRT18, HCT-8R) were studied. Only HT-29 cells grown in the absence of
glucose (HT-29 Glc~) and Caco-2 cells express an enterocytic differen
tiation with the presence of sucrase-isomaltase
on the apical surface of
the cells. Binding of anti-A antibodies to HT-29 Glc~ and of MAI
to
Caco-2 cells gave the same apical immunofluorescence pattern of staining
as did anti-sucrase-isomaltase
antibodies, whereas only a membrane
binding was observed in nondifferentiated cells. Sucrase-isomaltase
¡mmunoisolated from HT-29 Glc~ and Caco-2 cells reacted with anti-A
antibodies and Ulex europaeus agglutinin-I (UEA-I), respectively, at
sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblot. Immunoprecipitation of solubilized brush border-enriched fractions
from the same cells with UEA-I or anti-A antibodies resulted in an
inhibition of sucrase activity which reached —80%for Caco-2 cells with
UEA-I and =50% for HT-29 cells with anti-A antibodies. Similar results
were obtained in the corresponding tumors in nude mice: anti-A antibodies
in HT-29 and UEA-I in Caco-2 tumors bound to the same apical
structures as did anti-sucrase-isomaltase
antibodies; sucrase-isomaltase
immunoisolated from the tumors bound anti-A antibodies (HT-29) or
UEA-I (Caco-2). These results support the hypothesis that sucraseisomaltase from enterocyte-like differentiated human colon cancer cells
carries blood group antigens of the ABH system. These findings suggest
that colon cancers which have been shown to display an apical pattern of
expression of ABH antigens should be screened for their possible enter
ocytic differentiation.
INTRODUCTION
There is consistent evidence that ABH blood group sub
stances can be considered as developmentally regulated colon
cancer-associated antigens in humans. Their developmental
nature relies on the observation that during fetal life they are
evenly expressed in the gut, including the entire colon and
rectum (1, 2), whereas after birth and throughout adulthood,
they are present in the small intestine and in the upper part of
the colon but absent from its distal part and from the rectum
(1, 3-5). By contrast to the normal some cancers arising from
the distal part of the colon and from the rectum have been
shown to reexpress ABH antigens (6-15).
A similar pattern of expression has recently been described
for brush border-associated hydrolases such as sucrase-isomal
tase, aminopeptidase N, dipeptidylpeptidase-IV, and alkaline
phosphatase. These enzymes, which are normally associated
with the brush border membrane of the small intestinal villous
cells but absent from the adult colon (16), have been shown to
be present in the fetal colon from which they disappear at birth
(17-20). Recent studies have shown that these same enzymes
are associated with the enterocytic differentiation expressed by
the human colon carcinoma cell lines HT-29 (21-24) and Caco2 (25-27) in culture, the corresponding tumors in nude mice
(28, 29) and some tumors from patients (29).
The close parallelism between these two patterns of devel
opmentally regulated colon cancer-associated markers as well
as the fact that brush border hydrolases, like ABH substances,
are highly glycosylated glycoproteins (16), raises the question
of whether these apparently two distinct patterns could be in
some cases a unique pattern. This hypothesis is further sup
ported by the observation that ABH antigenic determinants are
integral parts of the molecules of brush border-associated hy
drolases from the normal small intestine (30-34). Whether this
is also true for hydrolases associated with the enterocytic dif
ferentiation of colon cancer cells has not yet been investigated.
The purpose of the present work was therefore to investigate
whether antigenic determinants of the ABH system were asso
ciated with brush border hydrolases from enterocyte-like differ
entiated HT-29 and Caco-2 cells in culture and the correspond
ing tumors in nude mice. The reason for selecting these two
cell lines is that they are so far the only human colon carcinoma
cell lines which have been shown to express an enterocytic
differentiation. In the case of Caco-2 cells in culture this differ
entiation occurs under standard conditions (25-27); in the case
of cultured HT-29 cells the differentiation occurs only when
the cells are grown in the absence of glucose, i.e., when glucose
is replaced by galactose (21, 23, 24), by inosine or uridine (23)
or when the cells are adapted to grow in an unsupplemented
hexose-free medium (22). Independently of the culture condi
tions both cell lines, when injected into nude mice, produce
enterocyte-like differentiated tumors (28-29). Undifferentiated
HT-29 cells, i.e., grown in the presence of glucose (21-24), as
well as four other undifferentiated human colon carcinoma cell
lines were used as negative in vitro controls.
The present study was focused on sucrase-isomaltase since,
among the different brush border hydrolases, it is the one which
has been shown to be the more specifically associated with the
enterocytic differentiation of human colon cancer cells in vitro
(21-23, 25-27) as well as in vivo (28, 29). Moreover the recent
production of monoclonal antibodies specific for human su
crase-isomaltase easily allows the immunopurification of the
enzyme (27).
MATERIALS
AND METHODS
Cell Lines, Tumors in Nude Mice, and Normal Adult Organs. The
cultured cells used were established lines derived from human colori-mi
adenocarcinoma. The cell lines HT-29 (35), Caco-2 (36), and SW-480
Received 4/29/86; revised 11/10/86; accepted 12/1/86.
The costs of publication of this article were defrayed in part by the payment
(37) were obtained from Dr. J. Fogh (Sloan Kettering Memorial Center,
of page charges. This article must therefore be hereby marked advertisement in
Rye, NY), HRT-18 (38), and HCT-8R (39) from Dr. W. A. F. Tompaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
kins (University of Illinois, Urbana, IL), and Co-115 (40) from Dr. B.
1This work was supported by the Association pour la Recherche sur le Cancer
Sordat (Swiss Institute for Experimental Cancer Research, Epalinges/
(ARC).
2To whom requests for reprints should be addressed.
Lausanne, Switzerland). The optimal culture and passage conditions of
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BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
the different cell lines have been previously described (22, 26, 41).
Differentiated HT-29 cells were obtained by growing the cells in glu
cose-free Dulbecco's modified Eagle's minimum essential medium sup
plemented with 2.5 IHMinosine (Sigma, St. Louis, MO) as reported
(23). Differentiated HT-29 cells grown in the absence of glucose and
undifferentiated HT-29 cells grown in the presence of glucose will be
referred to as HT-29 Glc" and HT-29 Glc+ cells, respectively. The
development of tumors in nude mice as well as their differentiation
characteristics has been previously reported (28, 42). Normal ileum
samples were obtained from irreversibly brain damaged kidney donors
as previously reported (43).
ABO Blood Group Typing. Confluent cells were harvested with 0.25%
trypsin in 3 mM EDTA in 0.13 M Ca2+- and Mg2+-free PBS3 washed
three times in ice-cold saline and tested for their ABO phenotype using
concurrently two different methods: (a) indirect immunofluorescence
with human anti-A and anti-B agglutinins and IT,.VI on cells in
suspension; and (b) direct hemagglutination of A, B, and O human
RBC with anti-A, anti-B, or UEA-I previously absorbed with each cell
line (five times l h at 4°C).Both methods gave concordant results
which were consistent with the blood group phenotypes of the original
donors, with HT-29, SW-480, and Co-115 being of blood group A and
Caco-2, HRT-18, and HCT-8R being of blood group O.
Electron Microscopy. Transmission electron microscopy was per
formed on cells grown for 30 days in 25-cm2 plastic flasks. The methods
used were as previously reported (25).
Antibodies. Monoclonal antibody HBB 2/614/88 specific forsucraseisomaltase from normal human small intestine (27) was obtained from
Dr. H. P. Hauri (Biocenter of the University of Basel, Basel, Switzer
land). Poh donai rabbit antibodies produced against sucrase-isomaltase
from human blood group O small intestine were obtained from Dr. N.
Triadou (Hôpital Necker-Enfants Malades, Paris, France) (44). Antiblood group A antibodies were produced in A" rabbits (45) selected by
immunofluorescence typing on rectal biopsies (46) and hyperimmunized with human blood group A RBC (47); the antisera were made
specific by serial absorptions with blood groups B and O RBC and had
a final anti-A hemagglutination titre of 1/4000. Whenever needed, antiA antibodies were further purified by precipitation with (NH4)2SO4
and chromatography on DE-52 (Whatman Chemical Separation, Ltd,
Maidstone, England). UEA-I labeled or unlabeled with fluorescein
isothiocyanate (Vector Laboratories, Burlingame, CA) was used for the
detection of H substance. Anti-rabbit sheep antiglobulins and antimouse rabbit antiglobulins labeled with horseradish peroxidase or fluo
rescein isothiocyanate were obtained from Institut Pasteur Production
(Marnes-la-Coquette, France).
Enzyme Assays. Sucrase activity was measured according to Messer
and Dahlqvist (48) in brush border-enriched fractions (P2) (49) from
cells and tumors as previously reported (21-23,25, 26, 28). The enzyme
activity is expressed as mil Iiun its per mg of proteins. One unit is defined
as the activity that hydrolyses 1 /¿mo\of sucrose/min under the exper
imental conditions.
Immunodetection Assays. Immunofluorescence was performed on
frozen tumor cryostat sections and on cell monolayers grown on glass
coverslips using the same methods as previously reported (22, 25, 28,
29). For immunoblotting analysis of sucrase isomaltase the same
method as previously reported for tissues (28) and cells (22) was used.
Briefly 60 Mgof brush border proteins (P2 fraction) from cells or tumors
were separated on 7.5% SDS-polyacrylamide gel electrophoresis in the
buffer system of Laemmli (50), and transferred to nitrocellulose sheets
by the method of Burnette (51). Sucrase-isomaltase was identified with
rabbit anti-sucrase-isomaltase antibodies and revealed with the peroxi
dase technique. The technique of Hauri et al. (27) was used for the
immunopurification of sucrase-isomaltase using monoclonal antibody
HBB 2/614/88. To investigate whether sucrase-isomaltase carries blood
group antigens the immunopurified enzyme was run on 7.5% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose
sheets as described above. Blood group A antigen was identified with
rabbit anti-A antibodies and the peroxidase technique using anti-rabbit
globulins labeled with horseradish peroxidase. H substance was identi
3 The abbreviations used are: PBS, phosphate-buffered
europaeus agglutinin-I; SDS, sodium dodecyl sulfate.
saline; UEA-I, Ulex
fied with Ulex europaeus agglutinin-I coupled with horseradish perox
idase type VI (Sigma) by the method of Gonatas and Avrameas (52).
Immunodepletion Experiments. P2 fractions of Caco-2 and HT-29
cells (4 fig) sol ubiI¡mlwith Triton X-100 (1% for 1 mg/ml of protein)
diluted in 25 ¿il
of PBS were incubated (2h, 4°C)with 150 ^1 of serial
2-fold dilutions of either UEA-I (starting solution = 4 mg/ml) or antiA rabbit 7-globulins (starting solution = 0.75 mg/ml) and 25 n\ of a
40% solution in PBS of polyethylene glycol 6,000 (Prolabo, Paris,
France) and centrifuged (10 min, 10,000 x g). The activity of sucrase
was measured in the supernatants. The same conditions were used for
controls except that UEA-I and anti-A antibodies were omitted.
RESULTS
Enterocytic Differentiation Characteristics of Cultured Cells.
Blood group phenotypes, presence of sucrase-isomaltase, su
crase activities, and morphological enterocytic differentiation
characteristics of cultured cells are summarized in Table 1.
Only HT-29 Glc" and Caco-2 cells exhibit as previously re
ported (21-27) a characteristic pattern of enterocytic differen
tiation with the cells organized into a polarized monolayer with
the presence of tight junctions and apical brush border regular
microvilli (Fig. 1, a and b). Sucrase activity is present in both
cell lines, with, however, much higher values in Caco-2 than in
HT-29 Glc" cells (Table 1). The presence of the enzyme was
confirmed by immunofluorescence and immunoblotting using
anti-sucrase-isomaltase antibodies. Immunofluorescence of cell
layers showed a typical apical membrane staining, correspond
ing to the apical brush border, in both Caco-2 (Fig. 2a) and
HT-29 Glc" cells (Fig. 2c). As previously shown (22, 27) im
munoblotting of P2 fractions from HT-29 Glc~ and Caco-2
cells showed the presence of only the high molecular weight
form of the enzyme which migrates faster than the normal
intestinal form (Fig. 3). Among the other four cell lines Co-115
(Fig. le) and HRT-18 (Fig. Id) are also polarized, with the
presence of tight junctions and, at phase contrast microscopy,
the formation of domes (not shown); however no brush border
could be found on their apical membrane which exhibits only
sparse irregular microvilli. HT-29 Glc* cells (Fig. le), SW-480
(Fig. ig), and HCT-8R (Fig. If) cells are not polarized; their
cell surface is covered with irregular microvilli, which are more
numerous in SW-480 than in Co-115, HCT-8R, and HT-29
Glc+ cells, but do not form a brush border. The absence of
morphological enterocytic differentiation of HT-29 Glc+, Co115, HRT-18, SW-480, and HCT-8R cells was associated with
Table 1 Blood group phenotypes and enterocyte-like differentiation
characteristics of cultured human colon carcinoma cell lines
Ultrastructural
differentiation
characteristicsCell
lines'Caco-2c
Tight
group
junctionsO
of
sucraseSucrase activity*
brush
isomaltase at
(milliunits/mg
border+Presence
immunoblotting
protein)+
780 ±26"
+
30 ±5
<0.2
<0.2<0.2
+A
HT-29 Glc- '
+
HT-29 Glc*
A
HRT-18
O
+
HCT-8R
0A
Co-115
+
SW-480Blood
AApical
* All cells were tested after 30 days in culture, except for Caco-2 cells which
were tested on day 16.
'Measured in a brush border-enriched fraction (P2) (see "Materials and
Methods").
' Passages 60 to 80.
d Mean ±SD of 3 to 5 different cultures.
' Grown for 5 to 10 passages in a glucose-free medium supplemented with 2.5
miviinosine (see "Materials and Methods").
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BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
Fig. 1. Transmission electron microscopy of postconfluent cultures of human colon carcinoma cell lines (day 16 for Caco-2 cells, day 30 for the other cell lines).
Sections are perpendicular to the bottom of the flasks, a, apical section of the monolayer of Caco-2 cells (X 6,000); b, apical section of the monolayer of HT-29 Ilk
cells (x 13,000); c, undifferentiated HT-29 Glc~ cells (x 2,000); d, HRT-18 cells (x 10,000); e, Co-115 cells (x 8,IOO);/ HCT-8R cells (X 2,500); g, SW-480 cells (X
3.000). Note the presence of apical regular brush border microvilli in Caco-2 and HT-29 Glc~ cells and of only irregular microvilli in the other cell lines.
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BLOOD GROUP ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
;
'
.
•
--Y '
'. •
•
"
4
»
>^
f*;-
Fig. 2. Immunofluorescence staining with rabbit anti-sucrase-isomaltase antibodies (a, <•.
e, g), rabbit anti-A antibodies (</,/). and UEA-I (/<.A) of monolayers (a,
b) of Caco-2 cells; c, d, differentiated HT-29 GIc~ cells; e, f, undifferentiated HT-29 GIc* cells; g, h, HRT-18 cells. There was no binding of anti-sucrase-isomaltase
antibodies to the monolayers of HCT-8R, Co-115, and SW-480 cells (not shown); the pattern of staining of Co-115 and SW-480 with anti-A antibodies, and of HCT8R cells with UEA-I were as in /and A, respectively. Caco-2 cells were tested on day 16 and the other cell lines on day 30 of the cultures, x 290.
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BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
an absence of expression of sucrase-isomaltase as substantiated
by the negative results obtained at both immunofluorescence
(Fig. 2, e and g) and Western blot analysis (Fig. 3) and the
absence of sucrase activity in the cell extracts (Table 1).
Immunofluorescence Binding of Anti-A Antibodies and UEAI to Cell Monolayers. The results shown in Fig. 2 demonstrate
that anti-A antibodies for HT-29 Glc" (Fig. 2d) and UEA-I for
small intestine reacting with anti-A antibodies and sucraseisomaltase from Caco-2 and blood group O small intestine
reacting with UEA-I. This was further analyzed by immunodepletion experiments using P2 fractions as a source of enzyme
and UEA-I and anti-A 7-globulins as immunoprecipitating
agents. Eighty % of sucrase activity disappears from the extracts
of Caco-2 cells incubated with UEA-I and 50% from HT-29
Caco-2 cells (Fig. 2Z>)react with the cell monolayers according
to the same pattern of apical staining as that observed with
anti-sucrase-isomaltase antibodies. The pattern of immunoreactivity of the undifferentiated cells was very different with the
antibodies corresponding to the blood group phenotype of the
cell lines, i.e., anti-A for HT-29 Glc+, SW-480, and Co-115
cells, and UEA-I for HRT-18 and HCT-8R cells showing only
a general membrane staining (Fig. 2,/and h).
Binding of UEA-I and Anti-A Antibodies to Sucrase-isomal
tase from Caco-2 and HT-29 Glc~ Differentiated Cells. The
5
123
—
6
I
strong similarity of the patterns of immunofluorescence stain
ing observed with anti-sucrase-isomaltase and anti-A antibodies
in HT-29 Glc" or UEA-I in Caco-2 cells suggested that both
types of antigens, i.e., A or H were associated with the same
histológica! structure, namely the brush border membrane, but
did not allow the conclusion that the same biochemical structure
was recognized. This was investigated by SDS-polyacrylamide
gel electrophoresis and immunoblotting with anti-A antibodies
and UEA-I of sucrase-isomaltase immunopurified from HT-29
Glc~ and Caco-2 cells. As shown in Fig. 4, anti-A antibodies
react with sucrase-isomaltase from HT-29 cells and UEA-I with
the enzyme from Caco-2 cells. In both cases the specificity is
similar to that observed with sucrase-isomaltase immunopuri
fied from blood groups A and O normal adult small intestine,
with sucrase-isomaltase from HT-29 and from blood group A
12345
200-
P
Fig. 4. Immunological detection of blood groups A and H antigens in sucraseisomaltase from HT-29 and Caco-2 cells in culture and tumors in nude mice and
from control normal small intestines. Sucrase-isomaltase extracted from P2
fractions was immunopurified with monoclonal antibody HBB 2/614/88, run on
SDS-polyacrylamide gel electrophoresis transferred onto nitrocellulose sheets,
and incubated with either rabbit anti-A antibodies (Lanes 1 to 3) or UEA-I (Lanes
4 to 6). Sucrase-isomaltase samples were from control blood group A normal
ileum (Lane 1), HT-29 Glc~ cultured cells (Lane 2), HT-29 tumors in nude mice
(Lane 3), control blood group O ileum (Lane 4), Caco-2 cultured cells (Lane 5),
and Caco-2 tumor in nude mice (Lane 6).
A —
100-,
116u
«3
92.5.
50U
Fig. 3. Immunological detection with monoclonal antibody HBB 2/614/88 of
sucrase-isomaltase in membrane fractions analyzed by SDS-polyacrylamide gel
electrophoresis transferred onto nitrocellulose sheets. Lane 1, control sucraseisomaltase complex from normal human ileum showing the high molecular weight
precursor (p) and the sucrase (s) and isomaltase (i) subunits; Lane 2, Caco-2
cells; Lane 3, HT-29 Glc~ cells; Lane 4, HT-29 Glc* cells; Lane 5, HRT-18 cells.
Results from Co-115, HCT-8R, and SW-480 cells (not shown) were as in Lanes
4 and 5. The cells were tested after 30 days in culture, except for Caco-2 cells
which were tested on day 16. Note the presence of only the high molecular weight
precursor in Caco-2 and HT-29 Glc" cells and the absence of detectable enzyme
in HT-29 Glc* cells and in the other cell lines. Left, place of migration of the
molecular weight markers: M, 200,000, myosin heavy chain; M, 116,000, ßgalactosidase; M, 92,500. phosphorylase b.
10
Fig. 5. Percentage of inhibition of sucrase activity in the supernatants of
sol ubili/ni P2 fractions of Caco-2 (•)and HT-29 cells (A) after a prior incubation
of P2 fractions with either UEA-I (for Caco-2 cells) or anti-A 7-globulins (for
HT-29 cells). Abscissa, 2-fold serial dilutions of inhibitors with 0 indicating the
starting solution (see "Materials and Methods").
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BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
Fig. 6. Immunofluorescent binding of rabbit anti-sucrase-isomaltase antibodies (a, c) and rabbit anti-A antibodies (ft), or UEA-I (</) on cryostat frozen tissue
sections from HT-29 (a, b) and Caco-2 tumors (c, d) in nude mice, x 120.
extracts incubated with anti-A 7-globulins (Fig. 5). No inhibi
tion was observed in Caco-2 cell extracts treated with anti-A yglobulins or HT-29 extracts treated with UEA-I (data not
shown).
Binding of Anti-A Antibodies and UEA-I to Sucrase-Isomaltase from HT-29 and Caco-2 Tumors. In order to check whether
the association of blood group antigenic determinants with
sucrase-isomaltase from HT-29 GIc~ and Caco-2 cells was not
restricted to cultured cells, but also present in vivo, further
investigations were performed with the corresponding tumors
in nude mice. The enterocytic differentiation pattern of HT-29
and Caco-2 tumors has been previously reported (28, 29). As
shown in Fig. 6 a similar pattern of immunoreactivity was
observed in tumor sections with anti-sucrase-isomaltase anti
bodies and either anti-A antibodies in the case of HT-29 or
UEA-I in the case of Caco-2 tumors. As with the corresponding
cultured cells, sucrase-isomaltase isolated from HT-29 and
Caco-2 tumors reacted in Western blot analysis with anti-A
antibodies and UEA-I, respectively (Fig. 4).
DISCUSSION
The present results show that, as in the normal small intestine
(31, 34), sucrase-isomaltase from enterocyte-like differentiated
HT-29 Glc~ and Caco-2 cells carries blood groups A and H
antigens corresponding to the blood group phenotypes of the
cell lines. This is true not only in cultured cells but also in the
corresponding tumors in nude mice. It should be noted that the
association of blood group antigens to the enzyme from both
the normal small intestine and colon tumor cells is most likely
independent of the molecular form of the enzyme which is
different in both situations: only the high molecular weight
form of the enzyme is present in malignant cells (22, 27, 28),
and this form, as in the fetal colon (53), shows a higher mobility
at SDS-polyacrylamide gel electrophoresis than the high mo
lecular weight form from the normal small intestine. Whether
all the molecules of sucrase-isomaltase carry the blood group
antigens is however questionable in view of the results of
immunodepletion experiments which show that the inhibition
of sucrase activity never reaches 100%. Whether the other
hydrolases associated with the enterocytic differentiation of
colon cancer cells also carry blood group antigens, as in the
normal intestine (30-34), has still to be investigated.
Other blood group antigens, different from ABH antigens,
have also been shown to be associated with colon cancers. This
is particularly the case of Lewis antigens (54-57). Whether
human brush border-associated hydrolases also carry Lewis
antigens is still unknown and has still to be investigated, not
only in enterocyte-like differentiated colon cancer cells but also
in the normal small intestine. In this regard cells such as HT29 Glc~ and Caco-2 should be ideal tools to help answer this
question.
It is of interest to note that so far the occurrence of ABH
antigens as colon cancer-associated markers has been mainly
an immunological observation and that nothing is known of its
physiological significance. The present results show that, at
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BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
least in the case of enterocyte-like differentiated colon cancer
cells, these antigens are integral parts of an enzyme whose
structure and function are perfectly known (16). What is less
known is the prognostic significance and the diagnostic interest
of this type of differentiation. In this regard the present results
raise the question of whether ABH antigens which have been
shown to be associated with some distal colon cancers (6-15)
are not, for some of them, markers of sucrase-isomaltase from
enterocyte-like differentiated tumors. They further suggest that
such tumors, and more particularly those in which blood group
antigens are restricted, as in Fig. 6, b and d, to apical structures
(11, 14), should be screened for their possible enterocytic dif
ferentiation.
22.
23.
24.
25.
26.
REFERENCES
1. Szulman, A. E. The histológica! distribution of the blood group substances
in man as disclosed by immunofluorescence. II. The H antigen and its relation
to A and B antigens. J. Exp. Med., 115:977-1005, 1962.
2. Szulman, A. E. The histológica! distribution of the blood group substances
in man as disclosed by immunofluorescence. III. The A, B and H antigens in
embryos and fetuses from 18 mm in length. J. Exp. Med., 119: 503-523,
1964.
3. Szulman, A. E. The histological distribution of blood group substances A
and B in man. J. Exp. Med.. ///: 785-807, 1960.
4. Oriol, R., Roussel, M., Zweibaum, A.. Dalix, A. M., Chevalier, G., Dussaulx,
E., and Strecker, G. Radioimmunoassay of the WZ polymorphic antigens of
normal human colon and their relationship with ABH antigenic determi
nants. Immunology, 32: 131-137, 1977.
5. Wiley, E. L., Murphy, P., Mendelsohn, G., and Eggleston, J. C. Distribution
of blood group substances in normal human colon. Use of the unlabeled
antibody (PAP) immunoperoxidase technic to identify A and B blood group
substances. Am. J. Clin. Pathol., 76: 806-809, 1981.
6. Denk, H., Tappeiner, G., and Holzner, Y. H. Blood group substances (BG)
as carcinofetal antigens in carcinomas of the distal colon. Eur. J. Cancer, III:
487-490, 1974.
7. Denk. H.. Tappeiner, G., Davidovits, A., Eckerstorfer, R., and Holzner, J.
H. Carcinoembryonic antigen and blood group substances in carcinomas of
the stomach and colon. J. Nati. Cancer Inst., 53:933-942, 1974.
8. Cooper, H. S., and Haesler, W. E. Blood group substances as tumor antigens
in the distal colon. Am. J. Clin. Pathol., 69: 594-598, 1978.
9. Abdelfattah-Gad, M., and Denk. H. Epithelial blood group antigens in human
carcinomas of the distal colon: further studies on their pathologic signifi
cance. J. Nati. Cancer Inst., 64: 1025-1028, 1980.
10. Wiley, E. L., Mendelsohn, G., and Eggleston, J. C. Distribution of carcinoembryonic antigens and blood group substances in adenocarcinoma of the
colon. Lab. Invest., 44: 507-513, 1981.
11. Yonezawa, S., Nakamura, T., Tanaka, S., and Sato, E. Glycoconjugate with
Ulex europaeus agglutinin-I binding sites in normal mucosa, adenoma and
carcinoma of the human large bowel. J. Nati. Cancer Inst., 69: 777-785,
1982.
12. Ernst, C, Thurin, J., Atkinson, B., Wurzel, H.. Herlyn, M., Stromberg, N.,
Civin. C., and Koprowski, H. Monoclonal antibody localization of A and B
isoantigens in normal and malignant fixed human tissues. Am. J. Pathol.,
117:451-461, 1984.
13. Schoentag, R., Williams, V., and Kuhns, W. The distribution of blood group
substance H and CEA in colorectal carcinoma. Cancer (Phila.), 53: 503-509,
1984.
14. Matshushita, Y., Yonezawa, S., Nakamura, T., Shimizu, S., Ozawa, M.,
Muramatsu, T., and Sato, E. Carcinoma-specific Ulex europaeus agglutininI binding glycoproteins of human colorectal carcinoma and its relation to
Carcinoembryonic antigen. J. Nati. Cancer Inst.. 75: 219-226, 1985.
15. Yuan, M., Itzkowitz, S. H., Palekar. A.. Shamsuddin, A. M., Phelps, P. C.,
Trump, B. J., and Kim, Y. S. Distribution of blood group antigens A, B, H
Lewis* and Lewis' in human normal, fetal, and malignant colonie tissue.
Cancer Res., 45:4499-4511, 1985.
16. Kenny, A. J., and Maroux, S. Topology of microvillar membrane hydrolases
of kidney and intestine. Physiol. Rev., 62:91-128, 1982.
17. Dahlqvist, A., and Lindberg, T. Development of the intestinal disaccharidases
and alkaline phosphatase activities in the human fetus. Clin. Sci., 30: 517528, 1966.
18. Koldovsky, O. Development of the Function of the Small Intestine of
Mammals and Man. Basel: S. Karger AG 1969.
19. \ ur¡ivi
iio. S., Stellato, A., and Devizia. B. Development of brush border
peptidases in human and rat small intestine during fetal and neonatal life.
Pediatr. Res., 15:991-995, 1981.
20. Lacroix, B., Kedinger, M., Simon-Assmann, P., Roussel, M., Zweibaum, A.,
and Haffen, K. Developmental pattern of brush border enzymes in the human
fetal colon. Correlation with some morphogenetic events. Early Hum. Dev.,
9:95-103, 1984.
21. Pinto, M., Appay, M. D., Simon-Assmann. P., Chevalier, G., Dracopoli, N.,
Fogh, J., and Zweibaum, A. Enterocytic differentiation of cultured human
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
colon cancer cells by replacement of glucose by galactose in the medium.
Biol. Cell, 44: 193-196, 1982.
Zweibaum, A., Pinto, M., Chevalier, G., Dussaulx, E., Triadou, N., Lacroix,
B., Haffen, K., Brun, J. L., and Roussel, M. Enterocytic differentiation of a
subpopulation of the human colon tumor cell line HT-29 selected for growth
in sugar-free medium and its inhibition by glucose. J. Cell. Physiol., 122:
21-29. 1985.
Wice, B. M., Trugnan, G., Pinto, M., Roussel, M., Chevalier, G., Dussaulx,
E., Lacroix, B., and Zweibaum, A. The intracellular accumulation of UDPA'-acetylhexosamines is concomitant with the inability of human colon cancer
cells to differentiate. J. Biol. Chem., 260: 139-146, 1985.
Robine. S., Huet, C., Moll, R., Sahuquillo-Merino, C., Coudrier, E., Zwei
baum, A., and Louvard, D. Can villin be used to identify malignant and
undifferentiated normal digestive epithelial cells? Proc. Nati. Acad. Sci. USA,
«2:8488-8492, 1985.
Pinto, M.. Robine-Léon,S., Appay. M. D., Kedinger, M., Triadou, N.,
Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., and
Zweibaum, A. Enterocyte-like differentiation and polarization of the human
colon carcinoma cell line Caco-2 in culture. Biol. Cell, 47: 323-330, 1983.
Roussel, M., Laburthe, M., Pinto, M., Chevalier, G., Rouyer-Fessard, C,
Dussaulx, E., Trugnan, G., Böige,N., Brun, J. L., and Zweibaum, A.
Enterocytic differentiation and glucose utilization in the human colon tumor
cell line Caco-2: modulation by forskolin. J. Cell. Physiol., 123: 377-385,
1985.
Hauri, H. P., Sterchi, E. E., Bienz, D., Fransen. J., and Marxer, A. Expression
and intracellular transport of microvillus membrane hydrolases in human
intestinal epithelial cells. J. Cell. Biol.. 101:838-851. 1985.
Zweibaum, A., Triadou, N., Kedinger, M., Augeron, C., Robine-Lèon, S.,
Pinto, M., Roussel, M., and Haffen, K. Sucrase-isomaltase: a marker of
foetal and malignant epithelial cells of the human colon. Int. J. Cancer, 32:
407-412, 1983.
Zweibaum, A., Hauri, H. P., Sterchi, E., Chantre!, 1., Haffen, K., Bamat, J.,
and Sordat, B. Immunohistological evidence, obtained with monoclonal
antibodies, of small intestinal brush border hydrolases in human colon
cancers and foetal colons. Int. J. Cancer, 34: 591-598, 1984.
Hermon-Taylor, J., Perrin, J., Grant, D. A. W., Appleyard, A., Bubel, M.,
and Magee, A. I. Immunofluorescent localization of enterokinase in human
small intestine. Gut, 18: 259-265, 1977.
Kelly, J. J.. and Alpers, D. H. Blood group antigenicity of purified human
intestinal disaccharidase. J. Biol. Chem., 248: 8216-8221, 1973.
Feracci, H., Bernadac, A., Gorvel, J. P., and Maroux, S. Localization by
immunofluorescence and histochemical labeling of aminopeptidase N in
relation to its biosynthesis in rabbit and pig enterocytes. Gastroenterology,
«2:317-324, 1982.
Gorvel, J. P., Wisner-Provost, A., and Maroux, S. Identification of glycoprotein bearing human blood group A determinants in rabbit enterocyte plasma
membranes. FEBS Lett., 143: 17-20, 1982.
Triadou, N., Audran, E., Roussel, M., Zweibaum, A., and Oriol, R. Rela
tionship between the secretor status and the expression of ABH blood group
antigenic determinants in human intestinal brush border membrane hydro
lases. Biochim. Biophys. Acta, 761: 231-236, 1983.
Fogh, J., and Trempe, G. New human tumor cell lines. In: J. Fogh (ed.),
Human Tumor Cells in Vitro, pp. 115-141. New York: Plenum Publishing
Corp., 1975.
Fogh, J., Fogh, J. M., and Orfeo, T. One hundred and twenty-seven cultured
human tumor cell lines producing tumors in nude mice. J. Nati. Cancer Inst.,
59:221-226, 1977.
Leibovitz, A., Stinson, J. C., McCombs, W. B., McCoy, C. E., Mazur, K. C,
and Mabry, N. D. Classification of human colorectal adenocarcinoma cell
lines. Cancer Res., 36: 4562-4569, 1976.
Tompkins, W. A. F., Watrach, A. M., Schmale, J. D., Schultz, R. M., and
Harris, J. A. Culture and antigenic properties of newly established cell strains
derived from adenocarcinomas of the human colon and rectum. J. Nati.
Cancer Inst., 52:1101-1110, 1974.
Rosenthal, K. L., Tompkins, W. A. F., Frank, G. L., McCulloch, P., and
Rawls, W. E. Variants of a human colon adenocarcinoma cell line which
differs in morphology and Carcinoembryonic antigen production. Cancer
Res., 37:4024-4030, 1977.
Carrel, S., Sordat, B., and Merenda, C. Establishment of a cell line (Co-115)
from a human colon carcinoma transplanted into nude mice. Cancer Res.,
36: 3978-3984, 1976.
Roussel, M., Chevalier, G.. Roussel, J. P., Dussaulx. E., and Zweibaum, A.
Presence and cell growth-related variations of glycogen in human colorectal
adenocarcinoma cell lines in culture. Cancer Res., 59: 531-534, 1979.
Roussel, M., Dussaulx, E.. Chevalier, G., and Zweibaum, A. Growth-related
glycogen levels of human intestine carcinoma cell lines grown in vitro and in
vivo in nude mice. J. Nati. Cancer Inst., 65:885-889, 1980.
Zweibaum, A., Oriol, R., Dausset, J., Marcelli-Barge, A., Ropartz, C., and
Lanset, S. Definition in man of a polymorphic system of the normal colonie
secretions. Tissue Antigens, 6: 121-128, 1975.
Triadou, N. Antigenic cross-reactions among human intestinal brush border
enzymes revealed by the immunoblotting method and rabbit anti-enzyme
sera. J. luminimi. Methods, 73: 283-291, 1984.
Zweibaum, A., and Steudler, V. Natural iso-antibodies specific for tissular
group antigens of the colon in dog and rabbit. Nature (Lond.), -'-'': 84-86,
1969.
46. Zweibaum, A., and Bouhou, E. Studies on digestive groups I. The A alloan-
1432
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1987 American Association for Cancer Research.
BLOOD GROUP
ANTIGENS
AS MARKERS OF SUCRASE-ISOMALTASE
tigen-alloantibody system in rabbits. Transplantation (Baltimore), 15: 291293, 1973.
47. Oriol, R., and Dalix. A. M. Differences in the maturation of the immune
response of A~ and A* rabbits, good and poor responders respectively for the
A antigenM.,Immunology
55-91-99,1977
48. Messer,
and Dahlqv'ist,
A. A one step ultramicromethod for the assay of
intestinal disaccharidases. Anal. Biochem., 14: 376-392, 1966.
49. Schmitz, J., Preiser, H., Maestracci. D., Ghosh, B. K., Cerda, J. J., and
Crane, R. K. Purification of the human intestinal brush border membrane.
_.
, : Biophys.
,
m os
iati
Biochim.
Acta, 323:
98-1in12 1973.
50. Laemmh, U. K. Cleavage of structural proteins during the assembly of the
head of bactenophage T4. Nature (Lond.), 227: 680-684, 1970.
5 1. Burnette, W. N. "Western blotting:" electrophoretic transfer of proteins from
sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and
radiographie detection with antibody and radioiodinated protein A. Anal.
Biochem., 112: 195-203, 1981.
52. Gonatas, N. K., and Avrameas, S. Detection of plasma membrane carbohy-
drates with lectin peroxidase conjugates. J. Cell Biol., 59: 436-443, 1973.
53. Triadou, N., and Zweibaum. A. Maturation of sucrase-isomaltase complex
in human fetal small and large intestine during gestation. Pediatr. Res., 19:
! 36- 138, 1985.
54- Fukushima,
P. I.,E.,Wakisaka,
A., Togashi,
H., Chia,
P- Suyama, K.,
N., Hirota,
Fukushi,M.,Y.,Terasaki,
Nudelman,
and Hakomori,
S. I. Character¡"0
«
'** " * "'" tumor-assoclated ant'8en- Cancer
„ „,es" , „ „,
„ „ ......
55' ^aa^.
M." Pakf' ,K" Y" Herlyn' M" SearS,' "¡ F" a"d StePlewskl-. Z;
Characterization
Lewis
in normal
and gastrointestinal
adenocarcinomas. ofProc.
Nati.antigens
Acad. Sci.
USA, 82:colon
3552-3556,
1985.
56 Fejzj T Demonstration by monoclonal antibodies that carbohydrate structures of giycoproteins and glycolipids are oncodevelopmental antigens. Na,ure (Lon(j )_}¡4:53-57, 1935.
57 Sakamoto, J., Furukawa, K., Cordon-Cardo. C., Yin, B. W. T., Rettig, W.
j„Oettgen, H. F., Old, L.J., and Lloyd, K. O. Expression of Lewis', Lewis',
X, and Y blood group antigens in human colonie tumors and normal tissue
and in human tumor-derived cell lines. Cancer Res., 46: 1553-1561, 1986.
1433
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1987 American Association for Cancer Research.
A and H Blood Group Antigens as Markers of
Sucrase-Isomaltase from the Enterocyte-like Differentiated
Human Colon Carcinoma Cell Lines HT-29 and Caco-2
Isabelle Chantret, Guillemette Chevalier, Elisabeth Dussaulx, et al.
Cancer Res 1987;47:1426-1433.
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