[CANCER RESEARCH 49, 4550-4556. August 15. 1989]
ABH Blood Group Antigen Expression, Synthesis, and Degradation in Human
Colonie Adenocarcinoma Cell Lines1
Rajvir Dahiya,2 Steven H. Itzkowitz,3 James C. Byrd, and Young S. Kim
Gastrointestinal Research Laboratory,
San Francisco, California 94121
Veterans Administration
Medical
Center, and Department
of Medicine,
University of California Medical
Center,
The synthesis of blood group ABH antigens is under genetic control,
where the primary gene products are glycosyltransferases. Several studies
have demonstrated cancer-associated alterations in ABH antigen expres
sion in human colon cancer tissues. However, the mechanism(s) respon
sible for these alterations has not been elucidated. Therefore, experiments
were conducted using nine established colon cancer cell lines (four type
O, three type A, and two type B) to examine ABH antigen expression by
immunocytochemistry and correlate this with activities of ABH biosynthetic (glycosyltransferase) and degradative (glycosidase) enzymes. The
products of the glycosyltransferase enzymes were characterized by high
performance liquid chromatography and paper chromatography, and
substrate affinities (apparent A,,, values) of the cancer cell-derived gly
cosyltransferases were analyzed.
The present data demonstrate: (a) all cell lines except H-498 (blood
type A) expressed the appropriate ABH glycosyltransferase as well as
all three glycusiduses; (b) product characterization and substrate depen
dence experiments suggested that the cancer cell-derived ABH glycosyl
transferase enzymes had properties that were similar to those of the
ABH enzymes in human serum; (c) H-498 cells exhibited A antigen
deletion with accumulation of H precursor substance, most likely due to
insufficient A transferase activity; (rf) SW1417 cells (blood type B)
demonstrated B antigen deletion without precursor accumulation, despite
adequate levels of B transferase and low a-galactosidase activity; and (c)
weak incompatible A antigen expression occurred in LoVo (type B) and
SW1116 (type O) cells, and weak incompatible B antigen expression
occurred in H-498 (type A) and SW1116 cells. However, since these
cells lacked incompatible A or B transferase activity, these incompatible
antigens are probably not the true A or B antigens.
Thus, the colon cancer cell lines used in this study exhibit all of the
ABH alterations previously described in colon cancer tissues and appear
to be useful experimental models for studying the molecular events
involved in cancer-associated ABH expression.
H antigen is synthesized by the addition of L-fucose to the
terminal galactosyl residues of blood group-type oligosaccharides on glycolipids or glycoproteins, the action of a-2-L-fucosyltransferase enzyme. The H substance can then serve as the
immediate precursor for the A and B antigens. The structural
alÃ-eles,A and B at the ABO locus, encode, respectively, an aW-acetyl-D-galactosaminyltransferase
and an a-o-galactosyltransferase, which catalyze the addition of jV-acetyl-D-galactosamine or o-galactose to H antigen and form the A and B
determinants (1,3, 7).
The blood group ABH antigens are expressed by colonocytes
throughout the entire colon during fetal life (8, 9) but only in
the proximal colon of the normal adult (4, 8-11). Because
adenomas and cancers of the distal colon frequently reexpress
the fetal phenotype, ABH antigens in this location are consid
ered as oncofetal tumor-associated antigens (9, 11-14). Studies
of colon cancer tissues have shown the deletion of ABH antigens
(9, 15, 16) and the accumulation of precursor determinants,
such as H and li substance (17), and of incompatible blood
group antigens, i.e., A antigen in patients with blood type O or
B (9, 18). The mechanisms underlying these alterations in
developmental and cancer-associated ABH antigen expression
are not clear. Cultured cells potentially offer a good experimen
tal model for studying regulation of expression of ABH anti
gens. There have been few reports of ABH antigen expression
in cultured colon cancer cell lines (19, 20). However, in those
studies, the blood group ABH-specific glycosyltransferases were
not assayed. Therefore, the present experiments were under
taken to investigate the expression of ABH antigens as well as
their synthetic and degradative enzymes in human colonie
adenocarcinoma cell lines.
INTRODUCTION
MATERIALS
Blood group ABH substances are carbohydrate structures,
which are formed by the sequential addition of specific monosaccharides to the carbohydrate side chains of glycolipids and
glycoproteins (1, 2). They are expressed not only in blood cells
(3) but also in a variety of epithelial cells (4). Their expression
is under genetic control, where the primary gene products are
glycosyltransferases (5). Classically, ABH blood group deter
minants reside on either a type 1 (Gal/81—>3GlcNAc-R)4or type
2 (Galßl—>4GlcNAc-R)chain (5), although recently type 3
(Gal/31-»3GalNAca-R) and type 4 (Gal/3l—3GalNAc/3-R)
chains have also been described (6).
Materials. UDP-D-[3H]galactose (specific activity, 17 Ci/mmol) was
purchased from New England Nuclear (Boston, MA), UDP-JV[l4C]Acetyl-D-galactosamine (specific activity, 23 mCi/mmol) was pur
chased from ICN (Irvine, CA), and GDP-L-[14C]fucose (specific activity,
ABSTRACT
Received 10/4/88; revised 3/27/89; accepted 5/9/89.
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.
1Supported by grants from the National Cancer Institute (CA42981 to
S. H. I. and CA47551 to Y. S. K.), a VA Research Associate award (to S. H. I.),
a VA Medical Investigator award (to Y. S. K.). and the Veterans Administration
Medical Research Service.
! To whom requests for reprints should be addressed, at GI Research Lab
(I51M2), VA Medical Center. 4150 Clement St., San Francisco, CA 94121.
3 Present address: Division of Gastroenterology, Box 1069, Mount Sinai
Medical Center, 1 Gustave Levy Place, New York, NY 10029.
* The abbreviations used are: Gal, galactose; GlcNAc, /V-acetylglucosamine;
GalNAc, A'-acetylgalactosamine; Fuc. fucose; PBS. phosphate buffered saline;
HPLC, high performance liquid chromatography.
AND METHODS
308 mCi/mmol) was obtained from Amersham (Arlington Heights,
IL). Phenyl /3-galactoside, 2'-fucosyllactose,/»-nitrophenyl galactoside,
p-nitrophenyl
a-fucoside, /»-nitrophenyl-a-A'-acetylgalactosaminide,
and biotinylated isolectin I-B4 from Bandeiraea simplicifolia were pur
chased from Sigma Chemical Co. (St. Louis, MO). Dowex 1-X8formate (100-200 mesh) was obtained from Bio-Rad Laboratories
(Richmond, CA). Histostain-S.P. kit was purchased from Zymed Lab
oratories (South San Francisco, CA).
Cell Culture. Human colonie adenocarcinoma cell lines LS174T,
SW1417, LoVo, SW1116, HT29, and SW480 were obtained from the
American Type Culture Collection (Bethesda, MD). The HM7 and
LM12 cell lines are high- and low-mucin variant clones established
from the LS174T cell line in our laboratory (21). The H498 cell line
was recently established from a blood type A individual and was
generously provided by Dr. A. Gazdar, National Cancer Institute. All
cell lines were grown in Dulbecco's modified Eagle's medium, with
high glucose, penicillin (50 units/ml), streptomycin (5 Mg/ml), and 10%
heat-inactivated fetal bovine serum, as described (18). All of the cell
lines were tested for Mycoplasma contamination using the Mycoplasma
T.C. II rapid detection system (Gen-Probe, Inc., San Diego, CA) and
were found to be negative. Cell cultures were incubated at 37°Cin a
4550
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH ANTIGENS IN ADENOCARCINOMA
CELL LINES
humidified atmosphere of 5% CO2 in air. Heat treatment (56"C for 1
['4C]fucose, as judged by using reference sugars, which were detected
h) of the fetal bovine serum destroyed almost all the glycosyltransferase
activity of the fetal bovine serum.
After the cells were confluent, the medium was decanted and the
cells were washed 3 times with PBS and scraped from plates into 8 ml
of PBS with a rubber policeman. The cells were centrifuged at 3000
rpm for 10 min and the cell pellet was resuspended in 2 ml of 20 HIM
cacodylate buffer (pH 6.8) and sonicated for 2 min. Aliquots (1 ml)
were frozen (—70°C)
immediately, until further use. Protein content
with silver nitrate reagent (25).
Glycosidase Assay. All glycosidase assays were performed as de
scribed previously, with 2 mM /7-nitrophenylglycoside in 0. l M acetate,
pH 4.0 (23). These reactions measure the ability of cell homogenates
to release any a-GalNAc, a-galactose, or a-fucose residue, including
those of the blood group A, B, and H substances, using p-nitrophenyla-glycoside substrates. Glycosidases were also assayed under the con
ditions of the glycosyltransferase assays (pH 6.8) with 2 mM p-nitrophenylglycosides but without nucleotide sugars.
Immunocytochemistry. The monoclonal antibodies used in this study
have been used by us previously (9) and were generously provided by
Dr. R. Murray Ratcliffe, Chembiomed, Ltd. (Edmonton, Alberta, Can
ada). The anti-A and anti-B monoclonal antibodies recognize their
respective epitopes on either type 1 or type 2 chains, whereas the antiH monoclonal antibody recognizes only H type 2 antigen.
Cells were seeded onto chamber slides (Intermountain Scientific Co.,
Bountiful, UT) at a density of 3 x IO5 cells/chamber and allowed to
was determined by the method of Lowry et al. (22).
Glycosyltransferase Assays. The details for glycosyltransferase assays
have been described earlier (23, 24). The assay mixture for al,3GalNAc
transferase (A transferase) contained 410 ¿IM
2'-fucosyllactose, 2.79
/Ã-M
UDP-Ar-['4C]acetyl-D-galactosamine (30,000-50,000 cpm), reaction
cocktail (0.1 M sodium cacodylate-HCl buffer, pH 6.8; 200 pM ATP;
20 mM MnCl2; 0.1% Triton X-100), and cell homogenate (25-100 pg).
The assay mixture for a 1,3-galactosyltransferase (B transferase) con
tained 410 MM 2'-fucosyllactose,
17-32 nM UDP-['H]galactose
(50,000-100,000 cpm), reaction cocktail, and cell homogenate (25-75
pg). The reaction mixture for al,2-fucosyltransferase (H transferase)
contained 39 mM phenyl-/3-galactoside, 1-1.8 pM GDP-['4C]fucose
(30,000-40,000 cpm), reaction cocktail, and cell extract (100-200 pg).
Final volumes of all assays were made up to 100 pi. All glycosyltrans
ferase assays were performed within the linear range of both the amount
of enzyme (cell homogenate) and incubation time. Results shown are
the median of triplicate tubes for A transferase and B transferase and
single assays for H transferase. Incubations were carried out at 37°C
for 90 min. The reactions were stopped by being placed on ice. The
reaction mixtures were then applied to Dowex 1-X8 (100-200 mesh)
formate columns (0.5 x 2 cm) and the products were separated by
washing each column with 2 ml water. The product of a 1,2-L-fucosyltransferase enzyme was further separated by descending paper chromatography (24). An aliquot of each eluate was mixed with ACS II
scintillation fluid and counted in a scintillation counter. The values
obtained without acceptor were taken as endogenous enzyme activity
and were subtracted from the total incorporation to give the acceptordependent activity.
Characterization of A and B Tetrasaccharides. For characterization
of A tetrasaccharide, cell homogenate (HT29 cell line), as well as
normal A blood type serum, was incubated with UDP-A'-['4C]acetyl-Dgalactosamine in the presence or absence of 2.2 m\i 2'-fucosyllactose
and the reaction mixtures were passed through Dowex 1-X8 formate
columns (0.5 x 2 cm) as for the standard assay. The unbound fractions
from Dowex 1-X8 formate columns were evaporated, chromatographed
on Sephadex G-15 columns (0.9 x 59 cm), and eluted with water at a
flow rate of 37 ml/h. In each case, there was a peak of I4C that eluted
at the position of stachyose standard which was absent in the controls
(without 2'-fucosyllactose). This peak was pooled, evaporated, and
examined by amine-phase HPLC. Two columns of 0.4- x 30-cm AX10 (Varian) were used in series with a reverse phase guard column. The
column was equilibrated with acetonitrile and 15 mM potassium phos
phate, pH 5.2 (in the ratio of 85:15, v/v), and eluted with an acetonitrile
gradient of 85 to 0% over the course of 170 min. Fractions of 0.6 ml
were collected and counted. For characterization of B tetrasaccharide,
cell homogenate (SW1417 cell line), as well as B blood type serum,
were incubated with UDP-['H]galactose in the presence or absence of
2.2 HIM2'-fucosylIactose. The reaction mixtures were passed through
Dowex 1-X8 formate columns and then chromatographed on Sephadex
G-15 as described for the A tetrasaccharide. The peak of apparent
tetrasaccharide size was pooled, evaporated, and analyzed by aminephase HPLC as described above.
Characterization of H Product. The product of «1.2-L-fucosyltransferase enzyme was characterized by using descending paper chromatography (24). The unbound Dowex 1-X8 column fractions were dried
under nitrogen and then spotted on Whatman No. 3 paper. Paper
chromatography was performed by using the solvent system of ethylacetate:pyridine:water (10:4:3, v/v) for 5 h. The paper strips were then
cut into 1.5-cm fractions and counted in a scintillation counter. The
radioactivity incorporated into phenylgalactoside acceptor was ex
pressed as a percentage of the total counts in the original reaction
mixture. The product was well separated from GDP-fucose and free L-
attach overnight. The cells were then washed 3 times with buffer (PBS/
1% bovine serum albumin), fixed in acid-ethanol for 20 min at -20°C,
and washed again 3 times with buffer. In pilot experiments, the acidethanol fixation was found to give lower background staining than
formaldehyde or glutaraldehyde fixation. Endogenous peroxidase activ
ity was blocked by incubating cells with 1% hydrogen peroxide in
methanol for 10 min. followed by three washes with buffer. Normal
rabbit serum (10%) was applied for 10 min and blotted off. Primary
antibody (2 pg/ml) was added for 60 min, the cells were washed 3 times
with buffer, and biotinylated rabbit anti-mouse IgG + IgA + IgM (10
Pg/ml) was applied for 20 min. The cells were then washed, reacted
with streptavidin-peroxidase conjugate (10 pg/ml) for 30 min, washed
again, and incubated with diaminobenzidine for 10 min. Slides were
counterstained with 1% methyl green, rinsed in tap water, dehydrated,
and mounted. For lectin binding the primary antibody was omitted and
the secondary antibody was replaced by biotinylated isolectin I-B4 of A.
simplicifolia.
Negative controls consisted of substituting normal mouse IgM for
primary antibody and PBS for the primary and secondary antibodies.
In all cases, when primary antibody was omitted, a completely negative
staining pattern was obtained. Cells were scored for antigen expression
based on percentage of positive cells as well as staining intensity: weak
(+), moderate (++), strong (+++).
RESULTS
Table 1 shows the expression of A antigen and a-/V-acetyl-Dgalactosaminyltransferase (A transferase) and aWV-acetylgalactosaminidase activities in the nine colonie adenocarcinoma cell
lines examined. Of the three cell lines derived from blood type
A individuals, only two, HT-29 and SW480, showed substantial
A transferase activity as well as A antigen expression (Fig. 1, a
and b). When unlabeled UDP-GalNAc (40 MM) was added,
there was a 2- to 3-fold decrease in net I4C incorporation for
HT-29 and SW480 and A transferase was still undetectable in
Table 1 Expression of A antigen, a 1,3-N-acetylgalactosaminyltransferase (A
transferase). and N-acetylgalactosaminidase in human colonie adenocarcinoma
cell lines
A antigen exprèso-W-AcetylgalactosaBlood sion (% of positive
A transferase
minidasc (nmol/mg/
Cell line type
cells)
(pmol/mg/90 min)
16h)
10.7°11.0
±
LS174THM-7LM-12SW1116HT-29SW-480H-498LoVoSW14170OoO
±8.74.5
2.725.2
±
15.1421.
±
32.8418.5
7 ±
37.83.9
±
3.935.9
±
(+)B19.0 5
±
15.70546103464337332420380850*46282636197568
70426±
±27
" Mean ±SE, five experiments except for H-498 (four experiments).
* Mean ±SE, four experiments.
c Numbers in parentheses, staining intensity scored as weak (+). moderate
(++). and strong (+++).
I0(+)rA
(++)A
60
100(+++)AB
4551
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH ANTIGENS IN ADENOCARCINOMA
the other cell lines. Interestingly, the H-498 cell line did not
express any A antigen, thereby representing an example of
blood group A antigen deletion (Fig. le). This antigenic deletion
most likely results from an absence of A transferase activity in
these cells (Table 1). Very weak expression of A antigen was
also found in SW1116 (blood type O) and LoVo (blood type
B), representing incompatible A antigen. However, since there
was very little or no activity of A transferase in any of the O or
B blood type cell lines, it is likely that this weak incompatible
A antigen represents an A-like or cross-reacting substance, ayV-Acetylgalactosaminidase activity measured at pH 4.0 was
similar in all of the cell lines, regardless of blood type (Table
Ar
CELL LINES
1). Under the conditions of the A transferase assay (pH 6.8),
a-yV-acetylgalactosaminidase was undetectable.
Using HT-29 cells as a prototype blood type A cell line, the
enzyme kinetics of A transferase were examined with different
concentrations of the acceptor, 2'-fucosyllactose, and the do
nor, UDP-GalNAc. The apparent Km for 2'-fucosyllactose was
0.050 HIM (Fig. 2b), which is comparable to the Km of the A
enzyme in human serum (Km = 0.03-0.16 mivi) (26-28). The
fucosyl moiety of 2'-fucosyllactose was required for acceptor
activity. When the nine cell lines were tested for transfer of A'acetylgalactosamine to lactose, there was less than 0.4 pmol/
mg/90 min in each. The apparent Km for UDP-GalNAc of the
HT-29 A transferase was 0.032 mM (Fig. 2a), which is also
similar to that of the serum enzyme (Km = 0.05-0.09 HIM)(2628). The apparent Km of the SW480 enzyme for UDP-GalNAc
was 0.024 mivi (data not shown). The A transferase of HT-29
cells was inhibited by UDP-galactose, with 65% inhibition at
50 /iM UDP-galactose in the presence of 64 /¿MUDP-[I4C]GalNAc. However, there was no detectable transfer of galactose
to 2'-fucosyllactose by the A transferase of HT-29 cells.
„•v*
*«'
••**••**•
In order to establish the product of the A transferase, reaction
mixtures were passed through Dowex 1-X8 columns, deionized,
and separated by gel nitration on Sephadex G-15 columns.
There was a peak of radioactivity corresponding to tetrasaccharide size which was formed only in the presence of 2'fucosyllactose. This peak was further examined by amine-phase
HPLC (Fig. 3). The tetrasaccharide-size peak was chromatographically similar to the product formed by the A transferase
of human serum, which has been previously characterized as
GalNAcal,3[Fucal,2]Gal/31,4Glc
(29).
Table 2 shows the expression of B antigen and «1,3-galactosyltransferase (B transferase) and galactosidase activities in
human colonie adenocarcinoma cell lines. Of the two blood
group B cell lines, only LoVo cells showed B antigen expression
(Fig. 4a) as well as B transferase activity. When unlabeled
UDP-galactose (2 /¿M)
was added, there was a 2-fold decrease
in net incorporation of 'H in SW1417 and LoVo cells, and B
*^H/*-*i.iî
.*
.lÃ- >
transferase was still undetectable in the other cell lines. Inter
estingly, the SW1417 cell line had a comparable level of B
*-:.v
100-
50E
O
20 -
1c
0.2
Fig. I. Blood group A antigen expression in blood type A cell lines, a, SW480. All of the cells express the antigen strongly on cell membranes and in
cytoplasm, x 62. 6. HT29. Antigen expression is localized more in the cytoplasm
with a granular staining pattern. Some cells are negative, x 62. e, H498. None of
the cells express the A antigen, x 50.
0.3
[Fuca2Galp4Glc],
0.4
mM
Fig. 2. Substrate dependence of A transferase. A homogenate of HT-29 (20
Mgprotein) was incubated with varying concentrations of UDP-[l4C]GalNAc (A)
and 2'-fucosyllactose (B). Linear regression analysis of V versus V'/S plots (insets)
was used to determine apparent Kmand I7â„¢.,
values.
4552
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABU ANTIGENS [N ADENOCARCINOMA CELL LINES
12
f
20
TT
T
40
60
fraction
4b), but there was also strong staining of LS174T, HM-7, SW480, and H-498 (Fig. 4d). Galactosidase activity at pH 4.0 was
present in all of the cell lines, regardless of blood type antigen
expression, but no «-galactosidase could be detected at pH 6.8.
The enzyme kinetics of the SW1417 B transferase was ex
amined with different concentrations of 2'-fucosyllactose and
UDP-galactose. The apparent K„,
for 2'-fucosyIlactose was 0.24
80
100
mM (Fig. 56), which is comparable to the serum B transferase
(Km = 0.05 HIM) (31). As for the A transferase, the fucosyl
moiety of 2'-fucosyllactose was required for B transferase ac
120
number
Fig. 3. Characteri/ation of A transferase and B transferasc products. Homogenates of HT-29, incubated with UDP-|MC]GalNAc and 2'-fucosyllactose (•).
and SW 1417. incubated with UDP-|'H[galactose and 2'-fucosyllactose (D), were
deionizcd on Dowex 1 formate, subjected to gel filtration on Sephadex G-l 5. and
then Mii.ih/>•<!
by HPLC as described in "Materials and Methods." Arrows, left to
right, show the elution positions of /V-acetylgalactosamine, galactose, and 2'fucosyllactose.
Table 2 Expression of H antigen, <\l,3-%alactosyltrunsferase (B transferasef, and
ga/actosidase in human colonie adenocarcinonta cell lines
antigenMAb"
transferase
Lectin60(++)40
lineLS174THM
Cell
typeOOooAAABBB
(pmol/mg/90
min)1.4
1.1*0.2
±
(nmol/mg/16
h)177
13.1'256.6
±
4a
4c
jrpr.
that of the serum enzyme (K,„
= 0.01 mM) (31). The B trans
ferase of LoVo cells had an apparent K,n for UDP-galactose of
0.0010 mM (data not shown). The B transferase of SW1417
cells was inhibited by UDP-GalNAc, with 65% inhibition at 10
MMUDP-GalNAc in the presence of 5 //M UDP-['H]galactose.
However, there was no detectable transfer of 7V-acetylgalactosamine to 2'-fucosyllactose by the B transferase of SW1417
cells.
The B-specific tetrasaccharide product was characterized in
a manner similar to that of the A tetrasaccharide (Fig. 3). A
single peak of tetrasaccharide size containing ['Hjgalactose was
formed only in the presence of 2'-fucosyllactose. This tetrasac
7LM-12SWI116HT-29SW-480H-498LoVoSW1417Blood
(++)NT30
±0.10.2
14146+
±
0.10.3
+
19.3114+
15.669.6
(+)40
0.30.3
±
0.10.3
+
9.8120.6
±
(+)10(+)
11.6150.6
±
0.30.2
±
80(++)70(++)
±0.18.8
±9.8141.6+
17.970.6
90(+++)5(+)B 1.99.4+
±
1.5(i-Galactosidase±12.5
" MAb, monoclonal antibody; NT, not tested.
* Mean ±SE, six experiments except for LoVo (five experiments).
' Mean ±SE, four experiments.
r>
ceptor activity. The nine cell lines all showed at most 2 pmol/
mg/90 min transfer of galactose to lactose. Using UDP-galac
tose, the B transferase of SW1417 colon cancer cells showed
an apparent A",,,of 0.0021 mM (Fig. 5a), which is lower than
charide product was chromatographically similar to that formed
by the serum B transferase, which has previously been charac
terized as Gal«l-3[Fuc«l-2]Gal/Jl-4Glc (32).
Expression of H antigen and «1,2-fucosyltransferase (H
transferase) and fucosidase activities in colonie adenocarcinoma
cell lines is shown in Table 3. All of the blood group O cell
lines expressed H antigen as well as H transferase activity. The
H antigen expression was less prevalent in LS174T and its
derivatives (HM-7, LM-12) than in SW1116 cells (Fig. 6). Of
the A and B cell lines, only H-498 reacted with anti-H antibody.
This represents accumulation of the precursor H substance in
this cell line, which is also deficient in A transferase activity.
There were significant amounts of H transferase activity in all
^
VT*.
4d
Fig. 4. Blood group B antigen expression, a, LoVo cells stained with mono
clonal anti-B antibody. Cells that react are stained strongly in the cytoplasm but
also on cell membranes, x 62. b, LoVo cells stained with B. simplicifolia I-B4
lectin. Staining is more intense and more prevalent than with monoclonal anti-B
antibody. X 62. c, H-498 (blood type A cell line) stained with monoclonal anti-B
antibody. There is faint, granular, cytoplasmic staining. X 62. d, H-498 cells
stained with B. simplicifolia-l lectin. Intense granular staining is shown. X 62.
transferase activity but did not react with anti-B antibody (Fig.
2Ä),thereby demonstrating B antigen deletion. This antigenic
deletion is not apparently due to excess degradation because, if
anything, a-galactosidase activity in SW1417 cells was lower
than in the other cell lines. Weak incompatible B antigen
expression was noted in SW1116 and H498 cell lines (Fig. 4c).
However, since there was little or no B transferase activity in
the blood type O and A cell lines, this incompatible antigen
expression must represent a B-like or cross-reacting substance.
Using B. simplicifolia isolectin I-B4, which is specific for agalactosyl residues (30), LoVo was very strongly stained (Fig.
10
is
[UDP-Gal],
20
MM
1.0-
V/S
I
0.2
0.4
0.6
[Fuco2G«lp4Glc],
'
0.8
mM
Fig. 5. Substrate dependence of B transferase. A homogenate of SW 1417 (20
Mgprotein) was incubated with varying concentrations of UDP-('H]galactose (A)
and 2'-fucosyllactose (B). Linear regression analysis of V versus VjS plots (insets)
was used to determine apparent A'mand Fm„
values.
4553
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH ANTIGENS IN ADENOCARC1NOMA CELL LINES
Table 3 Expression of H antigen, al,2-fucosyltransferase (H transferase), and
600fucosidase in human colonie adenocarcinoma cell lines
transferase
a-Fucosidase
H antigen
lineLSI74THM-7LM-12SW1116HT-29SW-480H-498LoVoSW1417Blood
Cell
type
expressionO
(pmol/mg/90
h)492.9452.4414.1241.8372.3211.9513.1399.1855.6248454046059177699865739881536345"4505059477442181101439
min) (nmol/mg/16
400-
10(++)O
10(+)O
5(+)O
80(+)AAA
20(-t-+)BBH
±202
CGDP-Fuc], MM
°Mean ±SE, four experiments.
2OO -
40
«0
80
[ph«nyl-ß-G«l], mM
Fig. 7. Substrate dependence of H transferase. A homogenate of LS 174T (50
jig protein) was incubated with varying concentrations of GDP-|'"C]fucose (A)
and phenyl-|8-galactoside (B). Linear regression analysis of V versus V/S plots
(insets) was used to determine apparent K,,,and I ',„.„
values.
.
Fig. 6. Blood group H antigen expression. I SI 741 cells stained with mono
clonal anti-H antibody. Cells are focally positive, with staining of cell membranes.
X50.
A
n
e
of the cell lines, irrespective of the blood type. a-Fucosidase
activity at pH 4.0 did not correlate with H antigen expression
but was higher than a-galactosidase or a-/V-acetylgalactosaminidase in all the cell lines. The activities of a-fucosidase at pH
6.8 were 1 to 3% of the activities at pH 4.0 (10 to 38 nmol/
mg/16 h).
The enzyme kinetics of H transferase was examined with
respect to different concentrations of acceptor, phenyl-/3-galactoside, and donor, GDP-fucose. The apparent Km for phenyl-/3galactoside was 8.65 mM (Fig. 7), which is similar to the Km of
the human serum enzyme (33). The apparent Km for GDPfucose was 39.7 Õ¿M.
This is lower than the GDP-fucose Km of
the human trachéalenzyme (34). Because it has been suggested
that ABH antigen expression is growth dependent (35), the
doubling times of seven of the cell lines were measured (21).
The doubling times of the two cell lines that failed to express
the expected antigen (H-498, 42 h; SW1417, 28 h) were within
the range of those that did express the appropriate antigen
(LS174T, 25 h; HT-29, 30 h; LoVo, 35 h; SW480, 38 h;
SW1116, 74 h).
In order to establish the product of H transferase, the reaction
mixtures were passed through Dowex 1-X8 columns and then
separated by descending paper chromatography. As shown in
Fig. 8, the reaction product from the fucosyltransferase reaction
was clearly separated from free fucose and phenyl-ß-galactoside.
This product was chromatographically similar to the product
formed by the H fucosyltransferase of human serum, which has
been previously characterized as Fucal-2Gal/3-phenyl (24).
DISCUSSION
Our findings can be summarized as follows, (a) Except for
the blood type A H-498 cell line, colon cancer cell lines derived
io
20
10
I
20
dittine«, cm
Fig. 8. Characterization of H transferase product. A homogenate of LS 174T
was incubated with GDP-[14C]fucose with (A) or without (B) 0.04 M phenyl-fîgalactoside. The reaction mixtures, after passing through Dowex 1 formate, were
subjected to paper chromatography. The first peak (a) has the mobility of free
fucose. The second peak (b) assumed to be Fuc-2Gal/3-phenyl, is taken as the H
transferase product.
from blood type A or B individuals expressed the appropriate
A or B gene-encoded glycosyltransferase. This was associated
with expression of the relevant antigen by each cell line, except
H-498 and SW1417, which manifested deletion of the A and B
antigens, respectively, (b) All cell lines, regardless of ABO type,
expressed the H gene-encoded fucosyltransferase. The H anti
gen was found on all blood type O cell lines. However, H-498
cells also expressed H antigen, thereby representing accumula
tion of precursor substance in a cell line which is deficient in A
transferase activity, (c) In general, the activity of the three aglycosidases did not correlate with blood group antigen expres
sion. (</) Demonstration that the measured A, B, and H glyco
syltransferase activities represented the action of the true geneencoded A, B, and H transferases was confirmed by HPLC,
revealing the predicted tetrasaccharide from the A and B trans
ferase reactions, and by paper chromatography, revealing the
expected disaccharide from the H transferase reaction. Fur
thermore, substrate affinities of the colon cancer-derived A, B,
4554
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH ANTIGENS IN ADENOCARC1NOMA CELL LINES
and H glycosyltransferases for the saccharide acceptors and
nucleotide sugar donors were similar to those of human serum
A, B, and H transferases.
Immunohistochemical
studies have demonstrated that nor
mal human colonocytes express A, B, and H antigens compat
ible with the host's blood type in the proximal but not distal
colon (4, 8-11). However, during fetal life, colonocytes along
the entire length of the colon bear the appropriate ABH antigen
(8, 9). Thus, there is a tight control of ABH expression that
occurs with development of this organ, with an apparent loos
ening of this control in malignancy, as manifested by ABH
reappearance in distal cancers, ABH deletion of proximal can
cers, and incompatible A and B expression (9, 11-18). Very
little is known about the mechanisms responsible for these
alterations in colonie ABH antigen expression. A few studies
have addressed this issue by measuring glycosyltransferases and
glycosidases in homogenates of normal and cancerous colonie
tissues (36-39). While such an approach is advantageous for
comparing normal and cancerous tissues from the same indi
vidual, it is limited by the fact that nonepithelial elements, such
as RBC, endothelial cells, and glycosidase-producing bacteria
might be contributing to the observed results. Therefore, to
obtain a better understanding of the mechanisms contributing
to ABH expression in malignant colonie epithelial cells, we
conducted the present study using well characterized colon
cancer cell lines established from individuals of known blood
type.
No study to our knowledge has investigated mechanisms of
ABH antigen biosynthesis in isolated colon cancer cells. In the
present study, there were two examples of blood group antigen
deletion. Because it is not known whether these cell lines were
established from cancers of the proximal or distal colon, it is
not clear whether the lack of expression of A and B antigens in
H-498 and SW1417 is analogous to the developmental loss of
antigen in normal distal colon or to the cancer-associated
deletion of antigen in the proximal colon. The H-498 cell line
failed to express the A antigen, most likely because of absent A
transferase activity. The deletion of A antigen was accompanied
by precursor accumulation of H substance in this case. The
SW1417 cell line failed to express the B antigen but, unlike in
H-498 cells, there appeared to be sufficient levels of the biosynthetic B transferase activity. Enhanced B-degrading enzyme
activity did not seem to explain the deletion. Interestingly, in
this cell line, there was no accumulation of precursor H antigen,
despite very high levels of H transferase activity. This suggests
that, despite adequate levels of B and H transferases, precursor
synthesis might be impaired. This could occur, for example, if
other glycosyltransferases, such as sialyltransferase or a 1,3fucosyltransferase, successfully competed with the H gene a 1,2fucosyltransferase for the type 1 or type 2 oligosaccharide side
chain. An alternate explanation for B antigen deletion in these
cells could be masking of the immunodeterminant in some way
as to make it inaccessible to bind antibody.
In terms of incompatible antigen expression, Drewinko and
Lichtiger (19) reported incompatible A and B antigen expres
sion in all seven colon cancer cell lines examined. However,
that study used human polyclonal anti-A and anti-B antisera
applied to cell suspensions analyzed by indirect immunofluorescence, and neither glycosyltransferase nor glycosidase activ
ities were determined. In the present study, we detected weak
binding of monoclonal anti-A to SW1116 (type O) and LoVo
(type B) cells, and weak monoclonal anti-B binding to SW1116
and H498 (type A) cells. However, since there was little or no
A or B gene-encoded glycosyltransferase activity in these cell
lines, this suggests that the antigens being detected are not the
products of the true ABO gene-encoded enzymes.
Although the mechanism(s) responsible for incompatible A
or B antigen expression is not known, a few possibilities exist.
First, the true A or B gene-encoded glycosyltransferases might
become activated in cancer cells. While this concept goes
against our understanding of classical immunogenetics of blood
group substances, one study did find A transferase activity in a
colon cancer from a blood type O individual whose tumor tissue
expressed incompatible A antigen (18). A second hypothesis to
explain incompatible A or B antigen expression might be a
loosening of substrate specificity of the A or B transferases. For
example, under certain in vitro experimental conditions, the
serum A gene-encoded transferase can synthesize the B antigen
(48), and the B transferase can synthesize the A antigen (29,
32, 40).
A third hypothesis is that the incompatible antigens are really
A-like and B-like structures and not the products of the geneencoded transferases. For example, substances with terminal
GalNAc residues, such as Forssman antigen (41-43) or Tn
antigen (44), behave as A-like structures. However, our mono
clonal anti-A antibody does not react with Forssman-positive
sheep RBC (9) and has a different staining pattern, compared
to specific anti-Tn reagents.5 A-like oligosaccharides have been
identified on glycoproteins from human rectal carcinoma (45).
Indeed, monoclonal antibodies raised against HT-29 cells react
not only with true A substances but also with A-like substances
(46). In other studies, naturally occurring antibodies which
recognize A-like determinants were found in the sera of two
healthy sisters belonging to a kindred at high risk for colon
cancer (47).
The A antigen can be converted into an "acquired" B antigen
by deacetylation of the terminal a-A'-acetylgalactosamine, an
uncommon event that has been reported for RBC (48). Alter
natively, B-like structures with terminal a-galactose residues,
such as fucose-less B antigen, might also account for incompat
ible B expression. The B4 isolectin of B. simplicifolia I has
greater affinity for B-like Galal,3Gal structures than for the
blood group B tetrasaccharide (30). In previous studies we found
that purified mucin from LS174T cells reacted strongly with B.
simplicifolia I (isolectins A4 and B4) (49) and, by cytochemistry,
all of the cell lines used in the present study, except HT-29 and
SW1116, bound the B4 isolectin of B. simplicifolia I. This
suggests the existence of B-like substances in our cell lines,
although we have not been able to demonstrate transfer of
galactose to lactose to form a fucosyl B antigen in these same
cell lines.
The A, B, and H transferases of human colon cancer cells
have not previously been characterized with respect to substrate
affinity. Our results demonstrate that the Km values for the
saccharide acceptors and nucleotide sugar donors of colon
cancer cell ABH enzymes were very similar to those reported
for these enzymes in human serum. Furthermore, using a sterile
cell culture system, our data illustrate the ABH-degrading gly
cosidases in the colon, which have been characterized as prod
ucts of enteric bacteria (50), may also be produced by colono
cytes.
ACKNOWLEDGMENTS
The authors wish to thank Annabelle Friera, Frank Fearney, Robert
Lagace, and Dr. Sam Ho for technical assistance and Rita Burns for
manuscript editing and preparation.
' Unpublished observations.
4555
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH ANTIGENS IN ADENOCARC1NOMA CELL LINES
REFERENCES
1. Watkins, M. W. Genetics and biochemistry of some human blood groups.
Proc. R. Soc. Lond. B Biol. Sci., 202: 31-53. 1978.
2. Sharon, N. Blood group substances. In: Complex Carbohydrates, Their
Chemistry, Biosynthesis, and Functions, N. Sharon (éd.),
pp. 215-257. MA:
Addison-Wesley, 1975.
3. Hakomori, S. Blood group ABH and li antigen of human erythrocytes:
chemistry, polymorphism and their developmental changes. Semin. Hematol., 18: 39-62, 1981.
4. Szulman. A. E. The histological distribution of blood group substances A
and B in man. J. Exp. Med., ///: 785-800, 1960.
5. Ginsburg, V. Enzymatic basis for blood groups in man. Adv. Enzymol.. 36:
131-148, 1972.
6. Clausen, H., Levery, S. B., Dabelsteen. E., and Hakomori, S. Blood group
ABH antigens: a new series of blood group A-associated structures (genetic
regulation and tissue distribution). Transplant. Proc., 19: 4408-4412, 1987.
7. Watkins, W. M., and Greenwell, P. Biosynthesis of blood group ABH
antigens: genetic regulation and tissue distribution. Transplant. Proc., 19:
4413-4415, 1987.
8. Szulman, A. E. The histological 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: 503-515,
1964.
9. Yuan, M., Itzkowitz, S. H., Palekar, A., Shamsuddin, A. M., Phelps, P. C,
Trump, B. F., and Kim, Y. S. Distribution of blood group antigens A, B, H,
Lewis", and Lewisb in human normal, fetal, and malignant colonie tissue.
Cancer Res., 45: 4499-4511, 1985.
10. Szulman, A. E. The histological distribution of 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.
11. Dabelsteen. E., Graem. N., Clausen, H., and Hakomori, S. Structural varialions of blood group A antigens in human normal colon and carcinomas.
Cancer Res., 48: 181-187, 1988.
12. Cooper, H. S., Cox, J., and Patehefsky, A. S. Immunohistologic study of
blood group substances in polyps of the distal colon. Am. J. Clin. Pathol.,
73: 345-350, 1980.
13. 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.
14. Itzkowitz, S. H., Yuan, M., Ferrell, L. D., Palekar, A., and Kim, Y. S.
Cancer-associated alteration of blood group antigen expression in human
coloréela!polyps. Cancer Res., 46: 5976-5981, 1986.
15. 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.
16. Schoentag, R., Primus, F. J., and Kuhns, W. ABH and Lewis blood group
expression in colorectal carcinoma. Cancer Res., 47:1695-1700, 1987.
17. Feizi, T., Turberville, C., and Westwood, J. H. Blood-group precursors and
cancer-related antigens. Lancet, 2: 391-393, 1975.
18. Clausen, H., Hakomori, S., Graem, N., and Dabelsteen, E. Incompatible A
antigen expressed in tumors of blood group O individuals: immunochemical,
immunohistologic, and enzymatic characterization. J. Immunol., 136: 326330. 1986.
19. Drewinko, B., and Lichtiger, B. Full expression of blood group-related,
transplantation-related, and carcinoembryonic antigens in human colorectal
cancer cells with different degrees of phenotypic differentiation. Cancer Res.,
27.
28.
29.
39
31
32.
33
34.
35
35
37
3g
39
49
41
42.
43
44
45: 1560-1564, 1985.
20. Chantret, I., Chevalier, G., Dussaulx, E., and Zweibaum, A. A and H blood
group antigens as markers of sucrase-isomaltase from the enterocyte-Iike
differentiated human colon carcinoma cell lines HT-29 and CaCo-2. Cancer
Res., 47: 1426-1433, 1987.
21. Kuan, S-F., Byrd, J. C., Basbaum. C. B., and Kim, Y. S. Characterization of
quantitative muchi variants from a human colon cancer cell line. Cancer
Res., 47:5715-5724, 1987.
22. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Protein
measurement with the Folin phenol reagent. J. Biol. Chem., 293: 265-275,
1951.
23. Whitehead, J. S., Fearney, F. J., and Kim, Y. S. Glycosyltransferase and
glycosidase activities in cultured human fetal and colonie adenocarcinoma
cell lines. Cancer Res., 39: 1259-1263, 1979.
24. Chester, M. A., Yates, A. D., and Watkins, W. M. Phenyl 0-D-galactopyranoside as an acceptor substrate for the blood group II gene-associated
guanosine diphosphate L-fucose: 0-D-galactosyl o-2-L-fucosyltransferase.
Eur. J. Biochem., 69: 583-592, 1976.
25. Trevelyan, W. E., Procter, D. P., and Harrison, J. S. Detection of sugars on
paper chromatograms. Nature (Lond.), 166:444-445, 1950.
26. Schachter, H., Michaels, M. A., Tilley, C. A., Crookston, M. C., and
Crookston, J. H. Qualitative differences in the /V-acetyl-n-galactosaminyl-
45
45.
47.
4g.
49.
50.
transferases produced by human A1 and A! genes. Proc. Nati. Acad. Sci.
USA, 70:220-224. 1973.
Whitehead, J. S., Bella, A., and Kim, Y. S. An ¿Y-acetylgalactosaminyltransferase from human blood group A plasma: purification and agarose binding
properties. J. Biol. Chem.. 249: 3442-3447, 1974.
Kobata, A., and Ginsburg, V. Uridine diphosphate iV-acetyl-n-galactosamine:
D-galactose «-3-A'-acetyl-D-galactosaminyltransferase, a product of the gene
that determines blood type A in man. J. Biol. Chem., 245:1484-1490.1970.
Yates. A. D., Feeney, J., Donald. A. R.. and Watkins. W. M. Characterization
of a blood group A active tetrasaccharide synthesized by a blood group B
gene-specified glycosyltransferase. Carbohydrate Res., 130: 251-260, 1984.
Wood. C., Kabat. E. A., Murphy, L. A., and Goldstein, I. J. Immunochemical
studies of the combining sites of two isolectins, V, and H.,. isolated from
Bandeiraea simplicifolia. Arch. Biochem. Biophys., 198: 1-11, 1979.
Carne, L. R., and Watkins, W. M. Human blood group B gene-specified «3-galactosyltransferase: purification of the enzyme in serum by biospecific
adsorption onto blood group O erythrocyte membranes. Biochem. Biophys.
Res. Commun., 77: 700-707. 1977.
Yates, A. D., and Watkins, W. M. The biosynthesis of blood group B
determinants by the blood group A gene-specified n-3-A'-acetyl-D-galactosaminyl transferase. Biochem. Biophys. Res. Commun., 109: 958-965, 1982.
Kumazaki, T., and Yoshida, A. Biochemical evidence that secretor gene. Se.
is a structural gene encoding a specific fucosyltransferase. Proc. Nati. Acad.
Sci. USA, 81:4193-4197, 1984.
Cheng, P. W., and DeVries, A. Mucin biosynthesis: enzymic properties of
human trachéalepithelial GDP-i.-fucose:/3-D-galactoside «-(
1,2)-L-fucosyltransferase. Carbohydrate Res., 149: 253-261, 1986.
Pann, C.. and Kuhns, W. J. Regulation of glycoconjugate expression on
cloned mammalian cells. Exp. Cell Res., 98: 73-78. 1976.
Kim, Y. S., Isaacs, R., and Perdomo, J. M. Alterations of membrane glycopeptides in human colonie adenocarcinoma. Proc. Nati. Acad. Sci. USA, 7:
4869-4873, 1973.
Kim, Y. S., and Isaacs, R. Glycoprotein metabolism in inflammatory' and
neoplastic diseases of the human colon. Cancer Res., 35: 2092-2097, 1975.
Kiang, D. T., Kennedy, B. J., and Edstrom, R. D. Levels of blood group
synthetic enzymes in human colonie carcinoma. Proc. Soc. Exp. Biol. Med..
157:411-414, 1978.
Yonezawa, S., Matsushita, Y., Muramatsu, H., Arila, Y., Nakamura. T..
Muramatsu, T., and Sato, E. Correlation between glycoconjugate expression
and fucosyltransferase activity in human colorectal carcinoma. Acta Pathol.
Jpn., 37: 1249-1261, 1987.
Greenwell, P., Yates, A. D., and Watkins, W. M. UDP-A'-acetyl-o-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene
at the human blood group ABO locus. Carbohydr. Res., 149:149-170,1986.
Hakomori, S., Wang, S. M., and Young, W. W., Jr. Isoantigenic expression
of Forssman glycolipid in human gastric and colonie mucosa: its possible
identity with "A-like antigen" in human cancer. Proc. Nati. Acad. Sci. USA,
74:3023-3027, 1977.
Hattori, H., Uemura, K., Ogata. H.. Katsuyama, T., Taketomi, T.. and
Kanfer, J. N. Characterization of glycolipids from the gastric cancer of a
patient of p.O, Le(a~,b*) blood type: presence of incompatible blood group
antigens in tumor tissues. Cancer Res., 47: 1968-1972, 1987.
Breimer. M. E. Adaptation of mass spectrometry for the analysis of tumor
antigens as applied to blood group glycolipids of a human gastric carcinoma.
Cancer Res., 40: 897-908, 1980.
Hirohashi, S,, Clausen, H., Yamada, T., Shimosato, Y., and Hakomori, S.
Blood group A cross-reacting epitope defined by monoclonal antibodies
NCC-LU-35 and -81 expressed in cancer of blood group O or B individuals:
its identification on Tn antigen. Proc. Nati. Acad. Sci. USA, 82: 7039-7043,
1985.
Kurosaka, A.. Nakajima, H., Funakoshi, I., Matsuyama, M., Nagayo, T., and
Yamashina, I. Structures of the major oligosaccharides from a human rectal
adenocarcinoma glycoprotein. J. Biol. Chem., 25«:11594-11598, 1983.
Gooi, H. C., Hounsell, E. F., Picard, J. K.. Lowe, A. D., Voak, D., Lennox,
E. S., and Feizi, T. Differing reactions of monoclonal anti-A antibodies with
oligosaccharides related to blood group A. J. Biol. Chem., 260: 1321813224, 1985.
Knuth, P., Lloyd, K. O., Lipkin, M., Oettgen. H. F., and Old. L. J. Natural
antibodies in human sera directed against blood-group-related determinants
expressed on colon cancer cells. Int. J. Cancer, 32: 199-204, 1983.
Stayboldt, C., Rearden, A., and Lane, T. A. B antigen acquired by normal A!
red cells exposed to a patient's serum. Transfusion (Phila.), 27:41-44, 1987.
Byrd, J. C., Nardelli, J., Siddiqui, B., and Kim, Y. S. Isolation and charac
terization of colon cancer mucin from xenografts of LS174T cells. Cancer
Res., 48: 6678-6685, 1988.
Hoskins, L. C., Agustines, M., McKee, W. B., Boulding, E. T., Kriaris, M.,
and Niedermeyer, G. Mucin degradation in human colon ecosystems: isola
tion and properties of fecal strains that degrade ABH blood group antigens
and oligosaccharides from mucin glycoproteins. J. Clin. Invest., 75: 944953, 1985.
4556
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
ABH Blood Group Antigen Expression, Synthesis, and
Degradation in Human Colonic Adenocarcinoma Cell Lines
Rajvir Dahiya, Steven H. Itzkowitz, James C. Byrd, et al.
Cancer Res 1989;49:4550-4556.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/49/16/4550
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.