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Int. J. Cancer:68,239-244 (1996)
0 1996 Wiley-Liss, Inc.
Publicatan of the International Union Against Cancer
Publication de I'Union lnternatlonale Contre le Cancer
Danuta DuS? and Maciej UGORSKI'
Deparfmentsof 'Immunochemistryand 2TumorImmunology, Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, Wroclaw,Poland
In a previous study we showed that tumorigenic and invasive
human uroepithelial cell lines are characterized by the presence of sialosyl le' ($Lee')ganglioside. Our data suggested that
expression of this glycolipid correlated with acquisition of the
malignant phenotype by human urothelial cells. To evaluate the
postulated adhesion function of $Lea antigen, we studied the
adherence of 6 human urothelial cell lines with different
expressions of this carbohydrate structure to E-selectinexpressing CHO cells. The only cell line that bound specifically
to E-selectin was Hu 1703He. which expressed the highest level
of sl& antigen. The involvement of carbohydrat+E-selectin
interaction in the adhesion of Hu 1703He cells was indicated by
the following facts: (i) anti-E-selectin monoclonal antibody
(MAb) completely abolished binding to E-selectin-expressing
CHO cells; (ii) removal of sialic acid from Hu 1703He cells highly
decreased the adhesion. Adhesion correlated with the presence
of several sLea-carrying glycoproteins, which was shown by
immunoblotting of Hu 1703He cell lysate with anti-sle. MAb
19-9. The bindingof antibody was abolishedwhen cell lysate was
treated with 0-sialoglycoprotein endopeptidase. suggesting
that sLea is present on 0-linked oligosaccharides. However,
incubation of Hu I703He cells with 0-sialogtycoprotease had no
effect on adhesion to E-selectinor on binding of 19-9 MAb to the
cell surface. Our data suggest that (I) protein-bound sLea
oligosaccharides represent only a minor portion of whole s h e '
anti en roduced by uroepithelial cells; (ii) effective binding to
E-sefecti occurs when s h . oligosaccharide present on cellsurface glycorphingolipidsis expressed in high density since the
cell lines with moderate expression of s l e ' ganglioside did not
bind to E-selectin-tramfectedCHO cells.
0 1996 Wiley-Lks, lnc.
The sLea and sLe" carbohydrate antigens, as well as their
sulfated equivalents, are the ligands for selectins, a newly
described family of adhesion molecules (Bevilacqua and Nelson, 1993; Feizi, 1993). Selectins function as lymphocytehoming and leukocyte-enrollment receptors or as activationdependent cell-surface receptors of platelets and endothelial
cells (Bevilacqua and Nelson, 1993). sLea and s L e x by themselves are not true physiological ligands for selectins (McEver
et al., 1995). It seems that specific glycoconjugates carrying
proper oligosaccharide chains are responsible for mediating
biologically relevant signals. Many of them have common
structural features. They are 0-glycosylated much-type glycoproteins (McEver et al., 1995).
Several lines of evidence suggest that sLea and s L e x structures, both representing typical tumor-associated carbohydrate antigens (TACA), are responsible for the adhesion of
human cancer cells to endothelium. E-selectin and P-selectin
present on endothelial cells mediate these interactions. Selectins and their carbohydrate ligands can thus play an important role in the selective homing of tumor cells during
metastasis (Honn and Tang, 1992). Involvement of the sLea
structure in E-selectin-mediated adhesion has been described
for colon and pancreatic cancer cells (Iwai et al., 1993; Majuri
et al., 1992; Takada et al., 1993). Takada et al. (1993) studied
the binding of 12 cultured human epithelial cancer cell lines
which express sLea and/or sLex antigens. Their data suggest
that sLea plays a major role in metastases of gastro-intestinal
cancers, whereas metastases of lung, liver and ovarian cancers
are mediated by sLex. However, human colon cancer cell lines
characterized by high expression of sLe" strongly adhere to
activated endothelial cells through E-selectin (Sawada et al.,
SLe"antigen is detected as ganglioside in cancerous tissues
of colon, stomach, pancreas and gall bladder (Magnani et al.,
1982). An elevated level of glycoproteins bearing sLea is found
in sera of cancer patients. Baeckstrom et al. (1991) have shown
that in colon carcinoma COLO 205 cells s k a is present on 2
different mucin-type glycoproteins. A larger mucin (600-800
kDa), named H-CanAg, is bound to the cell surface, and its
apoprotein was identified as MUC-1 much (Baeckstrom et al.,
1991). A smaller glycoprotein (150-300 kDa), called L-CanAg,
is secreted by cells, and its apoprotein is identical to that of
leukosialin (Baeckstrom et al., 1991, 1995). In a human
pancreatic cancer cell line, sLea and sLex oligosaccharides are
associated with MUC-1 apomucin (Ho et al., 1995). Takabayashi et al. (1993) analyzed sialosyl Lea-carrying glycoproteins in
human colorectal carcinomas and their surrounding nonneoplastic mucosa. They suggested that increased levels of
sLea-bearing glycoproteins of lower molecular mass are associated with a higher metastatic potential of tumor cells. The
major part of the sLeXantigen on colon carcinoma cells is
carried as well by high-molecular-mass mucins. According to
Hanski et al. (1999, MUC-2 is the major sLex-positive mucin
present in colonic tissue and colon carcinoma. The adhesion of
neuroblastorna and small-cell lung cancer cells t o P-selectin is
inhibited by treatment of cells with neuraminidase and trypsin
(Stone and Wagner, 1993). The inhibitors of N-glycosylation
had no effect on binding, suggesting that a carbohydrate ligand
is present on 0-linked chains. Taken together, these data give
evidence that rnucin-type glycoproteins of cancer cells are
primary ligands for E-selectin. However, the involvement of
N-linked oligosaccharides in E-selectin-mediated adhesion
was described for colon cancer cells by Sawada et af. (1993).
Adhesion of COLO 205 and HT-29 cells to E-selectinimmunoglobulin fusion protein is not affected by treatment
with 0-sialoglycoprotease, which specifically cleaves mucintype glycoproteins (Mannori ef al., 1995), suggesting that in
these cancer cells, adhesion to E-selectin is mediated by
glycolipids, which bear sLeXstructures.
We have shown previously that tumorigenic and invasive
human uroepithelial cell lines, representing grade of transformation 111 (TGr 111),are characterized by the presence of sLea
ganglioside (Ugorski et al., 1990). Our data suggested that
expression of this glycolipid is correlated with acquisition of
the malignant phenotype by human urothelial cell lines. Since
the specific biological role of the antigen was unknown at that
whom correspondence and reprint requests should be sent, at
Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Czerska 12, 53-114
Wroclaw. Poland. Fax: 48-71-67 91 11.
Abbmiotions: sLea,sialosyl Lea, sLex, sialosyl Lex,PBS, phos hate
buffered saline: TBS, tris-buffered saline; PMSF, phenylmethyfsulfo:
nyl fluoride: SDS-PAGE, sodium dodecyl olyacrylamide el electrophoresis; BSA, bovine serum albumin; J A b , monoclonaf antibody;
FITC, fluorescein-isothiocyanate
Received: January 3, 1996 and in revised form July 4, 1996.
time, the present study was undertaken to evaluate the role of
the sLea carbohydrate structure in adhesive properties of
uroepithelial cells.
Cells and cell culture
Cell lines of human urothelium origin were obtained from
Dr. J. Kieler (Copenhagen, Denmark). They were classified,
according to Christensen et al. (1984), as TGr I1 and 111. Cells
with an indefinite life span in vitro which are non-invasive for
chick heart fragments in in vitro assay and non-tumorigenic in
nude mice were classified as TGr 11. Cell lines invasive in in
vitro assay and tumorigenic in nude mice were classified as TGr
111. The following cell lines were used: HCV 29 (TGr 11). HCV
29T, Hu 609T, Hu 456, Hu 549 and Hu 1703He (TGr 111). Cells
were grown in Fib41 B medium, supplemented with 10% FCS
(GIBCO BRL, Grand Island, NY) and non-essential amino
acids (complete Fib41 B medium), at 37°C in 5% C02/95%
humidified air. Cells were passaged using 0.05%trypsin/0.02%
EDTA. Chinese hamster ovary cells (CHO, clone Pros),
obtained from the ATCC (Rockville, MD), were cultured in
a-minimum essential medium ((uMEM) supplemented with
10% FCS. 2 mM glutamine (GIBCO BRL) and antibiotics
(complete (uMEM). The E-selectin cDNA-transfected CHOPro5 cell line was maintained in complete aMEM in the
presence of geneticin ((3418, 0.25 mg/ml; GIBCO BRL).
Glycophorin A-expressing CHO cells (Remaley et al., 1991)
were grown in the same conditions.
Anti-carbohydrate antibodies
MAb 19-9 directed against sLea was kindly provided by Dr.
Z . Stqplewski (Wistar Institute, Philadelphia, PA). Two other
anti-sLe" antibodies, 9001/ 1B1 and 121-SLE, were obtained
from MonoCarb (Lund, Sweden) and Serotec (Oxford, UK),
respectively. The CSLEX and KM-93 antibodies to human
sLeX (CD 15s) were purchased from CAMFOLIO-BectonDickinson (San Jose, CA) and Serotec, respectively. For
inhibition of cell adhesion, 19-9 MAb IgG were purified from
ascitic fluid by ammonium sulfate precipitation and subsequent HPLC chromatography on a TSK DEAE-2SW column
(Toyo-Soda, Tokyo, Japan). Hybridoma cells producing 6A7
MAb to glycophorin A blood group M were obtained from the
Flow cytometry
Cells (2 x lo5) were detached with 0.2% EDTA, washed
and resuspended in TBS-BSA (50 mM TRIS-HCI, 180 mM
NaCI, 5 mM CaC12, 18 mM MgS04, containing 1% BSA and
0.1% NaNJ), then incubated with primary antibody for 1 hr on
ice. After washing, cells were incubated for another hour at
4°C with fluorescein-isothiocyanate (FITC)-conjugated rabbit
F(ab')z fragment against mouse immunoglobulins (DAKO,
Glostrup, Denmark), then analyzed for positive staining by
flow cytometry using a FACStar Plus model SOH apparatus
(Becton-Dickinson, Mountain View, CA) with 448 nm argon
laser equipment. Five thousand cells were acquired for each
data file. Data were processed using Becton-Dickinson PC
Lysys software, version 1.0.
Production of E-selectin cDNA-transfected CHO-Pro5 cell line
Plasmid containing complete cDNA sequence for E-selectin
(pSRa-E-selectin) was kindly provided by Dr. M. Fukuda (La
Jolla, CA) (Sawada et al., 1993). CHO cells (clone Pros) were
co-transfected with 10 kg of pSRa-E-selectin and 1 k g of
pSV2Neo by the calcium phosphate precipitation method as
described previously (Rcmaley et al., 1991). Geneticinresistant colonies were screened for E-selectin expression by
flow cytometry with anti-E-selectin MAb (Genzyme, Cambridge, MA).
Cell adhesion assay
Uroepithelial cells (4 x lo6) were labeled for 2 hr with 0.1
mCi of Naz'CrOp (Institute of Nuclear Research, Warsaw,
Poland) in 1 ml of complete Fib41 B medium. Cells were
washed with Fib41 B medium and incubated for another hour
in the same medium. 5'Cr-labeled cells, detached by 0.2%
EDTA or 0.05% trypsin/0.02% EDTA, were suspended in 1
ml medium, and 0.1 ml of 51Cr-labeledcells was added to the
monolayer of E-selectin cDNA-transfected CHO cells in a
24-well plate. After incubation at room temperature for 30
min, cells were washed twice with Hank's balanced salt
solution, lysed with 1% NP-40, and radioactivity was measured
in a gamma-counter (Beckman, Palo Alto, CA). Adherence of
cancer cells was corrected for binding to untransfected CHO
cells. For inhibition of cell adhesion, anti-sLea or anti-sLex
MAbs were pre-incubated with suspension of 5'Cr-labeled
cells for 1 hr at room temperature prior to application to the
monolayer of CHO cells. Purified MAb 19-9 was used up to
200 pg/ml or as ascitic fluid diluted 1:l; MAb 9001/1B1 and
MAb CSLEX were used as culture supernatants and MAb
121-SLE as ascitic fluid diluted 1:lO. Alternatively, a monolayer of CHO cells was pre-incubated with MAb anti-Eselectin (40 pg/ml).
Western blotting
Human urothelial cells were solubilized in lysis buffer (50
mM TRIS-HCL, 150 mM NaCl and 1 mM EDTA, pH 8.0,
containing 0.5% NP40, 1 mM PMSF, Aprotinin 2 pg/ml and
leupeptin 2 pg/ml; Sigma, St. Louis, MO), and protein content
was determined according to the Lowry method. Samples
containing 100 pg protein were separated by SDS-PAGE
(using gradient gel 5-15%), according to the method of
Laemmli (1970), and transferred to nitrocellulose (Schleicher
and Schuell, Dassel, Germany). To decrease non-specific
binding, nitrocellulose was pre-incubated with 1% casein
(Sigma) in TBS. Proteins were detected immunologically with
primary MAb 19-9 (anti-sLe') and secondary alkaline phosphatase-conjugated goat anti-mouse immunoglobulins (Bio-Rad,
Hercules, CA).
Treatment of cell lysate with 0-sialoglycoprotease, N-glycanase,
neuraminidase and fucosidase
To cleave sialomucins, solubilized glycoproteins (100 kg)
were incubated with 5 p1 of 0-sialoglycoprotein endopeptidase
from Pasteurella haemolytica (Cedarlane, Hornby, Canada) in
0.05 M TRIS-HCI, pH 7.4. To remove N-linked oligosaccharides, solubilized glycoproteins were incubated with N-glycanase
(5 units/100 pg protein; Genzyme), overnight at 37°C. The
effectiveness of N-glycanase action on release of N-glycans
from cellular glycoproteins was assayed by incubation of the
blots with biotinylated ConA (Duket al., 1994). Binding of the
lectin to the cell lysate after digestion with N-glycanase was
strongly diminished in comparison with untreated control.
Fucose residues were removed by incubation of cell lysates
with a-L-fucosidase from Charonia lampas (0.02 units/60 kg;
Seikagaku, Tokyo, Japan) overnight at 4°C. To remove sialic
acid, the glycoproteins transferred to nitrocellulose were
incubated with 25 mM sulfuric acid for 1 hr at 80°C or,
alternatively, before SDS-PAGE and Western blotting, cell
lysates (100 pg protein) were treated with neuraminidase from
Vibrio cholerae (0.25 units/ml; Boehringer Mannheim, Germany), overnight at 4°C. The enzyme-treated proteins were
electrophoretically separated and analyzed by irnmunoblotting.
Treatment of cells with neuraminidase, ttypsin
and 0-siafoglycoprorease
To remove sialic acid residues or cell-surface glycoproteins,
human uroepithelial cclls (4 x 106/ml) suspended in PBS,
supplemented with 3% BSA, were incubated with 0.1 units/ml
of V. cholerae neuraminidase (Boehringer Mannheim) or with
50 p.g/ml of trypsin (GIBCO BRL) for 1 hr at 37°C. To
specifically cleave cell-surface sialomucins, O-sialoglycoprotein endopeptidase from P. haemolytica (Cedarlane) was used.
Cells (4 x 106/440 p.1) were suspended in aMEM without FCS
and treated with 60 FI of enzyme for 1 hr at 37°C. For further
experiments, cells were washed twice with complete aMEM
and resuspended in the same mcdium.
Flow-cytometric analysis of surface sLea and sL& antigens
on human uroepithelial cell lines
The presence of sLea and sLex antigens on the surface of
human uroepithelial cell lines was evaluated by cytofluorimetric analysis. There was no binding of antibody 19-9 (anti-sLea)
to the surface of HCV 29 or HCV 29T cells. Three other cell
lines, Hu 609T, Hu 549 and Hu 456, were stained weakly, and
the strongest binding was observed for the cell line Hu 1703He
(Fig. 1).
Anti-sLeX antibodies (CSLEX and KM-93) did not bind
to cell lines HCV 29, HCV 29T, Hu 456 or Hu 549 (data not
shown) and bound weakly to 609T and Hu 1703He cells
(Fig. 2).
24 1
Adhesion of human uroepithelial cell lines to
E-selectin-erpressing CHO celki
The adhesion of human urothelial cell lines with different
expressions of surface sLea determinants to CHO cells transfected with cDNA for E-selectin was tested. The rationale for
selecting this experimental model was based on the following.
The adhesion of cancer cells to endothelium is a complex
process involving multiple adhesion molecules present on both
cell types. These multiple interactions can make difficult the
interpretation of data obtained. The use of transfected CHO
cells offers simplification of the experimental system. This
system is also devoid of additional effects caused by inflammatory mediators produced by activated endothelial cells. Human
colon carcinoma cells behave similarly in their adhesion to
activated human endothelial cells and to E-selectin-expressing
CHO cells (Sawada et al., 1993). We found that only Hu
1703He cells expressing a high level of sLea antigen bound
specifically to E-selectin-expressing CHO monolayers (Fig.
3a, b). Cells treated with a solution of 0.05% trypsin and 0.02%
EDTA for a few minutes showed lower non-specific binding to
non-transfected Pros cells (Fig. 3a) than cells harvested with
0.2% EDTA (Fig. 36). The short treatment with trypsin/
EDTA did not affect sLea structures present on the cell
surface, as shown by FACS analysis with antibody 19-9. It is
possible that trypsin removes from the cell surface components
involved in other types of interaction. Therefore, for adhesion
2 - Flow-cytometric analysis of sLex antigen in human
uroepithelial cell lines with CSLEX MAb. SLex antigen is slightly
expressed only on cell lines Hu 609T and Hu 1703He.
Hu 1703He
FIGURE 1 - Flow-cjtometric analysis of sLea antigen in human
uroepithelial cell lines with 19-9 MAb. Cell lines HCV 29 and
HCV 29T do not express sLeaantigen; cell lines Hu 609T, Hu 549
and Hu 456 contain moderate amounts of the structure; sLea is
strongly expressed only by Hu 1703He cells.
Hu 1703He
FIGURE 3 - Adhesion of human urothelial cell lines Hu 1703He,
HCV 29T and HCV 29 to E-selectin-expressing CHO-Pro5 cells.
Hu 1703He cells for the adhesion assay were harvested with 0.05%
sin/0.02% EDTA (a) or with 0.2% EDTA (b) HCV 29 and
29T cells were detached with 0.05% trypsin/0.02% EDTA
Open bars represent binding to untransfected Pro5 cells, solid
bars to E-selectin-expressingPro5 cells. Standard deviations from
triplicate assays are indicated at the top of each bar.
assays, cells were harvested with 0.05% trypsin/0.02% EDTA.
No specific adhesion was observed for other human uroepithelial cell lines, e.g., HCV 29 and HCV 29T (Fig. 3c), detached
with 0.2% EDTA as well as 0.05% trypsin/0.02% EDTA.
To evaluate the specificity of interaction between human
transitional cell carcinoma cells and E-selectin-transfected
C H O cells, E-selectin-expressing C H O cells were preincubated with the MAb anti-E-selectin. Incubation with
antibody completely abolished specific adhesion of Hu 1703He
cells to E-selectin (Fig. 4u). In contrast, pre-treatment of Hu
1703He cells with antibody 19-9 (anti-sLea) had only slight
effects on the adhesion to transfected C H O cells (Fig. 4a). The
same results were obtained with ascitic fluid (diluted 1 : l ) and
purified antibody (up to 200 pg/ml). Similar effects were
observed as well with 2 other anti-sle” antibodies (9001/1B1
and 121-SLE) and antibody CSLEX against s L e x (data not
shown). Treatment of urothelial cells with neuraminidase
decreased binding to E-selectin by 50% (Fig. 46), confirming
the involvement of an oligosaccharide moiety of Hu 1703He
cells in this binding. To establish the involvement of proteinlinked sLea oligosaccharide, additional digestion with trypsin
was performed. Incubation of Hu 1703He cells for 1 hr at 37°C
with 50 pg/ml of enzyme decreased adhesion by about 20%
(Fig. 46). However, trypsin had no effect on binding of MAb
19-9 to the cell surface, as shown by FACS analysis. These data
suggest that the slight decrease of adhesion is rather the result
of some unspecific action of enzyme on the cell membrane.
proteins, had no effect on binding of anti-sLea antibody (Fig.
5b), suggesting that sLedstructures are present on 0-linked
oligosaccharides. T h e nature of sLe”-bearing glycoproteins
was directly shown by the action of 0-sialoglycoprotease,
which selectively recognizes glycoproteins with 0-linked oligosaccharides. Enzyme treatment completely abolished the binding of 19-9 MAb (Fig. 5c), indicating that sialomucins are
carriers of sLea oligosaccharide.
Analysis ofglycoproteins canyitig SLP determinant
Lysates of human uroepithelial cell lines were subjected to
SDS-PAGE and blotting and analyzed with the anti-slea
antibody 19-9. The antibody reacted only with the extract from
Hu 1703He cells (Fig. 5a). It bound to several components
with apparent molecular masses of 10&250 kDa. The specificity of binding was confirmed by pre-treating cell lysates with
fucosidase and neuraminidase. Treatment with fucosidase
highly diminished the binding of 19-9 antibody, and in the case
of neuraminidase, binding was completely abolished (data not
shown). To further characterize the sLea-bearingglycoprotein,
the cell lysate was subjected to digestion with N-glycanase. The
enzyme, which removes N-linked oligosaccharides from glyco-
Treatment of cells with 0-sialoglycoprotease
To evaluate the participation of mucin-linked sLea determinant in adhesion, we checked the effect of O-sialoglycoprotease on binding of uroepithelial cells since this enzyme does not
cleave N-linked oligosaccharides and glycolipids. As seen in
Figure 6a,incubation of Hu 1703He cells with O-sialoglycoprotease did not decrease binding to E-selectin-expressing C H O
cells. We also found that binding of 19-9 MAb directed against
sLe” determinats was not affected by treating Hu 1703He cells
with this enzyme, as shown by FACS analysis (Fig. 66). The
effectiveness of 0-sialoglycoprotease action on cleavage of
mucin-like glycoproteins was assayed by incubating the enzyme with glycophorin A-expressing CHO cells. As expected,
we found that the binding of anti-glycophorin A 6A7 MAb to
glycophorin A-expressing C H O cells was highly decreased
(Fig. 6c).
Evidence suggests that sLea and sLex structures are not only
tumor-associated carbohydrate antigens but also cell-surface
components functionally associated with the expression of
malignant phenotype (Honn and Tang, 1992). Both antigens
play a role in the adhesion of cancer cells to activated en-
- 194 kDa
100 250 kDa
- 116
MAb anti-E-sel
MAb 19-9
Neuraminidase Trypsin
RCURE4 - ( a ) Effect of MAbs anti-E-selectin and anti-sLea on
adhesion of Hu 1703He cells to E-selectin-expressing CHO-Pro5
cells. Anti-E-selectin MAb (40 pg/ml) completely abolished
adhesion; anti-sLed MAb 19-9 (200 pg/ml) had no effect. (b)
Effect of neuraminidase and trypsin on adhesion of Hu 1703He
cells to E-selectin-expressiiig CHO-Pro5 cells. Hu 1703He cells
were incubated with neuraminidase from V. cholerae (0.1 units/
mi) for 1 hr at 37°C or with trypsin (SO pg/ml) for 1 hr at 37°C and
subjected to adhesion assay. Open bars represent binding of
untreated Hu 1703He cells; solid bars, Hu 1703He cells after
treatment with antibodies or enzymes.
3 4
5 - (a) Binding of 19-9 MAb (anti-sLea)to glycoproteins
from human uroepithelial cell line Hu 1703He, (6) effect of Nglycanase (5 units/ 100 pg cellular protein) and (c) O-sialoglycoprotease (5 p1 of enzyme solution/100 pg of cellular protein) on
binding of MAb 19-9 to glycoproteins from Hu 1703He cells.
Lanes 1 and 3, sham-treated cell lysates; lanes 2 and 4, Nglycanase- and O-sialoglycoprotease-treated cell lysates, respectively. Cell lysates (100 pg of cellular protein) were separated by
SDS-PAGE, under reducing conditions, in gradient gel (S-lS%)
and then electro horetically transferred onto nitrocellulose. Bound
antibody was Atected using alkaline phosphatase-conjugated
anti- oat IgG antibody. The positions of molecular mass standards
are skown on the ri ht side. On the left side the range of molecular
masses of 19-9 MAkbinding glycoproteins is indicated.
FIGURE6 - (a) Effect of 0-sialoglycoproteaseon adhesion of Hu
1703He cells to E-selectin-expressing CHO-Pro5 cells. To cleave
surface sialomucins, cells (4 x lo6) were treated with enzyme (60
pl) for 1 hr at 37T, and untreated and treated cells were subjected
to adhesion assay. Open bars represent binding to untransfected
Pro5 cells, solid bars to E-selectin-expressingPro5 cells. Standard
deviations from triplicate assays are indicated a t the top of each
bar. (b) Effect of 0-sialoglycoproteaseon binding of MAb 19-9 to
the surface of Hu 1703He. Cells, untreated and treated with
enzyme as described, were analyzed by flow cytometry with
anti-sLeaantibody and FITC-conjugated rabbit F(ab')* fragment
anti-mouse IgG/IgM. No differences between untreated and
treated cells were found. (c) Control glycophorin A blood group
M-expressing CHO cells di ested with 0-sialoglycoproteaseand
analyzed as above with MA% 6A7 anti-M. (1) Cells treated with
enzyme; (2) untreated cells.
dothelium, which can be one of the important factors in the
formation and localization of metastases (Takada et al., 1993).
The sLea antigen was identified in a ganglioside extract from
malignant human urothelial cell line Hu 1703He by fast atom
bombardment mass spectrometry and a thin-layer chromatogram-binding assay using the 19-9 MAb. The sLea structure
was further confirmed in other tumorigenic and invasive
uroepithelial cell lines. These data suggested that expression
of the sLe" ganglioside is connected with acquisition of the
malignant phenotype in human urothelial cell lines (Ugorski et
al., 1990).
To evaluate the biological role of sLea antigen present in
malignant human uroepithelial cell lines, we studied the
adhesion of 6 human uroepithelial cell lines with different
expressions of this carbohydrate structure. The only line in
which cells bound to E-selectin-expressing CHO cells was Hu
1703He, expressing the highest level of sLea.
The specificityof carbohydrate-E-selectin interaction in the
adhesion of Hu 1703He cells is indicated by the following: (i)
the anti-E-selectin antibody completely abolished binding to
E-selectin-expressing CHO cells; (ii) the removal of sialic acid
highly decreased adhesion. Unexpected results have been
obtained with MAbs directed against s L e a antigen. Incubation
of Hu 1703He cells with anti-sLea antibodies only slightly
decreased their interaction with E-selectin-expressing CHO
cells. Similar data have been obtained by Iwai et al. (1993), who
were unable to inhibit the E-selectin-mediated adhesion of
pancreatic carcinoma cells to activated endothelium by antisLeaantibody 1H4, while this interaction was partially blocked
by another anti-sLea antibody, 2D3. Similarly, Majuri et al.
(1992) were not able to completely inhibit the adhesion of
colon carcinoma cell line COLO 205 to recombinant E selectin
using the 19-9 antibody. The results obtained by us and others
(Iwai et al., 1993; Majuri et al., 1992) can be explained by the
antibody's inability to block efficiently epitopes recognized by
E-selectin. It seems that the fine specificity of anti-ska and
anti-sLexantibodies is the major factor affecting their ability to
inhibit the E-selectin-mediated adhesion of cancer cells. A
second possibility is the existence of an unknown sialylated
ligand for E-selectin, whose presence is closely associated with
the expression of sLea and sLeXantigens.
We have shown that in tumorigenic and invasive human
uroepithelial cell lines, an sLes structure is present in ganglioside extracts (Ugorski et al., 1990). We now have found that a
part of the sLe"antigen in Hu 1703He cells is carried by several
high-molecular-mass (100-250 kDa) glycoproteins. It is reasonable to assume that sLea is present on 0-linked oligosaccharides since digestion with N-glycanase to remove N-linked
oligosaccharides had no effect on binding of 19-9 MAb. Direct
evidence about the nature of sLea-bearing glycoprotein was
obtained by treating the cell lysate with 0-sialoglycoprotease,
which cleaves mucin-type glycoproteins with large numbers of
clustered 0-linked oligosaccharides but not N-linked oligosaccharides and glycolipids. This enzyme completely abolished
binding of 19-9 MAb to glycoproteins on the blot, showing that
a carbohydrate antigen is present on sialomucin-type glycoproteins.
Sialomucins are ligands for selectins in cells of hematopoetic
origin (McEver et a!., 1995). However, little is known about
ligands for selectins in cancer cells. The presence of a high
amount of sLea structure was found in cancers of the gastrointestinal tract. This antigen, first described as ganglioside
(Magnani et al., 1982), was subsequently found o n molecules
with mucin characteristics (Baeckstrom et al., 1991, 1995;
Magnani et al., 1982). To evaluate the role of cell-surface
mucin-like molecules carrying sLea determinant in adhesion,
Hu 1703He cells were treated with 0-sialoglycoprotease.
Adhesion of urothelial cells to E-selectin-expressing CHO
cells was essentially not affectcd by enzyme treatment. Flowcytometric analysis has shown that incubation of Hu 1703He
cells with 0-sialoglycoprotease did not decrease the binding of
anti-sLea 19-9 MAb, which is in striking contrast with data
obtained by immunoblotting with the same antibody. These
results suggest that (i) s L e d oligosaccharides present on sialomucins sensitive to 0-sialoglycoprotease constitute only a
minor component of the total cell-surface sLea antigen or (ii)
sialomucins present on the cell surface are not cleaved by the
enzyme. The second possibility is less likely as the binding of
anti-glycophorin MAb to control, enzyme-treated, glycophorinexpressing CHO cells was substantially diminished. Mannori et
al. (1995) have shown that adhesion of several colon cancer
cell lines to recombinant P- and L-selectin was highly inhibited
after treating the cells with 0-sialoglycoprotease but that this
enzyme had no effect on binding of COLO 205 and HT-29 cells
to E-selectin-immunoglobulin fusion protein. Furthermore,
E-selectin-immunoglobulin did not bind to 0-glycosylated
proteins isolated from HT-29 cells, suggesting that adhesion of
these colon cancer cells to E-selectin is mediated by glycolipids
as sLeX structures seem to be present in HT-29 cells on
glycolipids. Adhesion of neutrophils to E-selectin is resistant
to 0-sialoglycoprotease (Steininger et al., 1992), but neutrophi1 glycolipids bearing sLex structures as well as neoglycolipids with sLea determinants support E-selectin binding (Alon et
al., 1995; Tiemeyer et al., 1991). This is in agreement with our
data, showing that Hu 1703He cells, whose adhesion is
resistant to 0-sialoglycoprotease, express high levels of sLe"
In summary, we have shown that sLea oligosaccharide can be
involved in E-selectin-mediated adhesion of some urothelial
cell lines. Our data suggest that effective binding to E-selectin
occurs when sLea antigen present on glycosphingolipids is
expressed in a proper density on the surface of cancer cells.
This work was supported by grant P207 001 05 of the
Committee of Scientific Research (KBN), Warsaw, Poland.
The authors are grateful to Drs. E. Lisowska and C. Radzikowski for critical reading of the manuscript. We also acknowledge the excellent technical assistance of Ms. M. BanaS.
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