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Tumor Biology |
The Chicago Institute of Neurosurgery and Neuroresearch, Chicago, Illinois 60614 [H. Y., J. S., S. G., T. S., J. H., C. S., J. L., E. M., L. C., J. R. M.], and Department of Biochemistry, Osaka University Medical School, Osaka 565, Japan [A. N., Y. I., N. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Malignant gliomas, unlike tumors found outside of the central nervous
system, do not metastasize but are highly invasive. A number of reports
have strongly suggested that integrins play a key role in regulating
invasivity (8
, 9)
, and that the N-glycans of
integrins modulate the function of integrins (10
, 11)
. The
two most commonly observed aberrant N-glycosylations in
experimental tumor models are an increase in terminal sialylation
(2
, 12)
and a shift to more highly branched
N-linked oligosaccharides (1
, 13, 14, 15)
.
2,3-Linked sialic acids were shown to be expressed on malignant
glioma cell surfaces but were absent in normal human adult astrocytes
(16)
. When alterations in the glycosylation patterns of
the glioma-associated integrin, 3ß1, were introduced by
transfection of the 2,6-sialyltransferase gene into a
malignant glioma cell line, inhibition of invasivity was observed
in vitro (17)
. On the other hand, there have
been no studies examining whether highly branched N-linked
oligosaccharides play a role in glioma invasivity.
Recent studies demonstrate that branching of N-linked oligosaccharides is dependent upon two distinct enzymes: UDP-GlcNAc3 :ß-D-mannoside ß1,4-N-acetylglucosaminyl transferase III (GnT-III; EC 2.4.1.144) and UDP-GlcNAc:ß-D-mannoside ß1,6-N-acetylglucosaminyltransferase V (GnT-V; EC 2.4.1.155) (18, 19, 20) . GnT-III produces N-glycans with bisecting structures, whereas GnT-V increases ß1,6 branching to create tri- and tetra-antennary structures. Increased expression of tri- or tetra-antennary ß1,6-GlcNAc-bearing N-glycans has been correlated with metastatic potential in rodent tumor models (3 , 13) and also has been shown to be a marker of tumor progression in human breast and colon neoplasia (14 , 15) . GnT-V expression appears to be regulated at least in part by the Ets family of transcription factors because it has been shown that GnT-V expression is dependent upon Ets-1 in a human bile duct carcinoma cell line (21) and other cell lines (22) , and that increased expression of GnT-V by Src kinase stimulation was abolished by a dominant-negative mutant of Ets-2 in human hepatocarcinoma Hep G2 cells (23) .
On the basis of both the histochemical study of ß1,6-linked N-glycan expression in primary glioma specimens using L-PHA and Northern analyses of primary gliomas and glioma cell lines, we have examined the regulation of the branching of complex type N-glycans in glioma cells. We have also created GnT-III- and GnT-V-transfected glioma cells to directly evaluate the biological function of the branching of N-glycans in cell adhesion, migration, and invasion in vitro.
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MATERIALS AND METHODS |
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Northern Analysis.
Human GnT-V cDNA (1.24 kb) was isolated after EcoRI
restriction digestion and used as a cDNA probe for Northern analysis.
Human GnT-III cDNA (1.8 kb) was used after EcoRI and
XbaI restriction digestion. Human Ets-1 cDNA was cloned by
using the reverse transcription-PCR (RT-PCR) and poly(A)+ RNA from U-87
MG cells based on the sequence reported previously (25)
. A
sense primer 5'-TTGGGAAGAAAGTCGGATT-3' (bp -119 to -101) and an
antisense primer 3'-CAGGCTGAATTCATTCACAGC-5' (bp 270 to 250) were used
for reverse transcription-PCR. A 389-bp PCR product was cloned into pT7
Blue T vector (Novagen, Madison, WI), and the sequence of the insert
was confirmed by the dideoxy termination method (Sequenase, United
States Biochemical, Cleveland, OH). The cDNA coding for human ets-1 was
isolated from the gel after NdeI and BamHI
digestion and was used as the probe.
Surgical specimens were immediately frozen in liquid nitrogen upon resection. Total RNA was isolated from clinical glioma specimens and cultured brain tumor cells using guanidinium isothiocyanate, followed by CsCl2 centrifugation as described previously (16) . Thirty µg of total RNA per primary brain tumor and 20 µg of total RNA per tumor cell line per lane were electrophoresed in an agarose-formaldehyde gel and transferred to Duralon nylon membranes (Stratagene, La Jolla, CA). After UV cross-linking, the blots were hybridized with a 32P-radiolabeled cDNA probe synthesized by using a random priming kit (Stratagene) and ExpressHyb solution (Clontech, Palo Alto, CA). The blots were then exposed to X-OMAT film (Kodak, Rochester, NY), and the films were developed appropriately.
Lectin Histochemistry with L-PHA.
ß1,6-linked N-glycan expression in primary glioma
specimens was examined using L-PHA (26)
. Paraffin-embedded
sections (6 µm thick) of formalin-fixed specimens, derived from one
mixed glioma case, two cases of astrocytoma grade III, and two cases of
glioblastoma (astrocytoma grade IV), were processed at room temperature
unless otherwise mentioned. The sections were dewaxed and hydrated and
then soaked in Tris-buffered saline (TBS; 150 mM
NaCl, 50 mM Tris-HCl, pH 7.5) at 37°C for
1 h or 13 h (according to our preliminary studies with other
lectins) to unmask lectin binding sites. Then the sections were rinsed
with TBS for 10 min and incubated in 0.5% blocking reagent (Boehringer
Mannheim, Indianapolis, IN) in TBS for 45–60 min. After rinsing twice
with TBS and once with buffer 1 (TBS with 1 mM
MgCl2, 1 mM
MnCl2, and 1 mM
CaCl2, pH 7.5) for 10 min each, 10 µg/ml
digoxigenin-labeled L-PHA (Boehringer Mannheim) in buffer 1 with or
without 0.05% Tween 20 and 0.05% Triton X-100 was overlaid for 1 h. Rinsing with TBS (3 x 10 min) was followed by
incubation with anti-digoxigenin Fab fragments conjugated with 0.75
unit/ml alkaline phosphatase (Boehringer Mannheim) in TBS containing
0.05% Tween 20 and 0.05% Triton X-100 for 1 h. After rinsing
(TBS, 3 x 10 min), 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium solution (Sigma Chemical Co., St.
Louis, MO) was overlaid as chromogen in darkness up to 50 min and
rinsed with 10 mM Tris-HCl with 1
mM EDTA. The sections were lightly counterstained
with nuclear fast red and fixed with 10% buffered formalin to lessen
fading of reaction product during dehydration and clearing.
To check the specificity of lectin binding, each staining was performed simultaneously with labeled L-PHA that was preincubated in the presence of 9 µM bovine thyroglobulin (Sigma) for 90–120 min prior to lectin incubation as a negative control.
Western and Lectin Blot.
Cultured cells were rinsed twice with PBS and lysed in hot cell lysis
solution containing 1% SDS, 10 mM Tris-HCl (pH 7.4). To
detect ß1,6-GlcNAc N-glycans, 30 µg of cell lysates were
loaded on an 8% SDS-polyacrylamide gel. After electrophoresis,
proteins were transferred to a polyvinylidene difluoride membrane, and
the membrane was blocked with 5% BSA in PBS. It was then incubated
with 0.1 µg/ml horseradish peroxidase-conjugated L-PHA (EY
Laboratory, CA) in TBS containing 2% BSA and 0.1% Tween 20 for
1 h at room temperature. Next, the membrane was washed with TBS
containing 2% BSA and 0.1% Tween 20 for 10 min, followed by washing
twice with 0.1% Tween 20 in TBS. The blot was then developed with the
ECL Chemiluminescence detection system (Amersham, Buckinghamshire,
United Kingdom). Protein concentrations were determined using the BCA
reagent (Pierce). To detect Ets-1 protein expression in brain tumor
cell lines, 20 µg of protein cell lysates were loaded on a 8%
SDS-polyacrylamide gel immediately after boiling each sample in the
presence of 2% ß-mercaptoethanol. After electrophoresis, proteins
were transferred to a polyvinylidene difluoride membrane, and the
membrane was blocked with 5% BSA in PBS. It was then incubated with a
1: 10,000 dilution of monoclonal antihuman Ets-1 antibody (Clone 47;
Transduction Laboratory, KY) in Tris-buffered saline pH 7.4
(TBS) containing 2% BSA and 0.1% Tween 20 for 1 h at room
temperature. The membrane was then washed with TBS containing 2% BSA
and 0.1% Tween 20 for 10 min, followed by washing twice with 0.1%
Tween 20 in TBS. Next, it was incubated with a 1:10,000 dilution of
horseradish peroxidase-conjugated antimouse IgG (Amersham) for 1 h
at room temperature in 2% BSA in TBS containing 0.1% Tween 20. The
membrane was then washed as described above and developed with the ECL
Chemiluminescence detection system (Amersham) according to
manufacturer’s instructions.
Stable Transfection of Inducible ets-1 Gene into
Human Glioma SNB-19 Cells.
The 1.4-kb human c-ets-1 (full coding sequence) was inserted
into the pcDNA4/TO vector (Invitrogen, San Diego, CA). The
pcDNA4/TO/c-ets-1 and pcDNA6/TR (Invitrogen) vectors were
cotransfected into human glioma SNB-19 cells using the cationic
liposome system, DOTAP (Boehringer Mannheim). After 3 weeks of culture
in selection medium containing 10 µg/ml of Blasticidin and 1 mg/ml of
Zeocin, transfected cells were subcloned with cloning rings to isolate
individual clones. Individual clones were further cultured for 4 weeks
in the selection medium and then analyzed for the regulated gene
expression in the presence of 2 µg/ml tetracycline.
Stable Transfection of GnT-V into Human Glioma U-373MG Cells.
The 2.4-kb human GnT-V cDNA (full coding sequence) was inserted into
the pcDNA3 expression vector (Invitrogen) at the KpnI and
XbaI sites, and the orientation of the insert was confirmed
by HindIII restriction digestion. The pcDNA3/GnT-V was then
transfected into U-373 MG cells using the cationic liposome system
DOTAP, (Boehringer Mannheim) according to the methods described
previously (17)
. After 3 weeks of culture in selection
medium containing 800 µg/ml of G418, transfected cells were subcloned
with cloning rings to isolate individual clones. Individual clones were
further cultured for 4 weeks in the selection medium and then analyzed
for the gene expression by Northern analyses and L-PHA lectin blotting
to identify successful GnT-V transfectants. Stable transfection of
GnT-III gene into the same U-373 MG was reported previously
(27)
.
Invasion Assay.
Invasivity of the GnT-V-transfected subclones was examined using a
commercial membrane invasion culture system (9
, 28)
.
Biocoat Matrigel Invasion Chambers (Collaborative Research, Bedford,
MA) consist of two compartments separated by a filter precoated with
Matrigel (contains laminin, type IV collagen, entactin, and heparan
sulfate). Cell invasion, which is the result of cell adhesion to the
extracellular matrix, degradation of the matrix proteins, and cell
migration to the other side of the filter, is measured by counting the
number of cells passing to the opposite side of the filter via 8-µm
pores. Cells (4 x 104) were
plated into the upper compartment and incubated for 24 h. U-373 MG
cell conditioned medium (0.5 ml) was placed in the lower compartment to
facilitate chemoattraction (28)
.
Cells that migrated through the Matrigel and through the filter were fixed in 10% formalin and stained with hematoxylin. The membranes were mounted on glass slides, and the cells were counted (9) . Parental U-373 MG- and pcDNA3 vector-transfected cells were used as controls.
Immunofluorescence Microscopy.
To characterize the morphological change of GnT-V and GnT-III
transfectants, immunofluorescence microscopy was performed using
monoclonal antihuman vinculin antibody (Sigma; clone hVIN-1) and
monoclonal anti-VLA3 antibody (Chemicon; clone M-KD102). Anti-vinculin
antibody was used to visualize focal adhesion sites and anti-VLA3
antibody was used to visualized 3ß1 integrin in the transfectants.
Cells were plated on fibronectin-coated (10 µg/ml) coverslips and
incubated in DMEM supplemented with 10% FBS for 16 h. Cells were
gently washed twice with PBS, then fixed with 4% formalin in PBS for
30 min, followed by washing with PBS for 3 min. Cells were treated with
1% NP40 in PBS for 10 min, followed by washing with PBS three times.
After blocking with 10% normal goat serum for 15 min at room
temperature, cells were incubated with monoclonal antihuman vinculin
antibody (1:400 dilution) or monoclonal anti-3ß1 integrin antibody
(1:200 dilution) in PBS for 30 min at room temperature. They were then
washed three times with PBS (5 min each) and then incubated with
FITC-labeled goat antimouse immunoglobulin antibody (1:160 dilution;
Sigma) for 30 min at room temperature. The cells were washed with PBS
five times to remove unbound secondary antibody and were mounted with
Vectashield (Vector). Fluorescence microscopy was performed using a
Nikon Model 401 Fluorescence Microscope.
In Vitro Cell Migration Assays.
Directed cell migration on a solid-phase gradient of a fibronectin
substrate (haptotaxis) was measured using a Transwell (Costar,
Cambridge, MA), which consists of two compartments separated by 6.5-mm
inserts with 8-µm-pore polycarbonate filters in 24-well culture
plates. To establish a solid-phase gradient, only the underside of the
filter was coated with 10 µg/ml human plasma fibronectin (Life
Technologies, Grand Island, NY) in sodium bicarbonate buffer (pH 9.7)
overnight at 4°C. It was then blocked with 1% BSA (fatty acid free;
Sigma) in PBS for 45 min at room temperature and rinsed three times
with PBS.
GnT-V, GnT-III transfected U-373 MG and control cells were gently
treated with x 0.5 trypsin-EDTA (Life Technnologies,
Inc.) in PBS for 
5 min at 37°C and then neutralized with DMEM
containing 0.2% BSA. After washing with 0.2% BSA-DMEM, cells were
resuspended in protein-free DMEM and were plated 10,000 cells/100
µl/insert. The inserts were moved onto the lower wells, which
contained protein-free DMEM (0.5 ml), and were incubated for 6 h
at 37°C in a CO2 incubator. For inhibition of
cell migration by lectins, L-PHA or E-PHA (Vector Laboratory) at the
final concentration of 2 or 10 µg/ml was added to both upper and
lower compartments. Monoclonal anti-3 integrin antibody (Chemicon;
clone P1B5) was also used to inhibit 3ß1 integrin-mediated cell
migration. After thorough absorption of DMEM with cotton swabs, the
porous filter was dried with air blow and cut from the plastic
supports. Cells on both sides of the filter were fixed and stained with
DiffuQuick (Baxter, Chicago, IL). The filters were then mounted with
Permount (Fisher Scientific, Chicago, IL) on glass slides with 12-mm
coverslips. Under microscope, cells on both the topside
(i.e., nonmigrated) and underside (i.e.,
migrated) of the filters were counted in eight consecutive fields along
one filter diameter (10% of the entire surface was observed). The
percentage of migration (migrated cell count/total cell count) was
determined based on triplicate experiments.
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RESULTS |
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. On the basis of these results, no obvious correlation could be made
between tumor grade and mRNA expression, but there was a marked
difference in the GnT-III and GnT-V mRNA expression patterns in most
gliomas compared with normal brain.
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The expression of GnT-III and GnT-V mRNA was also studied in a panel of
five glioma and four neuroblastoma human brain tumor cell lines. These
data, along with the expression of the transcription factor ets-1, is
shown in Fig. 3
. Unlike in the clinical specimens examined, marked and consistent
GnT-III mRNA expression was found in all of the cell lines, whereas the
level of GnT-V mRNA expression varied from cell line to cell line. It
can be seen that the cell lines that highly expressed GnT-V mRNA also
expressed high levels of ets-1 mRNA (ets-2 mRNA
was undetectable in all of the cell lines examined; data not shown).
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. A major glycoprotein of
Mr 140,000 was revealed in all five
glioma cell lines but was absent or barely detectable in the
neuroblastoma cell lines. Moreover, the pattern of L-PHA-reactive
glycoprotein expression in the glioma cell lines was different from
that in the neuroblastomas.
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. The induction of
c-ets-1 mRNA in the glioma transfectants resulted in the
increased expression of GnT-V mRNA as shown in Fig. 5
.
|
. As shown in Fig. 6
, we have isolated five GnT-V stable transfectants that expressed the
3.0-kb GnT-V transcript in addition to the endogenous 9.5-kb
transcript.
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. The GnT-V-transfected cells also showed a fan-shaped cell morphology
with a distinct leading edge (Fig. 8)
, indicating directional cell migration of the transfectants.
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3ß1 integrin antibody to visualize the leading edge of the
cells. As shown in Fig. 9
, strong 3ß1 integrin staining, the predominant integrin in U-373
MG cells (17)
, was observed at the leading lamellipodia of
the GnT-V transfectants (Fig. 9F)
, whereas dendritic process
staining was observed in controls. Focal adhesion sites visualized by
anti-vinculin antibody staining radiate toward leading lamellipodia in
the GnT-V transfectants (Fig. 9E)
, whereas vinculin staining
was found along the edge of parental U-373 MG cells and
vector-transfected controls (Fig. 9, A and G)
. In
the GnT-III transfectants, focal adhesion sites were randomly
distributed (Fig. 9C)
.
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. The effect of E-PHA and L-PHA on cell migration was tested using
GnT-III- and GnT-V-transfected U-373 MG cells, their controls, as well
as the glioma cell lines D-54MG, SNB-19, SW1088, and U-87 MG.
GnT-III-transfected U-373 MG clone P9 overexpresses bisecting
ß1,4-GlcNAc oligosaccharides (27)
, and GnT-V-transfected
clone J13 expresses the highest level of GnT-V and ß1,6-GlcNAc
expression (data not shown). The GnT-III transfectants showed
significantly less cell migration on a fibronectin substrate compared
with parental U-373 MG or other transfectants, whereas the GnT-V
transfectants did not show a significant increase in cell migration
under our experimental conditions. Cell migration on a fibronectin
substrate was completely inhibited at 10 µg/ml E-PHA in all glioma
cell lines tested, whereas the inhibitory effect of L-PHA is much less
than 50% (data not shown). At 2 µg/ml, E-PHA showed strong
inhibition of cell migration, whereas L-PHA showed little effect (Fig. 10)
. The same levels of inhibition were observed by 2 µg/ml E-PHA and
10 µg/ml anti-3 monoclonal antibody (Chemicon; clone P1B5) in
parental U-373 MG cells. These results clearly demonstrate the
functional difference between the two different types of
N-glycan branching structures found in gliomas and are
consistent with previous observations that cells expressing highly
branched ß1,6-GlcNAc are less adhesive to extracellular matrices
(11
, 29)
.
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DISCUSSION |
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3ß1, was undertaken.
The importance of N-linked oligosaccharide branching in
tumor metastasis was demonstrated in a series of experiments reported
by Dennis and Laferté (14)
. Specifically, they
created a panel of glycosylation mutants in a highly metastatic murine
tumor cell line and showed a strong correlation between the increased
ß1,6-linked branching of complex type oligosaccharides and metastatic
potential. A number of more recent studies have also shown an increased
expression of highly branched ß1,6-GlcNAc-linked N-glycans
in a variety of tumor models. These have included experiments using
cells transformed by DNA viruses such as polyoma and Rous sarcoma,
oncogenes such as H-ras and src, as well as human
breast and colon cancers (3
, 15
, 16
, 22
, 30)
. Furthermore,
increased ß1,6-GlcNAc-linked N-glycans, brought about by
GnT-V gene transfection into premalignant mink lung
epithelial cells, resulted in increased tumorigenicity because of an
increase in cell motility by alterations in 5ß1 and vß3
integrins (11)
.
To address the question as to whether changes in N-glycan branching play a role in glioma invasivity, an examination of the expression of GnT-III and GnT-V mRNA was undertaken. In normal adult human brain, robust GnT-III mRNA expression was observed, whereas GnT-V mRNA expression was very low by comparison. This is not surprising in light of the fact that the predominant N-linked oligosaccharides found in normal brain are complex-type bisecting ß1,4-GlcNAc structures that are the product of GnT-III (31) . In the malignant gliomas examined, both GnT-III and GnT-V mRNAs were variably expressed. Lectin staining with L-PHA, which recognizes ß1,6-GlcNAc containing oligosaccharides, was undertaken to get a clearer picture of where these structures are expressed. L-PHA staining was found in malignant glioma cells, neovascular endothelial cells, and extracellular matrices surrounding the tumor cells but not in normal cells. Most of the clinical specimens used in this study were high-grade gliomas. Patients with these tumors have the shortest survival (6–12 months upon diagnosis). We did not find a statistically significant positive correlation between the levels of GnT-V mRNA expression and clinical outcome among the high-grade glioma patients whose tumors were used in these studies, and a larger number of low-grade gliomas than available would be required to evaluate GnT-V mRNA expression as a prognostic marker.
In glioma cell lines, GnT-III mRNA levels were uniformly high, whereas
GnT-V mRNA levels were quite variably expressed. An L-PHA lectin blot
revealed that most glioma cells express a major L-PHA-reactive
glycoprotein with a molecular weight of
Mr 140,000, whereas protein extracts
from neuroblastoma cells or normal brain showed different patterns of
L-PHA staining, and the Mr 140,000
glycoprotein was rarely found. The expression of the L-PHA-reactive
glycoprotein was high in SW1088 and U-87 MG glioma cell lines, which
show high levels of GnT-V expression, whereas a small amount of L-PHA
reactivity was found in U-118 MG glioma cells, despite its high GnT-V
mRNA expression. Furthermore, neuroblastoma cell lines with high GnT-V
mRNA expression (LAN-5) show little or no
Mr 140,000 staining. These results
suggest that the levels of ß1,6-GlcNAc-bearing N-glycans
in gliomas are controlled by mechanisms that regulate both GnT-V
expression and the availability of its protein substrates. Data
obtained from immunoprecipitation studies using anti-3 integrin
antibodies showed that the major glycoprotein recognized by L-PHA in
gliomas is 3ß1 integrin (data not shown), the most predominant
integrin found in clinical glioma specimens (8)
and the
U-373 MG glioma cell line used in these studies (17)
. A
very recent study has identified that 3 integrin mRNA expression
appears to be quantitatively correlated with the grade of malignancy of
gliomas and medulloblastomas (32)
.
Thus, ß1,6-GlcNAc-bearing oligosaccharides were found on the 3ß1
integrin and appeared to be associated specifically with gliomas and
not normal astrocytes. Furthermore, aberrant up-regulation of GnT-V
expression, as opposed to decreased GnT-III expression, appears to be
responsible for their expression. Because GnT-III and GnT-V are the two
enzymes that regulate the type of branching structures found within
N-linked oligosaccharides and compete for the same
substrates, the results suggest that a mechanism exists to shift the
integrin oligosaccharides from bisecting ß1,4-GlcNAc to highly
branched ß1,6-GlcNAc during the transformation of glia into gliomas
or noninvading glioma cells into invasive ones.
GnT-V expression appears to be regulated at least in part by the Ets family of transcription factors (21, 22, 23) . Ets-1 is expressed during neural crest cell migration (33) , which is a physiological example of cell invasion. Ets-1 is also found within neovascular endothelial cells to promote neovascularization and in stromal fibroblasts adjacent to carcinoma cells to promote tumor invasion (33) . We have found the expression of ß1,6-branched N-glycans in both glioma cells and neovascular endothelial cells. We also found Ets-1 protein in all glioma cells tested, whereas Ets-1 protein was reported to be absent within carcinoma cells (34) . This difference in terms of Ets-1 protein expression within tumors may be attributable to the fact that the stromal reaction by fibroblasts plays an important role in carcinoma invasion, whereas there is little stromal reaction in malignant gliomas. Unlike carcinomas, Ets-1 expression in glioma cells may play a direct role in promoting glioma invasion. It also has been reported that the expression of Ets-1 can be modulated by growth factors and protein kinase C activators (34) , such as phorbol ester, through its interaction with other transcription factors, such as AP-1 (35 , 36) . Those studies suggest that the mitogen-activated protein kinase pathway and Ets-1 play a role in the expression of GnT-V in glioma cells. To examine a molecular mechanism that increases the expression of GnT-V in gliomas, we chose to investigate the possible involvement of the Ets-1 transcription factor in glioma cells.
High GnT-V mRNA expression was found in brain tumor cell lines with
robust c-ets-1 mRNA expression, whereas no ets-2 mRNA was detected.
Induction of c-ets-1 resulted in the increased expression of GnT-V mRNA
in the glioma cells, suggesting that the Ets-1 transcription factor
directly controls the transcription of GnT-V in glioma cells. Thus, the
data presented here add further support to the idea that Ets-1 plays a
pivotal role in modulating glioma invasivity via coordinated expression
of aberrant ß1,6-GlcNAc N-glycans on the glioma-associated
3ß1 integrin and expression of metalloproteases (36)
.
To study the biological effects of aberrant ß1,6-GlcNAc-bearing
N-glycan in gliomas, the GnT-V gene was stably
transfected into U-373 MG glioma cells that express very low levels of
this mRNA. As predicted from the results discussed above,
GnT-V transfectants were more invasive than controls. These
transfectants showed the distinct fan-shaped morphologies indicative of
directional cell migration with a distinct leading edge. It has been
reported that small numbers of glycoproteins, particularly those
involved in adhesion, can be found at the leading lamellipodia in
locomoting cells (37)
. In the results reported here,
3ß1 integrin was found to be localized on the leading lamellipodia
of the GnT-V-transfected cells and focal adhesion sites radiated toward
leading lamellipodia, whereas parental cells or vector-transfected
controls did not show characteristics of migrating cells.
In contrast, GnT-III stable transfectants displayed decreased cell migration under the conditions described above (data not shown). Although the data were not presented, this is likely because of an increase in their adhesion to the fibronectin substratum used in these studies.
Thus, when all of the data presented here are taken in whole, it
suggests that: (a) cell surface expressed glycoproteins
bearing "brain-type" bisecting ß1,4-GlcNAc structures, the
products of GnT-III, may be directly involved in cell adhesion and
migration; and (b) the shift of N-glycans from
bisecting to highly branched ß1,6-GlcNAc structures on the
glycoproteins may function to reduce adhesivity and increase migration,
thus increasing cell invasivity. The increased invasivity found in
GnT-V-transfected clones may be attributable to altered interaction
between 3ß1 integrin and its laminin substrate, which is a matrix
component in the invasion assays. The interaction between 3ß1
integrin and appropriate substrata, such as laminin and fibronectin,
may be dependent on the N-glycans.
To test this hypothesis, in vitro migration assays were
performed using E-PHA and L-PHA lectins, which bind to bisecting
ß1,4-GlcNAc or highly branched ß1,6-GlcNAc-bearing
N-glycans on glycoproteins, respectively. We have reported
previously that E-PHA lectin had a marked effect on adhesion in U-373
MG cells (38)
. On the other hand, L-PHA lectin showed no
effect on either cell adhesion (38)
or cytotoxicity in
glioma cells; cytotoxicity was seen in highly metastatic tumor cell
lines (11
, 12)
. In solid phase cell migration (haptotaxis)
studies, E-PHA lectin completely abolished glioma cell migration on
fibronectin substrata, regardless of the levels of ß1,6-GlcNAc
expression in both U-373 MG transfectants and other glioma cell lines,
whereas migration of glioma cells with high levels of ß1,6-GlcNAc
N-glycans was weakly inhibited by L-PHA. Furthermore, the
inhibitory effect by E-PHA was comparable with that of anti-3
integrin monoclonal antibody. These data suggest that ß1,4-GlcNAc
N-glycans play a direct role in 3ß1 integrin-mediated
cell adhesion, whereas in gliomas, the observed shift to more highly
branched ß1,6-GlcNAc N-glycan reduces cell adhesivity and
increases invasivity by replacing functional ß1,4-GlcNAc-bearing
N-glycans on the adhesion molecules. The binding of E-PHA to
ß1,4-GlcNAc-bearing N-glycans interferes with cell
adhesion (38)
, thus inhibiting cell migration as shown in
this study. On the other hand, L-PHA binding to ß1,6-GlcNAc-bearing
N-glycans does not interfere with integrin function and,
therefore, has little effect on cell migration. The results presented
here are consistent with previous studies that: (a)
N-glycans on 5ß1 integrins are required for the
functional heterodimerization of integrin and ß subunits
(10)
; and (b) a shift of integrin
N-glycans to highly branched ß1,6-GlcNAc leads to
decreased cell adhesion, resulting in an increase in cell motility by
altering the function of 5ß1 and vß3 integrins
(11)
.
In conclusion, the data presented here show that a shift in the expression of normal "brain type" bisecting ß1,4-GlcNAc to highly branched ß1,6-GlcNAc N-glycans plays an important role in modulating the function of cell surface glycoproteins involved in glioma invasivity. A recent study suggests that the knock-out of the GnT-V gene results in the suppression of both breast tumor formation and lung metastases in the null mouse (39) . Likewise, the expression of bisecting ß1,4-GlcNAc N-glycans by GnT-III gene transfection has been reported to suppress lung metastasis of B16 melanoma (40) . It will be interesting to examine whether reversion from aberrant ß1,6-GlcNAc-expressing N-glycans to normal ß1,4-GlcNAc-bearing N-glycans can retard glioma invasivity in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
1 This work was supported in part by grants from
the Illinois division of The American Cancer Society (to H. Y.), The
Buchanan Foundation (to J. M.), The Brach Foundation (to J. M.), and
The Falk Foundation (to J. M.). 
2 To whom requests for reprints should be
addressed, at The Chicago Institute of Neurosurgery and Neuroresearch,
2515 North Clark Street, Suite 800, Chicago, IL 60614. Phone:
(773) 388-7880; Fax: (773) 935-2132; E-mail: cinnhiro{at}hotmail.com
3 The abbreviations used are: GlcNAc,
N-acetylglucosamine; GnT,
N-acetylglucosaminyltransferase; L-PHA, Phaseolus
vulgaris leukoagglutinating lectin; E-PHA, Phaseolus
vulgaris erythroagglutinating lectin; ATCC, American Type
Culture Collection.
Received 2/ 3/99. Accepted 10/18/99.
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