From the Sunnybrook & Women's College Health Sciences Centre and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M4N 3M5, Canada
Received for publication, January 23, 2001
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ABSTRACT |
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This study was designed to investigate the
mechanisms by which mutant versican constructs play a dominant-negative
effect on astrocytoma cell proliferation. Although a mini-versican or a
versican G3 construct promoted growth of U87 astrocytoma cells, a
mini-versican lacking epidermal growth factor (EGF) motifs
(versican Versican, a member of the large aggregating chondroitin sulfate
proteoglycan family, was initially detected in the limb bud of chick
embryo (1) and later cloned in human fibroblasts and chick embryo
(2-5). It is also expressed in normal human central nervous system and
brain tumors (6). RT-PCR1
reveals that transcripts of versican isoforms are present in astrocytomas, oligodendrocytomas, medulloblastomas, schwannomas, and
meningiomas (6). Versican expression levels are low, however, because
neuronal immunostaining for versican appears only pericellularly. Versican is highly expressed in the tissues flanking the regions where
neural crest cells migrate in chick embryos, but it is absent from the
actual migration pathways. Similar findings are noted for the
outgrowing sensory and motor axons of chick embryos, because versican
is notably absent in regions invaded by these axons (7). Versican is
known to associate with a number of molecules in the extracellular matrix such as hyaluronan, tenascin, and
fibronectin (8-10). In the central nervous system, versican has been
observed to co-localize with tenascin and hyaluronan (9). Tenascin
binds at the C-type lectin unit of versican (10, 11).
Structurally, versican is made up of an N-terminal G1 domain, a
glycosaminoglycan attachment region, and a C terminus containing a
selectin-like (or G3) domain. The latter contains two epidermal growth
factor (EGF)-like repeats, a lectin-like motif (also known as
carbohydrate recognition domain or CRD), and a complement binding protein (CBP)-like motif (3, 12, 13). Alternative splicing generates at
least four versican isoforms (14-16), and some of these are highly
expressed in brain tumors (6). The role of versican in brain tumor
formation and progression is not clear.
We have previously demonstrated that a mini-versican construct promoted
NIH 3T3 fibroblast proliferation through the G3 domain, and two
EGF-like motifs in the G3 domain are involved in this effect (17).
Deletion of the EGF-like motifs from the mini-versican construct
significantly reduced the effect of the mini-versican on cell
proliferation and differentiation (17, 18). Here we demonstrate that
the mini-versican construct promotes astrocytoma cell
proliferation through the G3 domain. To our surprise, deletion of these
EGF-like motifs produced a dominant-negative effect on astrocytoma cell
proliferation. We designed assays to uncover the mechanism associated
with this dominant-negative effect. A G3 construct lacking the EGF-like
motifs (G3 Materials and Cell Cultures--
Lipofectin, Geneticin (G418),
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS),
Hank's balanced salt solution, trypsin/EDTA were from Life
Technologies, Inc. ECL Western blot detection kit was from Amersham
Pharmacia Biotech. Horseradish peroxidase-conjugated goat anti-mouse
IgG and goat anti-rabbit secondary antibodies were from Sigma. Tissue
culture plates were from Nunc Inc. Oligonucleotides were synthesized by
BioBasic Inc. (Scarborough, Canada). All chemicals were from Sigma.
Astrocytoma cell line U87 and COS-7 cells were obtained from the
American Type Culture Collection (Manassas, VA). The cells were
cultured in DMEM supplemented with 10% (U87) or 5% (COS-7) FBS at
37 °C in a humidified incubator containing 5% CO2.
Construction of Recombinant Genes--
A mini-versican gene
consisting of a complete G1 domain, a partial CS domain (15% in size
of the entire sequence) and a complete G3 domain was constructed and
expressed in COS-7 cells. Briefly, the G1 domain encompasses
nucleotides 145-1182 of versican. The CS sequence is a 1242-base pair
cDNA (nucleotides 1183-2424 of versican). The G3 domain contains a
927-base pair cDNA corresponding to nucleotides 9904-10830 (3).
The recombinant mini-versican gene is 3.2 kilobases, which yields a
core protein of ~150 kDa. With the attachment of glycosaminoglycan
chains, the recombinant proteoglycan migrated on SDS-PAGE gel as a
smear, at around 200 kDa or higher. The preparation of three
constructs, a mini-versican lacking the EGF-like motifs
(versican Gene Expression--
The pcDNA1-mini-versican construct was
transiently expressed in COS-7 cells using Lipofectin as originally
described by Felgner et al. (21). Growth medium and cells
were harvested separately 3 days after transfection. Expression of
constructs was analyzed on Western blot probed with 4B6 (20). To obtain
stable expression, glioma cells were transfected with versican Analysis of Proteoglycans on Western Blot--
Cell lysate and
growth medium that contained recombinant gene products were each
subjected to SDS-PAGE electrophoresis and immunoblotting as described
previously (17, 18). Primary antibodies were used at 1:1000 dilution,
unless otherwise stated, and bound antibodies were visualized using an
ECL kit according to the manufacturer's instructions. Because of its
large size, electrophoresis of endogenous versican was performed in an
agarose gel (agarose-Western blot assay). The agarose gel (4 cm height
containing 1.5% agarose in a buffer containing 0.124 M
Tris-Cl, 27 mM barbituric acid, 1 mM EDTA, pH
8.7) was poured on top of a 1-cm conventional 10% polyacrylamide gel,
which served to seal the bottom of the casting apparatus. This buffer
was also used as a running buffer, and the electrophoresis was carried
out at 40 V for 5 h at room temperature. Molecules (up to 2 million daltons in size) were able to enter the agarose gel, as
shown by use of blue dextran 2000 as an internal control. Growth media
and lysate from the U87 cell line and human brain tumor tissues of
equal protein concentrations were analyzed in the gel. To allow
transfer of such large molecules onto the nitrocellulose membrane, the
blotting took place in Tris-glycine buffer at 20 V overnight at
4 °C. Western blotting was performed as above.
Protein Purification--
G3 recombinant proteins containing a
N-terminal His tag were purified using Ni-NTA affinity columns (17,
25). Briefly, the G3 domain was expressed in Escherichia
coli strain M15 using the bacterial expression vector pQE30
(Qiagen Inc., Chatsworth, CA; catalog number 32149) as shown in Fig.
1A. The G3 domain was amplified in a PCR using two primers,
5'-aaaggatccggacaggatccatgcaaa and 5'-aaagcatgcgcgccttgagtcctgccacgt.
The product was subcloned into pQE30, and the resulting construct
contained an N-terminal MRGSHis tag. Peptides were purified
on a Ni-NTA affinity column (Qiagen, catalog number 30230) according to
the manufacturer's instructions.
Proliferation Assays--
Growth media from COS-7 cells
transfected with different recombinant constructs were mixed in a 1:1
ratio with native culture medium (DMEM supplemented with 2.5% FBS),
and the mixture was introduced into glioma cells cultured in 96-well
tissue culture plates at a density of 2 × 103
cells/well. The cultures were maintained in an incubator for 3 days,
and cell number was counted using a cytometer. To test the effects of
the purified products on cell proliferation, glioma cells were plated
on 96-well dishes at a density of 2-4 × 103
cells/well, 200 µl/well. Purified products were added into each well
(50 µl of column eluate/well). Lysate from vector-transfected bacteria was loaded onto Ni-NTA purification columns, and the eluate
from these columns was used as a control. Cell proliferation was
determined after 3 days of incubation. To test the effects of
recombinant constructs on cell growth, glioma cells were transiently transfected with the constructs using the method described above. Four
days after transfection, cells were counted. Cell lysate and culture
medium were harvested for analysis of gene expression on Western blot.
Cell proliferation was also tested in glioma cell lines stably
transfected with versican Cell Cycle Analysis--
U87 cells were plated on 6-well tissue
culture plates at a density of 2 × 105 cells/well in
DMEM containing 10% FBS at 37 °C for 2 days. The cells were
analyzed by flow cytometry. Briefly, the cells were collected with
trypsin/EDTA, pelleted by centrifugation, and resuspended in 1 ml of
hypotonic propidium iodide solution (50 µg/ml) dissolved in 0.1%
sodium citrate plus 0.1% Triton X-100. The cells were analyzed using a
FACScan (Becton Dickinson).
Colony Formation Assay--
Glioma cells were seeded to six-well
plates at a density of 2 × 105 cells/well. The cells
were allowed to attach and grow overnight in DMEM supplemented with
10% FBS to reach 70% confluence. Cultures in each well were
transfected with 0.5-1 µg of G3 RT-PCR Assay--
Astrocytoma cells transfected with the mutant
G3 construct or the control vector (2.5 × 106 cells)
were harvested, and total RNA were extracted with Qiagen RNeasy mini
kit. RT-PCR assays were performed as previously described (18).
Briefly, 2 µg of total RNA was used to synthesize cDNA, a portion
of which (equal to 0.2 µg of RNA) was used in a PCR with two
appropriate primers. PCR products were analyzed in agarose gel
electrophoresis and detected using ethidium bromide staining. The
primers for endogenous versican were 5'-ccagccccctgttgtagaaaa and
5'-gcgcctcgactcctgccacct (producing a product of 297 base pairs). The
primers for G3 Cell Surface Binding and Competition Assays--
Growth medium
from COS-7 cells transfected with G3 or G3 Immunostaining of Versican in Astrocytoma Cell
Lines--
Astrocytoma cells U87 were cultured on glass slips to 80%
confluence. The cells were fixed with 4% paraformaldehyde and stained with rabbit anti-versican polyclonal antibody, which we generated to
recognize the CS sequence of the construct (17). The secondary antibody
was goat anti-rabbit IgG antibody conjugated with horseradish peroxidase. 3'-Amino-9-ethylcarbazole (Sigma) was used for color development according to the manufacturer's instructions. The stained
cells were examined with a light microscope and photographed.
Dominant-negative Effect on Cell Growth by Deletion of EGF-like
Motifs--
To examine the role of versican in glioma cell growth, we
generated a mini-versican gene and a number of mutants (Fig.
1A). We first confirmed the
expression of versican in human glioma sample using Western blot. Our
purified polyclonal antibody, originally raised against chicken
versican, recognized a proteoglycan migrating as a large smear in
agarose gel, characteristic of large aggregating chondroitin
sulfate proteoglycans (Fig. 1B). Versican expression was
also detected in the glioma cell line U87. The mini-versican gene was
expressed in COS-7 cells, and its expression and secretion were
confirmed by Western blot. Growth media from the mini-versican-, versican
Having determined that the effect of versican on glioma cell growth is
mediated, at least in part, by its G3 domain, we sought to further
characterize the molecular determinant(s) of the effect. Specifically,
we tested whether the EGF-like motifs in G3 might play a role. We
tested our hypothesis by using a G3 domain from which the EGF-like
motifs had been removed (G3
The results obtained from cell proliferation assays were confirmed by
analyzing cell cycle progression. Overexpression of G3 Interaction of G3 Domain with Glioma Cell Surface--
There are a
number of mechanisms by which the versican
To test whether G3
The finding that deletion of the EGF-like motifs from the mini-versican
and the G3 construct has a dominant-negative effect on cell growth
suggests that, at high concentrations, the products of the
versican Inhibition of Versican Secretion by the G3
To further confirm the effect of G3
The above G3 The Effect of CRD and CBP Expression on Cell Proliferation--
To
dissect the motif in the G3 Versican is highly expressed in various tissues where the cells
are metabolically active and proliferating, such as in the mesenchymal
tissues. In epidermis, versican is found only in the proliferating zone
(26, 27). In cultured cells, versican is expressed only when cells are
actively proliferating; once cells reach confluence, versican
expression decreases (27). Therefore, it has long been suspected that
versican is associated with the process of cell proliferation.
Immunohistochemical studies have revealed that versican is expressed in
brain tumors. This study was designed to investigate the role of
versican in tumor cell growth.
In the central nervous system, chondroitin sulfate proteoglycans
constitute the major proteoglycan component in the extracellular matrix. Versican is known to associate with a number of molecules in
the extracellular matrix such as hyaluronan (8), tenascin (9-11),
fibronectin (29), fibulin (30), and CD44 (31). Versican is excluded
from focal contact and its distribution is similar to hyaluronan, CD44,
and tenascin. Interestingly, this same study demonstrated that tracks
left by migratory fibroblasts on culture plates exhibited versican
immunoreactivity (32). Thus, versican may be involved in cell invasion.
In astrocytomas, however, the expression of versican is not restricted
to the invasive borders but is present in all grades of astrocytomas,
indicating that all the tumors examined possess some invasive
potential. Whereas low grade tumors contain scattered individual cells
with versican expression, high grade tumors have large clusters of
versican-immunoreactive cells, probably the result of clonal expansion
of cells with invasive potential. Previous studies (33) of astrocytoma
cell lines have not been able to correlate the invasiveness of
astrocytomas with their proliferative activity. But, clinically, grade
III and IV astrocytomas with high proliferative activity are definitely
highly invasive tumors that can infiltrate proximal tissue as well as the distant regions such as brain stem, the leptomeninges, and even the
contralateral cerebral hemisphere through structures such as the corpus
callosum. Our study suggests that there may be a correlation between
astrocytoma proliferative activity and density of versican expression
within these tumors, because we showed that versican can stimulate
glioma cell growth. High histological grades or MIB-1 labeling index
would be associated with a greater propensity to invade. One other
finding is the expression of versican in areas surrounding tumor
necrosis. This suggests that the up-regulation of versican expression
could be either associated with factors produced by necrotic tumor
tissue such as cytokines (34) or linked to tumor necrogenesis.
Our studies made use of an in vitro system by using glioma
cells transfected with a mini-versican construct. We demonstrated that
the mini-versican construct promoted glioma cell proliferation and that
this occurred through the G3 domain in the mini-versican gene. We have
previously demonstrated that two EGF-like motifs in the mini-versican
construct were involved in enhancing NIH 3T3 fibroblast proliferation.
In this report, we found that deletion of the EGF-like motifs not only
abolished the effect of mini-versican on cell growth, but the resultant
mutant also exerted a dominant effect on glioma cell proliferation. The
G3 We have previously demonstrated that a mini-versican construct also
enhanced the growth of NIH 3T3 cells (17) and chicken chondrocytes
(35). Deletion of two EGF-like motifs from the mini-versican
significantly reduced the effect of the mini-versican on cell
proliferation but did not completely abolish this effect. A
dominant-negative effect on NIH 3T3 cell proliferation was not seen.
Perhaps in NIH 3T3 cells, the endogenous versican has only a minimal
effect on cell growth. Consequently, expression of exogenous mini-versican enhanced cell proliferation, but deletion of the EGF-like
motifs did not result in a dominant-negative effect. In the studies
reported here, we demonstrated that deletion of the EGF-like motifs
from the mini-versican or the G3 construct produced a dominant-negative
effect on glioma cell proliferation. Thus, this effect is apparently
specific to the glioma U87 cell line. In glioma cells, endogenous
versican probably plays an important functional role in enhancing cell
proliferation, and the mutant constructs likely interfere with a
process that is crucial for cell proliferation. In other cell types
this process may be less crucial or nonexistent, and so the effect of
the mutant versican is less profound.
In cell surface binding assays, we demonstrated that the products of
mutant G3 construct (G3 Another possible explanation for the dominant-negative effect is that
the G3 Our studies have demonstrated two possible mechanisms that may underlie
the dominant-negative effect of G3EGF) and a G3 mutant (G3
EGF) exerted a dominant-negative
effect on cell proliferation. G3
EGF-transfected cells formed smaller
colonies, arrested cell cycle at G1 phase, inhibited
expression of cell cycle proteins cdk4 and cyclin D1, and
contained multiple nucleoli. In cell surface binding assays, G3
products expressed in COS-7 cells and bacteria bound to U87 cell
surface. G3
EGF products exhibited decreased binding activity, but
higher levels of G3
EGF products were able to inhibit the binding of
G3 to the cell surface. G3
EGF expression inhibited secretion of
endogenous versican in astrocytoma cells and also inhibited the
secretion of mini-versican in COS-7 cells co-transfected with the
mini-versican and G3
EGF constructs. The effect seems to depend on
the expression efficiency of G3
EGF, and it occurred via the
carbohydrate recognition domain.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EGF) binds to the astrocytoma cell surface. This may have
competed with endogenous versican for binding sites on the cell
surface and blocked the function of versican in cell growth.
Furthermore, we demonstrate that the mutant construct inhibited
secretion of endogenous versican in glioma cells and the mini-versican
in COS-7 cells. These two mechanisms may account for the
dominant-negative effect of the mutant on cell growth. It appears that
the effects of the G3
EGF on cell growth are
concentration-dependent and occurred via the CRD motif.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EGF), a G3 construct, and a G3 lacking the EGF-like motifs
(G3
EGF), was described in detail earlier (17, 18). In all constructs
used in this study, the link protein leading peptide was attached at
the N terminus to allow product secretion (17, 19). This leading
peptide contains an epitope recognized by the monoclonal antibody 4B6
(20). To generate CBP construct, CBP was synthesized using CBPN
(5'-aaactcgaggttgcctgtggtcaacct) and G3C
(5'-aaaaaagcatgcgcgccttgagtcctgccacgtcct) as primers in a PCR, and the
product was digested with XhoI and SphI. The
leading peptide was synthesized with LPN
(5'-aaaaaagaattcctaagtctactctttctggtgctg) and LP60b
(5'-aaactcgagaggcagtgtgacgttgcc) to generate an EcoRI restriction site at the N terminus and an XhoI site at the C
terminus of the leading peptide. Thus, the leading peptide and the CBP fragment were inserted into EcoRI- and
SphI-digested pcDNA1 plasmid. To produce the CRD
construct, CRD was synthesized using CRDN (5'-aaactcgagcaagacacagagact) and CRDC (5'-aaatctagatgttcctttcttgcaggt) as primers in a PCR. The PCR
products were digested with XhoI and XbaI and
purified. The purified products were inserted into XhoI- and
XbaI-digested CBP construct, in which the XhoI
site was situated between the leading peptide and the CBP fragment,
whereas the XbaI site was located at 3' of the
SphI site.
EGF as
previously described (22-24). Geneticin was introduced into the growth
medium (0.5 or 1.5 mg/ml) 24 h after transfection, and the cells
were maintained in this medium until individual colonies were large enough for cloning. The selected cell lines were stored in liquid nitrogen or maintained in growth medium containing 0.5 mg Geneticin/ml for subsequent gene expression assays and functional studies. Cell
lines were monitored to ensure expression of the transgene for the
duration of functional studies. In co-transfection assays, equal
amounts of the two constructs were mixed and used for the transfection.
EGF construct and the control vector.
Briefly, cells were seeded to 96-well tissue culture plates at a
density of 2 × 103 cells/well in DMEM containing 10%
FBS. The cultures were maintained in an incubator at 37 °C for 3 days, and cell number was counted as above.
EGF plasmid or a control vector
(pcDNA3) accompanied by 8 µl of Lipofectin as described above.
Two days after transfection, Geneticin was added to the culture media
at a final concentration of 1.5 mg/ml. The media were changed every 5 days or earlier if necessary. Colonies of transfected cells were
observed after 2 weeks.
EGF transgene were 5'-aaactcgaggttgcctgtggtcaacct and
5'-aaatctagagcgccttgagtcctgcca (complementary to nucleotides 10519-10831 of versican encoding the CBP motif and the tail). The
control primers were 5'-ccagagcaagagaggcatcc and
5'-ccgtggtggtgaagctgtag (complementary to
-actin nucleotides
247-683).
EGF was incubated with
glioma cells, which had been pretreated with hyaluronidase (165 units/ml) at 37 °C for 1 h. The cells were incubated at
37 °C with gentle shaking for 3 h. The medium was removed after
centrifugation (at 1000 × g), and the cells were washed with 10 ml of PBS with gentle shaking to prevent nonspecific interaction. The cells were collected and lysed in lysis buffer. Cell
extract was analyzed on Western blot probed with 4B6 to detect the
binding of G3 product and G3
EGF product to glioma cells. To test
whether one product could compete with another for binding to glioma
cells, 50 µl of peptides purified from bacteria was mixed with
different amounts of competing medium (from G3- or G3
EGF-transfected
COS-7 cells). Culture medium from vector-transfected COS-7 cells was
used to bring the final volume to 2 ml. The mixture was incubated with
glioma cells at 37 °C for 2 h. The cells were washed as above,
and cell lysate was prepared. Equal amounts of proteins from each
treatment were analyzed on Western blot to estimate the binding of the
above products to glioma cells. As well, culture media from COS-7 cells
expressing G3 (200 µl) were mixed with 0, 500, or 1500 µl of
culture media from G3
EGF-transfected COS-7 cells, and the
competition assay was performed as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EGF-, and vector-transfected COS-7 cells were collected and
introduced into U87 glioma cell cultures. After 3 days, cell count
indicated that the mini-versican enhanced cell proliferation compared
with the control, whereas growth medium from versican
EGF-transfected cells exerted a dominant-negative effect, producing inhibition of
glioma cell growth compared with the control (Fig. 1C). The dominant-negative effect of the mutant was further confirmed in cell
lines stably transfected with versican
EGF; three cell lines expressing the versican
EGF construct exhibited a dominant-negative effect on cell growth compared with the vector control (Fig.
1D). One of the cell lines was cultured and incubated with
growth medium from the mini-versican- and vector-transfected COS-7
cells. Addition of exogenous growth medium from the
mini-versican-transfected cells reversed this effect somewhat but not
completely (Fig. 1E).
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Fig. 1.
Deletion of the EGF-like motifs from versican
(versican EGF) results in a dominant-negative
effect on cell proliferation. A, recombinant constructs
used in this study are as follows: mini-versican (containing a complete
G1 domain, an abbreviated CS domain, and a complete G3 domain),
versican
EGF (a mini-versican lacking EGF-like motifs), G3, G3
EGF
(the G3 construct lacking the EGF-like motifs), and pQE30 (G3 domain
subcloned in bacterial expression vector). The leading peptide from
link protein was added to the N terminus of each mammalian expression
construct for product secretion. IgG, immunoglobular domain;
TR, tandem repeat. Numbers above schematic
correspond to nucleotides in the sequence of full-length versican.
B, human brain tumor tissue and U87 cell homogenate were
analyzed on Western blot probed with polyclonal antibodies against
versican. The smear is a characteristic of proteoglycans. Growth medium
collected from COS-7 cells transiently transfected with versican
EGF,
control vector, or the mini-versican construct was also analyzed on
Western blot probed with 4B6. Deletion of the EGF-like motifs resulted
in a smaller core protein, and this proteoglycan migrated slightly
faster than the recombinant mini-versican. C, growth medium
collected from COS-7 cells transfected with the versican
EGF
construct was mixed with DMEM containing 2.5% FBS, and the mixture was
introduced into glioma cultures that had been seeded into 96-well
tissue culture plates at a cell density of 2 × 103
cells/well, 200 µl/well. Media collected from the vector-transfected
cells were used as controls. After 3 days, cells were counted. Data
represent the mean ± S.D. of four separate experiments
(n = 4; *, p < 0.05). D,
versican
EGF was stably expressed in glioma cells. Three such cell
lines and three cell lines stably transfected with a control vector
were seeded in tissue culture plates for cell proliferation assay. Data
represent the means ± S.D. of four separate experiments
(n = 4; **, p < 0.01). E,
cells stably transfected with versican
EGF or the control vector were
seeded in 96-well tissue culture plates. Growth media from COS-7 cells
transfected with the mini-versican construct or the control vector were
introduced into the glioma cultures as indicated. The cultures were
maintained in an incubator for 3 days, and cell number was determined.
The dominant-negative effect of the versican
EGF construct on cell
proliferation was significantly reduced by addition of growth medium
containing mini-versican products. Data represent the means ± S.D. of four separate experiments (n = 4; **,
p < 0.01).
EGF). G3
EGF construct was expressed in
COS-7 cells (Fig. 2A), and
culture medium containing G3
EGF products was shown to have a weak
inhibitory effect on cell growth (Fig. 2B), whereas purified
G3
EGF product produced a significant inhibitory effect on cell
growth (Fig. 2C). The effect of G3
EGF on cell
proliferation was also obtained from colony formation assays. U87 cells
transfected with G3
EGF and a control vector were treated with
Geneticin (1.5 mg/ml), and the cultures were maintained in this medium
until individual colonies were formed. G3
EGF transfection resulted
in the formation of smaller colonies (Fig. 2E) than did
transfection with control vector (Fig. 2D).
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Fig. 2.
The effects of G3 EGF
on cell proliferation and colony formation. A, culture
medium from COS-7 cells transfected with G3, G3
EGF, or the vector
was analyzed on Western blot. Deletion of the EGF-like motifs resulted
in a smaller band compared with the G3 product. The G3
EGF product
was also purified over a Ni-NTA affinity column, and the purified
product is shown. B, growth media from COS-7 cells
transfected with G3
EGF and the vector were incubated for 3 days with
glioma cell cultures that had been seeded on 96-well tissue culture
plates at a cell density of 2 × 103 cells/well. The
growth medium containing the G3
EGF products exerted a
dominant-negative effect on cell proliferation compared with the
control (n = 4). C, the purified G3
EGF
products and the control elute were added to the glioma cultures as
above. The purified G3
EGF products also exerted a dominant-negative
effect on cell growth compared with the control elute
(n = 3; *, p < 0.05). Glioma cells in
6-well tissue culture plates at 70% confluence were also transfected
with G3
EGF or the vector and selected with Geneticin (1.5 mg/ml).
Formation of colonies was monitored under a light microscope. Typical
examples of a vector-transfected colony (D) and a mutant
G3-transfected colony (E) are shown. The entire colony of
the vector-transfected cells was not included in the picture. The
mutant G3-transfected colony was smaller than the vector-transfected
colony, and the cells exhibited a shortened morphology.
EGF caused
arrest of a greater number of cells in G1 phase. A typical
G3
EGF-transfected cell line and a vector-transfected cell line are
shown in Fig. 3A, in which
88% of G3
EGF-transfected cells were arrested in G1
phase (7% in G2 phase and 4.7% in S phase). Only 65.5%
of control vector-transfected cells were detected in G1
phase (24.8% in G2 phase and 9.7% in S phase). The
effects of G3
EGF expression on two cell cycle proteins are shown in
Fig. 3B. Cell lysate harvested from transfected cells was
analyzed on Western blots probed with antibodies against cdk4 and
cyclin D1 (Santa Cruz). Levels of cyclin D1 and cdk4 decreased
dramatically in G3
EGF-transfected cell lines. The structure of
nuclei was then examined, and it was observed that each nucleus of the
vector-transfected cells contained one or two nucleoli (Fig.
3C), whereas each nucleus of the G3
EGF-transfected cells
contained multiple nucleoli (Fig. 3D).
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Fig. 3.
Expression of
versican EGF alters cell cycle and expression
of cell cycle proteins and nucleoi. Cell cycle was analyzed in
cell lines stably transfected with versican
EGF and the vector
(A). The proportion of cells in each phase, determined using
a FACScan, is indicated in each figure. The G3
EGF-transfected cell
line exhibited an altered cell cycle pattern. Its cycle was arrested in
G1 phase (88% of total cells). In the vector-transfected
cell line, only 65.5% of cells were detected in G1 phase.
Cell lysate from each cell line was analyzed on Western blot probed
with antibodies against cyclin D1 and cdk4 (B). The levels
of cyclin D1 and cdk4 were reduced in the G3
EGF-transfected cells as
compared with the control. The structure of nuclei from cells
transfected with the vector (C) and G3
EGF (D)
was examined. G3
EGF-transfected cells contained multiple nucleoli
within each nucleus, whereas vector-transfected cells contained only
one or two. Each insert is the enlargement of one nucleus.
EGF and G3
EGF
constructs could exert a dominant-negative effect on cell growth. The
simplest explanation is that these mutant gene products are able to
bind to sites on the glioma cell surface and thus successfully block
the proliferative effects of endogenous versican. Because the G3
EGF
construct was alone sufficient to exert this effect, we used it in
these studies to minimize complications arising from potential cell
surface binding sites present in other versican domains
(e.g. G1). We first demonstrated that the full-length G3
products were able to bind to the cell surface. Glioma cells were
incubated in growth media from G3- and vector-transfected COS-7 cells.
After extensive washing, cell lysate was harvested and analyzed on
Western blot. G3 bound to the glioma cell surface, resulting in
detection of a G3 band in the cell lysate (Fig.
4A). Using the same methods,
we demonstrated that G3 produced by bacteria and added exogenously to
U87 cells was also able to bind to the cell surface (Fig.
4A).
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Fig. 4.
The interaction of G3 and
G3 EGF with glioma cells. A,
growth medium from G3-transfected COS-7 cells and cell lysate from
G3-pQE-transformed bacteria were incubated with glioma cells for 2 h. The products that were retained by the cell surface were analyzed on
Western blot. G3 and G3-pQE interacted with glioma cells resulting in
the detection of G3 from both sources. B, growth medium from
COS-7 cells transfected with G3, G3
EGF or the control vector was
analyzed on Western blot (medium). The medium was then incubated with
glioma cells to test the binding activities of these products to glioma
cells. The protein band of G3 was significantly stronger than that of
G3
EGF. C, cell lysate from G3-pQE30-expressing bacteria
(50 µl) was mixed with medium from G3-transfected COS-7 cells
(upper panel) or from G3
EGF-transfected COS-7 cells
(lower panel) in various amounts (0, 25, 250, 500, and 1500 µl), and the mixture was incubated with glioma cells for binding
assays. The interaction of G3-pQE with glioma cells was inhibited by G3
and G3
EGF in a dose-dependent manner. At high
concentrations, some degraded G3-pQE migrated slightly faster.
D, growth medium (200 µl) from G3-transfected COS-7 cells
was mixed with growth medium from G3
EGF-transfected COS-7 cells (0, 500, and 1500 µl), and the mixture was incubated with glioma cells to
test the binding of these products to glioma cells. High concentrations
of G3
EGF reduced the binding of G3 to the cells.
EGF products were able to bind to glioma cells,
U87 cells were incubated with growth media from COS-7 cells transfected
with either G3
EGF or G3 construct, both of which were well expressed
(Fig. 4B). The cells were washed extensively and lysed for
Western blot analysis. The G3 signal was significantly more intense
than that of G3
EGF, indicating that G3 had a higher affinity for the
glioma cell surface (Fig. 4B). The media from G3- and
G3
EGF-transfected cells were mixed with the cell lysate of bacteria
expressing G3. The mixture was incubated with glioma cells, and the
amount of His-tagged bacterial G3 product remaining on the cells was
assessed. G3 and G3
EGF from COS-7 cells inhibited the binding of
bacterial G3 product in a dose-dependent manner (Fig.
4C).
EGF and G3
EGF constructs were able to compete with
endogenous versican for binding sites on glioma cells, although their
binding is apparently weaker. We tested whether G3
EGF could compete
with G3 to bind to glioma cell surface. G3 was mixed, at a fixed
concentration, with varying amounts of G3
EGF, and the mixtures were
incubated with glioma cells. High concentration of G3
EGF inhibited
G3 binding to glioma cell surface (Fig. 4D).
EGF Construct--
To
further characterize the mechanism of dominant-negative effect of
G3
EGF on cell growth, we examined the expression of endogenous
versican in cell lines stably transfected with the G3
EGF construct
and the control vector. Interestingly, we observed that cells stably
transfected with G3
EGF had a higher level of versican in their
cytoplasm, as revealed by labeling with polyclonal anti-versican
antibody (Fig. 5A) as compared
with cells transfected with the control vector (Fig. 5B).
This finding raised the possibility that the G3
EGF construct had no
effect on the transcription and translation of endogenous versican, but
in fact inhibited its post-translational processing. To test this, we
analyzed culture media from cells stably transfected with G3
EGF or
the vector on Western blot. Cells transfected with the vector did
indeed secrete higher levels of versican into the growth medium as
compared with the G3
EGF-transfected cells (Fig. 5C). It
was then necessary to examine whether expression of G3
EGF had any
effect on transcriptional regulation of the endogenous versican gene.
RT-PCR was performed using RNA from astrocytoma cell lines transfected
with G3
EGF or the vector. Levels of RT-PCR products were similar in
both cell lines, implying that G3
EGF had no effect on versican
transcription (Fig. 5D).
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Fig. 5.
Secretion of endogenous versican was
inhibited by G3 EGF-expression. Cell lines
stably transfected with G3
EGF (A) or the vector
(B) were cultured on glass slides until subconfluent. The
cultures were fixed with 4% paraformaldehyde and probed with
polyclonal antibody against versican. Cell lines transfected with
G3
EGF had a higher level of staining in the cytoplasm as compared
with control. C, growth media from these cells were analyzed
using agarose-Western blot assay probed with the polyclonal antibody
against versican. The cell line transfected with G3
EGF secreted
reduced levels of versican to the growth medium as compared with vector
control. D, cell lysate was prepared from both the vector-
and G3
EGF-transfected cell lines to analyze mRNA expression
using RT-PCR. The amounts of RT-PCR products (297 base pairs)
were similar in both cell lines. The loading dye migrated in the front
of the gel.
EGF on proteoglycan secretion, we
co-transfected COS-7 cells with a mini-versican construct and one of
the following three constructs: G3
EGF construct, CD44, or a control
vector. Cell lysate and culture media were analyzed on Western blot
probed with 4B6, which recognizes an epitope present in the G3
EGF
and mini-versican constructs. Cells co-transfected with the
mini-versican/CD44 or mini-versican/vector secreted much higher levels
of mini-versican than did those co-transfected with mini-versican/G3
EGF (Fig.
6A). However, cells expressing
mini-versican and G3
EGF had higher levels of mini-versican staining
in their cell lysate (Fig. 6A). Secretion of the G3
EGF
product was unaffected because it was detected in the culture medium
(Fig. 6B). Expression of CD44 was confirmed in cell lysate
probed with a monoclonal antibody against CD44 (Fig. 6C).
These findings indicate that co-transfection of mini-versican and
G3
EGF results in a specific retention of mini-versican in the
cell.
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Fig. 6.
Secretion of the mini-versican products was
inhibited by co-transfection of mini-versican and
G3 EGF. A, COS-7 cells were
co-transfected with a mini-versican construct and one of the following:
G3
EGF, CD44, or a control vector using equal amounts of plasmid DNA.
Cell lysate and culture media were analyzed on Western blot (5% gel of
SDS-PAGE) probed with 4B6. The levels of mini-versican in the culture
media from cells co-transfected with the mini-versican/CD44 or
mini-versican/control vector were much higher than that in cells
co-transfected with mini-versican/G3
EGF. However, the levels of
mini-versican in the cell lysate were opposite: cells co-transfected
with the mini-versican and G3
EGF had higher level of mini-versican
staining than the others. (Because of its small size, G3
EGF had run
out of the gel.) B, expression and secretion of the G3
EGF
product to the culture medium was confirmed on Western blot probed with
4B6. C, expression of CD44 was confirmed in cell lysate
probed with a monoclonal antibody against CD44.
EGF-transfceted cell lines were selected with high level
of Geneticin (1.5 mg/ml), and we only used those cell lines expressing
high levels of G3
EGF. It is obvious that the effect of G3
EGF on
cell proliferation depends on the levels of G3
EGF expression. To
further confirm this, cell lines expressing low levels of G3
EGF were
selected with low levels of Geneticin (0.5 mg/ml). Most of these cell
lines expressed low levels of G3
EGF. Three cell lines expressing low
levels of G3
EGF and three cell lines expressing high levels of
G3
EGF (shown in Fig. 7A with Western blot assay and Fig. 7B with RT-PCR) were used
for cell proliferation assay. Cell lines expressing high levels of G3
EGF had higher levels of inhibitory effect on cell proliferation as compared with the vector control, whereas cell lines expressing low
level of G3
EGF had moderate inhibitory effect on proliferation (Fig.
7C). The former also had a significant inhibitory effect on
cell elongation compared with the control, whereas the latter had a
median effect (Fig. 7D).
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Fig. 7.
The effect of G3 EGF
levels on cell morphology and proliferation. Western blot analysis
(A) and RT-PCR (B) of three cell lines expressing
high levels of G3
EGF (E1, E2, and E3), three cell lines expressing
low levels of G3
EGF (E4, E5, and E6), and three cell lines
transfected with the control vector (V1, V2, and V3). The amounts of
proteins loaded in E4, E5, and E6 were 3-fold that of E1, E2, and E3.
Cell proliferation (C) and cell morphology (D)
were assayed in these nine cell lines (n = 3; **,
p < 0.01).
EGF construct that inhibited cell
proliferation, constructs containing either CRD motif or CBP motif were
produced as shown in Fig. 1A. Cell lines expressing CRD and
CBP were selected, and their effects on cell proliferation were
examined. Expression of CRD and CBP constructs were tested on Western
blot probed with 4B6 (Fig.
8A). The cell lines expressing CRD had an inhibitory effect on cell proliferation as compared with the
vector control, whereas expression of CBP had little effect on
proliferation (Fig. 8C). Similarly, only the cell lines expressing CRD had a moderate effect on the alteration of cell morphology (Fig. 8D).
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Fig. 8.
The effect of CRD and CBP expression on cell
proliferation. A, Western blot analysis of four cell
lines expressing CRD, four cell lines expressing CBP, and one cell line
transfected with the control vector (V). B, cell
proliferation were assayed in these nine cell lines. C, cell
morphology of cell lines transfected with CRD (D1, D2, D3, and D4) and
CBP (P1, P2, P3, and P4) (n = 3; **, p < 0.01).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EGF construct had the same effect. This indicated that motifs in
G3 other than the EGF-like motifs were important in producing this
effect. This result allowed us to use mutant G3 construct (G3
EGF) to
investigate the mechanisms by which deletion of the EGF-like motifs
from the mini-versican generated a dominant-negative effect on glioma
cell proliferation. The products of the G3
EGF construct was small enough to analyze even trace amount of G3
EGF product in Western blot
assay. This has made the cell surface binding assay possible because
the levels of protein bound to the cell surface was very low in some
cases. Low levels of mini-versican, which migrated as a smear in the
gel of SDS-PAGE, were impossible to be detected on Western blot. As
well, in the assay that the G3
EGF construct inhibited endogenous
versican secretion, the small G3
EGF products were easily separated
from the endogenous versican. Otherwise, we would see the
versican
EGF products overlapped by the endogenous versican. In the
COS-7 cell transfection assays, we also benefited from the fact that
the small mutant construct G3
EGF was able to inhibit the secretion
of the mini-versican products.
EGF) were bound to the glioma cell surface.
High levels of G3
EGF were able to inhibit the interactions of native
G3 products with the cell. Thus, the G3
EGF mutant may suppress the
role of endogenous versican in enhancing cell proliferation by
hindering its interaction with the cell surface. Other G3 motifs such
as CRD and/or CBP may bind to the cell surface, and this binding may
promote interaction of the EGF-like motifs with molecules on the cell
surface such as signal transduction molecules (e.g. the EGF
receptor EGFR). The mutant G3
EGF, which still contains CRD and CBP
regions, would retain a binding ability but be inactive, because it
lacks EGF-like motifs. G3
EGF would thus compete with endogenous
versican for binding. This represents a potential molecular mechanism
to account for the dominant-negative effect of G3
EGF.
EGF products might inhibit the production of endogenous
versican. To test this, we analyzed the secretion of endogenous
versican and observed that less versican was secreted from cells
transfected with G3
EGF, compared with control. Immunostaining revealed that the endogenous versican was synthesized at similar levels
in both types of cells. Thus, it appears that G3
EGF expression does
not inhibit the synthesis of endogenous versican but does suppress its
secretion. This was further confirmed in co-transfection studies. In
COS-7 cells co-transfected with G3
EGF and mini-versican, secretion
of the mini-versican was inhibited, but synthesis was not affected.
These results strongly suggested that the mutant G3
EGF construct
plays a dominant-negative effect on cell proliferation through
suppressing the secretion of endogenous versican, and this represents a
second mechanism for dominant-negative effect of the mutant G3
EGF.
This was further confirmed in our study that only those cell lines
expressing high levels of G3
EGF had a lower rate of proliferation
and shortened cell morphology. On the other hand, the cell lines
expressing low levels of G3
EGF had little effect on cell
proliferation and morphology. Because the major motifs in the G3
EGF
construct are CRD and CBP, their effect on cell proliferation was
investigated, and our studies suggested that the inhibitory effects of
G3
EGF on cell proliferation and morphology occurred via the CRD
motif. The effect of CBP motif on cell activity was not clear. Our
previous study indicated that CBP plays a role in glycosaminoglycan
chain attachment and product secretion (28, 36).
EGF on glioma cell proliferation:
competition from cell surface binding sites and suppression of
secretion of endogenous versican. We cannot exclude a third
possibility: that G3
EGF binds to tenascin-C, a molecule that is
believed to play a role in cell proliferation. It has been shown that
the CRD motif can bind to tenascin-C (10, 11). The effect of this
binding on cell proliferation awaits further investigation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Paul F. Goetinck for the 4B6 monoclonal antibody and Dr. Yeqi Yao for technical help.
![]() |
FOOTNOTES |
---|
* This work was supported by The Cancer Research Society Inc, and Grant MOP-13730 from the Canadian Institutes of Health Research (to B. B. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ On sabbatical from the University of Calgary.
¶ Scholar of the Arthritis Society of Canada. To whom correspondence should be addressed: Research Bldg., Sunnybrook & Women's College Health Sciences Centre, 2075 Bayview Ave., Toronto, ON M4N 3M5, Canada. Tel.: 416-480-5874; Fax: 416-480-5737; E-mail: Burton.Yang @swchsc.on.ca.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M100618200
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ABBREVIATIONS |
---|
The abbreviations used are:
RT, reverse
transcriptase;
PCR, polymerase chain reaction;
EGF, epidermal growth
factor;
CRD, carbohydrate recognition domain;
CBP, complement binding
protein;
DMEM, Dulbecco's modified Eagle's medium;
PAGE, polyacrylamide gel electrophoresis;
Ni-NTA, nickel-nitrilotriacetic
acid;
CS, chondroitin sulfate;
G3, selectin-like domain;
versicanEGF, a mini-versican construct lacking two EGF-like motifs;
G3
EGF, versican G3 domain lacking two EGF-like motifs;
FBS, fetal
bovine serum..
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