1 Department of Pharmacology, Center of Excellence of Neurodegenerative Disease,
University of Milan, IN CNR, Cellular and Molecular Pharmacology Section,
Italy
2 Department of Pharmacology, Center of Excellence of Neurodegenerative Disease,
University of Milan, Italy
* Author for correspondence (e-mail: graziap{at}csfic.mi.cnr.it )
Accepted 16 May 2002
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Summary |
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Key words: Adherens junctions, Astrocytes, Migration, LIN-7 PDZ protein
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Introduction |
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Cell-cell adhesion plays a central role in complex biological processes,
including cell growth, proliferation, differentiation and survival.
Alterations in epithelial cadherin (E-cadherin)-mediated cell-cell adhesion
are associated with an increase in carcinoma cell motility, invasiveness and
metastasis (Behrens et al.,
1993). Conflicting results have been obtained concerning the
involvement of neuronal N-cadherin in the acquisition of invasive properties.
An increase in the expression of N-cadherin, or its exogenous expression, is
associated with greater invasive potential in breast cancer cell lines
(Hazan et al., 1997
;
Hazan et al., 2000
), whereas
no correlations have been found between N-cadherin expression and the invasive
behaviour of astrocytomas or cell migration in a series of glioblastoma cell
lines (Shinoura et al.,
1995
).
As the process of cell migration and invasion into surrounding tissue
probably requires the coordinated and dynamic assembly and disassembly of
cell-cell adhesions, the acquisition of invasive properties by tumour cells
may depend on changes in the stability and organisation of cell-cell contacts
rather than on modifications to the total expression of the molecular
components of cell-cell adhesion. We therefore characterised the composition,
maturity and organisation of the cadherin-mediated adhesion system in primary
cultured astrocytes and in the T98G and U373MG glioblastoma cell lines, which
come from highly invasive human tumours [grade III and IV according to the
World Health Organisation (WHO), (Kleihues
et al., 1993)] but have different migration and invasion
capacities depending on the substrate in which they are cultured
(Giese et al., 1994
;
Nakagawa et al., 1996
;
Belot et al., 2001
). In this
study, we tested the capacity of the glioblastoma cell lines to migrate on
poly-L-lysine or invade a Matrigel matrix, and established a relationship
between glioma cell aggressiveness and the degree of maturation and
organisation of their cell-cell junctions.
The maturity of the junctional domain was evaluated on the basis of the
reorganisation of the actin cortical cytoskeleton along the cell-cell contacts
using morphological [filamentous (F) actin staining] and biochemical analysis
[resistance of the E-cadherin-ß-catenin complex to Triton X-100 (TX-100)
extraction (Nathke et al.,
1994; Adams et al.,
1996
]. The maturation of cadherin-mediated junctions is also
accompanied by a reduction in tyrosine-phosphorylated junctional proteins
(Daniel and Reynolds, 1997
;
Lampugnani et al., 1997
), and
so morphological and biochemical analyses were made to measure the
phosphorylation level of the structural junctional components.
In order to characterise the process of maturation and organisation of
cadherin-mediated cell-cell adhesion, we followed the junctional accumulation
of the LIN-7 PDZ protein. Together with LIN-2/CASK and LIN-10/Mint/X11, this
protein (also called Veli or MAL) forms a functional scaffold complex that is
involved in the assembly of junctional components in neurons and epithelial
cells (Borg et al., 1998;
Butz et al., 1998
;
Kaech et al., 1998
;
Perego et al., 1999
;
Kamberov et al., 2000
). The
association of LIN-7 with the cadherin-catenin adhesion system is mediated by
its physical interaction with the PDZ target sequence of ß-catenin
(Perego et al., 2000b
). As the
recruitment of LIN-7 in ß-catenin-containing cell-cell contacts is a
progressive process that is fully competed only in mature junctions, its
accumulation was used to mark the degree of maturation of cadherin-mediated
adhesion.
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Materials and Methods |
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Primary antibodies and immunocytochemistry
The polyclonal anti--catenin, monoclonal and polyclonal
anti-ß-catenin, monoclonal anti-Pan-cadherin (CH-19) and monoclonal
anti-E-cadherin (Uvomorulin clone DECMA-1) came from Sigma. LIN-7 was detected
using a specific rabbit polyclonal anti-peptide antibody
(Perego et al., 2000b
) or a
rabbit polyclonal antiserum raised against amino acids 44-207 of mouse LIN-7A
(Histidin-LIN7A fusion protein) that was recently generated in our laboratory.
As similar results were obtained with both antibodies, all of the figures in
this paper were obtained using the LIN-7 anti-peptide antibody. Fluorescein
isothiocyanate (FITC)-labelled phalloidin (Sigma) was used to detect
filamentous actin and mouse anti-phosphotyrosine antibody (Transduction
Laboratories, Lexington, KY) to detect tyrosine-phosphorylated proteins.
The cells were fixed in 4% paraformaldehyde and permeabilised with 0.5% Triton X-100 (TX-100). Immunostaining with primary antibodies was followed by incubation with rhodamine-conjugated anti-rabbit, FITC-conjugated anti-mouse antibodies from Jackson Immunoresearch (West Grove, PA). The confocal images were obtained using a Bio-Rad MRC-1024 confocal microscope.
Scanning electron microscopy
Subconfluent T98G and U373MG cells grown on round coverslips coated with
poly-L-lysine were fixed as a monolayer with 2.5% glutaraldehyde in 0.1M
cacodylate buffer pH 7.4 for two hours at 4°C, post-fixed in 1%
OsO4 in the same buffer, dehydrated using an ethanol series, and
then bathed in hexamethyldisilazane (HMDS, Sigma) and allowed to dry overnight
under the fume hood. The samples were mounted on stubs and gold coated
(Balzers CED 100) before being examined using a Philips SEM505 scanning
electron microscope.
Transmission electron microscopy
Confluent T98G and U373MG cells were fixed in Petri dishes with 2.5%
gluteraldehyde in 0.1M cacodylate buffer for 2 hours, post-fixed in 1%
OsO4 in the same buffer, stained en bloc using a saturated solution
of uranyl acetate in 20% ethanol, dehydrated using an ethanol series and
embedded in EPON 812 (Fluka, Buchs, Switzerland).
Ultrathin monolayer sections were obtained using an ultramicrotome Ultracut E (Reichert-Jung) equipped with a diamond knife (Diatome), counterstained with uranyl acetate and lead citrate and examined using a Philips CM10 transmission electron microscope.
Triton X-100 extraction and western blot analysis
Cells grown to confluence on Petri dishes were extracted using Triton X-100
as previously described (Perego et al.,
2000b). The same volumes of the TX-soluble and TX-insoluble
fractions were solubilised in solubilisation buffer
(Perego et al., 1999
), loaded
onto 10% SDS-PAGE and immunostained with the indicated antibodies followed by
anti-IgG or Protein A conjugated to peroxidase (Sigma). The immunoreactive
bands were revealed using Supersignal West femto maximum sensitivity
substrate, Pierce Chemical Co.).
Immunoprecipitation experiments
The brain and cell lysates were obtained as previously described
(Perego et al., 2000b). The
immunocomplexes were separated by SDS-PAGE and analysed by means of
immunoblotting with the appropriate antibodies.
In order to measure the level of tyrosine phosphorylation, 5x105 cells were plated in 100 mm Petri dishes and cultured for 72 hours before cell lysis. In order to extract ß-catenin efficiently and maintain the phosphorylated tyrosine residues, 0.02% SDS and 100 µM pervanadate were added to the lysis buffer.
Cell surface biotinylation
Cells grown to confluence on 100 mm Petri dishes were starved for 30
minutes in Dulbecco's modified Eagle's medium and biotinylated with
NHS-ss-biotin (Pierce Chemical Co.) according to a previously published
protocol (Sargiacomo et al.,
1989). They were then lysed, and the biotinylated N-cadherin was
recovered using 150 µl Ultra-link Streptavidin beads (Pierce Chemical Co.).
The proteins were released from the beads by boiling the samples in SDS
solubilisation buffer and then analyzed on 10% SDS-PAGE.
Affinity chromatography assay
Glioblastoma cell line or cortical astrocyte lysates were incubated with
immobilised GST-mLIN-7A fusion protein
(Perego et al., 1999). The
bound material was resolved by SDS-PAGE, transferred to nitrocellulose
membrane and probed with the ß-catenin antibody.
Cell migration and invasion assay
Cell migration through poly-L-lysine-coated filters or cell invasion
through Matrigel-coated filters was measured in a Boyden chamber
(Albini et al., 1987), with 12
µm nuclepore polyvinylpyrrolidine (PVP)-free polycarbonate filters (Neuro
Probe, Gaithersburg, MD) being coated on the side facing the cells with 200
µl of Matrigel (0.5 mg/ml) or 200 µl of poly-L-lysine (1 mg/ml).
Conditioned media, obtained by incubating the T98G and U373MG cells for 48
hours in serum-free medium, were used as attractants in the bottom chamber
(stimulated) (Yamamoto et al.,
1997
; Dai et al.,
2001
) and a serum-free medium containing 0.1% fatty acid-free
bovine serum albumin (BSA) as a negative control (basal). Suspended in MEM
containing 0.1% fatty acid-free BSA, the T98G and U373MG cells were added to
the upper chamber at a density of 30x106cells/well. After 6
hours of incubation at 37°C, the non-migrated cells on the upper surface
of the filter were removed by scraping. The cells that had migrated to the
lower side of the filter were stained with Diff-quick stain (VWR Scientific
Products, NJ), and five to eight unit fields per filter were counted using a
microscope (Zeiss) at 160x magnification. The assays were run in
triplicate.
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Results |
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To verify whether LIN-7 is a component of the cadherin-catenin complex in cultured astrocytes, as it is in neuron and epithelia, we tested the ability of a GST-mLIN-7A fusion protein to retain ß-catenin from a lysate of cortical astrocytes. A fraction (3%) was specifically retained by the GST-mLIN-7A fusion protein (Fig. 1B) but not by immobilised GST (data not shown).
The in vivo association of LIN-7 with ß-catenin was revealed by co-immunoprecipitation experiments (Fig. 1C). The polyclonal LIN-7 antibody (but not a pre-immune serum) co-immunoprecipitated ß-catenin from the cultured astrocyte lysate.
The organisation of the cadherin adhesion system in astrocytes was
investigated using confocal microscopy. Astrocyte cultures mainly contained
type I glial fibrillary acidic protein-positive astrocytes (data not shown)
that grew as a monolayer of flat epithelium-like cells when cultured to
confluence (Perego et al.,
2000a). In recently confluent astrocytes, the LIN-7 staining was
predominantly cytosolic (Fig.
1D), whereas ß-catenin and N-cadherin (data not shown) were
enriched in irregular structures along the cell-cell contacts. The confluent
astrocytes showed continuous adhesion throughout the cell-cell contact
surfaces in which LIN-7 and ß-catenin colocalised. LIN-7 can therefore be
considered to be a marker of junctional maturity in astrocytes, as previously
shown in MDCK cells and neurons (Perego et
al., 2000b
).
Biochemical and morphological characterisation of the adherens
junctions in T98G and U373MG glioblastoma cell lines
In order to explore the role of cadherin-mediated cell-cell adhesion in
astrocytes and determine whether its is affected in glial tumors, we analysed
the expression of its components and its maturity and organisation in
glioblastoma cell lines with different in vitro migration properties. U373MG
and T98G cells come from highly invasive human tumours but, when we tested
their ability to migrate and penetrate 12 µm porous Transwell filters
coated with poly-L-lysine in a Boyden-modified chamber
(Albini et al., 1987), we found
that only U373MG permeated the poly-L-lysine barrier
(Fig. 2A).
|
We then tested whether the motility of U373MG cells might be explained by the altered expression of the molecular components of the cadherin system. Immunoblot analysis of the cell homogenates revealed similar levels of ß-catenin and LIN-7 in the T98G and U373MG cells, but the expression of total N-cadherin was lower in the former (Fig. 2B). No E-cadherin expression was detected by the specific antibody in either cell line (data not shown).
Given that only surface-expressed cadherin is involved in cell-cell
contacts, we investigated the amount of functional cadherin in the
glioblastomas by means of a surface biotinylation assay
(Fig. 2C), which revealed a
lower surface expression of cadherin in T98G cells. The
N-cadherinß-catenin association is maintained, as indicated by the
presence of ß-catenin in the streptavidin immunocomplex; however, the
T98G cells contained a considerably higher amount of ß-catenin associated
with surface N-cadherin. As catenins are thought to participate in
strengthening cell-cell adhesion by interacting with the actin cytoskeleton
(Adams et al., 1996), an
altered level of cadherin-associated ß-catenin may indicate reduced
adhesive strength. Alternatively, although undetectable by the available
antibodies, the differences in ß-cateninN-cadherin stoichiometry
might be explained by interactions with other isoforms of cadherin (in T98G
cells) or catenin (in U373MG cells).
We next tested whether LIN-7 formed a complex with the cadherinß-catenin system in T98G and U373MG cells. Affinity chromatography experiments showed that ß-catenin was specifically retained on GST-mLIN7A sepharose beads in both cell lines (Fig. 2D), thus suggesting that the ß-catenin in T98G and U373MG cells is equally capable of binding to LIN-7. However, ß-catenin was co-immunoprecipitated by LIN-7-specific antibodies only in the T98G cells (Fig. 2E), which excludes the possibility that the two proteins are truly associated in U373MG cells.
Taken together, the results of these experiments suggest an altered organisation of the cadherin-mediated system in migrating U373MG cells. In order to analyse the degree of maturation and organisation of cell-cell adhesion in the glioblastoma cell lines, we followed the morphological phases of cell-cell contact formation in cells grown at different densities and stained for N-cadherin and ß-catenin. Confocal analysis showed colocalisation of ß-catenin and N-cadherin on the cell surfaces of both cell lines, regardless of the cell density (Fig. 3). However, the morphology and genesis of the cell-cell contacts were different. In the subconfluent T98G cells, neighbouring cells appeared to be connected by filopodia enriched in ß-catenin and cadherin (arrowheads); these cultures often contained neighbouring cells sporadically enriched in ß-catenin and cadherin along their juxtaposed surfaces (arrows). In the confluent cell cultures, neighbouring cells were sealed together by continuous contacts along their entire surfaces, in which ß-catenin and N-cadherin accumulated.
|
In the subconfluent U373MG cells, ß-catenin and N-cadherin accumulated at high levels in lamellae and lamellipodia-like structures (arrows), connecting adjacent cells, and these markers remained irregularly distributed along the cell-cell contacts, even in the cells that had been confluent for a long time.
To investigate the detailed structure of the cell-cell contacts, we used scanning electron microscopy to analyse non-confluent T98G and U373MG cells (Fig. 4a,b). The T98G cells (a) grew flat on the substratum, were polygon-shaped and had many parallel, long and thin filopodial processes concentrated in the regions facing neighbouring cells (arrows), which they contact using the tips of the filopodia. The filopodia contained ß-catenin and N-cadherin and seemed to be rich in actin filaments, as shown by FITC-phalloidin staining and transmission electron microscopy (data not shown). The U373MG cells (b) were irregularly shaped and had borders characterised by two main features: broad lamellae whose edges had lamellipodia-like structures (arrows) and sparse, thin and branched processes (arrowheads) making contacts with the substratum but without any significant cytoskeletal organisation (data not shown). The U373MG intercellular contacts appeared to be mediated by the lamellipodia.
|
The detailed ultrastructure of the contact sites in confluent cells was analysed using a transmission electron microscopy (Fig. 4c-f). Ultrathin sections cut perpendicularly to the substratum showed that the T98G cells formed a monolayer with every cell lying adjacent to the other in an epithelia-like organisation (c); as a result, the cell edges in the sections parallel to the substratum are very sharp, and it is possible to observe many adherens junctions along the plasma membranes (e, arrows). On the other hand, the confluent U373MG cells tended to crawl over each other (d) and, because of this, the plasma membranes of two cells in thin sections cut parallel to the substratum are barely visible and no adherens junctions can be seen (f).
Taken together, these data indicate stronger cell-cell adhesion in
non-migrating T98G than in migrating U373MG cells. To characterise the state
of maturation and organisation of the cell-cell contacts in both types, we
used immunofluorescence staining to follow the junctional recruitment of LIN-7
and -catenin and the organisation of F-actin
(Fig. 5).
|
As in cultured astrocytes, LIN-7 in T98G cells colocalised with
ß-catenin in areas with continuous cell-cell contacts (arrowheads) but
was absent from regions characterised by irregular ß-catenin staining
(arrows). No surface colocalisation of LIN-7 with ß-catenin was ever
observed in the U373MG cells, regardless of the cell density (data not shown).
Both T98G and U373MG cells were rich in -catenin at the
ß-catenin-containing cell-cell contacts (arrowheads and arrows).
Phalloidin staining revealed the presence of organised filaments of F-actin
running parallel to the cell-cell contact areas and colocalising with
ß-catenin at the junctional domain of the T98G cells (arrowheads). On the
contrary, the U373MG cells had untidily distributed short filaments of
F-actin, and no colocalisation of F-actin with ß-catenin was observed at
the cell-cell contacts (arrows).
The inability of the U373MG cell line to form mature and organised
cell-cell adhesion was tested biochemically by measuring the distribution of
ß-catenin and LIN-7 in the Triton X-100 soluble and insoluble fractions
after the detergent extraction of cells cultured to confluence for four days
(Fig. 6A). Fifty percent of
ß-catenin and LIN-7 were found in the Triton-X-100-insoluble fraction of
T98G cells, but only 20% were in the insoluble fraction of U373MG cells. This
result is similar to that described during junctional maturation in MDCK
cells, when the amount of LIN-7 and ß-catenin recovered from the
Triton-X100-insoluble fraction increases from 20% in subconfluent cells to 50%
in fully polarised cells (Perego et al.,
2000b). The lack of LIN-7 junctional recruitment and cytoskeleton
reorganisation in U373MG cells strongly suggests a defect in the maturation
process of their cadherin-mediated cell-cell contacts.
|
Given that junctional maturation is accompanied by dephosphorylation of
components of the adhesion system (Daniel
and Reynolds, 1997; Lampugnani
et al., 1997
), and that the tyrosine phosphorylation of junctional
molecules is frequently associated with pathological alterations in
cadherin-mediated cell-cell adhesion
(Behrens et al., 1993
), we
expected to find the immature cell-cell contacts of the U373MG cells highly
phosphorylated in tyrosine residues.
P-tyrosine (P-tyr) staining revealed greater total tyrosine phosphorylation
in cell lysates and phosphorylated - and ß-catenin in the
immunoprecipitates of U373MG cells (Fig.
6B-D). No detectable tyrosine phosphorylation of N-cadherin was
found in either cell line (data not shown). Further confirmation of the
inability of U373MG cells to form mature junctions came from the confocal
analysis of cells double-stained for P-tyr and ß-catenin. Phosphorylated
proteins accumulated in the `free' surfaces of both cell lines, but only the
U373MG cell-cell contacts were clearly positive for P-tyr
(Fig. 5, arrows).
Invasive behaviour of glioblastoma cell lines correlates with
alterations in their adherens junctions
Taken together, our data suggest that only cells with immature adherens
junctions are capable of migrating and penetrating the poly-L-lysine barrier.
To validate the relationship between immature junctions and cell
aggressiveness in the same cellular context, we took advantage of the reported
ability of glioma cells to acquire invasive properties when plated on
particular extracellular matrix components
(Nakagawa et al., 1996;
Belot et al., 2001
). As
expected, the T98G cells showed highly invasive properties when plated on
Matrigel (Albini et al., 1987
)
(Fig. 7A), but the acquisition
of this invasive behaviour was not promoted by an increase of N-cadherin
expression. Western blot analysis revealed unchanged levels of cadherin,
ß-catenin and LIN-7 regardless of the substrate in which the cells were
plated (Fig. 7B).
|
Morphological analysis of the intercellular contact maturity and organisation of T98G cells cultured on Matrigel-coated glass coverslips (20-50 µg/cm2) (Fig. 7C,D) revealed a dramatic change from a non-invasive epithelia-like morphology when grown on poly-L-lysine to an invasive fibroblastoid morphology when grown on Matrigel. The cells formed branching invasive colonies and had a tendency to grow over each other (Fig. 7C). Immunostaining for ß-catenin and N-cadherin revealed their colocalisation on the cell surface, where they mainly accumulated in irregular intercellular contacts (Fig. 7D) which, in most cases, seemed to be caused by overlap rather than the juxtaposition of neighbouring cell surfaces. LIN-7 was found intracellularly, but virtually no LIN-7 staining was observed even in the cell-cell contacts that showed regular ß-catenin staining (arrows).
Similarly, phalloidin staining revealed the presence of untidily distributed filaments of F-actin in the cells and no colocalisation of F-actin with ß-catenin at the cell-cell contacts (arrows). These contacts had a high P-tyr content (arrows), thus further confirming their immaturity.
The culturing of T98G cells on Matrigel therefore induced the disorganisation of the actin cytoskeleton and cell-cell adhesion, modifications that seemed to drive their invasive behaviour.
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Discussion |
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The expression of adherens junction components has been previously
characterised in human glioblastomas and in a number of astrocytoma and
glioblastoma cell lines (Shinoura et al.,
1995; Roth et al.,
2000
); however, no data were reported concerning the organisation
of their junctions, and no correlation was found between N-cadherin expression
and the migration/invasion properties of the glioblastoma cell lines. We show
that alterations in the organisation of cadherin-mediated junctions rather
than in the expression of N-cadherin are associated with the migration and
invasive properties of glioblastoma cell lines. Despite their lower expression
of N-cadherin, the T98G cells formed mature and organised cadherin-mediated
junctional domains, as assessed by morphological and biochemical criteria,
whereas the alterations in the status of the cell-cell adhesion of U373MG
cells were associated with their higher cell motility in the poly-L-lysine
migration assay. Similarly, the T98G cells showed altered cell-cell adhesion
and invasive properties without any alteration in the total expression of
junctional components when cultured on a Matrigel matrix
(Fig. 7B). These data also
exclude the hypothesis that an increase in N-cadherin expression is associated
with the acquisition of invasive properties by glioblastoma cells, as has been
shown to be the case with breast cancer cells
(Hazan et al., 1997
;
Hazan et al., 2000
) and thus
further suggest that cell-cell adhesion alterations rather than changes in the
expression of cadherin system components are required to promote cell motility
and invasiveness.
Our data demonstrate that the LIN-7 PDZ protein is a component of the
mature cadherin-based junctional domain of primary cultured astrocytes and
non-migrating T98G glioblastoma cells (cultured in poly-L-lysine), whereas the
immature junctions of migrating U373MG and invasive T98G cells (cultured in
Matrigel) fail to accumulate LIN-7. As the absence of LIN-7 marks immature
contacts of migrating and invasive cells, its presence can be considered to be
a marker of junctional maturity and invasiveness. It is still not known
whether LIN-7 recruitment to intercellular adhesion is the cause or a
consequence of the stabilisation of this domain. Together with LIN-2 and
LIN-10, LIN-7 may be required to accumulate signalling molecules with crucial
junctional stabilisation functions, as in the case of the PTEN tumour
suppressor protein, whose PDZ-mediated recruitment into protein complexes
enhances its lipid phosphatase activity
(Vazquez et al., 2001) and is
critical for stabilising intercellular junctions and reverting invasiveness
(Kotelevets et al., 2001
).
T98G cells derive from a grade IV human glioblastoma multiform tumour and
do not grow when implanted subcutaneously or intracerebrally in nude mice
unless they are suspended on reconstituted basement membrane Matrigel
(Rubenstein et al., 1999). Our
results indicate a similar in vitro behaviour, which suggests that these cells
have a more selective integrin-dependent invasive phenotype than U373MG cells
rather than defects in the intracellular machinery involved in cell migration
and invasion. Furthermore, they had more invasive properties than U373MG cells
when cultured in Matrigel, which can be explained by their greater secretion
of the specific proteases required to remodel the surrounding ECM component of
the matrix (C.P, C.V., S.M. et al., unpublished). It is not known whether the
cell-substrate-dependent behaviour of T98G cells is a typical feature of the
original tumour or an acquired characteristic of the cell line.
Different ECM-dependent migration capacities have been previously
documented in various astrocytoma and glioblastoma cell lines
(Giese et al., 1994), and we
found that invasion properties and cell-cell adhesions can also be modulated
by modifying the extracellular matrix. Our results are in line with the in
vivo behaviour of astrocytoma and glioblastoma tumours, whose characteristic
features include the ability to permeate brain regions and the infrequency of
extracranial metastases (Berens et al.,
1990
).
Cell-cell and cell-ECM adhesion proteins influence cell locomotion through
the reorganisation of the actin cytoskeleton
(Bissel and Nelson, 1999;
Hemler and Rutishauser, 2000
).
It is therefore possible that competitive pathways mediate the organisation of
the cytoskeleton and that, when cell-substrate interactions prevail, the
cytoskeleton is organised in order to allow locomotion, and this affects the
strength of cell-cell adhesions. In line with this possibility, a progressive
reduction in the interaction of adherens junction proteins with the
cytoskeleton has been shown during integrin-induced epithelial tubule
formation in MDCK cells (Ojakian et al.,
2000
), and we found that migrating U373MG
(Fig. 5) and invasive T98G
cells (Fig. 7D) have immature
cell-cell adhesions and a disorganised cortical actin cytoskeleton. Further
experiments will be required in order to establish whether extracellular
matrix receptors are involved and to elucidate how the cell-substrate and the
cell-cell adhesion systems are integrated.
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Acknowledgments |
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