From the Department of Neurobiology and Civitan International Research Center, University of Alabama, Birmingham, Alabama 35294
Received for publication, June 7, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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Primary brain tumors (gliomas) have the unusual
ability to diffusely infiltrate the normal brain thereby evading
surgical treatment. Chlorotoxin is a scorpion toxin that specifically
binds to the surface of glioma cells and impairs their ability to
invade. Using a recombinant His-Cltx we isolated and identified the
principal Cltx receptor on the surface of glioma cells as matrix
metalloproteinase-2 (MMP-2). MMP-2 is specifically up-regulated in
gliomas and related cancers, but is not normally expressed in brain. We
demonstrate that Cltx specifically and selectively interacts with MMP-2
isoforms, but not with MMP-1, -3, and -9, which are also expressed in
malignant glioma cells. Importantly, we show that the anti-invasive
effect of Cltx on glioma cells can be explained by its interactions
with MMP-2. Cltx exerts a dual effect on MMP-2: it inhibits the
enzymatic activity of MMP-2 and causes a reduction in the surface
expression of MMP-2. These findings suggest that Cltx is a specific
MMP-2 inhibitor with significant therapeutic potential for gliomas and other diseases that invoke the activity of MMP-2.
Malignant gliomas are the most common primary intracranial tumors
in man. They constitute a heterogeneous group of tumors associated with
significant morbidity and mortality (1). Gliomas are characterized by a
high invasive potential and display a wide diversity of histological
features. They are derived from glial support cells in the brain and
the vast majority are thought to be of astrocytic origin (2). Even
low-grade gliomas infiltrate the entire brain, a feature that precludes
their successful therapy (3). Although systemic spread is a rarity in
malignant gliomas, invasion of tumor cells has been tracked
predominantly in the basement membrane of blood vessels, the
subependymal space, the glia limitans externa, and parallel and
intersecting nerve fiber tracts in the white matter (4). Significant
progress has been made in understanding pathways of glioma migration
and invasion. Molecular mechanisms of brain tumor invasion are complex.
They involve modification of receptor-mediated adhesive properties of
tumor cells, degradation and remodeling of extracellular matrix (ECM)1 by tumor-secreted
metalloproteinases, and creation of an intercellular space for invasion
of tumor cells (5). Spatial and temporal regulation of ECM proteolytic
degradation is maintained to ensure successful invasion of tumor cells
through the ECM. It has been established through in vitro
and in vivo glioma models that the deposition of ECM
components occurs at the confrontation zone between glioma cells and
normal brain tissue (6). In addition, paths of invading tumor cells are
also enriched in ECM molecules that promote cell motility (7, 8). Cells
interact with the ECM via integrins that play a major role in cell
invasion. Indeed, functional blocking anti-integrin antibodies (9) and
disintegrins, a group of snake venom toxins, can block migration and
invasion via an RGD-sequence dependent interaction (10-12).
In addition to these interactions with the extracellular brain
environment, invading glioma cells undergo dramatic shape and cell
volume changes allowing them to make their way through the narrow
extracellular spaces (13). This process appears to require the
secretion of fluid along with Cl The unusual specificity of Cltx for glioma tissues and its remarkable
anti-invasive effect prompted us in the current study to isolate and
identify the membrane receptor for Cltx. Affinity purification with a
recombinant His6-Cltx followed by mass spectroscopy and
sequencing identified the putative Cltx receptor as the matrix metalloproteinase-2 (MMP-2). MMP-2 co-purified as a major component of
a stable macromolecular complex comprised of MMP-2,
Cell Lines and Cell Culture
Human glioma cell lines D54-MG (obtained from Dr. D. D. Bigner, Duke University, Durham, NC) and CCF-STTG-1 (ATCC, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 2 mM L-glutamine
(Invitrogen) and 7% heat inactivated fetal bovine serum
(Hyclone). Cortical astrocytes were isolated from Sprague-Dawley rat
pups at postnatal day 0 and cultured as described earlier (19).
Antibodies and Reagents
Polyclonal antibodies directed against MMP-2, MT1-MMP, TIMP-2,
actin, as well as Synthesis and Purification of Recombinant Chorotoxin
As a first step toward biochemical isolation of the receptor for
Cltx, a recombinant fusion protein, His-Cltx, was synthesized in
Escherichia coli. To produce His-Cltx, we cloned Cltx into HindIII and EcoRI sites of pGHBE (a modified
pGEX2T vector in which the EcoRI, SmaI, and
BamHI sites were replace with HindIII, BamHI, and EcoRI). The cloned Cltx was then
subcloned into the BamHI and EcoRI sites in
pRSETB vector (Invitrogen). pRSETB has an N-terminal polyhistidine tag
(His6), which allowed the purification of His-Cltx by
immobilized metal affinity chromatography (Talon Resin,
Clontech Labs). His-tagged fusion protein was
purified as a 8-kDa protein using buffer conditions as described by the manufacturer.
Biological Activity of His-Cltx Was Assessed by Two Assays
Matrigel Invasion Assay--
Matrigel invasion chamber (BD
Biosciences) inserts of 8 µm pore size were rehydrated
following the manufacturer's recommendations. These chambers were then
coated on the lower surface with vitronectin and used for invasion
assay as described earlier for transwell migration assays (13).
Briefly, glioma cells were plated at a density of 5 × 104 cells/filter. Cells were treated with 30 nM
to 3 µM of either commercial peptide Cltx
(Alomone, Jerusalem, Israel) or His-Cltx and further incubated
at 37 °C for 24 h. Cells which remained in the upper chamber
were scrubbed off the inserts and the invaded cells were fixed and
stained with Diff-Quik stain kit (Dade Behring, VWR Scientific,
Switzerland). The relative inhibition of invasion was calculated as
decrease in the number of migrated cells in the presence of Cltx
compared with untreated vitronectin-coated control. Dose-response
curves were obtained from six samples each and data were averaged.
Half-maximal inhibition of matrigel cell invasion was achieved with
~200 nM Cltx and did not differ between His-Cltx and
commercial Cltx (Alomone).
Chloride Flux Measurements Using MEQ--
Glioma cells plated in
a 96-well plate at 5000 cells/well and loaded with MEQ and
Cl Overlay Assays (Far Westerns)
Briefly, proteins processed from membrane fractions, cytosolic
fractions, or total cell lysates were analyzed by Far Western blots using protocols described before (20). Membranes were probed with 300-500 nM His-Cltx followed by detection of
the bound proteins using an anti-His monoclonal antibody (Clontech,
1:5000).
Cell Lysis and Fractionation
Cultured cells were washed twice with cold PBS, scraped with
cell scrapers, and pelleted at 2000 × g for 5 min at
4 °C. The cell homogenates were prepared by resuspending the cell
pellet in 1.0 ml of homogenization buffer (10 mM Tris-Cl,
pH 7.5, 0.32 M sucrose, 1 mM MgCl2,
5 mM CaCl2 supplemented with 10 µl/ml
protease inhibitor mixtures I and II (mixture I: 1 mg/ml leupeptin, 1 mg/ml antipain, 5 mg/ml aprotinin, 10 mg/ml benzamidine hydrochloride, 10 mg/ml soybean trypsin inhibitor; and mixture II: 1 mg/ml pepstatin, 30 mM phenylmethanesulfonyl fluoride in dimethyl
sulfoxide)) and homogenizing in tissue grinders for 1 min with
incubations on ice at 1-min intervals. Cell debris was spun down at
2000 × g for 5 min at 4 °C and the supernatant was
collected and fractionated and membrane fractions were processed for
analysis as described earlier (21). Western blot analysis was performed
as described (22). For immunoprecipitation analysis, samples were
prepared and precleared as described earlier (22). The precleared
lysate was incubated with Protein A beads already pre-reacted with
rabbit serum (60 min at room temperature) for 2 h at room
temperature. Beads were then centrifuged gently and supernatant was
collected. Protein A beads were pre-reacted with primary antibody (1:1,
v/v) and followed procedures for immunoprecipitation as described
(22).
Biotinylation of Cell Surface Proteins
Cell surface proteins were biotinylated as previously described
(21). Cells at 80% confluence in a 100-mm dish were incubated with 3.8 ml of a 1.5 mg/ml sulfo-NHS biotin (Pierce) in
PBS/calcium/magnesium solution for 30 min at 4 °C with gentle
shaking. Following quenching of biotinylation in PBS/calcium/magnesium
plus 100 mM glycine, and further incubation for 30 min at 4 °C with gentle shaking, glycine solution was aspirated and
rinsed twice with PBS/calcium/magnesium. Cells were then lysed and
biotinylated cell surface fractions and intracellular fractions were
separated and analyzed by immunoblotting as described (21).
Coupling Recombinant Cltx to Actigel-ALD
Recombinant chlorotoxin was coupled to Actigel-ALD (Sterogene,
Carlsbad, CA) and stored following the manufacturer's recommendations. His-Cltx was added to Actigel-ALD (0.5 mg/ml of resin) followed by
ALD-coupling solution (1 M NaCNBH3) to a final
concentration of 0.1 M (0.2 ml/ml resin).
Affinity Purification
Four gorilla flasks of D54-MG cells were grown to confluence,
yielding about 20 mg of total protein. Cell debris was removed by
centrifugation at 2000 × g for 5 min at 4 °C and
the supernatant was collected and re-centrifuged at 100,000 × g in a Beckman T70.1 rotor for 60 min at 4 °C. Pellets,
representing the total cell membrane fraction, were resuspended in
homogenization buffer (supplemented with protease inhibitors)
containing 1% SDS followed by addition of a 7-fold excess volume of
1% Triton X-100. The samples were then heated to 48 °C for 5 min.
This lysate was then precleared with unconjugated Actigel-ALD beads
with end-over-end rotation (100-µl bead slurry per 1-ml lysate) for
4 h at 4 °C. Following a brief centrifugation at 100 × g, the supernatant was removed and incubated with the
His-Cltx-conjugated Actigel-ALD beads (250 µl/ml of precleared
lysate) for 4 h at 4 °C or overnight. The beads were then
centrifuged for 2 min at 100 × g and unbound material was removed. The beads were extensively washed with homogenization buffer (supplemented additionally with 0.1% Nonidet P-40 and 0.01% Tween). The bound proteins were then eluted by boiling with Laemmli SDS
sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue, and 600 mM 2-mercaptoethanol)
for 5 min. Samples were separated on denaturing 8, 10, or 4-15%
gradient gels by SDS-PAGE and further analyzed by Western blots and
overlay assays. The identity of the receptor was determined following electrophoresis of the affinity purified fractions on a 4-15% gradient polyacrylamide gel, staining with Bio-Safe Coomassie (Bio-Rad), and excising the band of interest. The proteins were then
destained and trypsinized and the protein digest extract was analyzed
by a MALDI-TOF mass spectrometer (PE Biosystems, Framingham, MA). The
peptide masses were entered into MASCOT to identify the protein by
searching the NCBI data base. Sequence information was obtained with a
Micromass Q-TOF-2 mass spectrometer.
Gelatin Zymography
Gelatin zymography was performed using 10% polyacrylamide gels
containing 0.1% gelatin (Bio-Rad). Briefly, eluates from the affinity
purification column were separated by SDS-PAGE on the gel, and
following electrophoresis the gel was washed with 2.5% Triton X-100
for 1 h to remove SDS and incubated at 37 °C for 24 h in a
buffer containing 50 mM Tris-Cl (pH 8.0), 5.0 mM CaCl2, and 1 µM
ZnCl2, and 0.005% Brij buffer. The gel was then stained with Coomassie for 30 min and destained quickly for 5 min to reveal gelatinolytic activity as opaque unstained bands.
Gelatinase Activity Assay for Analysis of Modulation of Enzymatic
Activity by Cltx
Gelatinase activity was analyzed utilizing a MMP gelatinase
activity assay kit (Chemicon) provided with recombinant human MMP-2
following the manufacturer's recommendations. Briefly, purified human
MMP-2 (Chemicon) was diluted in reaction buffer (50 mM
Tris-Cl, pH 7.5, 0.15 M NaCl, 5 mM
CaCl2) at concentrations ranging from 2.8 to 280 nM. 50-µl samples of these different concentrations of
MMP-2 were transferred to a rehydrated biotin-binding plate and
incubated for 30 min with 160 µl of 30-1000 nM Cltx in a
96-well plate in the presence of biotinylated gelatin. After incubation at 37 °C for 15 or 30 min, 100 µl of each sample was analyzed for
MMP-2 activity (n = 8) as described by the manufacturer (Chemicon).
In Situ Zymographic Analysis of Cell Surface Gelatinolytic
Activity in Vitro
To localize net gelatinolytic activity of MMPs by in
situ zymography, fluorescein isothiocyanate-labeled DQ gelatin
that is intramolecularly quenched (Molecular Probes) was used as a
substrate for degradation by gelatinases as reported earlier (23).
Proteolysis by gelatinases yields cleaved fluorescein
isothiocyanate-gelatin peptides and the localization of this
fluorescence indicates the sites of net gelatinolytic activity.
Briefly, glioma cells were plated on 12-mm coverslips. After 24 h
incubation, cells were treated with 30 nM Cltx, 300 nM Cltx, or 50 µM 1,10-phenanthroline for 30 min at 37 °C. Untreated cells served as negative control for this
experiment. Cells were washed with PBS and then incubated with
zymography reaction buffer (0.05 M Tris-HCl, 0.15 M NaCl, 5 mM CaCl2, and 0.2 mM NaN3, pH 7.6, the high concentration of azide prevented the gelatin from being phagocytosed and thus allowing cell surface gelatinolytic activity to occur) containing 100 µg/ml DQ
gelatin at 37 °C overnight. At the end of the incubation period, without fixation or further washes, gelatinolytic activity of the MMPs
was localized and photographed by fluorescence microscopy and images
were acquired by Spot digital camera.
Immunofluorescence Studies of Glioma Cells
Glioma cells were plated at 40% confluency onto 12-mm round
coverslips in appropriate tissue culture medium. After overnight incubation at 37 °C, medium was aspirated and replaced by
serum-free medium containing irrelevant His-protein (pRsetB-His) for
untreated controls and serum-free media containing His-Cltx (300 nM) and further incubated at 37 °C for 30 min. Cells
were then washed with PBS and fixed for 10 min at room temperature in
PBS containing 4% paraformaldehyde. Cells were rinsed, after fixation,
with PBS (3 × 10 min). After a 30-min incubation at room
temperature with blocking buffer (PBS containing 3% goat serum, 0.1%
azide, and 0.3% Triton X-100 (for permeabilized samples), Internalization Assay
Glioma cells were plated at a density of 5 × 105 cells in 100-mm tissue culture dishes in serum
containing media. After overnight incubation at 37 °C, cells were
washed and then incubated with serum-free media containing 300 nM His-Cltx, irrelevant His-protein (pRsetB-His), or 50 µM 1,10-phenanthroline for 30 min at 37 °C to allow
sufficient internalization of His-Cltx. Cells were then washed and cell
surface biotinylation was carried out as described above. Biotinylated
cell surface fractions and intracellular fractions were analyzed using
Western blots for quantitative levels of Cltx. Alternatively, cells
were treated with filipin at 5 µg/ml for 15 min at 4 °C to flatten
the caveolae prior to treatments with Cltx. Cells were then processed
for cell surface biotinylation as described above to analyze the level
and route of internalization of His-Cltx and associated MMP-2.
Statistical Analysis
Data were analyzed with Origin (version 6.0, MicroCal,
Northampton, MA). All curve fitting was performed using curve fitting routine provided by this software. Analysis of variance was used for
multiple comparisons of data. All values are reported as mean ± S.E. and percentages calculated from mean values.
The Cltx Receptor on Glioma Membranes Is MMP-2--
To
isolate and characterize the putative Cltx receptor, we synthesized
recombinant Cltx with a His6 tag (His-Cltx) in E. coli. The biological activity of the recombinant His-Cltx was
indistinguishable from that of commercially available peptide (see
"Experimental Procedures"). Total cell lysates as well as crude
membrane preparations of glioma cells were probed with His-Cltx and
showed binding to a prominent protein with a corresponding molecular
weight of ~70,000. This protein was not present in cultured
rat astrocytes (Fig. 1a).
To demonstrate that the expression of the Cltx-binding protein is on
the cell surface, we used a cell surface biotinylation approach to
distinguish between cell surface proteins and proteins contained in
intracellular membranes. In Far Western blots with His-Cltx, we found
that the Cltx-binding protein was present both in the cell surface
membrane fractions in two glioma cell lines as well as intracellullar
fractions (D54-MG and CCF-STTG1; Fig. 1b). Actin
immunoreactivity, which served as a control, was only found associated
with intracellular fractions (Fig. 1b).
For isolation and further purification of the Cltx receptor, His-Cltx
was immobilized to Actigel ALD beads by chemical conjugation. Affinity
purification of glioma membrane fractions (20 mg) using this column
revealed that the putative Cltx receptor could be purified together
with a number of other proteins that were mostly insoluble in 1%
Triton X-100 (Fig. 3a). To disrupt this complex and
selectively purify only Cltx-binding proteins, membrane fractions were
sequentially treated with buffers containing 1% SDS and then a 7-fold
excess volume of 1% Triton X-100. Samples were subsequently heated to
48 °C for 5 min before binding to the affinity column. These
conditions were able to disrupt the complex and allow the isolation of
a ~70-kDa protein, which retained binding to His-Cltx as assessed by
Far Western blots (Fig. 1c). MALDI-TOF mass spectrometric analysis of this affinity purified 70-kDa protein following tryptic digestion of the excised protein band from SDS-PAGE revealed its identity to be the matrix metalloproteinase, MMP-2 (see mass profile Fig. 1d). Sequence information obtained with a Micromass Q
TOF-2 mass spectrometer further confirmed the identity of the Cltx
receptor as MMP-2 (Fig. 3b).
The overlay assays of affinity purified membrane fractions in Fig.
1c suggest that the Cltx receptor, most likely MMP-2, is only expressed in glioma cell membranes but not in astrocytes. To
demonstrate that this lack of Cltx binding in astrocytes is because of
the absence of MMP-2, we performed Western blot analysis of astrocyte
and glioma cell lysates with rabbit
MMP-2 can exist in several molecular weight forms including a latent
proenzyme of 72,000, a ~64,000 activation intermediate, a ~62,000
mature protease (24), and a 45,000 process active form (25, 26). In
Western blots, MMP-2 antibodies recognized three bands at these
molecular weights in total cell lysates from glioma cells (Fig.
2a). Following affinity purification of glioma membranes
with His-Cltx, MMP-2 antibodies recognize primarily a 72-kDa band (Fig.
2b). This is consistent with previous reports showing this
species to be the major form of MMP-2 on glioma membranes (27). The
ability of Cltx to interact with MMP-2 in glioma cells and not
astrocytes is supported by overlays demonstrating that His-Cltx binds
to recombinant MMP-2, as well as bands of the same mobility in D54-MG
and CCF-STTG1 glioma cell lines but not astrocytes (Fig.
2c). Proteins affinity purified from D54 membranes with His-Cltx were found to exhibit gelatinase activity in gelatin zyomograms (Fig. 2d). The mobility of the zymographically
active bands appeared between ~60,000 and 75,000 and ~45,000, which
are molecular weights consistent with the presence of the various MMP-2
species. Whether Cltx inhibits MMP-2 catalytic activity will be
examined below.
Given that glioma cells express several MMPs including MMP-1, MMP-2,
MMP-3, and MMP-9 (28) we sought to evaluate whether the zymographically
active bands in Fig. 2d were because of an association of
Cltx with MMP-2 or one of these other MMPs. To do this, purified MMP-1,
-3, and -9 were run on gels and probed with His-Cltx. Our data show
that Cltx only interacts with MMP-2 (Fig. 2e). Taken
together, these findings suggest that MMP-2 is the principal receptor
for Cltx on the surface of glioma cells.
Characterization of the Cltx Receptor Protein Complex--
As
mentioned above, His-Cltx affinity purification of its receptor also
led to the isolation of additional protein bands (Fig. 3a) that could be separated
from MMP-2 by heating to 48 °C in SDS and Triton X-100. These data
indicate that in the plasma membrane of glioma cells, the Cltx receptor
is present in a macromoelcular complex with other proteins. To better
understand the nature of this protein complex, membrane samples
solubilized in Triton X-100 and affinity purified with His-Cltx were
separate by SDS-PAGE, stained with Coomassie Blue, excised, and
processed for mass spectrometry. Of the additional six major proteins
bands seen by Coomassie Blue staining (Fig. 3a), mass
spectrometric analysis was able to determine the identity of three as
MT1-MMP, Cltx Binds to MMP-2 and Inhibits Its Catalytic Activity--
Tumor
invasion, metastasis, and angiogenesis require controlled degradation
of ECM. Increased expression of MMPs has been associated with these
processes in malignant tumors of different histogenetic origin (31). In
gliomas, up-regulation of MMP-2, MMP-9, and MT1-MMP characterizes high
grade gliomas (glioblastoma multiformae) as opposed to low grade
gliomas or nontransformed control brain tissues (27, 32, 33). Moreover,
MMP-2 activity also modulates glioma cell migration and contributes
significantly to their invasive potential (34). Consequently (35)
several MMP inhibitors including 1,10-phenanthroline, cyclic peptides, and hydroxamate derivatives have been found to effectively block migration and invasion of tumor cells (36).
To gain a better understanding of how Cltx inhibits glioma cell
invasion, we investigated whether Cltx could bind to and modulate the
metalloprotease activity of MMP-2. To assess binding of His-Cltx to
MMP-2, we used an enzyme-linked immunosorbent based binding assay with
purified MMP-2 (Chemicon) immobilized on the plate. When we compare the
efficiency of binding of His-Cltx versus an irrelevant
control His-protein (Fig. 4a,
pRsetB-His) to MMP-2, we found that His-Cltx binds MMP-2 in a
dose-dependent manner with 50% binding achieved at ~115
nM (Fig. 4a).
We next examined the effect of Cltx on the catalytic activity of MMP-2.
In the presence of 500 nM Cltx, a concentration reported to
inhibit glioma cell invasion (13), gelatinase activity of MMP-2 was
markedly reduced, as shown for MMP-2 concentrations of up to 70 nM in Fig. 4b. To examine the
dose-dependent inhibition of MMP-2 by Cltx, we used a fixed
concentration of MMP-2 (0.7 nM) that was within the linear
range of the relative MMP-2 activity (Fig. 4b). Under these
conditions, Cltx showed dose-dependent inhibition of MMP-2,
which saturated at 1 µM Cltx (Fig. 4c). Both the enzymatic activity of recombinant MMP-2 and the inhibitory effect
of Cltx on its activity were potentiated by in vitro
activation of MMP-2 with 1 mM aminophenylmercuric acetate
but without a change in relative affinity (data not shown).
Cltx Inhibits Cell Surface Gelatinase Activity--
We next
investigated the effect of Cltx on the cell surface gelatinolytic
activity by in situ zymography using fluorescein isothiocyanate-labeled DQ gelatin. Untreated glioma cells exhibited significant cell surface gelatinolytic activity. A significant decrease
in surface gelatinolytic activity was observed following treatment with
30 nM Cltx, with complete inhibition in the presence of 300 nM Cltx (Fig. 5). The
inhibition by 300 nM Cltx was comparable with that achieved
with 50 µM 1,10-phenanthroline, a well established MMP-2
inhibitor (37), for which we determined an IC50 of ~8 µM for MMP-2 inhibition (data not shown). These data
suggest that Cltx is much more potent in inhibiting cell surface
gelatinolytic activity than suggested by our in vitro enzyme
assay with purified MMP-2 (Fig. 5). This suggests that perhaps,
secondarily, Cltx may also affect the surface expression of MMP-2.
Cltx Alters Surface Expression of MMP-2--
MMP-2 is a major
matrix metalloproteinase in glioma cells (27) and the vast majority of
MMP-2 is thought to stay in association with the cell surface (34). It
has previously been demonstrated that certain toxins, MT1-MMP, and
integrins can be internalized via caveolae (38). We therefore examine
the possibility that Cltx may cause an internalization of cell surface
MMP-2. To this end, we treated D54-MG glioma cells for 30 min with 500 µM Cltx at 37 °C. Cells were then fixed and stained
under either unpermeabilized or permeabilized conditions (Fig.
6a). The former only detects cell surface MMP-2, whereas the latter reveals the distribution of both
surface and intracellular MMP-2. Prominent expression of MMP-2 was
observed in untreated glioma cells both at the cell surface and
intracellularly (Fig. 6a). However, following a 30-min treatment with Cltx, surface staining for MMP-2 was essentially absent
whereas intracellular staining remained. These data suggest that
His-Cltx does indeed reduce the surface expression of MMP-2, perhaps by
inducing an increased internalization of MMP-2. This was also examined
biochemically using a cell surface biotinylation approach. Therefore,
cell surface membrane proteins were labeled with biotin and isolated
using avidin beads, from either untreated control cells or cells
treated with Cltx for 30 min at 37 °C. The majority of
membrane-associated MMP-2 disappeared from the cell surface upon Cltx
exposure (Fig. 6b).
Receptor-mediated endocytosis has been shown to occur either by a
clathrin-mediated pathway (39) or by caveoli (40). To assess whether
this observed MMP-2 internalization by Cltx is via caveolae, we
examined cell surface expression of MMP-2 in the presence and absence
of filipin, a sterol binding drug known to disrupt caveolae
(41). In the presence of filipin, a significantly larger fraction of
His-Cltx as well as MMP-2 remained on the cell surface, whereas in the
absence of filipin, a significant reduction in membrane-associated
His-Cltx and MMP-2 was observed (Fig. 6b). These data
suggest that a filipin-sensitive mechanism, possibly caveolae, is
involved in the internalization of MMP-2 following binding of Cltx.
Cltx Inhibits Matrigel Invasion of Glioma Cells by Its Interactions
with MMP-2--
It has previously been demonstrated that MMP-2
activity is essential for matrigel invasion of glioma cells (42).
Hence, one would expect, based on the above data, that Cltx binding to MMP-2 should reduce glioma cell invasion either by the inhibition of
its enzymatic activity or by decreasing the surface expression and/or
release of MMP-2. We examined the inhibitory properties of Cltx on
glioma matrigel invasion by directly comparing the effect of His-Cltx
to the commercially available peptide. Both peptides
inhibited invasion in a concentration-dependent manner with
an IC50 of about 200 nM (Fig.
7a), a value consistent with the inhibition of MMP-2 activity by Cltx (see Fig. 4c). The
maximal inhibition obtained with Cltx was between 70 and 80% as
compared with untreated control cells. Interestingly, addition of
1,10-phenanthroline yielded essentially identical inhibition of
invasion as did Cltx (Fig. 7b). However, when we examined
the effect of either Cltx or 1,10-phenanthroline in the presence of
filipin, the effect of Cltx was reduced by over 50%, suggesting that a
significant component of the Cltx effect on matrigel invasion
presumably involves the endocytosis of MMP-2 via caveolae. Consistent
with this concept, we find that the inhibition of glioma invasion by
1,10-phenanthroline, which specifically inhibits the enzymatic activity
of MMP-2, was only minimally affected by treatment with filipin.
Taken together, these findings suggest a novel mechanism of action for
this scorpion toxin, wherein Cltx regulates invasion of glioma cells by
modulating the surface expression of enzymatically active MMP-2.
Cltx is a 36-amino acid peptide that belongs to a large family of
insect toxins. Several recent studies have suggested that Cltx is a
highly specific ligand for malignant human gliomas, which shows no
significant binding to normal brain (17). Cltx selectively targets
xenografted gliomas in scid mice and remains associated with
the tumor for at least 24 h (17). Moreover, Cltx also inhibits
glioma cell invasion in vitro (13). However, the receptor
for Cltx and the mechanism underlying its anti-invasive effect have
been unknown.
In this report, we show that MMP-2 is the primary receptor for Cltx on
the surface of glioma cells. MMP-2 belongs to a superfamily of
zinc-dependent endopeptidases. It is secreted as a latent
zymogen requiring activation. Once activated, the latent 72-kDa MMP-2 is converted via a ~64-kDa activation intermediate to a ~62-kDa mature protease (24, 26). Efficient activation of the pro-enzyme to the
mature enzyme has been shown to contribute to the invasive potential of
tumor cells (43). MMP-2 has been demonstrated to be expressed in
numerous tumor types (31) and also in diseases that involve tissue
remodeling (44). However, MMP-2 appears to be a major matrix
metalloproteinase in gliomas (27) and is absent from normal brain
tissue (45, 46). Hence, the finding that MMP-2 is the principal
receptor for Cltx is consistent with the recent finding that the Cltx
receptor is primarily expressed in glioma and tumors of neuroectodermal
origin (18). Our data suggests that Cltx does not interact with MMP-1,
MMP-3, or MMP-9, which are also expressed by glioma cells (28).
Importantly, we show that the anti-invasive effect of Cltx on glioma
cells is mediated predominantly by its interactions with MMP-2. Our
data, discussed sequentially below, suggest that Cltx exerts a dual
effect on MMP-2: it inhibits the enzymatic activity of MMP-2 and it
causes a reduction in surface expression.
Our in vitro affinity purification and binding assays
indicate that Cltx can interact with both the latent 72 kDa and the activated forms of MMP-2. The 72-kDa form of MMP-2 is generally referred to as a latent form of the enzyme, but it constitutes the
dominant MMP-2 species in glioma cells, which, when activated exhibits
significant gelatinolytic activity in these cells. Interestingly this
activation is thought to occur on the cell surface of glioma cells in
an MT1-MMP-specific manner (34).
Our biochemical and cell biological assays indicate that Cltx binding
to MMP-2 inhibits its catalytic activity. For example, Cltx inhibits
MMP-2 activity in vitro in a dose-dependent
manner, and both the catalytic activity and the inhibition by Cltx are enhanced upon activation of recombinant MMP-2 with aminophenylmercuric acetate. In addition, we found that Cltx can completely inhibit the
cell surface gelatinolytic activity. Taken together, this data
suggests that the binding of Cltx to the surface of glioma cells occurs
through its direct association with MMP-2 and that this binding
inhibits the catalytic activity of MMP-2.
MMP-2 plays a crucial role in degradation and remodeling of the ECM
(34) thereby allowing the penetration of normal and tumor cells through
tissue barriers. The mechanism of activation and regulation of MMP-2 is
tightly regulated by several other proteins that form a macromolecular
complex (47). Specifically, this involves interactions with
membrane-associated MT1-MMP and Based on these macromolecular interactions, it is not surprising that
Cltx, by binding to MMP-2, would indirectly interact with this entire
protein complex. Our data suggests that the anti-invasive effects of
Cltx occur at least at two levels. In the first, Cltx inhibits the
enzymatic activity of the enzyme in vitro. Second, our
studies suggest that treatment of cells with Cltx also causes a
reduction of surface expression of MMP-2 from glioma cells. Although
not investigated in detail, our studies using filipin, a sterol binding
drug known to disrupt caveolae (41), suggests that endocytosis of the
MMP-2·Cltx complex via caveolae may be one of the underlying
pathways that regulates the surface expression of MMP-2. The
endocytosis of receptor-ligand complexes is generally because of
dynamin-mediated endocytosis via clathrin coated pits (39). However,
caveolin-1 expression has been demonstrated in several rat and human
astroglioma cell lines (40) and the entry of certain pathogens and
toxins into host cells has also been shown to occur via caveolae
(53-55). Indeed, the internalization of cholera toxin into CACO-2
cells can be inhibited by filipin (39) a finding that is consistent
with our findings on Cltx.
Our results from matrigel invasion assays suggest that the interaction
of Cltx with MMP-2 and the resulting decrease of surface expression are
sufficient to inhibit glioma cell invasion. Although the enzymatic
activity of MMP-2 was directly inhibited by Cltx, internalization of
MMP-2 appears to be an important step in the functional inhibition,
because Cltx looses much of its effectiveness in the presence of filipin.
We currently do not know which site on the MMP-2 molecule interacts
with Cltx. We performed preliminary competition experiments to examine
two well characterized sites, namely that for TIMP-2 and the integrin
binding site. Neither recombinant TIMP-2 nor functional blocking
The findings presented in this study have significant therapeutic
implications. The anti-invasive effects of Cltx on glioma cells suggest
that this drug may be highly useful in the treatment of malignant
gliomas. Indeed, Cltx has passed preclinical safety studies and has
recently won FDA approval for use in a phase I/II clinical trial.
Several embryologically related tumors, including melanomas, have also
been shown to express MMP-2 and to bind Cltx (18). Clinical use of Cltx
may thus be expanded to include these tumors as well. Importantly,
however, potentially being a specific MMP-2 inhibitor, Cltx may have
even broader utility. MMP-2 is involved in a range of diseases that
involve tissue remodeling in disease progression. Several chemical
inhibitors of MMP-2 are in various stages of clinical testing but most
have failed because of toxicity or lack of specificity. Cltx may be a
safer and more specific drug, worthy of further exploration in this context.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ions, as several
Cl
channel inhibitors including DIDS and tamoxifen have
been shown to inhibit glioma cell invasion in vitro (13).
Chlorotoxin (Cltx) is a 36-amino acid peptide that was originally
isolated from Leiurus quinquestriatus venom (14) and
has been shown to inhibit small conductance Cl
channels
in colonic epithelial cells (14, 15). Cltx also inhibits
Cl
fluxes across glioma membranes (13, 16).
Immunohistochemical studies show that Cltx specifically and selectively
binds to glioma cells (17) and radiolabeled Cltx targets tumor cells in
mice bearing xenografted glioma tumors. Glioma cell migration and
invasion into fetal brain aggregates is significantly reduced by Cltx
(13). A recent survey of over 200 tissue biopsies from patients with various malignancies suggests that Cltx binds to the surface of gliomas
and other embryologically related tumors of neuroectodermal origin (18)
but not to normal brain.
v
3 integrin, MT1-MMP, and TIMP-2, which
are proteins involved in cell migration and invasion. Cltx inhibited
the enzymatic activity of MMP-2 in vitro and reduced the
surface expression of MMP-2 in glioma cells. Cltx was found to
specifically bind to MMP-2 but not to MMP-1, MMP-3, and MMP-9, which
are also expressed by glioma cells. These findings explain the
specificity of Cltx for glioma in the intracranial animal model studies
and also the previously documented anti-invasive potential of Cltx.
Cltx may thus be a highly effective drug of therapeutic potential for
gliomas and possibly other diseases that invoke the activity of
MMP-2.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-rabbit and
-mouse horseradish
peroxidase-conjugated secondary antibodies, pure MMP-1, MMP-3, MMP-9,
and filipin were purchased from Sigma. Monoclonal
-MMP-2 antibody
was obtained from RDI Inc. (Flanders, NJ). Monoclonal antibodies for
v
3 integrin and recombinant human MMP-2
were purchased from Chemicon (Temecula, CA). Alexa Fluor (488) goat
anti-mouse IgG2a and Alexa red (546) goat anti-rabbit conjugated
secondary antibodies (Molecular Probes) were used for
immunofluorescence at the manufacturers recommended dilutions.
fluxes across glioma membranes were assayed in the
presence of 30 nM to 3 µM His-Cltx or native
peptide Cltx (Alomone) using previously described methods (13) with
minor modifications in preparation of MEQ dye. Briefly, MEQ was reduced
to a yellow oil by 12% sodium borohydride under constant flow of
nitrogen. When yellow oil separated out, the oil was subjected to two
consecutive extractions in a 50% ether:water (v/v) mixture and then
further dried under nitrogen. Experiments were performed at room
temperature with gluconate-based buffers as described earlier.
Inhibition of Cl
fluxes was directly compared for
His6-Cltx and commercially available Cltx and found to
be indistinguishable.
-MMP-2
primary antibodies were added to the cells (1:500) and incubated
overnight at room temperature in blocking buffer with or without Triton X-100. Cells were then rinsed (3 × 10 min) with PBS containing blocker and then incubated with Alexa red (1:750) secondary antibodies in blocker for 1 h at room temperature. Coverslips were mounted using gel mount following another 10-min wash in PBS.
Immunofluorescence study was carried out using a Leica epifluorescence
microscope (×40 oil and ×100 oil objectives), and images were
captured using a digital spot camera.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The putative Cltx receptor is MMP-2, which is
expressed in glioma cells but not in astrocytes. a, far
Western blot of cell lysates from glioma cells and cortical astrocytes
were probed with His-Cltx (500 nM) showing specific binding
to a 72-kDa protein in D54-MG cells but not in astrocytes.
b, expression of a Cltx receptor in plasma membrane and
intracellular fractions of glioma cells. Immunoblot of cell
surface-biotinylated D54-MG (WHO grade IV) and CCF-STTG1 (WHO grade
III) glioma cell lines was initially probed with an anti-actin
monoclonal antibody, stripped, and then overlaid with His-Cltx. The
actin immunoreactive band is a control to positively identify the
intracellular fractions. The 72-kDa band, bound by His-Cltx, is
detected in both the plasma membrane and intracellular fractions.
c, far Western blot of glioma cell membranes with His-Cltx.
Cell membranes were affinity purified using His-Cltx-conjugated
Actigel-ALD beads under stringent conditions (see "Experimental
Procedures") to break interactions between proteins. Far Western blot
with His-Cltx (500 nM) shows specific binding to a 72-kDa
protein in D54-MG cell membranes but not to astrocyte membranes.
d, mass spectrometric analysis and mass profile of the
affinity purified receptor, in which the mass profile of the Cltx
receptor was compared with internal bovine serum albumin
standards.
-MMP-2 antibodies (Fig.
2a). MMP-2 immunoreactivity
was only seen in glioma cells but not in astrocytes explaining the lack
of His-Cltx binding to astrocytes.
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Fig. 2.
Cltx specifically binds to MMP-2.
a, gliomas express the various species of MMP-2 whereas
astrocytes do not. Whole cell lysates from D54 glioma cells and primary
astrocyte cultures were probed with MMP-2 antibodies showing binding to
several molecular weight bands consistent with the 62,000, 68,000, and
72,000 species of MMP-2 in glioma cells but not in astrocytes. A
~20-kDa band that binds MMP-2 antibodies in astrocytes was not
characterized. b, immunoblot with MMP-2 antibodies
demonstrates binding to a 72-kDa band in His-Cltx affinity purified
membrane fractions. This is the same molecular weight as that
identified by His-Cltx in Western blots of glioma extracts.
c, His-Cltx interacts with recombinant MMP-2, and shows the
same immunoreactivity observed in membrane fractions from two glioma
cells lines. His-Cltx Far Western blot of recombinant purified MMP-2
shown side-by-side with D54-MG and CCF-STTG1 cell membranes show
protein bands at ~72,000 (arrow), the same molecular
weight as recombinant MMP-2. Specific binding with astrocytic membranes
was not observed. d, gelatin zymography of the His-Cltx
affinity purified fractions show 3 opaque gelatinolytic protein bands.
The most prominent two bands, between ~60,000 and ~75,000, and
~45,000 have similar molecular weights to that of various species of
MMP-2 isoforms (45,000, 62,000, 65,000, 68,000, and 72,000) that have
gelatinase activity. Left lane contains protein markers
only. e, Cltx interacts specifically with MMP-2. Far Western
blot with His-Cltx of purified recombinant MMP-2, MMP-9, MMP-1, and
MMP-3 resulted in binding only to MMP-2. Coomassie-stained gel
(bottom) indicates that the comparable amount of each
protein was loaded on the SDS gel.
v integrin, and TIMP-2 (Fig. 3b).
Details pertaining to the identification by mass spectrometry are given
in Fig. 3b. The presence of these proteins in
His-Cltx-isolated material was confirmed by Western blot analysis with
antibodies against
v
3 integrin, MT1-MMP,
and TIMP-2 (Fig. 3c). The co-existence of MMP-2 (Fig. 2) in
a complex with MT1-MMP,
v
3 integrin, and TIMP-2 is intriguing as it has previously been shown that MMP-2 is
docked on the surface of cells via a protein complex that includes MT1-MMP,
v
3 integrin, and TIMP-2 (29).
MT1-MMP is presumed to play an important role in mediating the docking
of MMP-2 proenzyme on the plasma membrane for efficient activation of
the protease (24, 26, 30). To assess whether in glioma cells this
protein complex occurs naturally or whether it is induced to form by
His-Cltx, we immunoprecipitated glioma membranes with MMP-2 antibodies
and probed the purified fractions with antibodies to
v
3 integrin, MMP-2, MT1-MMP, or with
His-Cltx (Fig. 3d). These immunoblots demonstrate that
MMP-2,
v
3 integrin, and MT1-MMP are
indeed present in a complex on the surface of glioma cells in the
absence of His-Cltx.
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Fig. 3.
Cltx binds to a protein complex containing
several proteins implicated in glioma cell invasion.
a, Coomassie Blue-stained SDS gel of the His-Cltx affinity
purified glioma membrane fraction obtained under stringent conditions
that break apart the interactions between the purified proteins (see
text) shows several prominent protein bands. The molecular weight of
these bands is consistent with their identity being integrin
v, MT1-MMP, TIMP-2, and MMP-2. b, mass
spectrometric analysis of His-Cltx affinity purified macromolecular
complexs. The table lists a mascot match score for protein bands
affinity purified with His-Cltx from glioma membrane fractions and
characterized by mass spectrometric analysis. Accession numbers and
Swiss protein data bank identity numbers are also shown in the table.
c, His-Cltx affinity purified complex contains integrin,
MT1-MMP, and TIMP-2. Immunoblots of His-Cltx affinity purified D54-MG
cell membrane fractions incubated with antibodies directed against
v
3 integrin, MT1-MMP, and TIMP-2
confirming the presence of these proteins in the affinity purified
Cltx-complex. D, the macromolecular complex of MMP-2,
MT1-MMP, and integrin exist in the absence of His-Cltx. Immunoblots of
D54-MG membranes after immunoprecipitation with MMP-2 antibodies were
probed with antibodies to
v
3 integrin,
then stripped and re-probed with MMP-2, MT1-MMP, or His-Cltx
(consecutively), to show existence of this protein complex in the
absence of His-Cltx.
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Fig. 4.
Cltx binds to MMP-2 and inhibits its
catalytic activity. a, His-Cltx binds to pure recombinant
MMP-2. Concentration dependent increase in binding of His-Cltx (3 nM to 3 µM) to MMP-2 was detected in a
enzyme-linked immunosorbent based assay. y axis represents
percent binding compared with irrelevant His-protein (pRsetB-His). 50%
binding was achieved at 115 nM Cltx. b, Cltx
inhibits gelatinase activity of purified MMP-2. Quantitative analysis
of gelatinase activity of recombinant MMP-2 as a function of varying
MMP-2 concentration (0.7 to 280 nM) in the presence or
absence of 500 nM Cltx. c,
dose-dependent inhibition of MMP-2 activity following
treatment with Cltx. Dose-response curve of gelatinase activity of
recombinant MMP-2 (0.7 nM) treated for 30 min at 37 °C
with increasing concentrations of Cltx. Cltx inhibits MMP-2 in a
dose-dependent manner.
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Fig. 5.
Cltx inhibits cell surface gelatinase
activity. Representative fluorescent micrographs of D54-MG cells
plated on coverslips coated with DQ-gelatin in the presence or absence
of 30 nM Cltx, 300 nM Cltx, or 50 µM 1,10-phenanthroline. Cell surface gelatinolytic
activity was assayed as a measure of fluorescence emitted by degrading
gelatin. Untreated cells exhibited significant cell surface gelatinase
activity, which was completely inhibited in the presence of 300 nM Cltx or 50 µM 1,10-phenanthroline. An
intermediate effect was observed with 30 nM Cltx.
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Fig. 6.
Cltx reduces the surface association of MMP-2
and Cltx/MMP-2. a, surface expression of MMP-2 is regulated
by Cltx. Representative fluorescent micrographs of D54-MG cells that
have been treated (bottom panels) with His-Cltx for
30 min at 37 °C. Untreated cells are shown in the top
panels. Nonpermeabilized cells (left panels
top and bottom) reveal the surface expression of
MMP-2, whereas cells permeabilized (right panels
top and bottom) reveal the total amount of MMP-2
expressed by D54-MG cells. Magnification ×40. Cltx treatment reduced
the surface expression of MMP-2. b, filipin prevents
internalization of MMP-2 induced by Cltx. Immunoblots from cell
surface-biotinylated D54-MG glioma cells either treated with Cltx (30 min at 37 °C) or untreated in the presence or absence of filipin (+)
(4 °C for 10 min). Blots were probed with antibodies to MMP-2 or
anti-His antibodies to detect the presence of MMP-2 and His-Cltx,
respectively. Treatment with Cltx enhances the internalization of MMP-2
in the absence of filipin resulting in less membrane-associated MMP-2.
In the presence of filipin, the membrane-associated MMP-2 is much
enhanced and Cltx fails to reduce surface localization of MMP-2.
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Fig. 7.
Cltx inhibits matrigel invasion of glioma
cells. a, His-Cltx inhibits glioma cell migration.
Dose-response curve of D54-MG cells treated with His-Cltx or commercial
Cltx peptide (Alomone) and analyzed by matrigel invasion assay at
24 h post-treatment. Half-maximal inhibition (IC50)
for Cltx was 184 nM. Percent inhibition was calculated as
the decrease in the number of migrated cells normalized to control.
b, His-Cltx inhibition of matrigel invasion is
filipin-sensitive. Normalized percent inhibition of glioma cell
invasion following filipin (5 µg/ml) treatment, with/without His-Cltx
(500 nM) as well as filipin (5 µg/ml) treatment
with/without 1,10-phenanthroline (30 µM). The data
suggest that the reduction in matrigel invasion by Cltx was at least in
parts mediated by a filipin-sensitive mechanism, possibly involving
internalization of MMP-2 via caveoli.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3
integrin, matrix proteins, and its endogenous inhibitor TIMP-2. These
proteins are stoichiometrically balanced in equimolar ratios to
facilitate tumor cell migration, invasion, and control and maintenance
of ECM proteolysis (34, 48, 49). MT1-MMP, a membrane-type MMP activates
MMP-2, and
v
3 integrin promotes the
maturation and release of MMP-2 (49). It has also been demonstrated that MT1-MMP forms a homophilic complex through the hemopexin domain
keeping the MT1-MMP molecules together facilitating pro-MMP-2 activation (50). Integrins have been suggested to localize proteases to
invadopodia (51), cell extensions in the active process of invasion. As
mentioned above, activation of MMP-2 and other MMPs and expression of
MT1-MMP and
v
3 have been shown to
correlate with tumor invasion, neovascularization, and metastasis of
glioma (43) melanoma cells both in vivo and in
vitro (29) and breast cancer (52).
v
3 integrin antibodies were able to
significantly compete for binding of Cltx to recombinant MMP-2. Clearly
future studies need to examine these molecular interactions in greater detail.
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ACKNOWLEDGEMENTS |
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We acknowledge the valuable contributions made by Xiaojin Liu and Joon Wook Chung toward fusion protein constructs. We also acknowledge Lori Coward (Mass spectrometry Facility, University of Alabama at Birmingham) for expertise in mass spectrometric analysis of proteins.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 2RO1-NS-36692 and American Cancer Society Grant RPG-97-083-04-CDD. Operation of the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility is supported in part by National Institutes of Health NCI Core Research Support Grant P30 CA-13148 to the UAB Comprehensive Cancer Center. The mass spectrometer was purchased by funds from National Institutes of Health shared instrumentation Grant S10RR11329.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.
Current address: Dept. of Psychiatry and Behavioral Science,
Stanford University, Palo Alto, CA 94304-5485.
§ To whom correspondence should be addressed: 1719 6th Ave., South CIRC 589, Birmingham, AL 35294-0021. Tel.: 205-975-5805; Fax: 205-975-5518; E-mail: hws@nrc.uab.edu.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M205662200
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; Cltx, chlorotoxin; MMP-2, matrix metalloproteinase-2; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TIMP-2, tissue inhibitor of matrix metalloproteinase-2.
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1. | Prados, M. D., and Levin, V. (2000) Semin. Oncol. 27, 1-10 |
2. | Linskey, M. E. (1997) J. Neuro-Oncol. 34, 5-22[Medline] [Order article via Infotrieve] |
3. | Rooprai, H. K., Rucklidge, G. J., Panou, C., and Pilkington, G. J. (2000) Br. J. Cancer 82, 52-55[CrossRef][Medline] [Order article via Infotrieve] |
4. | Tonn, J. C., Wunderlich, S., Kerkau, S., Klein, C. E., and Roosen, K. (1998) Anti-cancer Res. 18, 2599-2605[Medline] [Order article via Infotrieve] |
5. | Giese, A., and Westphal, M. (1996) Neurosurgery 39, 235-250[Medline] [Order article via Infotrieve] |
6. | Giese, A., Laube, B., Zapf, S., Mangold, U., and Westphal, M. (1998) Anti-cancer Res. 18, 2435-2447[Medline] [Order article via Infotrieve] |
7. | Gladson, C. L. (1999) J. Neuropathol. Exp. Neurol. 58, 1029-1040[Medline] [Order article via Infotrieve] |
8. | Haugland, H. K., Tysnes, B. B., and Tysnes, O. B. (1997) Anti-cancer Res. 17, 1035-1042[Medline] [Order article via Infotrieve] |
9. |
Gutheil, J. C.,
Campbell, T. N.,
Pierce, P. R.,
Watkins, J. D.,
Huse, W. D.,
Bodkin, D. J.,
and Cheresh, D. A.
(2000)
Clin. Cancer Res.
6,
3056-3061 |
10. | Ritter, M. R., Zhou, Q., and Markland, F. S., Jr. (2000) J. Cell. Biochem. 79, 28-37[CrossRef][Medline] [Order article via Infotrieve] |
11. | Schmitmeier, S., Markland, F. S., and Chen, T. C. (2000) Anti-cancer Res. 20, 4227-4233[Medline] [Order article via Infotrieve] |
12. | Sheu, J. R., Yen, M. H., Kan, Y. C., Hung, W. C., Chang, P. T., and Luk, H. N. (1997) Biochim. Biophys. Acta 1336, 445-454[Medline] [Order article via Infotrieve] |
13. |
Soroceanu, L.,
Manning, T. J., Jr.,
and Sontheimer, H.
(1999)
J. Neurosci.
19,
5942-5954 |
14. | DeBin, J. A., and Strichartz, G. R. (1991) Toxicon 29, 1403-1408[CrossRef][Medline] [Order article via Infotrieve] |
15. |
DeBin, J. A.,
Maggio, J. E.,
and Strichartz, G. R.
(1993)
Am. J. Physiol.
264,
C361-C369 |
16. |
Ullrich, N.,
and Sontheimer, H.
(1996)
Am. J. Physiol.
270,
C1511-C1521 |
17. | Soroceanu, L., Gillespie, Y., Khazaeli, M. B., and Sontheimer, H. (1998) Cancer Res. 58, 4871-4879[Abstract] |
18. | Lyons, S. A., O'Neal, J., and Sontheimer, H. (2002) Glia 39, 162-173[CrossRef][Medline] [Order article via Infotrieve] |
19. | Ye, Z. C., and Sontheimer, H. (1996) Neuroreport 7, 2181-2185[Medline] [Order article via Infotrieve] |
20. | Fenster, S. D., Chung, W. J., Zhai, R., Cases-Langhoff, C., Voss, B., Garner, A. M., Kaempf, U., Kindler, S., Gundelfinger, E. D., and Garner, C. C. (2000) Neuron 25, 203-214[Medline] [Order article via Infotrieve] |
21. |
Ye, Z. C.,
Rothstein, J. D.,
and Sontheimer, H.
(1999)
J. Neurosci.
19,
10767-10777 |
22. |
MacFarlane, S. N.,
and Sontheimer, H.
(2000)
J. Neurosci.
20,
5245-5253 |
23. |
Oh, L. Y.,
Larsen, P. H.,
Krekoski, C. A.,
Edwards, D. R.,
Donovan, F.,
Werb, Z.,
and Yong, V. W.
(1999)
J. Neurosci.
19,
8464-8475 |
24. | Sato, H., and Seiki, M. (1996) J. Biochem. (Tokyo) 119, 209-215[Abstract] |
25. | Fridman, R., Toth, M., Pena, D., and Mobashery, S. (1995) Cancer Res. 55, 2548-2555[Abstract] |
26. |
Strongin, A. Y.,
Collier, I.,
Bannikov, G.,
Marmer, B. L.,
Grant, G. A.,
and Goldberg, G. I.
(1995)
J. Biol. Chem.
270,
5331-5338 |
27. | Sawaya, R. E., Yamamoto, M., Gokaslan, Z. L., Wang, S. W., Mohanam, S., Fuller, G. N., McCutcheon, I. E., Stetler-Stevenson, W. G., Nicolson, G. L., and Rao, J. S. (1996) Clin. Exp. Metastasis 14, 35-42[Medline] [Order article via Infotrieve] |
28. | Nakagawa, T., Kubota, T., Kabuto, M., Sato, K., Kawano, H., Hayakawa, T., and Okada, Y. (1994) J. Neurosurg. 81, 69-77[Medline] [Order article via Infotrieve] |
29. |
Hofmann, U. B.,
Westphal, J. R.,
Waas, E. T.,
Becker, J. C.,
Ruiter, D. J.,
and Van Muijen, G. N.
(2000)
J. Invest. Dermatol.
115,
625-632 |
30. |
Nakahara, H.,
Howard, L.,
Thompson, E. W.,
Sato, H.,
Seiki, M.,
Yeh, Y.,
and Chen, W. T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7959-7964 |
31. | Kahari, V. M., and Saarialho-Kere, U. (1999) Ann. Med. 31, 34-45[Medline] [Order article via Infotrieve] |
32. | Ellerbroek, S. M., and Stack, M. S. (1999) BioEssays 21, 940-949[CrossRef][Medline] [Order article via Infotrieve] |
33. | Friedberg, M. H., Glantz, M. J., Klempner, M. S., Cole, B. F., and Perides, G. (1998) Cancer 82, 923-930[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Deryugina, E. I.,
Bourdon, M. A.,
Luo, G. X.,
Reisfeld, R. A.,
and Strongin, A.
(1997)
J. Cell Sci.
110,
2473-2482 |
35. | Planchenault, T., Costa, S., Fages, C., Riche, D., Charriere-Bertrand, C., Perzelova, A., Barlowatz-Meimon, G., and Tardy, M. (2001) Neurosci. Lett. 299, 140-144[CrossRef][Medline] [Order article via Infotrieve] |
36. | Hidalgo, M., Pierson, A. S., Holden, S. N., Bergen, M., and Eckhardt, S. G. (2001) Adv. Intern. Med 47, 159-190[Medline] [Order article via Infotrieve] |
37. | Sawicki, G., Salas, E., Murat, J., Miszta-Lane, H., and Radomski, M. W. (1997) Nature 386, 616-619[CrossRef][Medline] [Order article via Infotrieve] |
38. | Puyraimond, A., Fridman, R., Lemesle, M., Arbeille, B., and Menashi, S. (2001) Exp. Cell Res. 262, 28-36[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Orlandi, P. A.,
and Fishman, P. H.
(1998)
J. Cell Biol.
141,
905-915 |
40. | Cameron, P. L., Liu, C., Smart, D. K., Hantus, S. T., Fick, J. R., and Cameron, R. S. (2002) Glia 37, 275-290[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Pol, A., Lu, A.,
Pons, M.,
Peiro, S.,
and Enrich, C.
(2000)
J. Biol. Chem.
275,
30566-30572 |
42. | Deryugina, E. I., Luo, G. X., Reisfeld, R. A., Bourdon, M. A., and Strongin, A. (1997) Anti-cancer Res. 17, 3201-3210[Medline] [Order article via Infotrieve] |
43. |
Bello, L.,
Lucini, V.,
Carrabba, G.,
Giussani, C.,
Machluf, M.,
Pluderi, M.,
Nikas, D.,
Zhang, J.,
Tomei, G.,
Villani, R. M.,
Carroll, R. S.,
Bikfalvi, A.,
and Black, P. M.
(2001)
Cancer Res.
61,
8730-8736 |
44. | Deryugina, E. I., Bourdon, M. A., Reisfeld, R. A., and Strongin, A. (1998) Cancer Res. 58, 3743-3750[Abstract] |
45. | Kachra, Z., Beaulieu, E., Delbecchi, L., Mousseau, N., Berthelet, F., Moumdjian, R., Del Maestro, R., and Beliveau, R. (1999) Clin. Exp. Metastasis 17, 555-566[CrossRef][Medline] [Order article via Infotrieve] |
46. | Pagenstecher, A., Stalder, A. K., Kincaid, C. L., Shapiro, S. D., and Campbell, I. L. (1998) Am. J. Pathol. 152, 729-741[Abstract] |
47. |
Overall, C. M.,
Tam, E.,
McQuibban, G. A.,
Morrison, C.,
Wallon, U. M.,
Bigg, H. F.,
King, A. E.,
and Roberts, C. R.
(2000)
J. Biol. Chem.
275,
39497-39506 |
48. | Brooks, P. C., Stromblad, S., Sanders, L. C., von Schalscha, T. L., Aimes, R. T., Stetler-Stevenson, W. G., Quigley, J. P., and Cheresh, D. A. (1996) Cell 85, 683-693[Medline] [Order article via Infotrieve] |
49. | Deryugina, E. I., Ratnikov, B., Monosov, E., Postnova, T. I., DiScipio, R., Smith, J. W., and Strongin, A. Y. (2001) Exp. Cell Res. 263, 209-223[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Itoh, Y.,
Takamura, A.,
Ito, N.,
Maru, Y.,
Sato, H.,
Suenaga, N.,
Aoki, T.,
and Seiki, M.
(2001)
EMBO J.
20,
4782-4793 |
51. |
Mueller, S. C.,
Ghersi, G.,
Akiyama, S. K.,
Sang, Q. X.,
Howard, L.,
Pineiro-Sanchez, M.,
Nakahara, H.,
Yeh, Y.,
and Chen, W. T.
(1999)
J. Biol. Chem.
274,
24947-24952 |
52. | Deryugina, E. I., Bourdon, M. A., Jungwirth, K., Smith, J. W., and Strongin, A. Y. (2000) Int. J. Cancer 86, 15-23[Medline] [Order article via Infotrieve] |
53. | Montesano, R., Roth, J., Robert, A., and Orci, L. (1982) Nature 296, 651-653[Medline] [Order article via Infotrieve] |
54. | Tran, D., Carpentier, J. L., Sawano, F., Gorden, P., and Orci, L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7957-7961[Abstract] |
55. |
Skretting, G.,
Torgersen, M. L.,
van Deurs, B.,
and Sandvig, K.
(1999)
J. Cell Sci.
112,
3899-3909 |