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Department of Pathology, Miyazaki Medical College, Miyazaki 889-1692 [K. N., T. I., Y. S., M. K.]; Department of Pathology, Keio University School of Medicine, Tokyo 160-0016 [Y. O.]; and Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo 108 [Y. I., M. S.], Japan
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Active migration (translocation) of tumor cells includes both single
cell locomotion and cohort-type migration (6, 7, 8, 9)
. In
vitro studies of cell migration are predominantly based on the
concept of the single cell locomotion using Boyden chamber-type assays
and scattering assays. However, in human surgical specimens, carcinoma
cells, especially those of well to moderately differentiated types,
frequently invade the stroma as coherent cell nests rather than single
cells (9
, 10)
. A time-lapse videomicroscopic study
in vitro also demonstrated the movement of carcinoma cells
as coherent cell clusters in collagen gels (11)
. We have
called this type of carcinoma cell movement "cohort migration" and
developed an in vitro model to examine the mechanisms
involved in this type of migration (6, 7, 8, 9
, 12
, 13)
. Thus
far, in our model of cohort migration induced by
12-O-tetradecanoylphorbol-13-acetate or HGF/SF, carcinoma
cells migrated keeping close cell-cell contact with one another in the
upper portion of cells, and intercellular gaps became wider in the
lower portion of cells. This gap formation enabled the cells to extend
their leading edges forward onto the substrate beneath the preceding
cells and move (8
, 13)
. The process was also associated
with increased tyrosine phosphorylation of the E-cadherin/catenin
complex, including ß-catenin, in
12-O-tetradecanoylphorbol-13-acetate-induced migration
(6)
, and with decreased amounts of 
-catenin
complexed with E-cadherin in HGF/SF-induced migration
(8)
. In addition, production of FN, especially EDA+FN, by
migrating carcinoma cells themselves was essential for this type of
migration (7)
. Although degradation and remodeling of ECMs
are generally thought to be necessary for cell migration, the role of
MMPs in this cohort migration model has never been explored.
In this study, we examined the expression patterns of MMPs, expecially MT1-MMP and MMP-2, which play a major role in the pericellular degradation of ECMs and their activity during the cohort migration. The data suggest that gelatin matrix is reorganized to suit cell migration via leading-edge-of-front-cell-specific localization of MT1-MMP and MMP-2 during the migration and that the reorganization is essential for this type of migration.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Cell Cultures.
A highly metastatic subline to the liver (L-10) of the human colon
adenocarcinoma cell line RCM-1 obtained by in vivo selection
in nude mice was maintained in GM (for L-10), a 1:1 mixture of RPMI
1640 and Ham’s F-12 (Nissui Seiyaku, Tokyo, Japan) supplemented with
5% FCS (12)
. Another human colon adenocarcinoma cell
line, SW837, obtained from Dainihon Seiyaku (Osaka, Japan), was
maintained in DMEM supplemented with 10% FCS (GM for SW837).
Cell Motility on a Lab-Tek Chamber Slide.
Motility assay was run as described (13)
, with some
modifications. Briefly, L-10 or SW837 cells were seeded into
compartments of an 8-well Lab-Tek tissue culture chamber slide (Nunc,
Naperville, IL; 1.2 x 105 cells
in 0.4 ml of GM) and allowed to attach for 40 h (1 day and
overnight) in standard culture conditions. The cells formed
interlinked, piled-up cell islands on the tissue culture glass
substrate of the Lab-Tek chamber slide. The cells were then exposed to
test medium (serum-free medium or GM with or without 20 ng/ml HGF/SF)
for various periods up to 24 h. Stimulated cells migrated outward
from the cell islands into interisland spaces as coherent cell sheets
one-cell thick. Migration was quantified by counting the migrated cells
in 10 randomly selected high-power fields (x400) after the cells were
fixed with 3.7% formaldehyde in PBS and stained with hematoxylin. All
experiments were performed in duplicate and repeated three times. Means
and SEs of the mean were calculated, and statistical differences were
analyzed using Student’s t test for nonpaired samples.
In experiments on gelatin-coated substratum, the glass substrate of each compartment of 8-well Lab-Tek chamber slides was coated with 200 µl of 1 µg/ml gelatin for 48 h at 4°C (7) . Diffuse coating was confirmed by protein staining. Using these coated chamber slides, cell migration was determined as described above.
In experiments with BB-94 (British Biotech Pharmaceuticals, Oxford, United Kingdom), 10 or 50 µM BB-94 was included in the gelatin solution (1 µg/ml, 200 ml) and used to coat the glass substrate of chamber slides. Coating was done for 48 h at 4°C. Cell migration was induced as described above, except for a 1-h preincuation of cells with 10 or 50 µM BB-94 prior to HGF/SF addition and presence of BB-94 during the whole assay period.
In experiments with TIMP-1 and -2 and the GelA PEX, cells were pretreated with TIMPs (10 µg/ml) or PEX-2 (25 or 50 µg/ml) for 1 h, followed by a 24-h incubation with HGF/SF (20 ng/ml) in the presence of TIMPs or GelA PEX.
Expression and in Vitro Folding of the GelA PEX
Protein.
Expression vector GelA PEX/pET was constructed by cloning a PCR product
of GelA PEX into protein expression vector pET-3a (Stratagene, La
Jolla, CA). The PCR product corresponded to bases 1001–1939
(14)
, and start and stop codons were added at its 5' and
3' ends, respectively. The nucleotide sequences of the GelA PEX product
were confirmed by the dideoxy chain-termination method before cloning.
The vector GelA PEX/pET was transfected into an Escherichia
coli BL21(DE3)pLyS host (Stratagene), and expression and in
vitro folding of the GelA PEX protein were done according to Huang
et al. (15)
. The obtained GelA PEX protein was
concentrated using Diaflo ultrafiltration membranes (YM5; Amicon,
Beverly, MA) and Centriprep 10 (Amicon) and was stored at -20°C
until use.
Scattering Assays.
L-10 and SW837 cells were added to Lab-Tek tissue culture chamber
slides at 5.0 x 103 cells/well in
the presence of 50 µg/ml anti-E-cadherin mAb (HECD-1; Takara) and
allowed to grow for 7 days, after which loosely aggregated cell islands
were obtained. The cells of these loose islands showed scattering in
response to treatment with HGF/SF (20 ng/ml) for 24 h.
Immunofluorescent Staining.
After cell motility assays were done as described above,
immunofluorescent staining of cells was performed as described
(7)
using mouse mAb against human MT1-MMP (114-6G6; Fuji
Chemical Industries), MMP-2 (75-7F7; Fuji Chemical Industries),
E-cadherin or vinculin (VG61110; Affinity Research Products,
Nottingham, United Kingdom) as primary antibodies. For E-cadherin
immunostaining, all of the solutions contained 1 mM
CaCl2.
Biotin Labeling of Coated Gelatin (Double Staining with
Phalloidin).
Gelatin-coating of Lab-Tek chamber slide substratum was done as
described above. After rinsing with cold PBS twice, the gelatin was
biotinylated by incubating in PBS containing water-soluble
sulfo-N-hydroxysuccinimide-biotin (2 mg/ml; Pierce
Chemical Co., Rockford, IL) for 2 h at 4°C, followed by washing
with PBS three times and then rinsing in PBS overnight. L-10 cells
(1.2 x 105 cells/well) were added
and allowed to attach for 40 h, and migration assays were run as
described above. After the cells were fixed with 2%
paraformaldehyde/0.1 M PB for 30 min at 4°C and
permeabilized with 0.1% Triton X-100/PBS for another 30 min at 4°C,
the biotinylated gelatin was visualized with FITC-conjugated
streptavidin (Sigma Chemical Co.) for 2 h at room
temperature. After rinsing with PBS four times, the cells were
stained with rhodamine-phalloidin in PBS (Molecular Probes, Eugene, OR)
for 20 min at room temperature.
To obtain footprints of migrating cell sheets, after cohort migration was induced on gelatin-coated substratum, cells were treated with 0.5 mM EDTA/PBS, followed by tapping of chamber slides by fingers. About 30% of cell islands with migrating cell sheets were removed by the treatment, and the remaining footprints were demonstrated by visualization of biotinylated gelatin matrix, as described above.
RT-PCR.
mRNA expression for MT1-MMP and MMP-2 in L-10 cells was analyzed using
RT-PCR, as described (8)
. Annealing was done at 60°C for
MT1-MMP and 58°C for MMP-2. The sequences of the forward and reverse
primers for MMP-2 are 5'-ACCTGGATGCCGTCGTGGAC-3' (1800–1819) and
5'-TGTGGCAGCACCAGGGCAGC-3' (2228–2247), respectively, and the PCR
product size is 447 bp (14)
. The sequences of the forward
and reverse primers for MT1-MMP are 5'-CCCTATGCCTACATCCGTGA-3' and
5'-TCCATCCATCACTTGGTTAT-3', respectively, and are complementary to
bases 598–617 and 1129–1148 (3)
. The PCR product size is
550 bp.
Preparation of CM and Zymography.
After L-10 cells (5.0 x 105 cells
in 2 ml of GM) were added and cultured in 35-mm tissue culture dishes
(Becton Dickinson, Lincoln Park, NJ) for 24 h, the cells were
rinsed in serum-free media three times, then incubated in 1.0 ml of
serum-free GM with or without HGF/SF (20 ng/ml) for another 24 h.
CM harvested from these cultures was centrifuged twice (110 x g, 5 min; and 4000 x g,
20 min) to remove cellular debris, concentrated by 8-fold using
centricon 10 (Amicon) and subjected to zymography using gelatin as a
substrate, as described previously (16)
.
Immunoblotting.
After L-10 cells were treated with HGF/SF (20 ng/ml) for 24 h,
they were then lysed by boiling for 5 min in 2% SDS, 0.1 M
dithiothreithol/1 M Tris-HCl (pH 6.8), followed by
centrifugation at 11,000 x g for 5 min.
These cell extracts and CM obtained as above were subjected to
immunoblotting, as described (8)
, using mouse mAb to human
MMP-2 (75-7F7; Fuji Chemical Industries) or MT1-MMP (114-1F2; Fuji
Chemical Industries) as primary antibodies.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. On immunoblots, stimulation of MMP-2
production with HGF/SF treatment (20 ng/ml) was shown, whereas the
MT1-MMP expression was not altered. Zymograpy of L-10 cell CM revealed
the presence of both pro- (Mr
68,000) and active (Mr 62,000)
forms of MMP-2 (Fig. 1C)
. The amounts of samples loaded for
zymography were the same as those for immunoblotting, but gel lysis in
zymography was so sensitive that a less amount of pro- and active MMP-
2 in L-10 cell CM without HGF/SF treatment could cause lysis of gelatin
substrate like that by CM with HGF/SF treatment.
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, a). When stimulated with HGF/SF (20 ng/ml,
24 h), L-10 cells migrated outward from the cell islands as
coherent cell sheets one-cell thick (Fig. 2A
, c and e), as described previously (14)
. In the
migrating cell sheets, MT1-MMP was demonstrated only at their fronts,
including the frontmost first to second rows of cells, with the
following migrating cells being negative (Fig. 2A
, d and f). At a higher magnification, expression
of MT1-MMP was observed predominantly in the leading edges of the
frontmost cells, accompanied by some staining in perinuclear regions
(Fig. 2A
, b). Negative control with nonimmune
mouse IgG did not show any immunofluorescent staining (data not shown).
MMP-2 was also demonstrated specifically in the frontmost cells of
migrating cell sheets, especially at their leading edges (Fig. 2A
, g and h).
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, i and j). Vinculin also
successfully demonstrated focal adhesion plaques in both front and
following cells of the migrating cell sheets (data not shown). These
results clearly showed that mAbs can reach to not only frontmost cells
but also following cells of the migrating cell sheets.
Expression of MT1-MMP and MMP-2 in Scattering L-10 Cells.
As another control, we induced scattering of L-10 cells by stimulating
cell migration in the presence of anti-E-cadherin antibody and examined
how MT1-MMP and MMP-2 were expressed in the scattering cells.
Individual scattering L-10 cells expressed both MT1-MMP and MMP-2 in
their leading edges and also within the cytoplasm, especially
perinuclear regions (Fig. 2B
, a–d).
The front cell-specific pattern of expression observed during cohort
migration was lost.
Expression of MT1-MMP and MMP-2 in SW837 Cells.
To examine whether front cell-specific expression of MT1-MMP and MMP-2
is unique only to L-10 cells or can be seen in other cell lines,
another human colon carcinoma cell line (SW837) was used for an
immunolocalization study. This cell line also shows cohort migration in
response to HGF/SF treatment (8)
, and its expression of
MT1-MMP and MMP-2 was demonstrated by RT-PCR (data not shown). When
cohort migration was induced with HGF/SF treatment, MT1-MMP (Fig. 2C
, a and b) and MMP-2 (Fig. 2C
, c and d) were demonstrated
predominantly in the frontmost cells or sometimes in the first and
second rows of cells at the front of the migrating cell sheets. In
contrast, when scattering was induced by treament with HGF/SF in the
presence of anti-E-cadherin antibody, all scattering SW837 cells
expressed MT1-MMP (Fig. 2C
, e and f)
and MMP-2 (Fig. 2C
, g and h), without
preferential localization in the front cells.
Degradation of Gelatin Matrix by Migrating Cells.
Using gelatin as a substrate, we studied whether MT1-MMP and MMP-2
expressed at the front of the migrating cell sheets cause ECM
degradation. Gelatin labeled with biotin was visualized with
avidin-conjugated FITC, and, at the same time, cytoplasmic actin
filaments were demonstrated with rhodamine-phalloidin. At the sites of
leading edges of the front cells of migrating cell sheets, limited
gelatinolysis and consequent partial removal of gelatins occurred,
causing radially arrayed lysis of gelatin matrix at the outermost
portions (Fig. 3, a–d
). Thin, also radially arrayed gelatin
matrix was left in between the lysed portions. At the base of the
radially arrayed gelatinolysis, an arcuate band-like gelatin matrix was
also left beneath the leading edges.
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An Essential Role of Gelatin Degradation in Cohort Migration.
We finally investigated whether the above organized degradation of
gelatin matrix was necessary for cohort migration of carcinoma cells.
BB-94, a synthetic inhibitor for MMPs, inhibited HGF/SF-induced cohort
migration of L-10 cells in a dose-dependent manner: approximately 34%
and 78% inhibition at 10 µM and 50 µM,
respectively (Fig. 4)
. Both TIMP-1 and TIMP-2 also inhibited the migration, although TIMP-2
seemed to be more effective than TIMP-1: approximately 72% inhibition
by TIMP-1 and 82% inhibition by TIMP-2. Furthermore, GelA PEX
effectively inhibited the migration: approximately 60% inhibition at
25 µg/ml and 74% inhibition at 50 mg/ml. Taken together, these
results indicate that MMP-2 activated on the cell surface by MT1-MMP
plays a major role in cohort migration on gelatin matrix.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Although the precise mechanisms involved in the front cell-specific
localization of MT1-MMP and MMP-2 are currently unknown, there are two
possibilities: their localization is regulated (a) at the
gene expression level or (b) at the protein level. In the
former, only front cells may predominantly express mRNAs for MT1-MMP
and MMP-2. Gene expression of MT1-MMP is reported to decrease in
confluent cultures of mouse mammary gland epithelial cells
(17)
, suggesting the regulation of MT1-MMP expression by
cell-cell contact in normal epithelial cells. They also suggested the
presence of translational control mechanisms for MT1-MMP expression
because MT1-MMP immunoreactivity disappeared very early in confluency
whereas MT1-MMP mRNA levels started to decline a few days later when
cell cultures reached confluence. These possibilities are now under
investigation using in situ hybridization. Another
possibility, regulation of MT1-MMP localization at the protein level,
is suggested in single-cell locomotion: invading human melanoma cells
show the accumulation of MT1-MMP at the invadopodia, which are
specialized membrane extensions into the FN-coated gelatin matrix
(5)
. However, cytoplasmic docking systems supporting this
localization are still unknown. MMP-2 is reported to bind to vß3
integrin (18)
or MT1-MMP (2
, 19
, 20)
on cell
surfaces, and in breast carcinoma cells the cellular binding of MMP-2
reduces in confluent cultures compared with that in sparse cultures
(21)
. Taken together, the presence of abundant cell-cell
contact as in confluent cultures seems to suppress MT1-MMP expression
and MMP-2 binding, whereas specialized cell-ECM contact sites
facilitate localization of these enzymes. In our study, it is possible
that immunolocalization of MT1-MMP and MMP-2 was detectable positively
only when they were aggregated to some extents and the enzymes that
were sparsely distributed on the cell surface could not be detected.
Thus, the two enzymes may be concentrated in the large leading edges of
the front cells of migrating cell sheets but not in short cell
protrusions of the following migrating cells that are extended forward
beneath the preceding cells (13)
. This difference may be
caused by cell-cell contact all around the following cells and more
abundant cell-ECM interactions in the front cells. Because MT1-MMP was
distributed diffusely as small dots in the leading edges, its
localization was different from that of focal adhesion plaques that
were shown as vinculin-positive staining. Radial array of linear
gelatinolytic zones beneath the leading edges of front cells was
similar to the arrangement of actin in the leading edges that we
previously showed (12)
. Localization of the MT1-MMP/MMP-2
complex might somehow be related to actin distribution.
In general, ECM is thought to be a kind of barrier for tumor cell invasion, and more or less its cleavage and removal are necessary for tumor cells to migrate (1) . At the same time, however, ECM components provide cells with good substrate to move on. In our study, removal of gelatin matrix at the leading edges of the front cells of migrating cell sheets was not random or complete. Instead, it was performed in a very coordinate and organized manner, leaving radially arrayed gelatin matrix at the frontmost part. This limited and organized clearing of gelatin matrix was essential for cell migration because MMP inhibitors efficiently inhibited migration. Thus, an important role of MMP is not just to remove ECM but to rearrange it to suit cell migration. We previously showed that synthesis and deposition of FN, especially EDA+FN, by migrating carcinoma cells were important for cohort-type migration (7) , and production of this EDA+FN was stimulated by HGF/SF treatment (22) . Because EDA+FN deposition occurs around and beneath the leading edges of front migrating cells (7) and gelatin is removed orderly, rearrangement of ECM by cells for cell migration may include both partial removal of preexisting matrix and the addition of newly synthesized matrix components. MMPs may play a role in the reorganization of the preexisting and newly deposited matrix components to suit cell migration.
The proteinases, which can cleave ECM components on the cell surfaces, may be the most suitable enzymes to rearrange the pericellular matrix. The MT1-MMP/MMP-2 and urokinase-type plasminogen activator/plasmin systems are the most potent examples of those. In our study, the inhibitors specific for MMPs inhibited cohort migration on the gelatin matrix by 70–80%, suggesting the predominant involvement of MMPs in the migration. MT1-MMP not only activates proMMP-2 but also degrades ECM components such as gelatin, FN, and collagen (23 , 24) , and digestion of collagen matrix by MT1-MMP rather than a MMP-2-mediated effect is reported to play a predominant role in branching tubule formation by Madin-Darby canine kidney epithelial cells in the three-dimensional cultures in collagen matrix (25) . Chinese hamster ovary cells transfected with MT1-MMP caused subjacent proteolysis of a gelatin film due to cell surface MT1-MMP rather than via activated MMP-2 (24) . In our study, however, GelA PEX effectively inhibited cohort migration on gelatin matrix. GelA PEX, the carboxyl-end domain of MMP-2, is involved in the trimolecular complex formation of MT1-MMP, TIMP-2, and MMP-2 on the cell surfaces (19 , 20) and competitively inhibits MMP-2 activation by MT1-MMP (2 , 19) . Thus, MMP-2 that is activated by MT1-MMP on the cell surfaces seems to play a major role during the cohort migration. Effective inhibition by both TIMP-1 and TIMP-2 also supports this hypothesis because MMP-2 is known to be inhibited by these TIMPs, whereas MT1-MMP is inhibited by TIMP-2 but not by TIMP-1 (24) . As shown in our study, in human glioma and fibrosarcoma cells transfected with cDNA encoding MT1-MMP, their ability to contract collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of proMMP-2 and cell surface association of activated MMP-2 (2) . Soluble MMP-2 failed to affect gel contraction. Furthermore, in human melanoma cells, the presence of active MMP-2 just on the cell surfaces was not enough, but more specialized localization of MT1-MMP and MMP-2 to the invasion front of cells (invadopodia) was essential for their invasion (5) . In cohort migration, the spacial organization of MT1-MMP/MMP-2 into the invasion front of the whole migrating cell sheets occurs. The disturbance of this organization might lead to prevention of efficient tumor invasion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, Sports and
Culture, Japan (10670212) and a Grant-in-Aid for Research Project from
Miyazaki Medical College. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Miyazaki Medical College,
Miyazaki 889-1692, Japan.
3 The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane-type 1 MMP; MMP-2, Mr 72,000 type IV collagenase/gelatinase A; TIMP, tissue inhibitor of metalloproteinase; HGF/SF, hepatocyte growth factor/scatter factor; CM, conditioned medium; GM, growth medium; FN, fibronectin; EDA, extradomain A of FN; EDA+FN, EDA-containing FN; GelA PEX, the hemopexin-like domain of MMP-2; RT-PCR, reverse-transcription-PCR; ECM, extracellular matrix; mAb, monoclonal antibody.
Received 12/28/99. Accepted 5/11/00.
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REFERENCES |
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vß3. Cell, 85: 683-693, 1996.[Medline]
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M. Toth, M. M. Bernardo, D. C. Gervasi, P. D. Soloway, Z. Wang, H. F. Bigg, C. M. Overall, Y. A. DeClerck, H. Tschesche, M. L. Cher, et al. Tissue Inhibitor of Metalloproteinase (TIMP)-2 Acts Synergistically with Synthetic Matrix Metalloproteinase (MMP) Inhibitors but Not with TIMP-4 to Enhance the (Membrane Type 1)-MMP-dependent Activation of Pro-MMP-2 J. Biol. Chem., December 22, 2000; 275(52): 41415 - 41423. [Abstract] [Full Text] [PDF]
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D. V. Rozanov, E. I. Deryugina, B. I. Ratnikov, E. Z. Monosov, G. N. Marchenko, J. P. Quigley, and A. Y. Strongin Mutation Analysis of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP). THE ROLE OF THE CYTOPLASMIC TAIL CYS574, THE ACTIVE SITE GLU240, AND FURIN CLEAVAGE MOTIFS IN OLIGOMERIZATION, PROCESSING, AND SELF-PROTEOLYSIS OF MT1-MMP EXPRESSED IN BREAST CARCINOMA CELLS J. Biol. Chem., July 6, 2001; 276(28): 25705 - 25714. [Abstract] [Full Text] [PDF]
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A. M. Belkin, S. S. Akimov, L. S. Zaritskaya, B. I. Ratnikov, E. I. Deryugina, and A. Y. Strongin Matrix-dependent Proteolysis of Surface Transglutaminase by Membrane-type Metalloproteinase Regulates Cancer Cell Adhesion and Locomotion J. Biol. Chem., May 18, 2001; 276(21): 18415 - 18422. [Abstract] [Full Text] [PDF]
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