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From the Department of Molecular Immunology and
Pathology*
and the Department of Virology and
Oncology,¶
Cancer Research Institute, and the
Department of Neurosurgery,
School of
Medicine, Kanazawa University, Kanazawa, the Department of
Pathology,
School of Medicine, Keio
University, Tokyo, the Biopharmaceutical
Department,§
Fuji Chemical Industries, Ltd.,
Takaoka, and Department of Cancer Cell Research,||
Institute of Medical Science, University of Tokyo, Tokyo, Japan
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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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A characteristic of malignant astrocytic tumors is their ability to infiltrate and invade the surrounding normal brain tissue. Glioma invasion involves cell adhesion and proteolytic degradation of the ECM,17 and MMP-2 has been shown to correlate with the invasive activity of the human glioma cell lines.18,19 Active MMP-2 species are also detected in human malignant astrocytic tumor tissues,12,20 and MT1-MMP expression is known in the tumors.12 However, these studies determined neither the relationship between the expression and pro-MMP-2 activation nor the expression of MT2-MMP and MT3-MMP in the tumors. Thus, the question of which MT-MMP is responsible for pro-MMP-2 activation in astrocytic tumors remains unanswered. In addition, tissue localization of the activity has not been studied in the astrocytic tumor tissues.
In the present study, we examined the expression of MT1-, MT2-, and MT3-MMPs, correlation between their expression and pro-MMP-2 activation, and tissue localization of these MMPs and gelatinolytic activity in the human astrocytic tumor tissues. The results suggest that overexpression of pro-MMP-2 and its activation mediated by MT1-MMP and MT2-MMP are important in the invasive behavior of the malignant astrocytic tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh human brain tumor tissues were obtained from 35 patients with astrocytic tumor and 4 patients with metastatic brain tumor (metastatic lung adenocarcinoma) who underwent therapeutic removal of brain tumors. Normal brain tissues were obtained from 5 patients undergoing temporal lobectomy for the epilepsy. The samples were snap-frozen in liquid nitrogen immediately after surgical removal and stored at -80°C to obtain total mRNA and proteins. They were also fixed with periodate-lysine-paraformaldehyde or 4% paraformaldehyde fixative for 18 to 24 hours at 4°C for immunohistochemical study. Histological diagnosis was made by standard light-microscopic evaluation of the sections stained with hematoxylin and eosin (H&E). The classification of human brain tumors used in this study is based on the revised World Health Organization criteria for tumors of the central nervous system.21 A total of 35 astrocytic tumors consisted of 9 low-grade astrocytomas, 9 anaplastic astrocytomas, and 17 glioblastomas. All of the tumor tissues were obtained at primary resection, and none of the patients had been subjected to chemotherapy or radiation therapy before resection.
Tissue Homogenates and Sandwich Enzyme Immunoassay for MMP-2
Tissue samples of the brain tumors (35 astrocytic tumor and 4 metastatic brain tumor cases) and control normal brain (5 cases) stored at -80°C were homogenized in 50 mmol/L Tris/HCl buffer, pH 7.5, containing 0.15 mol/L NaCl, 10 mmol/L CaCl2, and 0.05% Brij35 on ice. The homogenates were then centrifuged at 4°C for 20 minutes at 10,000 x g, and protein concentrations in the supernatants were determined by the dye-binding method according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Concentrations of MMP-2 in the tissue homogenates were measured in the corresponding sandwich enzyme immunoassay (EIA) system for MMP-2 as described previously.22 The EIA system measures pro-MMP-2 and its complex form with tissue inhibitor of metalloproteinases-2 (TIMP-2) but not active MMP-2 species. The values, nmol/g of protein, were determined by using the molecular weight of pro-MMP-2, 70,930, which was calculated by amino acid sequence.
Gelatin Zymography
Gelatinolytic activity in the above-mentioned tissue homogenates was examined by gelatin zymography. The supernatants (50 µg of protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 0.2% gelatin-containing gels as described previously.23 The supernatants were incubated at 37°C for 30 minutes in SDS sample buffer without reducing agent and then electrophoresed on 9% polyacrylamide gels at 4°C. After electrophoresis, gels were washed in 2.5% Triton X-100 to remove SDS, incubated for 36 hours at 37°C in 50 mmol/L Tris/HCl, pH 7.5, containing 0.15 mol/L NaCl, 10 mmol/L CaCl2, and 0.02% NaN3, and then stained with 0.1% Coomassie brilliant blue R250. Ratios of pro-MMP-2 activation were estimated by computer-assisted densitometric scanning of Mr 62,000 and Mr 68,000 proteolytic bands, which correspond to active and latent species of MMP-2, respectively.23
RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated from surgical specimens by ISOGEN (Nippon Gene, Toyama, Japan). RNA extracted was treated with RNAse-free DNAse (Boehringer Mannheim, Mannheim, Germany) to eliminate DNA contamination in the samples and converted to a single-stranded cDNA using a random hexamer of oligonucleotide (Takara, Otsu, Japan). Randomly primed cDNAs were prepared from 5 µg of total RNA by M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) followed by PCR amplification. Nonradioisotopic quantitative RT-PCR was done as previously described.24 cDNAs obtained from surgical specimens were amplified using specific primers of human MT1-MMP (forward primer 5'-TCGGCCCAAAGCAGCAGCTTC-3', reverse primer 5'-CTTCATGGTGTCTGCATCAGC-3'), MT2-MMP (forward primer 5'-CAG-CCCAGCCGCCATATGTC-3', reverse primer 5'-CTTTCACTCGTACCCCGAAC-3'), MT3-MMP (forward primer 5'-ACAGTCTGCGGAACGGAGCAG-3', reverse primer 5'-GTCAATTGTGTTTCTGTCCAC-3'), and GAPDH (forward primer 5'-CCACCCATGGCAAATTCCATGGCA-3', reverse primer 5'-TCTAGACGGCAGGTCAGGTCCACC-3'). PCR conditions for MT-MMP amplifications were 20 to 36 cycles at 2-cycle intervals, at 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute, followed by incubation at 72°C for 3 minutes. The conditions for GAPDH amplifications were the same as those for MT-MMPs except for 1 minute of annealing at 60°C. The products were electrophoresed on 3% agarose gels, including 0.1 µg/ml ethidium bromide. The intensity of ethidium bromide fluorescence was measured using a charge-coupled device imaging system (FAS II, TOYOBO, Tokyo, Japan) and Digital Image File Fujix DF-20 (Fuji Photo Film, Tokyo, Japan). The reaction cycle/PCR product of each reaction mixture was plotted on semilogarithmic graphs for each sample. To obtain the control curves, serial dilutions of MT-MMP and GAPDH plasmid cDNAs6-8 were prepared, and PCR was performed in the same way as surgical specimens, as we have previously described.16 The sample concentration/PCR products of each reaction mixture were plotted on semilogarithmic graphs. To standardize the condition of gel staining, a constant amount of control DNA marker (Promega, Madison, WI) was electrophoresed every time. The PCR procedure was performed at least three times for each sample.
Northern Blot Analysis
To confirm the quantification of RT-PCR, six specimens of glioblastoma and three specimens of normal brain were used for Northern blotting. The RNA samples (30 µg/lane) were electrophoresed on 1% agarose gels containing 2.2 mol/L formaldehyde and transferred onto Hybond N+ membranes (Amersham International, Tokyo, Japan). The membranes were hybridized with 32P-labeled probes for MT1-MMP (a 1.2-kb cDNA fragment corresponding to nucleotides 1647 to 2889),6 MT2-MMP (a 1.2-kb fragment to nucleotides 273-1526),7 MT3-MMP (a 2.1-kb fragment to nucleotides 1–2107)8 and GAPDH as previously described.6,8 As a control, total RNA was extracted from OSC-19 cells (a highly metastatic oral squamous cell carcinoma cell line), which are known to express MT-MMPs,8,15 and the samples were processed in a similar way. The blotted membranes were scanned by Bioimage analyzer BAS 1000 (Fuji Photo Film).
In Situ Hybridization
To verify the origin of cells expressing MT1-MMP and MT2-MMP mRNA, the glioblastoma samples (five cases) that showed MT1-MMP and MT2-MMP expression by Northern blotting were used for in situ hybridization by modification of the methods previously described.25 Briefly, the cDNA fragments encoding MT1-MMP nucleotides 2483 to 2884 (401 bp) and MT2-MMP nucleotides 1249 to 1716 (467 bp) were subcloned into Bluescript KS (Stratagene, La Jolla, CA), and sense and antisense digoxigenin-labeled RNA probes were prepared with T3 or T7 RNA polymerase using DIG RNA labeling kit (Boehringer Mannheim). Paraffin sections of the tissues, which were treated with 10 µg/ml proteinase K (Promega Biotec, Oakland, CA) and 0.0025% acetic anhydride (Eastman Kodak, Rochester, NY) in 0.1 mol/L triethanolamine, pH 8.0 (Eastman Kodak), were hybridized with antisense RNA or sense RNA for ~12 hours at 50°C. The slides were then treated with 20 µg/ml RNAse A and washed under stringent conditions (2X SSC, 0.5X SSC, and 0.1X SSC, twice for 30 minutes each at 50°C). They were incubated with alkaline-phosphatase-conjugated Fab fragments from sheep anti-digoxigenin antibody (1:1000 dilution; Boehringer Mannheim) at room temperature for 1 hour. Color was developed with nitro blue tetrazolium chloride (Boehringer Mannheim) and 5-bromo-4-chloro-3-idolyl phosphate (DIG nucleic acid detection kit, Boehringer Mannheim). Counterstaining was performed with safranin O. The regions of sequence used to produce riboprobes were selected carefully to avoid stretches of sequence that might cross-hybridize. A computer scan for regions of homology to the MT1-MMP and MT2-MMP probes to the published cDNA sequences for other MMPs, including MT-MMPs, showed that the MT1-MMP and MT2-MMP probes had the highest homology to MT2-MMP (45%) and MT1-MMP (36%), respectively. However, no cross-hybridization to these MT-MMPs was observed using the hybridization protocol described in the present study.
Antibodies, Immunohistochemistry, and Immunoblotting
The monoclonal antibodies to MT1-MMP and MT2-MMP were developed by use of the synthetic peptides, REVPYAYIREGHEK (corresponding to the amino acids at positions 160 to 173 in human MT1-MMP) and DTDNFQLPEDDLRG (corresponding to the amino acids at positions 281 to 294 in mouse MT2-MMP), respectively, and provided by Dr. Kazushi Iwata at Fuji Chemical Industries (Takaoka, Japan). The monospecific reactivity of the antibodies and their applicability to immunohistochemistry were determined previously by us.15 The paraffin sections were immunostained using the monoclonal antibodies to MT1-MMP (30 µg/ml; clone 114-6G6) and MT2-MMP (30 µg/ml; clone 162-22G5) or nonimmune mouse IgG (30 µg/ml). After reactions with biotinylated horse IgG to mouse IgG (Vector Laboratories, Burlingame, CA) and an avidin-biotin-peroxidase complex (DAKO, Glostrup, Denmark), the color was developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co., St. Louis, MO). Immunostaining for MMP-2 was performed in a similar way using a monoclonal antibody to MMP-2 (2 µg/ml; clone 75-7F7) that recognizes both zymogen and active forms.22 Counterstaining was performed with hematoxylin.
Supernatants of the homogenates (50 µg/lane) from six glioblastomas and three normal brains were resolved by SDS-PAGE (10% total acrylamide) under reduction and transferred onto nitrocellulose filters (Amersham International, Little Chalfont, UK). The filters were reacted with 20 µg/ml monoclonal antibodies to MT1-MMP (clone 114-6G6) and MT2-MMP (clone 162-22G5) or 20 µg/ml nonimmune mouse IgG. After reactions with biotinylated horse IgG to mouse IgG (Vector Laboratories) and an avidin-biotin-peroxidase complex (DAKO), the color was developed with 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) as described previously.2
In Situ Zymography
The fresh specimens of the glioblastoma and normal brain tissues (five and two cases, respectively) were embedded without fixation in Tissue-Tek OCT compound (Miles, Elkhart, IN). Serial frozen sections were made by a cryostat (MICROM, Walldorf, Germany) and mounted onto the gelatin films that were coated with 7% gelatin solution (Fuji Photo Film) or slide glasses. The films with sections were incubated for 24 hours at 37°C in a moisture chamber and stained with 1.0% Amido Black 10B. The gelatin in contact with the proteolytic areas of the sections was digested, and thus zones of enzymic activity were indicated by negative staining. The digested areas in the sections were compared with the serial sections stained with H&E. As a control, glioblastoma tissues were incubated in Dulbecco's modified Eagle's medium containing 0.2% lactalbumin hydrolysate with or without 50 µmol/L BB94 (British Biotech Pharmaceuticals, Oxford, UK) for 3 hours at 37°C, and the frozen sections were treated in a similar way as described above.
Statistics
Statistical analyses were performed using the 
2
test and the two-tailed Mann-Whitney U test. P
values less than 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To measure the amounts of pro-MMP-2 produced by the tumor tissues,
the EIA system was applied to the supernatants of the tissue
homogenates. As shown in Figure 1
, the
production levels of pro-MMP-2 in the anaplastic astrocytomas
(0.021 ± 0.010 nmol/g weight; n = 9) and
glioblastoma samples (0.038 ± 0.023; n = 17) were
remarkably higher than those in the low-grade astrocytomas (0.012
± 0.008; n = 9; P < 0.05 and
P < 0.01, respectively), control normal brain tissues
(0.006 ± 0.003; n = 5; P <
0.01), and metastatic brain tumors (0.010 ± 0.004;
n = 4; P < 0.05).
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Pro-MMP-2 activation in the supernatants was analyzed by gelatin
zymography. Pro-MMP-2 of Mr 68,000 was detected
in all of the samples examined, and the levels in glioblastomas and
anaplastic astrocytomas appeared to be higher than those of other
samples (Figure 2A)
, confirming the EIA
data described above. On the other hand, the active species of
Mr 62,000 was found in all of the glioblastoma
and metastatic brain tumor samples (17/17 cases and 4/4 cases,
respectively) and in 67% of the anaplastic astrocytomas (6/9 cases),
but it was absent in the normal brain and low-grade astrocytoma samples
(Figure 2A)
. Computer-assisted image analyses of the proteolytic bands
indicated that the activation ratio of pro-MMP-2 (the ratio of the
active form to pro-MMP-2 and active forms) is significantly higher in
the glioblastomas (15 ± 6%; n = 17) than in the
anaplastic astrocytomas (5 ± 4%; n = 9;
P < 0.01) or metastatic brain tumors (6 ± 3%;
n = 4; P < 0.01) (Figure 2B)
.
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To evaluate the expression levels of each MT-MMP, quantitative
analyses of the mRNA expression of MT-MMPs and GAPDH were performed
according to the modification of the methods reported by our recent
study.16
When RT-PCR for MT-MMPs and GAPDH was carried out
using total RNA from glioblastomas by running for 20 to 36 cycles at an
interval of 2 cycles, the products emerged between 22 and 26 cycles,
increased exponentially with cycles up to 30 to 34, and then reached a
plateau (data not shown). Thus, PCR amplification was set at 28 cycles,
and calibration lines for cDNA concentrations of MT-MMPs and GAPDH were
obtained using serial dilutions of plasmid cDNAs for MT-MMPs and GAPDH
(Figure 3A)
. Fluorescence intensity of
each PCR product was proportional to the amounts of cDNAs used as
templates (Figure 3B)
, and calibration lines obtained for cDNAs of
MT-MMPs and GAPDH were used for further calculation of MT-MMP/GAPDH
cDNA molar ratios, which represent MT-MMP/GAPDH mRNA ratios in surgical
specimens. We analyzed each sample at least three times by this method,
and the difference in the obtained values was less than 2%.
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), whereas no hybridization was
detected in the normal brain samples (data not shown). MT2-MMP mRNA was
also found in glioblastomas (5/6 cases, Figure 4A
). However, MT3-MMP
mRNA was undetectable in all of the specimens examined (0/6 cases)
except for the positive control (data not shown). As shown in Figure 4, B and C
, direct correlations were obtained between the results from
RT-PCR and Northern blot for MT1-MMP/GAPDH and MT2-MMP/GAPDH. Thus, we
used the PCR method for the quantification of mRNA expression for
MT-MMPs in the tumor and normal brain tissues.
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.
MT2-MMP was also expressed in 71% of the glioblastomas (12/17 cases)
and in 100% of the metastatic brain tumors (4/4 cases), but it was
detected in only 22% of the anaplastic astrocytomas (2/9 cases) and
0% of the low-grade astrocytomas (0/9 cases) and normal brains (0/5
cases) (Figure 5)
. On the other hand, MT3-MMP mRNA was detectable in
71% of the glioblastomas (12/17 cases), 44% of the anaplastic
astrocytomas (4/9 cases), 67% of the low-grade astrocytomas (6/9
cases), and 100% of the normal brains (5/5 cases), but not in
metastatic brain tumors (0/4 cases) (Figure 5)
. The quantitative RT-PCR
indicated that the levels (MT-MMP/GAPDH ratios) of both MT1-MMP and
MT2-MMP are significantly higher in the glioblastoma samples (0.49
± 0.27 and 0.26 ± 0.26, respectively; n = 17)
than in the anaplastic astrocytomas (0.07 ± 0.14,
P < 0.01, and 0.06 ± 0.12, P <
0.05, respectively; n = 9), low-grade astrocytomas (0
and 0, respectively; n = 9; P < 0.01)
and normal brain tissues (0, P < 0.01, and 0,
P < 0.05, respectively; n = 5) (Figure 6, A and B)
. However, the mean expression
level of MT3-MMP was at least 50-fold lower compared with that of
MT1-MMP and MT2-MMP, and there was no correlation between the MT3-MMP
expression and histological grades of the astrocytic tumors (Figure 6C)
.
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When the activation ratio of pro-MMP-2 was plotted against mRNA
expression levels of MT1-MMP or MT2-MMP in each case, it showed direct
correlations with the expression of MT1-MMP (r =
0.893, P < 0.01) and MT2-MMP (r
= 0.792, P < 0.01) (Figure 7, A and B)
. In addition, the correlation
was stronger when the activation ratio was compared with the expression
level of MT1-MMP plus MT2-MMP in each case (r =
0.953, P < 0.01) (Figure 7C)
. However, no correlation
was observed between the activation and MT3-MMP expression (data not
shown).
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Cells expressing MT1-MMP and MT2-MMP mRNA in the glioblastomas
were identified by in situ hybridization. The signals for
MT1-MMP and MT2-MMP were observed with the antisense RNA probes mainly
in the neoplastic astrocytes (5/5 cases and 5/5 cases, respectively)
(Figure 8, A and C)
and some endothelial
cells. The sense probes gave only a background signal in the
glioblastoma tissues (Figure 8, B and D)
.
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MT1-MMP was immunolocalized predominantly to the neoplastic
astrocytes in all of the glioblastoma cases (17/17 cases; Figure 9A
). In anaplastic astrocytomas and
metastatic brain tumors, some atypical cells were weakly immunostained,
but no staining was seen in the normal brains or low-grade astrocytomas
(data not shown). MT2-MMP was also immunostained predominantly in the
glioma cells (12/17 cases; Figure 9B
). Endothelial cells of blood
vessels in the glioblastoma tissues were occasionally immunostained for
MT1-MMP and MT2-MMP. MMP-2 was also localized in the neoplastic
astrocytes and endothelial cells in all of the glioblastoma samples
(Figure 9C)
. In normal brain tissues, some endothelial cells reacted
with the monoclonal antibody against MMP-2 (data not shown). No
staining was observed with nonimmune mouse IgG (Figure 9D)
.
|
. Immunoblotting of the normal brain
samples was negative (Figure 10)
.
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By in situ zymography using gelatin films, strong
gelatinolytic activity was detected in the glioblastoma tissues (Figure 11, A and B)
, but no activity was
recognized in the normal brain tissues (Figure 11, E and F)
. The
activity was completely blocked in the glioblastoma tissues that had
been incubated with BB94 (Figure 11, C and D)
. Distributions of the
activity were consistent with the immunolocalization of MT1-MMP,
MT2-MMP, and MMP-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MT3-MMP mRNA expression was observed by RT-PCR in the human malignant astrocytic tumors, although it was undetectable by Northern blotting. The quantitative RT-PCR indicated that the mean expression level in the brain tumors is at least 50-fold lower than that of MT1-MMP and MT2-MMP. Interestingly, however, the expression surely exists in various human brain samples, including normal brains, whereas no expression is present in the metastatic lung adenocarcinomas. This brain-specific expression agrees with the previous finding that the brain is one of the organs where MT3-MMP is selectively expressed.8 However, our data demonstrate that the expression level of MT3-MMP is not significantly different between the normal brain and astrocytic tumor tissues without correlations with the pro-MMP-2 activation. Thus, it seems likely that MT3-MMP may have a different role from the activation in the normal brain or human astrocytic tumors. Recent studies on MT3-MMP showed that this MMP has proteolytic activity against ECM components such as type III collagen.26 We have also demonstrated that recombinant MT3-MMP can digest proteoglycan as well as interstitial collagens (Shimada et al, manuscript submitted). It may be possible to speculate that MT3-MMP is involved in the turnover of ECM in the normal brain and astrocytic tumor tissues.
Previous studies reported the mRNA expression and protein production of MMP-2 by human astrocytic tumors in a small number of samples.12,20 The present studies have demonstrated that pro-MMP-2 production is enhanced significantly as tumor grade increases from low-grade astrocytomas to glioblastomas in 35 astrocytic tumor samples. The increase in pro-MMP-2 level in the tumor tissues is ascribed to enhanced production by the astrocytic tumor cells, as MMP-2 was immunolocalized predominantly to the tumor cells. In contrast, the production level of pro-MMP-2 and its activation in the metastatic lung adenocarcinomas were significantly lower than those in the glioblastomas and primary lung carcinomas.6,10 The expression level of MT1-MMP also tended to be lower in the metastatic carcinomas than in the glioblastomas12 and primary lung adenocarcinomas.6,10 This may be explained by the lack of stromal cells originated from the lung tissue in the metastatic adenocarcinomas of the brain, as pro-MMP-2 is produced mainly by stromal fibroblasts in the primary lung carcinomas,23,27,28 and MT1-MMP is detected in both carcinoma cells and stromal fibroblasts.10 Another possibility is, however, the presence of the mechanism by which expression of MMP-2 and MT1-MMP is reduced in the metastatic tumors of the brain. Regardless of the mechanisms, suppression of pro-MMP-2 production and activation might be related to a less invasive character of the metastatic lung adenocarcinomas in the brain29 compared with the highly invasive malignant astrocytic tumors.
In situ hybridization indicated that neoplastic astrocytes and some endothelial cells are responsible for the expression of MT1-MMP and MT2-MMP in the glioblastoma tissues. Immunohistochemically, both MT1-MMP and MT2-MMP were localized to the glioblastoma cells and endothelial cells of blood vessels. In carcinomas of the breast and head and neck, however, the discrepancy in the localization patterns of MT1-MMP by in situ hybridization and immunohistochemistry is argued.15,28 The former showed predominant distribution of MT1-MMP mRNA in the stromal cells,28 but the latter indicated that MT1-MMP is localized in the carcinoma cells, although some staining is also observed in the stromal cells.15 However, our data indicate that this is not the case in glioblastomas.
One interesting finding in the present studies is that, with in situ zymography, gelatinolytic activity was detected for the first time in the glioblastoma tissue but not in normal brains. This method was originally developed by Galis et al.30 However, because of the simplicity and better resolution, our method using gelatin films is considered to be better than the original, in which frozen sections mounted on the slide glasses were dipped in emulsion containing gelatin.30 As the gelatinolytic activity was abolished by the treatment of the tissues with BB94, a hydroxamate MMP inhibitor, the activity is ascribed to MMP(s). Gelatin is a nonspecific substrate susceptible to many MMPs.3 Among the MMP gene family members, however, MMP-2 and MMP-9 have the highest activity to gelatins,3 and the major gelatinolytic MMPs produced in the glioblastomas are MMP-2 and MMP-9.12,17,20 Thus, it is reasonable to speculate that these MMPs are mainly responsible for the activity.
In addition to the activator function for pro-MMP-2, recent studies on the characterization of MT1-MMP have demonstrated that it has ECM-degrading activities, including collagenolytic activity.31-33 Like MT1-MMP, MT2-MMP has also been reported to exhibit ECM-degrading activity as well as activation of pro-MMP-2.34,35 As MT1-MMP, MT2-MMP, and MMP-2 are co-localized in the glioblastoma cells, the combination of these MMPs may function as a powerful machinery for the pericellular ECM digestion in glioblastomas, which facilitates invasion of the glioma cells in the brain.
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Acknowledgements |
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Footnotes |
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Supported by a grant-in-aid for Cancer Research (10152255) from the Ministry of Education, Science, and Culture of Japan and Health Sciences Research Grants from the Ministry of Health and Welfare of Japan (to Y. Okada).
Accepted for publication October 19, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Y. Zhai, K. B. Hotary, B. Nan, F. X. Bosch, N. Munoz, S. J. Weiss, and K. R. Cho Expression of Membrane Type 1 Matrix Metalloproteinase Is Associated with Cervical Carcinoma Progression and Invasion Cancer Res., August 1, 2005; 65(15): 6543 - 6550. [Abstract] [Full Text] [PDF]
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 L. J. Kaufman, C. P. Brangwynne, K. E. Kasza, E. Filippidi, V. D. Gordon, T. S. Deisboeck, and D. A. Weitz Glioma Expansion in Collagen I Matrices: Analyzing Collagen Concentration-Dependent Growth and Motility Patterns Biophys. J., July 1, 2005; 89(1): 635 - 650. [Abstract] [Full Text] [PDF]
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 T. Kodama, E. Ikeda, A. Okada, T. Ohtsuka, M. Shimoda, T. Shiomi, K. Yoshida, M. Nakada, E. Ohuchi, and Y. Okada ADAM12 Is Selectively Overexpressed in Human Glioblastomas and Is Associated with Glioblastoma Cell Proliferation and Shedding of Heparin-Binding Epidermal Growth Factor Am. J. Pathol., November 1, 2004; 165(5): 1743 - 1753. [Abstract] [Full Text] [PDF]
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K. Kaikita, T. Hayasaki, T. Okuma, W. A. Kuziel, H. Ogawa, and M. Takeya Targeted Deletion of CC Chemokine Receptor 2 Attenuates Left Ventricular Remodeling after Experimental Myocardial Infarction Am. J. Pathol., August 1, 2004; 165(2): 439 - 447. [Abstract] [Full Text] [PDF]
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, MT1-MMP; 
,
MT2-MMP; 
, MT3-MMP; 
, GAPDH.
