Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors

Full access

✓ The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.

Abstract

✓ The role of matrix metalloproteinases (MMP's) and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), in human brain tumor invasion was investigated. Gelatinolytic activity was assayed via gelatin zymography, and four MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunolocalized in human brain tumors and in normal brain tissues using monoclonal antibodies. The tissue was surgically removed from 44 patients: glioblastoma (five cases), anaplastic astrocytoma (six cases), astrocytoma (four cases), metastatic tumor (six cases), neurinoma (10 cases), meningioma (10 cases), and normal brain tissue (three cases). Glioblastomas, anaplastic astrocytomas, and metastatic tumors showed high gelatinolytic activity and positive immunostaining for MMP's; TIMP-1 was also expressed in these tumors, but some tumor cells were negative for the antibody. Astrocytomas had low gelatinolytic activity and the tumor cells showed no immunoreactivity for MMP's and TIMP-1. Although neurinomas and meningiomas had only moderate proteinase activity and exhibited positive immunoreactivity for MMP-9, intense expression of TIMP-1 was simultaneously observed in these tumor cells. These findings suggest that MMP's play an important role in human brain tumor invasion, probably due to an imbalance between the production of MMP's and TIMP-1 by the tumor cells.

Local tissue invasion by brain tumors renders treatment of the lesion extremely difficult. Tumor invasion is a complex phenomenon that involves disruption of the extracellular matrix and penetration by the tumor cells into the normal adjacent tissues. Degradation of the extracellular matrix is mediated by tumor cell-derived proteolytic enzymes, such as heparinase, serine proteinases, cathepsins, and matrix metalloproteinases (MMP's).24,28 The MMP gene family, in particular, has been the subject of much study.2,4,8,24,25,28,30,49 Several authors have reported the production of MMP's and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMP's), by human brain tumors in vitro.1,15 However, to our knowledge, no studies have focused on MMP and TIMP activity or in situ localization in human brain tumors. In the current study, we investigated metalloproteinase activity in six different types of brain tumor and in normal brain. This activity was determined by gelatin zymographic analysis and immunolocalization of MMP-1 (interstitial collagenase), MMP-2 (72-kD gelatinase/type IV collagenase/gelatinase A), MMP-3 (stromelysin-1), MMP-9 (92-kD gelatinase/type IV collagenase/gelatinase B), and TIMP-1, using monoclonal antibodies. The nomenclature used for MMP's follows the numbering system proposed by Nagase, et al.31

Materials and Methods

Tissue Samples

Samples of brain tumors were obtained from 44 patients at surgery performed at the Fukui Medical School Hospital. The tumors included five glioblastomas, six anaplastic astrocytomas, four astrocytomas, six metastatic adenocarcinomas, 10 meningiomas, and 10 neurinomas. Normal brain tissue specimens were obtained from three patients undergoing lobectomy for deep-seated tumors. The specimens were treated as follows. Contaminating blood and necrotic regions were removed, after which the specimens were snap-frozen in liquid nitrogen and stored at −80°C until use. They were subsequently thawed, weighed, minced, and homogenized at a ratio of 50 mg to 1 ml of 50 mM Tris-HCl (pH 7.5), containing 75 mM NaCl and 1 mM phenylmethyl sulfonyl fluoride (PMSF). The homogenate was then centrifuged at 4°C for 20 minutes at 10,000 G and the supernatant was used for zymographic study. The protein concentrations of each sample were determined by the dye-binding method using a protein assay kit* with bovine serum albumin as a standard. For immunohistochemical study, freshly excised samples were embedded in a compound without fixation, and 8-µm cryostat sections were prepared. Histological diagnoses were made from adjacent tissue samples fixed in formalin and embedded in paraffin.

Zymographic Study

The gelatinolytic activity was determined by gelatin-substrate gel electrophoresis (7.5% total acrylamide) according to the method described by Hibbs, et al.18 Samples of supernatant were diluted to a 500-µg/ml protein concentration and mixed with an equal volume of 80 mM Tris-HCl (pH 6.8) containing 4% sodium dodecyl sulfate (SDS) and 10% glycerol, then 25-µl aliquots of this mixture were loaded on the gel. The samples were not boiled or reduced. After electrophoresis at 4°C, the gels were rinsed with distilled water and washed three times in 300 ml of 2.5% (vol/vol) Triton X-100 solution for 20 minutes each time to remove the SDS. They were then incubated for 16 hours at 37°C in 500 ml of 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl2 and 0.02% NaN3. In some experiments, 1 mM 1,10-phenanthroline, 10 mM ethylenediamine tetra-acetic acid (EDTA), or 2 mM PMSF was added to the incubation buffer. The gels were stained with 0.1% amido black in a 1:3:6 ratio of acetic acid:methanol:water and destained with the same ratio of acetic acid:methanol:water. Proteolytic activity appeared as clear bands on a blue background. Molecular weight standards were myosin (200 kD), beta-galactosidase (116.25 kD), phosphorylase b (97.4 kD), and bovine serum albumin (66.2 kD).

Quantitative analysis of gelatinolytic activity was achieved by scanning densitometry of the zymograms as previously described by Sawaya and Highsmith.40 Values were expressed as lysis per milligram of protein.

Immunohistochemical Study

The MMP's (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 were immunostained by the labeled streptavidin biotin method, according to the manufacturer's protocol. Mouse monoclonal antibodies against human MMP's and TIMP-1§ were used. The monoclonal antibodies against MMP-1 (41—1E5), MMP-2 (42—5D11), MMP-3 (55—2A4), and MMP-9 (56—2A4) have been characterized previously.12,32,33,50 These were found suitable for immunohistochemical studies of paraffin-embedded sections from a rheumatoid synovium and of metastatic human tumor cells implanted in chickens.46 To develop the monoclonal antibody, TIMP-1 was purified from the homogenates of human placental tissues and used as an antigen. The monospecificity of the antibody to TIMP-1 (50—2F6) was determined by immunoblotting, and its availability for immunostaining was confirmed by applying the antibodies to paraffin-embedded sections of a rheumatoid synovium (Y Okada, et al., in preparation). Frozen sections were washed free of fixative with phosphate-buffered saline (PBS, pH 7.4), exposed to 0.3% H2O2 in PBS for 5 minutes to inactivate endogenous peroxidase, then incubated with normal goat serum for 5 minutes. The sections were then reacted with primary antibodies for 90 minutes in a humidity chamber at room temperature. Dilutions of primary antibodies were 0.32 µg/ml for anti-MMP-1 immunoglobulin G (IgG), 0.9 µg/ml for anti-MMP-2 IgG, 0.5 µg/ml for anti-MMP-3 IgG, 2 µg/ml for anti-MMP-9 IgG, and 22.5 µg/ml for anti-TIMP-1 IgG. Biotinylated goat anti-mouse IgG antibody, as the secondary antibody, was applied to the sections for 10 minutes. The slides were then incubated with horseradish peroxidase-labeled streptavidin solution for 10 minutes, and the color was developed with 0.02% 3-3′ diaminobenzidine tetrahydrochloride and 0.006% H2O2 in PBS. The slides were counterstained with Mayer's hematoxylin. Between steps, the slides were washed twice in PBS. Sections incubated with normal mouse serum in place of the primary antibody served as negative controls.

Statical Analysis

Statistical significance was assessed using Student's unpaired t-test. Probability values of less than 0.05 were considered significant.

Results

The gelatinolytic activity and immunohistochemical findings in the 44 tumor specimens are summarized in Tables 1 and 2, respectively. The data were also analyzed in sample groups classified according to the histological diagnosis (Tables 3 and 4). Gelatinolytic activity detected by gelatin zymography was inhibited by EDTA and 1,10-phenanthroline (metal chelating agents), but not by PMSF (a serine proteinase inhibitor), indicating that the activity was that of a class of metalloproteinases. Representative digestion profiles of each group are shown in Fig. 1.

TABLE 1

Gelatinolytic activity in 44 tissue samples*

Sample No.Patient Age (yrs), SexTissue HistologyGelatinolytic Activity (lysis/mg protein)
3950, F normal6.03
5842, F normal13.04
6049, M normal8.83
439, M GM276.91
866, M GM167.88
1166, M GM134.59
1842, M GM320.78
2268, F GM283.32
528, M AA85.33
645, F AA152.33
1469, M AA115.38
1914, M AA176.51
2338, M AA132.95
3029, F AA36.40
78, F astrocytoma3.00
953, F astrocytoma11.75
1030, F astrocytoma55.13
2437, F astrocytoma3.51
2560, M met tumor87.34
2662, M met tumor326.30
3182, M met tumor107.71
3260, F met tumor286.53
4662, M met tumor261.50
5949, F met tumor106.27
4132, F neurinoma52.54
4561, M neurinoma161.32
4737, M neurinoma105.37
4927, F neurinoma15.71
5168, M neurinoma168.57
5248, F neurinoma11.92
5362, F neurinoma99.63
5456, M neurinoma35.06
5549, F neurinoma37.04
5650, F neurinoma30.68
1549, F meningioma112.70
1667, M meningioma47.90
1724, M meningioma25.14
3454, M meningioma59.76
3667, M meningioma289.55
3780, F meningioma163.97
3874, F meningioma71.05
4244, F meningioma51.14
4352, M meningioma45.61
4848, M meningioma107.63

GM = glioblastoma multiforme; AA = anaplastic astrocytoma; met = metastatic.

TABLE 2

Immunoreactivity for MMP's and TIMP-1 in 44 tissue samples*

Sample No.Tissue HistologyImmunoreactivity
MMP-1MMP-2MMP-3MMP-9TIMP-1  
39normal+†+++†
58normal+†+++†
60normal+†+++†
4GM++++++++++
8GM++++
11GM+++++++
18GM++++++
22GM++++++
5AA++++
6AA++++++
14AA++++
19AA++++++
23AA++++++
30AA++++
7As
9As
10As
24As
25Met+++++++
26Met+
(+++‡)(+++‡)
31Met+++++++++
32Met++++
46Met++++
59Met+++++
41Neu+++++++
45Neu+++++++
47Neu+++++++
49Neu++++++
51Neu+++++++
52Neu+++++++
53Neu+++++++
54Neu++++++
55Neu++++++
56Neu++++++
15Men++++++
16Men++++++++
17Men++++++++
34Men++++++
36Men+++++++
37Men+++
38Men++++++
42Men++++
43Men++++
48Men++++++

Abbreviations: MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinases; GM = glioblastoma multiforme; AA = anaplastic astrocytoma; As = astrocytoma; Met = metastatic tumor; Neu = neurinoma; Men = meningioma. Symbols: − = no immunoreactivity; + = occasional faint immunoreactivity; ++ = focal immunoreactivity; +++ = diffuse immunoreactivity; † = positive immunoreactivity of endothelial cells; ‡ = positive immunoreactivity of stroma cells.

TABLE 3

Summary of gelatinolytic activity in each histological group

Sample GroupNo. of SamplesGelatinolytic Activity (lysis/mg protein)
Mean*Median  
normal brain39.30 ± 3.53 8.83
glioblastoma5236.80 ± 80.73 276.91
anaplastic6116.48 ± 50.09 124.17
 astrocytoma 
astrocytoma426.57 ± 23.72 7.63
metastatic6195.94 ± 106.88 184.61
neurinoma1069.08 ± 61.01 44.75
meningioma1097.45 ± 79.08 65.41

Mean ± standard deviation.

TABLE 4

Summary of immunoreactivity in each histological group*

Sample GroupPercent of Immunoreactive Specimens
MMP-1MMP-2MMP-3MMP-9TIMP-1 
normal brain0.0 0.00.0100* 100* 
glioblastoma100 60.040.0100 100 
anaplastic66.7 66.733.3100 100 
 astrocytoma   
astrocytoma0.0 0.00.00.0 0.0 
metastatic100 50.050.066.7 50.0 
neurinoma30.0 30.00.0100 100 
meningioma0.0 40.010.0100 100 

Positive immunoreactivity of endothelial cells. Abbreviations: MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinases.

Fig. 1.
Fig. 1.

Zymographic analysis of extracts of various brain tumors and of normal brain: normal brain (lane 1), glioblastoma (lane 2), anaplastic astrocytoma (lane 3), astrocytoma (lane 4), metastatic tumor (lane 5), neurinoma (lane 6), and meningioma (lane 7). Molecular weight (MW) is shown at left in kilodaltons.

Normal Brain Group

Normal brain samples were obtained from three patients (Table 1). There were two cerebral tissue specimens and one cerebellar tissue specimen, consisting of both gray and white matter. The specimens contained no gliotic or edematous regions. Gelatin zymography showed that all normal brain samples had faint gelatinolytic activity at 87 kD. Immunohistochemically, however, no reactivity for MMP's or TIMP-1 was found in neuronal or glial cells, except for positive immunostaining for MMP-9 and TIMP-1 in the endothelial cells.

Glioblastoma Group

Samples of glioblastoma were obtained from five patients (Table 1). This group demonstrated the highest degree of gelatinolytic activity, as determined by zymography. The activity was significantly higher than that observed in normal brain (p = 0.003), anaplastic astrocytoma (p = 0.027), astrocytoma (p = 0.005), neurinoma (p = 0.007), and meningioma (p = 0.016) (Table 3). Gelatinolytic bands migrated at 87 and 64 kD in all samples, at 250 kD in two, at 130 kD in three, at 84 kD in two, and at 61 kD in one. Immunohistochemistry showed that the tumor cells exhibited positive immunostaining in all samples for MMP-1, MMP-9, and TIMP-1, in three for MMP-2, and in two for MMP-3 (Table 2 and Fig. 2). Endothelial cells, especially those showing proliferation, had strong immunoreactivity for MMP-9 and TIMP-1. Most inflammatory cells were positively stained by antibodies to MMP-1 and MMP-9.

Fig. 2.
Fig. 2.

Photomicrographs in glioblastoma tissue demonstrating immunoreactivity for matrix metalloproteinase (MMP)-1 (A), MMP-2 (B), MMP-3 (C), MMP-9 (D), and tissue inhibitor of metalloproteinases (TIMP)-1 (E). A and B: Intense immunoreactivity for MMP-1 and MMP-2 is demonstrated in the tumor cells of Sample 4. C: Only faint immunoreactivity for MMP-3 is demonstrated in Sample 4. D: Both endothelial and tumor cells express MMP-9 (arrow) in Sample 11. E: Some tumor cells show no immunoreactivity for TIMP-1 (arrows) in Sample 22. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 180.

Anaplastic Astrocytoma Group

Anaplastic astrocytoma samples were obtained from six patients (Table 1). Gelatinolytic bands migrated at 87 and 64 kD in all samples, at 250 kD in two, at 130 kD in four, and at 61 kD in one. The gelatinolytic activity was significantly higher than that observed in normal brain (p = 0.003) and astrocytoma (p = 0.007) (Table 3). Immunohistochemically, the tumor cells exhibited positive immunostaining in four samples for MMP-1, in four for MMP-2, in two for MMP-3, and in all samples for MMP-9 and TIMP-1 (Table 2).

Astrocytoma Group

The astrocytoma group consisted of tissue from four patients (Table 1). Pilocytic astrocytomas were not included. The mean level of gelatinolytic activity was higher than that observed in normal brain, but the difference was not significant (p = 0.247) (Table 3). On gelatin zymography, weak gelatinolytic bands were seen at 87 kD in all samples, at 250 kD in three, at 130 kD in three, at 84 kD in one, at 64 kD in three, and at 61 kD in one. Immunohistochemistry revealed no immunoreactivity for MMP's or TIMP-1 in the tumor cells, whereas endothelial cells were labeled by antibodies to MMP-9 and TIMP-1 (Table 2).

Metastatic Tumor Group

The metastatic tumor specimens comprised six metastatic adenocarcinomas, from the lung in three patients, the rectum in two, and an unidentified organ in one. The mean gelatinolytic activity for this group was significantly higher than that seen in normal brain (p = 0.008), astrocytoma (p = 0.013), and neurinoma (p = 0.04) (Table 3). All samples had lytic bands at 130 and 87 kD. Other gelatinolytic bands were seen at 250 kD in four samples, at 84 kD in one, at 78 kD in one, at 64 kD in three, and at 61 kD in three. Metastatic tumor cells were positively immunostained for MMP-1 in all samples, for MMP-2 in three, for MMP-3 in three, for MMP-9 in four, and for TIMP-1 in three (Table 2). Stroma cells also showed intense immunoreactivity for MMP-2 and MMP-9 in one sample.

Neurinoma Group

Neurinoma specimens were obtained from 10 patients: one with accessory nerve neurinoma and nine with acoustic neurinoma. The gelatinolytic activity was significantly higher than that observed in normal brain (p = 0.008) (Table 3). All samples had definite gelatinolytic bands at 130 and 87 kD and a faint band at 64 kD. Two samples had a lytic band at 61 kD. Immunohistochemically, the tumor cells were almost exclusively stained with antibodies to MMP-9 and TIMP-1 (Fig. 3). Faint immunoreactivity was observed for MMP-1 in three samples and for MMP-2 in three. No immunoreactivity for MMP-3 was found (Table 2).

Fig. 3.
Fig. 3.

Photomicrographs demonstrating immunoreactivity for matrix metalloproteinase (MMP)-9 (A) and tissue inhibitor of metalloproteinases (TIMP)-1 (B) in neurinoma tissue (Sample 45). Almost all the tumor cells show immunoreactivity for MMP-9 and TIMP-1. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 270.

Meningioma Group

The meningioma specimens consisted of three meningotheliomatous, three fibroblastic, two transitional, and two anaplastic meningiomas. The mean level of gelatinolytic activity was significantly higher than that seen in astrocytoma (p = 0.026) and normal brain (p = 0.007) (Table 3). In addition to the gelatinolytic bands at 87 and 64 kD found in all samples, a band was noted at 250 kD in one, at 130 kD in two, at 84 kD in one, and at 61 kD in four. Immunoreactive profiles for MMP's and TIMP-1 were similar to those of the neurinoma group. All samples were positively immunostained for MMP-9 and TIMP-1 (Fig. 4). Positive immunostaining for MMP-2 was observed in four samples and for MMP-3 in one sample. No immunoreactivity for MMP-1 was found (Table 2). There were no differences in gelatinolytic activity, digestion profiles of gelatin, and immunoreactivity for MMP's and TIMP-1 among the histological subtypes.

Fig. 4.
Fig. 4.

Photomicrographs demonstrating immunoreactivity for matrix metalloproteinase (MMP)-9 (A) and tissue inhibitor of metalloproteinases (TIMP)-1 (B) in meningioma tissue (Sample 15). Almost all the tumor cells exhibit immunoreactivity for MMP-9 and TIMP-1. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 270.

Discussion
Proteinases and Proteinase Inhibitors in Brain Tumors

Rapidly mounting evidence has shown that proteinases and proteinase inhibitors derived from tumor cells play an important role in tumor invasion.8,13,20,24,36,49 Previous studies of human brain tumor tissues and cells have reported the production of plasminogen activators5,11,22,40,41 and MMP's.1,15 Plasminogen activators, which belong to a class of serine proteinases, activate plasminogen to its proteolytically active enzyme, plasmin, a serine proteinase with broad substrate specificity. Recent studies, both in vitro and in vivo, have shown that urokinase-type plasminogen activator is closely linked to the malignant phenotype of human brain tumors.5,11,40,41 Activity of the serine proteases is inhibited by a group of plasma proteinase inhibitors named “serpins.” Human brain tumors are known to produce alpha-1-antitrypsin, a member of the serpin family,42 and plasminogen activator inhibitor-1.22

Matrix metalloproteinases represent a gene family of zinc metalloproteinases that are capable of degrading almost all extracellular matrix macromolecules. Seven of these MMP's have now been well characterized.48 These enzymes have different substrate specificities: MMP-1 degrades collagen types I, II, and III; MMP-2 degrades denatured collagens (gelatins), collagen types IV and V, fibronectin, and laminin; MMP-3 digests collagen type IV, gelatins, fibronectin, laminin, and proteoglycans; and MMP-9 cleaves gelatins, collagen types III, IV, and V, and the alpha-2 chain of collagen type I. In addition to these MMP's, MMP-7 (matrilysin/pump-1), MMP-8 (neutrophil collagenase), and MMP-10 (stromelysin-2) have also been reported.22,48 This enzymatic activity is regulated by natural inhibitors in the extracellular milieu, of which two related but distinct inhibitors, designated TIMP-1 and TIMP-2, are considered to be major regulators of MMP's.10,44,48 Apodaca, et al.,1 reported that human glioma cell lines in culture secreted MMP-1, MMP-2, and MMP-9, as well as TIMP's. Our present study demonstrated that human brain tumors in vivo produced MMP-1, MMP-2, MMP-3, and MMP-9, as well as TIMP-1. Tumor cell-derived metalloproteinases have also been shown to be responsible for the invasion of cultured brain tissues by rat glioma cells.26,35

Recent studies suggest that more than one proteinase could be involved in the process of invasion and metastasis, since inhibitors of either serine proteinases or metalloproteinases can block tumor cell invasion in vitro.6,7,24,28,37,43 This occurs, at least in part, because of interactions between the two classes of proteinases. Plasmin activates the zymogens of MMP-1 and MMP-3 to take the active enzyme form, which can degrade serpins such as alpha-1-antiproteinase.9,27,34,45 Reith and Rucklidge38 reported that the rat glioma cell line BT5C secretes both urokinase-type plasminogen activator and 72-kD metalloproteinase, and that the former activates the latter. Although it is difficult to determine which of various proteinases is responsible for the cellular invasion of human brain tumors, our demonstration of the in vivo production of MMP-1, MMP-2, MMP-3, and MMP-9 as well as TIMP-1 by brain tumors in humans suggests that these MMP's may play a significant role in the degradation of the extracellular matrix that is associated with brain tumor invasion.

Detection of MMP Activity

Gelatin zymography is a useful means of analyzing the activity of many MMP's.1,4,19,20,26,29,49 In this study, zymography showed that most human brain tumor tissues had proteinase activity of 87 and 64 kD. According to their molecular weights, the 87-kD proteinase is considered to be MMP-9 and the 64-kD enzyme to be MMP-2. The gelatinolytic activity at 250 and 130 kD shown in this study may correspond to the dimeric form of pro-MMP-9 and the pro-MMP-9/TIMP-1 complex, respectively.14,18,19,33 Since MMP-9 was localized by immunohistochemistry not only in the tumor cells but also in the endothelial and inflammatory cells in the tumor tissues, it is conceivable that the MMP-9 activity detected by zymography could be derived at least in part from these cells.18,19,21 Gelatin zymography enables detection of the activity of many MMP's; however, it is possible that the activity measured in the present study may have been derived mainly from MMP-2 and MMP-9, since MMP-1, MMP-3, MMP-8, and MMP-10 exhibit weak gelatinolytic activity and MMP-7 is a 28-kD enzyme that cannot be analyzed on 7.5% SDS-polyacrylamide gel electrophoresis.4,48 Therefore, the MMP activity in our study might be under-represented.

Growth Patterns of Brain Tumors and Expression of MMP's and TIMP-1

Glioblastomas and anaplastic astrocytomas infiltrate and invade adjacent brain structures in a diffuse manner while neurinomas and meningiomas generally show expansive “pushing” growth. Metastatic tumors in the brain, whatever their origin, are usually circumscribed on macroscopic examination. However, light microscopy reveals groups and trabecular masses of tumor cells penetrating the adjacent brain tissue, with frequent infiltration of the perivascular sheaths of neighboring vessels.39 Our immunohistochemical localization studies demonstrated that glioblastoma and anaplastic astrocytoma cells synthesized MMP-1, MMP-2, MMP-3, and MMP-9 in vivo, in contrast to astrocytoma and normal brain tissue cells, which did not. The zymographic findings indicated that gelatinolytic activity in glioblastomas and anaplastic astrocytomas was much higher than that in astrocytomas and normal brain tissues. Although TIMP-1 expression was observed in all samples in the glioblastoma and anaplastic astrocytoma groups, some tumor cells were not immunoreactive for TIMP-1, unlike the findings in the neurinoma and meningioma groups. This was particularly true in the metastatic tumor group, where only a few tumor cells were weakly labeled with anti-TIMP-1 antibody. These findings suggest that there is a local imbalance of MMP and TIMP-1 activity in favor of proteolysis in glioblastomas, anaplastic astrocytomas, and metastatic tumors.

We found no significant difference in gelatinolytic activity between the metastatic tumor and meningioma specimens (p = 0.086), between the anaplastic astrocytoma and meningioma specimens (p = 0.568), or between the anaplastic astrocytoma and neurinoma specimens (p = 0.131), although these tumors do vary clinically in degree of brain invasion and patient survival times. This lack of significant difference could be because metastatic tumor tissue contain necrotic microfoci with potentially lower gelatinolytic activity. It is possible, therefore, that the actual gelatinolytic activity in viable metastatic tumor tissues is higher than was detected in this study. Alternatively, the lack of significant difference could reflect the limitation of the assay used in our study. The activity we measured was derived mainly from MMP-2 and MMP-9, as discussed above. Thus, it is possible that other MMP activity in the metastatic tumor and anaplastic astrocytoma groups could be higher than in the meningioma and neurinoma groups. Furthermore, it is also possible that the amounts of TIMP produced at the local site are a determinant of tumor invasiveness when certain levels of MMP's are present. Our data, showing that TIMP-1 immunoreactivity was markedly lower in the metastatic tumor specimens and higher in the meningioma and neurinoma specimens, support this idea. Further work to measure levels of MMP's and TIMP's in tumor tissue is necessary to better understand the role of these proteinases in human brain tumor invasion. Such a project, using sandwich enzyme immunoassays, is now underway in our laboratories.

We found that MMP-9 was expressed markedly in all neurinoma and meningioma samples. The role of MMP-9 in these tumors remains unclear. Since neurinomas and meningiomas produce various extracellular matrix macromolecules,3,23 it is possible that MMP-9 is important for their replication. However, it is also possible that the enzyme activity of MMP-9 in these tumors is controlled at a very low level by TIMP-1, since the tumor cells were also intensely labeled with anti-TIMP-1 antibody in our study. Halaka, et al.,15 reported that invasive meningiomas produce significantly lower levels of TIMP-1 than noninvasive meningiomas. In this study, however, we found no difference in the gelatinolytic activity and immunoreactivity of MMP's and TIMP-1 between anaplastic astrocytomas versus ordinary meningiomas.

Role of MMP's and TIMP-1 in Neovascularization in Brain Tumors

In the initial stage of neovascularization, it is thought that capillary endothelial cells degrade the basement membrane surrounding intact capillaries and migrate through the extracellular matrix toward the source of the angiogenic stimulus. It is likely that the induction and arrest of capillary endothelial cell migration during neovascularization require precise regulation of extracellular matrix synthesis and degradation. Endothelial cells produce MMP's and TIMP's in vitro.16,17,21 Vaithilingam, et al.,47 recently reported that the growth of C6 rat glioma cells in a spheroid implantation model was associated with an increase in general protease and type IV collagenase activity in serum; they hypothesized that the activity of these enzymes played an important part in the neovascularization of tumor tissues. In our study, we found that most capillary endothelial cells, especially those proliferating in the glioblastoma tissues, exhibited immunoreactivity for both MMP-9 and TIMP-1, suggesting that these molecules are involved in the neovascularization of brain tumor tissues.

Acknowledgments

We are grateful to Mr. Hishima and Miss Matsuda for technical assistance and to Miss Fujibe for secretarial assistance.

References

  • 1.

    Apodaca GRutka JTBouhana Ket al: Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells. Cancer Res 50:232223291990Cancer Res 50:

  • 2.

    Barsky SHTogo SGarbisa Set al: Type IV collagenase immunoreactivity in invasive breast carcinoma. Lancer I:2962971983Lancer I:

  • 3.

    Bellon GCaulet TCam Yet al: Immunohistochemical localisation of macromolecules of the basement membrane and extracellular matrix of human gliomas and meingiomas. Acta Neuropathol 66:2452521985Acta Neuropathol 66:

  • 4.

    Brown PDLevy ATMargulies IMKet al: Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res 50:618461911990Cancer Res 50:

  • 5.

    Caccamo DVMcKeever PE: Plasminogen activators and inhibitors in gliomas: an immunohistochemical study. J Neuropathol Exp Neurol 51:3321992 (Abstract)J Neuropathol Exp Neurol 51:

  • 6.

    DeClerck YAPerez NShimada Het al: Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases. Cancer Res 52:7017081992Cancer Res 52:

  • 7.

    DeClerck YAYean TDChan Det al: Inhibition of tumor invasion of smooth muscle cell layers by recombinant human metalloproteinsae inhibitor. Cancer Res 51:215121571991Cancer Res 51:

  • 8.

    D'Errico AGarbisa SLiotta LAet al: Augmentation of type IV collagenase, laminin receptor, and Ki67 proliferation antigen associated with human colon, gastric, and breast carcinoma progression. Mod Pathol 4:2392461991Mod Pathol 4:

  • 9.

    Desrochers PEJeffrey JJWeiss SJ: Interstitial collagenase (Matrix metalloproteinase-1) expresses serpinase activity. J Clin Invest 87:225822651991J Clin Invest 87:

  • 10.

    Docherty AJPLyons ASmith BJet al: Sequence of human tissue inhibitor of metalloproteinase and its identity to erythroid-potentiating activity. Nature 318:66691985 (Letter)Nature 318:

  • 11.

    Franks AJEllis E: Immunohistochemical localisation of tissue plasminogen activator in human brain tumours. Br J Cancer 59:4624661989Br J Cancer 59:

  • 12.

    Fujimoto NMouri NIwata Ket al: A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2(72-kDa gelatinase/type IV collagenase) using monoclonal antibodies. Clin Chim Acta 221:911031993Clin Chim Acta 221:

  • 13.

    Fukuda YImoto MKoyama Yet al: Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers. Gastroenterol Jpn 26:37411991Gastroenterol Jpn 26:

  • 14.

    Goldberg GIStrongin ACollier IEet al: Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem 267:458345911992J Biol Chem 267:

  • 15.

    Halaka ANBunning RADBird CCet al: Production of collagenase and inhibitor (TIMP) by intracranial tumors and dura in vitro. J Neurosurg 59:4614661983in vitro. J Neurosurg 59:

  • 16.

    Herron GSBanda MJClark EJet al: Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J Biol Chem 261:281428181986J Biol Chem 261:

  • 17.

    Herron GSWerb ZDwyer Ket al: Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity. J Biol Chem 261:281028131986J Biol Chem 261:

  • 18.

    Hibbs MSHasty KASeyer JMet al: Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J Biol Chem 260:249325001985J Biol Chem 260:

  • 19.

    Hibbs MSHoidal JRKang AH: Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest 80:164416501987J Clin Invest 80:

  • 20.

    Hicks NJWard RVReynolds JJ: A fibrosarcoma model derived from mouse embryo cells: growth properties and secretion of collagenase and metalloproteinase inhibitor (TIMP) by tumour cell lines. Int J Cancer 33:8358441984Int J Cancer 33:

  • 21.

    Kalebic TGarbisa SGlaser Bet al: Basement membrane collagen: degradation by migrating endothelial cells. Science 221:2812831983Science 221:

  • 22.

    Keohane MEHall SWVandenBerg SRet al: Secretion of α2-macroglobulin, α2-antiplasmin, and plasminogen activator inhibitor-1 by glioblastoma multiforme in primary organ culture. J Neurosurg 73:2342411990α2α2J Neurosurg 73:

  • 23.

    Kubota TNakagawa THosotani Ket al: Immunohistochemical study of extracellular matrix in schwannomas: in vivo and in vitro observations. Brain Tumor Pathol 9:23311992in vivo and in vitro observations. Brain Tumor Pathol 9:

  • 24.

    Liotta LASteeg PSStetler-Stevenson WG: Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64:3273361991Cell 64:

  • 25.

    Liotta LATryggvason KGarbisa Set al: Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67681980Nature 284:

  • 26.

    Lund-Johansen MRucklidge GJMilne Get al: A metalloproteinase, capable of destroying cultured brain tissue isolated from rat glioma cells. Anticancer Res 11:100110061991Anticancer Res 11:

  • 27.

    Mast AEEnghild JJNagase Het al: Kinetics and physiologic relevance of the inactivation of αl-proteinase inhibitor, α1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin). J Biol Chem 266:15810158161991ααJ Biol Chem 266:

  • 28.

    Mignatti PRobbins ERifkin DB: Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell 47:4874981986Cell 47:

  • 29.

    Moll UMYoungleib GLRosinski KBet al: Tumor promoter-stimulated Mr 92,000 gelatinase secreted by normal and malignant human cells: isolation and characterization of the enzyme from HT1080 tumor cells. Cancer Res 50:616261701990Cancer Res 50:

  • 30.

    Monteagudo CMerino MJSan-Juan Jet al: Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue. Am J Pathol 136:5855921990Am J Pathol 136:

  • 31.

    Nagase HBarrett AJWoessner JF Jr: Nomenclature and glossary of the matrix metalloproteinases. Matrix Suppl 1:4214241992Matrix Suppl 1:

  • 32.

    Obata KIwata KOkada Yet al: A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies. Clin Chim Acta 211:59721992Clin Chim Acta 211:

  • 33.

    Okada YGonoji YNaka Ket al: Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. J Biol Chem 267:21712217291992J Biol Chem 267:

  • 34.

    Okada YHarris ED JrNagase H: The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts. Purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate. Biochem J 254:7317411988Biochem J 254:

  • 35.

    Paganetti PACaroni PSchwab ME: Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease. J Cell Biol 107:228122911988in vitro: involvement of a metalloprotease. J Cell Biol 107:

  • 36.

    Ponton ACoulombe BSkup D: Decreased expression of tissue inhibitor of metalloproteinases in metastatic tumor cells leading to increased levels of collagenase activity. Cancer Res 51:213821431991Cancer Res 51:

  • 37.

    Reich RThompson EWIwamoto Yet al: Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells. Cancer Res 48:330733121988Cancer Res 48:

  • 38.

    Reith ARucklidge GJ: Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase. Biochem Biophys Res Commun 186:3483541992Biochem Biophys Res Commun 186:

  • 39.

    Russell DSRubinstein LJ: Pathology of Tumours of the Nervous Systemed 5. Baltimore: Williams & Wilkins1989834837

  • 40.

    Sawaya RHighsmith R: Plasminogen activator activity and molecular weight patterns in human brain tumors. J Neurosurg 68:73791988J Neurosurg 68:

  • 41.

    Sawaya RRämö JShi MLet al: Biological significance of tissue plasminogen activator content in brain tumors. J Neurosurg 74:4804861991J Neurosurg 74:

  • 42.

    Sawaya RZuccarello MHighsmith R: Alpha-1-antitrypsin in human brain tumors. J Neurosurg 67:2582621987J Neurosurg 67:

  • 43.

    Schultz RMSilberman SPersky Bet al: Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells. Cancer Res 48:553955451988Cancer Res 48:

  • 44.

    Stetler-Stevenson WGKrutzsch HCLiotta LA: Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J Biol Chem 264:17374173781989J Biol Chem 264:

  • 45.

    Suzuki KEnghild JJMorodomi Tet al: Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 29:10261102701990Biochemistry 29:

  • 46.

    Tsuchiya YEndo YSato Het al: Expression of type IV collagenases in human tumor cell lines that can form liver colonies in chick embryos. Int J Cancer 56:46511994Int J Cancer 56:

  • 47.

    Vaithilingam ISMcDonald WBrown NKet al: Serum proteolytic activity during the growth of C6 astrocytoma. J Neurosurg 77:5956001992J Neurosurg 77:

  • 48.

    Woessner JF Jr: Matrix metalloproteinase and their inhibitors in connective tissue remodeling. FASEB J 5:214521541991Woessner JF Jr: Matrix metalloproteinase and their inhibitors in connective tissue remodeling. FASEB J 5:

  • 49.

    Yamagata SIto YTanaka Ret al: Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis. Biochem Biophys Res Commun 151:1581621988Biochem Biophys Res Commun 151:

  • 50.

    Zhang JFujimoto NIwata Ket al: A one step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies. Clin Chim Acta 219:1141993Clin Chim Acta 219:

Protein assay kit obtained from Bio-Rad, Richmond, California.

Tissue-Tek OCT compound obtained from Miles Scientific, Naperville, Illinois.

Streptavidin biotin immunostaining kit obtained from DAKO, Carpinteria, California.

Monoclonal antibodies provided by Dr. Kazushi Iwata, Fuji Chemical Industries, Ltd., Takaoka, Japan.

Article Information

Address for Dr. Hayakawa: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.Address for Dr. Okada: Department of Pathology, School of Medicine, Kanazawa University, Kanazawa, Japan.Address reprint requests to: Takao Nakagawa, M.D., Department of Neurosurgery, Fukui Medical School, Matsuoka, Fukui 910–11, Japan.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Zymographic analysis of extracts of various brain tumors and of normal brain: normal brain (lane 1), glioblastoma (lane 2), anaplastic astrocytoma (lane 3), astrocytoma (lane 4), metastatic tumor (lane 5), neurinoma (lane 6), and meningioma (lane 7). Molecular weight (MW) is shown at left in kilodaltons.

  • View in gallery

    Photomicrographs in glioblastoma tissue demonstrating immunoreactivity for matrix metalloproteinase (MMP)-1 (A), MMP-2 (B), MMP-3 (C), MMP-9 (D), and tissue inhibitor of metalloproteinases (TIMP)-1 (E). A and B: Intense immunoreactivity for MMP-1 and MMP-2 is demonstrated in the tumor cells of Sample 4. C: Only faint immunoreactivity for MMP-3 is demonstrated in Sample 4. D: Both endothelial and tumor cells express MMP-9 (arrow) in Sample 11. E: Some tumor cells show no immunoreactivity for TIMP-1 (arrows) in Sample 22. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 180.

  • View in gallery

    Photomicrographs demonstrating immunoreactivity for matrix metalloproteinase (MMP)-9 (A) and tissue inhibitor of metalloproteinases (TIMP)-1 (B) in neurinoma tissue (Sample 45). Almost all the tumor cells show immunoreactivity for MMP-9 and TIMP-1. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 270.

  • View in gallery

    Photomicrographs demonstrating immunoreactivity for matrix metalloproteinase (MMP)-9 (A) and tissue inhibitor of metalloproteinases (TIMP)-1 (B) in meningioma tissue (Sample 15). Almost all the tumor cells exhibit immunoreactivity for MMP-9 and TIMP-1. Immunostaining by labeled streptavidin biotin, counterstained with Mayer's hematoxylin, × 270.

References

1.

Apodaca GRutka JTBouhana Ket al: Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells. Cancer Res 50:232223291990Cancer Res 50:

2.

Barsky SHTogo SGarbisa Set al: Type IV collagenase immunoreactivity in invasive breast carcinoma. Lancer I:2962971983Lancer I:

3.

Bellon GCaulet TCam Yet al: Immunohistochemical localisation of macromolecules of the basement membrane and extracellular matrix of human gliomas and meingiomas. Acta Neuropathol 66:2452521985Acta Neuropathol 66:

4.

Brown PDLevy ATMargulies IMKet al: Independent expression and cellular processing of Mr 72,000 type IV collagenase and interstitial collagenase in human tumorigenic cell lines. Cancer Res 50:618461911990Cancer Res 50:

5.

Caccamo DVMcKeever PE: Plasminogen activators and inhibitors in gliomas: an immunohistochemical study. J Neuropathol Exp Neurol 51:3321992 (Abstract)J Neuropathol Exp Neurol 51:

6.

DeClerck YAPerez NShimada Het al: Inhibition of invasion and metastasis in cells transfected with an inhibitor of metalloproteinases. Cancer Res 52:7017081992Cancer Res 52:

7.

DeClerck YAYean TDChan Det al: Inhibition of tumor invasion of smooth muscle cell layers by recombinant human metalloproteinsae inhibitor. Cancer Res 51:215121571991Cancer Res 51:

8.

D'Errico AGarbisa SLiotta LAet al: Augmentation of type IV collagenase, laminin receptor, and Ki67 proliferation antigen associated with human colon, gastric, and breast carcinoma progression. Mod Pathol 4:2392461991Mod Pathol 4:

9.

Desrochers PEJeffrey JJWeiss SJ: Interstitial collagenase (Matrix metalloproteinase-1) expresses serpinase activity. J Clin Invest 87:225822651991J Clin Invest 87:

10.

Docherty AJPLyons ASmith BJet al: Sequence of human tissue inhibitor of metalloproteinase and its identity to erythroid-potentiating activity. Nature 318:66691985 (Letter)Nature 318:

11.

Franks AJEllis E: Immunohistochemical localisation of tissue plasminogen activator in human brain tumours. Br J Cancer 59:4624661989Br J Cancer 59:

12.

Fujimoto NMouri NIwata Ket al: A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 2(72-kDa gelatinase/type IV collagenase) using monoclonal antibodies. Clin Chim Acta 221:911031993Clin Chim Acta 221:

13.

Fukuda YImoto MKoyama Yet al: Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers. Gastroenterol Jpn 26:37411991Gastroenterol Jpn 26:

14.

Goldberg GIStrongin ACollier IEet al: Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin. J Biol Chem 267:458345911992J Biol Chem 267:

15.

Halaka ANBunning RADBird CCet al: Production of collagenase and inhibitor (TIMP) by intracranial tumors and dura in vitro. J Neurosurg 59:4614661983in vitro. J Neurosurg 59:

16.

Herron GSBanda MJClark EJet al: Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J Biol Chem 261:281428181986J Biol Chem 261:

17.

Herron GSWerb ZDwyer Ket al: Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity. J Biol Chem 261:281028131986J Biol Chem 261:

18.

Hibbs MSHasty KASeyer JMet al: Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J Biol Chem 260:249325001985J Biol Chem 260:

19.

Hibbs MSHoidal JRKang AH: Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages. J Clin Invest 80:164416501987J Clin Invest 80:

20.

Hicks NJWard RVReynolds JJ: A fibrosarcoma model derived from mouse embryo cells: growth properties and secretion of collagenase and metalloproteinase inhibitor (TIMP) by tumour cell lines. Int J Cancer 33:8358441984Int J Cancer 33:

21.

Kalebic TGarbisa SGlaser Bet al: Basement membrane collagen: degradation by migrating endothelial cells. Science 221:2812831983Science 221:

22.

Keohane MEHall SWVandenBerg SRet al: Secretion of α2-macroglobulin, α2-antiplasmin, and plasminogen activator inhibitor-1 by glioblastoma multiforme in primary organ culture. J Neurosurg 73:2342411990α2α2J Neurosurg 73:

23.

Kubota TNakagawa THosotani Ket al: Immunohistochemical study of extracellular matrix in schwannomas: in vivo and in vitro observations. Brain Tumor Pathol 9:23311992in vivo and in vitro observations. Brain Tumor Pathol 9:

24.

Liotta LASteeg PSStetler-Stevenson WG: Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64:3273361991Cell 64:

25.

Liotta LATryggvason KGarbisa Set al: Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67681980Nature 284:

26.

Lund-Johansen MRucklidge GJMilne Get al: A metalloproteinase, capable of destroying cultured brain tissue isolated from rat glioma cells. Anticancer Res 11:100110061991Anticancer Res 11:

27.

Mast AEEnghild JJNagase Het al: Kinetics and physiologic relevance of the inactivation of αl-proteinase inhibitor, α1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin). J Biol Chem 266:15810158161991ααJ Biol Chem 266:

28.

Mignatti PRobbins ERifkin DB: Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell 47:4874981986Cell 47:

29.

Moll UMYoungleib GLRosinski KBet al: Tumor promoter-stimulated Mr 92,000 gelatinase secreted by normal and malignant human cells: isolation and characterization of the enzyme from HT1080 tumor cells. Cancer Res 50:616261701990Cancer Res 50:

30.

Monteagudo CMerino MJSan-Juan Jet al: Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue. Am J Pathol 136:5855921990Am J Pathol 136:

31.

Nagase HBarrett AJWoessner JF Jr: Nomenclature and glossary of the matrix metalloproteinases. Matrix Suppl 1:4214241992Matrix Suppl 1:

32.

Obata KIwata KOkada Yet al: A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 3 (stromelysin-1) using monoclonal antibodies. Clin Chim Acta 211:59721992Clin Chim Acta 211:

33.

Okada YGonoji YNaka Ket al: Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. J Biol Chem 267:21712217291992J Biol Chem 267:

34.

Okada YHarris ED JrNagase H: The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts. Purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate. Biochem J 254:7317411988Biochem J 254:

35.

Paganetti PACaroni PSchwab ME: Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease. J Cell Biol 107:228122911988in vitro: involvement of a metalloprotease. J Cell Biol 107:

36.

Ponton ACoulombe BSkup D: Decreased expression of tissue inhibitor of metalloproteinases in metastatic tumor cells leading to increased levels of collagenase activity. Cancer Res 51:213821431991Cancer Res 51:

37.

Reich RThompson EWIwamoto Yet al: Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells. Cancer Res 48:330733121988Cancer Res 48:

38.

Reith ARucklidge GJ: Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase. Biochem Biophys Res Commun 186:3483541992Biochem Biophys Res Commun 186:

39.

Russell DSRubinstein LJ: Pathology of Tumours of the Nervous Systemed 5. Baltimore: Williams & Wilkins1989834837

40.

Sawaya RHighsmith R: Plasminogen activator activity and molecular weight patterns in human brain tumors. J Neurosurg 68:73791988J Neurosurg 68:

41.

Sawaya RRämö JShi MLet al: Biological significance of tissue plasminogen activator content in brain tumors. J Neurosurg 74:4804861991J Neurosurg 74:

42.

Sawaya RZuccarello MHighsmith R: Alpha-1-antitrypsin in human brain tumors. J Neurosurg 67:2582621987J Neurosurg 67:

43.

Schultz RMSilberman SPersky Bet al: Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells. Cancer Res 48:553955451988Cancer Res 48:

44.

Stetler-Stevenson WGKrutzsch HCLiotta LA: Tissue inhibitor of metalloproteinase (TIMP-2). A new member of the metalloproteinase inhibitor family. J Biol Chem 264:17374173781989J Biol Chem 264:

45.

Suzuki KEnghild JJMorodomi Tet al: Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin). Biochemistry 29:10261102701990Biochemistry 29:

46.

Tsuchiya YEndo YSato Het al: Expression of type IV collagenases in human tumor cell lines that can form liver colonies in chick embryos. Int J Cancer 56:46511994Int J Cancer 56:

47.

Vaithilingam ISMcDonald WBrown NKet al: Serum proteolytic activity during the growth of C6 astrocytoma. J Neurosurg 77:5956001992J Neurosurg 77:

48.

Woessner JF Jr: Matrix metalloproteinase and their inhibitors in connective tissue remodeling. FASEB J 5:214521541991Woessner JF Jr: Matrix metalloproteinase and their inhibitors in connective tissue remodeling. FASEB J 5:

49.

Yamagata SIto YTanaka Ret al: Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis. Biochem Biophys Res Commun 151:1581621988Biochem Biophys Res Commun 151:

50.

Zhang JFujimoto NIwata Ket al: A one step sandwich enzyme immunoassay for human matrix metalloproteinase 1 (interstitial collagenase) using monoclonal antibodies. Clin Chim Acta 219:1141993Clin Chim Acta 219:

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 6 6 6
Full Text Views 59 59 21
PDF Downloads 22 22 15
EPUB Downloads 0 0 0

PubMed

Google Scholar