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From the Abteilung Neuropathologie,*
Neurozentrum and
Abteilung für Rheumatologie und Klinische
Immunologie,
Medizinische
Universitätsklinik, Universität Freiburg, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For reasons that are poorly understood, most primary brain tumors do not metastasize systemically.3 However, they spread locally through extensions of infiltrating tumor cells in normal brain or, more rarely, through cerebral spinal fluid dissemination along the neuroaxis.4 Local invasiveness is an important characteristic of gliomas, contributing substantially to the failure of curative treatments. Even low-grade cerebral gliomas are infiltrative, whereas malignant gliomas are widely invasive. The seeding of tumor cells in the neuroaxis is a common feature of PNETs, whereas distant metastases are found in about 15% of the cases.5 These characteristics common to different brain tumors make their complete surgical resection difficult and focal therapy often ineffective. Relapses and recurrences occur not only at the primary site, but also in distant locations. Thus, the understanding of the mechanisms involved in tumor invasiveness within the brain is of high importance to develop new strategies to control local tumor growth.
Clues to the process of tumor invasion have been ascertained through an increased understanding of key roles played by the extracellular matrix, cell adhesion molecules, and proteases. Two families of proteases, the serine proteases of the plasminogen activator/plasmin system and the matrix metalloproteinases (MMPs), have been implicated in tumor invasion.6-10 MMPs are a family of structurally related enzymes that degrade macromolecules of the extracellular matrix. The gene family of human MMP comprises at least 15 members. They have been classified into five different classes based on their substrate specificities (collagenases, gelatinases, stromelysins, and others) or on their transmembrane localization (such as the recently described membrane-type MMP class, mt-MMP).11 With the exception of stromelysin 3, all members of the substrate-specific classes of MMP are secreted as inactive proenzymes that require enzymatic cleavage of the propeptide domain for activation.9,12 Once secreted and activated, they are inhibited by a family of endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).13,14 The balance between the levels of activated MMP and free inhibitors determines the overall MMP activity.15 Maintenance of this critical equilibrium is essential, because a disturbed balance or ratio of MMP and TIMP affects the invasive process. Decreased TIMP gene expression results in increased tumor invasiveness, whereas overexpression leads to reduced invasive growth in vivo.
A positive correlation between tumor malignancy and MMP levels has been documented in brain tumors.16 Previous studies have also shown that glioma cells are able to secrete a number of metalloproteinases and TIMP in vitro.17 The production of matrilysin and stromelysin 1 has been reported in glioma cell lines.18 Gelatinase A and B production has been shown to be increased in human malignant gliomas.19
Although the literature on this subject has grown in recent years, there are only very few systematic studies involving the most representative members of the MMP and TIMP families. The present study was performed to examine the expression pattern of all MMP genes in gliomas and PNETs to determine whether their expression correlates to the invasive potential and to histological grade of these tumors. Similarly, the role played by TIMP in the invasion process and its relationship to secreted MMP was studied at the RNA and protein levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twelve samples of previously untreated cerebral gliomas (low-grade astrocytoma (n = 3), anaplastic astrocytoma (n = 2), glioblastoma multiforme (GBM; n = 4,) and samples of medulloblastoma (n = 3) were obtained during surgical removal at the Department of Neurosurgery of the Freiburg University Hospital. Histopathological diagnoses were based on the criteria from the latest World Health Organization classification.20
Two samples of nonneoplastic brain tissue used as control were obtained during surgical removal of deeply localized meningiomas. Samples were immediately snap frozen in liquid nitrogen and stored at -80°C until analysis.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted from tissue samples using a two-step protocol. Frozen tissues were pulverized and RNA was prepared in the first step using the guanidinium thiocyanate method according to Chomczynski and Sacchi.21 After precipitation with isopropanol, the RNA was redissolved in lysis buffer from the RNeasy mini kit (Qiagen, Hilden, Germany) and prepared according to the manufacturer's instructions. Total RNA (15 µg) was separated in a 1.4% denaturing agarose gel containing 2.2 mol/L formaldehyde, transferred overnight by capillary blot onto Duralon ultraviolet membrane (Stratagene, La Jolla, CA) and cross-linked with an ultraviolet cross-linker (Stratagene). The membranes were hybridized for 1 hour in Quick Hyb (Stratagene) at 68°C with randomly primed 32P-labeled cDNA along with the following DNA probes: the 2.1-kb collagenase 1, a 469-bp collagenase 2 fragment, the 1.9-kb collagenase 3,22 the 2.1-kb gelatinase A,23 a 1.7-kb gelatinase B fragment,24 the 1.8-kb stromelysin 1, the 1.6-kb stromelysin 2,22 the 1.8-kb stromelysin 3,25 a 1.1-kb matrilysin fragment,26 a 408-bp human metalloelastase fragment, a 735-bp RASI fragment,27 the 1.2-kb mt1-MMP,28 a 495-bp mt2-MMP fragment, a 429-bp mt3-MMP fragment, a 317-bp mt4-MMP fragment,29 a 780-bp TIMP 1 fragment (kindly provided by S. Gay), a 357-bp TIMP-2 fragment, a 341-bp TIMP-3 fragment, and the 1.27-kb glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membranes were washed twice for 15 minutes at 60°C in 2x standard saline citrate, 0.1% sodium dodecyl sulfate (SDS), and finally in 0.1x standard saline citrate and 0.1% SDS for 30 minutes and exposed to Biomax MR film (Kodak) with intensifying screen for 18 to 48 hours at -80°C. The mRNA levels were quantified by densitometric analysis using an Image Master System DTS (Pharmacia, Uppsala, Sweden). GAPDH was used as loading control.
Polymerase Chain Reaction Analysis
Total cellular RNA was extracted as described above. A reverse transcription system (Promega, Madison, WI) was used to generate the first cDNA strand according to the instructions of the manufacturer. Subsequently, a polymerase chain reaction (PCR) was performed using 2 µl of the reverse transcription reaction and Taq polymerase (high fidelity; Boehringer Mannheim, Mannheim, Germany) with the following thermocycle parameters: 4 minutes at 94°C; 31 cycles of 45 seconds at 94°C, 1 minute at 55°C, and 1 minute at 72°C; and 7 minutes at 72°C. The following oligonucleotide primers were used for PCR: collagenase 1: 5-ATTCTACTGATATCGGGGCTTTGA-3 and 5-ATGTCCTTGGGGTATCCGTGTAG-3 (+683 to +1092); collagenase 2: 5-ACGGGAAGCCAAATGAGGA-3 and 5-GAGTGAGCGAGCCCCAAAGAAT-3 (+222 to +680); collagenase 3: 5-CCTGGCTGCCTTCCTCTTCTTGA-3 and 5-AACCCCGCATCTTGGCTTTTTC-3 (+15 to +294); stromelysin 1: 5-ATGCCCACTTTGATGATGATGAAC-3; 5-CCACGCCTGAAGGAAGAGATG-3 (+581 to +1022); stromelysin 2: 5-ATGCGCAAGCCAAGGTGTG-3 and 5-GGAGGGGGAGGTCCGTAGAGA 3 (+257 to +871); stromelysin 3: 5-GCCGCCGGACGTCCACCACCTC-3 and 5-CAGCGCCCGCCAGAAAGCACGAAC-3 (+103 to +315); matrilysin: 5-TGTTAAACTCCCGCGTCATAGAAA-3 and 5-CTCCTCGCGCAAAGCCAATCAT-3 (+218 to +486); human metalloelastase: 5-GACCGGGCAACTGGACACATCTA-3 and 5-CACGGGCAAAAACCACCAAAAT-3 (+225 to +496); RASI: 5-GCTGGGCCGCTGGAGAAAGAA-3 and 5-CGAGGCGAGTAGACAGCAGCATCC-3 (+300 to +1035); and TIMP-3: 5-TTGGCTCGGGCTCATCGTGCTC-3 and 5-GCCCCGTGTACATCTTGCCATCAT-3, (+9 to +349).
GAPDH primers were used for loading control. For negative control, reverse transcription-PCR was carried out in the absence of RNA. The amplification products were separated in 1.5% agarose gels.
Substrate Gel Electrophoresis
Human brain tumor tissues were thawed, weighed, homogenized in Tris buffer (50 mmol/L Tris-HCl and 75 mmol/L NaCl, pH 7.5) and centrifuged for 20 minutes at 5000 x g. The pellets were discarded, the supernatants were aliquoted, and the protein content was determined using the BCA Protein Assay Reagent (Pierce, Rockford, IL). Samples were mixed with SDS sample buffer in the absence of a reducing agent and incubate for 20 minutes to denature MMP and dissociate MMP-TIMP complexes. Electrophoresis proceeded on 10% polyacrylamide gels containing 0.1% SDS and gelatin at a final concentration of 0.12%. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 1 hour to remove the SDS. During this process, proenzymes are activated autocatalytically. Gels were then incubated for 24 hours at 37°C in reaction buffer (50 mmol/L Tris-HCl, pH 7.5, 0.15 mol/L NaCl, 10 mmol/L CaCl2, and 0.02% NaN3) and stained with 0.1% Coomassie Brilliant Blue R 250. After adequate destaining, gelatinolytic activity is detectable as a clear band against blue background.
Immunohistochemistry
Staining was carried out using immunoperoxidase staining kits for mouse and for rabbit immunoglobulin (Vector Laboratories, Burlingame, CA). The following primary antibodies and corresponding concentrations were used: anti-mt-MMP (114-1F2), 5 µg/ml; anti-gelatinase A (42-5D11), 5 µg/ml (both mouse monoclonal, Calbiochem, Bad Soden, Germany); anti-MMP-9, 10 µg/ml (rabbit polyclonal, Quartett, Berlin, Germany); and anti-TIMP-2, 10 µg/ml (rabbit polyclonal, Biogenesis, Poole, UK).
Surgical specimens were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. After incubation with citrate buffer, endogenous peroxidase activity was blocked with 0.3% H2O2 for 30 minutes. After several washes with phosphate-buffered saline, sections were incubated with 2% normal serum for blocking of nonspecific binding, followed by incubation with the primary antibody in a humid chamber at 4°C overnight. After washing in phosphate-buffered saline, the sections were incubated with biotinylated anti-immunoglobulin of the appropriate species specificity followed by avidin-biotin-horseradish peroxidase complex; between each incubation step the slides were washed three times in phosphate-buffered saline. Immunoperoxidase reaction was visualized with 3,3'-diaminobenzidine-HCl (3,3'-diaminobenzidine buffer tablets; Merck, Darmstadt, Germany) and 0.006% H2O2. The slides were briefly counterstained with hematoxylin and mounted and examined under a Leitz Dialux 20 EB microscope. Normal serum 2% was used as control.
For immunofluorescence, an anti-mt-MMP antibody was used at a dilution of 10 µg/ml and counterstained for 3 hours at room temperature with a goat anti-mouse-immunoglobulin G conjugated with Cy3 (2 µg/ml) (Rockland Lab, Gilbertsville, PA). Mounted slides were evaluated under a Leica TCS NT microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test the expression of MMP and TIMP mRNAs in gliomas,
medulloblastomas, and normal brain, we performed Northern blot analysis
(Table 1)
. We found elevated levels of
expression of 7 out of 15 MMPs tested and of 2 out of 3 TIMP. The
transcripts shown in Figure 1A
correspond
at 2.9 kb to gelatinase A, at 2.8 kb to gelatinase B, at 0.9 kb to
TIMP-1, at 4.5 kb to mt1-MMP, at 4.2 kb to mt2-MMP, at 5.2 kb to
mt3-MMP, and at 1.4 kb to GAPDH. TIMP-2 transcripts were detected in
brain tissues and tumors as a major 3.5-kb band with additional minor
bands at 2.3, 1.5, and 1.0 kb (Figure 1A)
.
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. The levels of gelatinase B were increased in three
of four GBM. In medulloblastomas mt1-MMP and gelatinase A were
overexpressed, but to a far lesser extent than observed in GBM.
Moreover, in two of three medulloblastoma samples, mt2-MMP was
dramatically up-regulated as compared with other tumors examined. In
the third sample (a desmoplastic variant), an up-regulation of
gelatinase A was detected. Only a weak expression of mt3-MMP and of
mt4-MMP (data not shown) was found in all tissues examined.
Interestingly, in none of the tumors tested in this study did we
observe detectable levels by Northern blot analysis of collagenases,
stromelysins, matrilysin, metalloelastase, and RASI (data not shown). PCR Analysis Demonstrated Very Low Levels of Expression of Further MMP Genes
Using PCR analysis, we did not find any detectable level of mRNA
for collagenase 2, collagenase 3, and stromelysin 2 (data not shown).
Very weak expression of collagenase, metalloelastase, stromelysins 1
and 3, and RASI was observed in different probes without any
correlation to the tumor grading or histology (data not shown). Weak
matrilysin expression was seen in two GBM (Figure 2)
; TIMP-3 expression was seen in all
tissues tested without any significant correlation to tumor grading
(Figure 2)
. Results of PCR analysis are summarized in Table 1
.
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We analyzed the secretion of gelatinases by substrate gel
electrophoresis (zymography). With this method one can distinguish
between the latent progelatinases and the activated enzyme forms. The
respective progelatinases with a molecular weight of 92 kd for
progelatinase B and 72 kd for progelatinase A are indicated in Figure 3
. Below the latent forms appear the
proteolytically activated species as a prominent complex of two or
three bands. The zymography demonstrated a constitutive secretion of
the latent forms of gelatinases A and B in every tumor examined, even
in normal brain; however, in low-grade astrocytomas and in normal
brain, we did not find detectable amounts of the activated enzymes. In
contrast, in GBM, an increase amount of active forms of gelatinases was
clearly demonstrable as indicated by a second and even a third band
(Figure 3)
. In medulloblastomas, the level of proenzyme forms was high,
with only barely detectable active forms of both gelatinases. The
results of zymography are summarized in Table 1
.
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Based on the results of Northern blots, we performed
immunostaining of gliomas and medulloblastomas with anti-mt1-MMP,
anti-gelatinase B, anti-gelatinase A, and anti-TIMP-2. The findings of
the immunostaining corroborate those of the Northern blot analysis.
Positive staining for mt1-MMP was restricted to tumor cells; it was
localized in the cell membrane and displayed a heterogeneous pattern
with focal increase of immunoreactivity. Similarly, gelatinase A was
identified in tumor cells, however, intracytoplasmatically and in
partial co-localization with mt1-MMP. In low-grade astrocytomas, few
cells displayed a sporadic, positive staining for gelatinase A and
mt1-MMP, whereas malignant gliomas and medulloblastomas showed an
intense immunoreaction (Figure 4)
. One
case of medulloblastoma was a desmoplastic variant and exhibited a
distinct feature: the high cellular trabeculae of tumor cells, which
were marked by reticulin deposition, showed an intense immunostaining
for gelatinase A, whereas the reticulin-free islands stained
predominantly for mt1-MMP (Figure 7)
.
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. Immunofluorescence with
anti-mt1-MMP antibody and confocal laser microscopy clearly showed
immunostaining on the cell membrane of tumor cells (Figure 6)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Gelatinase A and B differ from other MMPs by their ability to interact, as latent proenzymes, with endogenous TIMP-2 and TIMP-1, respectively.31 The complex of TIMP-2 with the progelatinase A seemed to be essential for activation of progelatinase A, because this enzyme in the presence of TIMP-2 binds to and can be activated by a membrane-bound MMP (mt1-MMP) on the cell surface.28,32-34 It has been shown that activated gelatinase A itself is able to activate the progelatinase B.35 Also, stromelysin-1 and plasmin can activate gelatinase B.36 However, in some tumors cells, these enzymes are not always co-expressed. In contrast, co-expression of gelatinase A and B was detected in different tumor tissues.35,37 The activation of progelatinase B by gelatinase A may be significant in tissues in which stromelysin-1 may not be present. Thus, it is highly plausible that the following proteolytic cascade may enhance extracellular matrix degradation in malignant gliomas: increased expression of mt-MMP leading to activation of gelatinase A, which is able to activate gelatinase B. Our results support this hypothesis and are consistent with a recent report on increased expression of mt1-MMP and gelatinase A in malignant gliomas: the increased expression of mt-MMP is correlated with the expression and activation of gelatinase A and with histological progression of human gliomas in vivo.16
In Northern blot analysis, we could not detect mRNA of collagenases, stromelysins, matrilysin, metalloelastase, and RASI in human brain tissues. Using PCR analysis, we found very low expression of stromelysin 3. This enzyme is mainly associated with human breast cancer stroma cells, although its substrate specificity has not yet been defined.38 We also detected very low levels of matrilysin mRNA, which is found overexpressed in human breast and colonic carcinomas.38,39 Stromelysin 2 and interstitial collagenase have been detected in epidermoid carcinomas of the head and neck40 but were expressed only at very low basal levels in the tumors included in this report. These studies suggest that there may be organ- or cell type-specific expression of certain members of the MMP gene family in malignant tumors. From our present results, it can be speculated that the mt1-MMP/gelatinase A system may play a much more important role in malignant gliomas than other MMP. Corroborating this hypothesis, Deryugina et al41 have recently shown that transfection of glioma cells with cDNA encoding mt1-MMP resulted in increased cell surface activation of mt1-MMP and TIMP-2, constitutive activation of gelatinase A proenzyme, increased collagen degradation, and cell migration in tumor spheroid outgrowth assay.
Several studies have emphasized that the balance between MMP and their inhibitors is critical for control of proteolysis.40 In the literature, conflicting results are found regarding the expression of TIMP in gliomas. Mohanam et al15 reported that TIMP-1 and TIMP-2 were expressed in normal brain and in tumor tissues but were significantly lower in highly invasive glioblastomas. Low-grade central nervous system tumors may thus produce proportionately more TIMP than high-grade tumors, which would then explain the different degrees of invasiveness. However, corroborating the results of Nakano et al,19 we found that TIMP-1 overexpression is closely related to the degree of malignancy in a given glioblastoma and to the overexpression of mt1-MMP/gelatinase A. It is thus tempting to speculate that the overexpression of TIMP-1 may prevent the excess action of MMP.
We detected TIMP-2-increased levels in all tissues examined. In neoplastic brain tissue, TIMP-2 was detectable by immunohistochemistry in a high percentage of tumor cells. In normal brain, TIMP-2 localized to endothelial cells and sporadically occurred in astrocytes and neurons. The constitutive expression of TIMP-2 in normal brain is still poorly understood.
Recently, a third member of the TIMP family has been described: TIMP-3 inhibits, as do TIMP-1 and TIMP-2, collagenase-1, stromelysin-1, and gelatinase A and B.42,43 In gliomas, medulloblastomas, and normal brain, we found very low expression of TIMP-3. This finding suggests that TIMP-3 is probably not involved in the regulation of MMP in brain tumors.
One characteristic of medulloblastomas is their tendency of leptomeningeal invasion and subsequent spread within the cerebrospinal fluid.44 Although this property is of considerable clinical relevance, the underlying mechanism is poorly understood. The present study demonstrates for the first time that medulloblastomas express high levels of MMP. An interesting aspect was observed in a desmoplastic medulloblastoma: the high cellular trabecula showed intense immunostaining for gelatinase A, whereas the islands of more differentiated cells exhibited strong reactivity for mt1-MMP. This particular localization pattern is intriguing and suggests that different populations of cells may interact to activate the cascade of gelatinase A. The two other medulloblastomas included were the only tumors that exhibited a high expression of mt2-MMP. This membrane-bound enzyme has been shown to activate like mt1-MMP, the progelatinase A, and progelatinase A-TIMP-2 complexes.45 Although the small number of cases does not allow us to draw a definitive conclusion, these preliminary results suggest that MMP may play an important role in determining the biological behavior of medulloblastomas. Additional studies on the expression of MMP in medulloblastoma are certainly warranted.
In conclusion, the results of this study indicate that the mt1-MMP/gelatinase A system may play a critical role in the mechanism of local tumor invasion observed in malignant gliomas. The overexpression of MMP is accompanied by a concomitant up-regulation of TIMP-1. Future attempts to specifically block MMP enzyme activity or increase TIMP-1 local expression may provide a novel means to inhibit brain tumor invasiveness.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Zentrum für Klinische Forschung Universitätsklinik Freiburg (to WC and HHP) and SFB 505-A5 (to BV).
K. Lampert and U. Machein contributed equally to this study.
Accepted for publication May 20, 1998.
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|
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) and mean
(+) of mt1-MMP and
gelatinase A mRNAs in normal brain
(NB), low-grade
astrocytoma (II),
anaplastic astrocytoma
(III), GBM
(IV), and medulloblastoma
(PNET). The mRNA levels
were quantified by densitometric analysis using an Image Master System
DTS
(Pharmacia). GAPDH
was used as loading control. The relative hybridization signal numbers
were calculated by ascribing an arbitrary value of 1 to the least
intense signal.
