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Advances in Brief |
Departments of Neurosurgery [S. K., S. M., S. K. C., Y. K., C. N., S. S. L., Y. A., F. A-O., R. S., J. S. R.], Neuro-Oncology [S. L. J., A. P. K.], and Neuropathology [G. N. F.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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The specific mechanisms facilitating the invasive behaviors of brain cancers remain obscure; however, the interactions between cancer cells and the surrounding normal cells and ECM4 are thought to be key aspects in tumor cell invasion (3) . To invade and spread through surrounding normal tissue, tumor cells must degrade multiple elements of the ECM, including fibronectin, laminin, and type IV collagen (4) . Several different proteases, including serine proteases, MMPs, and cysteine proteases, are thought to potentiate the invasiveness and metastatic ability of a variety of malignant tumors (5) .
MMPs are neutral proteinases encoded by a multigene family. These proteinases mediate ECM degradation and tissue turnover during physiological and pathological processes (6) . These enzymes have been grouped into four broad categories on the basis of their substrate specificity: (a) interstitial collagenases (MMP-1, MMP-8, and MMP-13) that degrade fibrillar collagens; (b) type IV collagenases (MMP-2 and MMP-9) that degrade basement membrane collagens, gelatin, and elastin; (c) stromelysins (MMP-3, MMP-10, and MMP-11) that degrade proteoglycans, fibronectin, laminin, gelatin, and the globular portions of type IV collagen; and (d) MT-MMPs (MT1-MMP or MMP-16, MMP-15, MMP-16, and MMP-17) that contain a unique transmembrane domain in their COOH terminus that localizes these MMPs at the cell surface (7) . All of these enzymes are secreted in latent forms and are activated by cleavage of NH2-terminal propeptides at a conserved sequence. This cleavage can be mediated by proteases such as plasmin, trypsin, kallikreins, and cathepsins and by organomercury compounds (7) . The activities of the MMPs are regulated by gene expression (8) , proenzyme activation, and inhibition of the active enzymes by their specific tissue inhibitors (TIMPs; Ref. 9 ). Levels of MMP-2, MMP-9, and MT1-MMP are reportedly elevated in various cancers (10 , 11) . Evidence that MMPs are involved in invasion and angiogenesis in gliomas comes from observations that MMP-2, MMP-9, and MT1-MMP have been found in several glioma cell lines and surgical specimens (12, 13, 14, 15, 16, 17) , that MMP-2 or MMP-9 protein is localized to the tumor neovasculature (16) , and that the extent of MMP overexpression correlates with glioma grade (16 , 17) .
In this study, we down-regulated MMP-9 by transfecting a human glioma cell line with an antisense vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Cells transfected with this antisense MMP-9 showed inhibition of MMP-9 at the mRNA, protein expression, and enzymatic activity levels as well as subsequent reduction of invasiveness in vitro and in vivo.
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Materials and Methods |
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Cell Culture.
SNB19, an established human glioma cell line, was routinely grown in
high-glucose DMEM/F-12 medium (1:1) supplemented with 10% fetal bovine
serum, 20 mM HEPES, 100 units/ml penicillin, and 100
µg/ml streptomycin in a humidified atmosphere containing 5%
CO2 at 37°C. Preliminary experiments revealed
that at 50 ng/ml, PMA had a maximal stimulatory effect on MMP-9
expression without affecting cell viability (as assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay).
Preparation of MMP-9 Constructs.
A 528-bp cDNA fragment of MMP-9 was amplified by PCR with synthetic
primers and subcloned into the pcDNA3 vector (Invitrogen, Carlsbad, CA)
between the HindIII and XhoI polycloning sites in
the sense and antisense orientations. The following primers were used:
(a) for sense MMP-9, the forward primer was
5'-AATAAGCTTAGACACCTCTGCCCTCACCATGAG-3', and the reverse
primer was 5'-AATCTCGAGAACTGGATGACGATGTCTGCGTCC-3; and
(b) for antisense MMP-9, the forward primer was
5'-AATCTCGAGAGACACCTCTGCCCTCACCATGAG-3', and the reverse
primer was 5'-AATAAGCTTAACTGGATGACGATGTCTGCGTCC-3' (enzyme
recognition sites are underlined). Sequence analysis
of the 528-bp internal sequence verified its 100% homology with the
published sequence of MMP-9 cDNA (18)
.
Transfection of SNB19 Cells.
SNB19 cells were transfected with the MMP-9 cDNA constructs in sense
and antisense orientations by using LipofectAMINE (Life Technologies)
according to a previously described procedure (19)
. Stable
transfectants were selected by growing cells in 800 µg/ml G418; the
cells that survived were then expanded in the absence of G418 for
additional studies. Stable transfectants were screened on the basis of
MMP-9 expression in the presence of 50 µg/ml PMA.
Northern Blot Analysis.
Total cellular RNA was extracted from confluent cultures as described
previously (19)
. Aliquots of 10 µg of RNA were separated
by electrophoresis on 1.2% agarose-formaldehyde gels, transferred to a
nylon membrane overnight, and cross-linked with UV irradiation. The
filters were hybridized at 65°C with a MMP-9 cDNA probe labeled with
[32P]dCTP by random primer labeling
(16)
. The filters were washed in SSC (3 M
sodium chloride, 0.3 M sodium citrate) and 0.1% SDS for 20
min at room temperature and then washed in 0.5x SSC, 0.1% SDS for 15
min at 65°C and exposed to X-ray film at -70°C. The membranes were
then stripped and rehybridized with GAPDH cDNA to verify equal loading
in all of the lanes.
Gelatin Zymography.
MMP-2 and MMP-9 expression was analyzed on SDS polyacrylamide gels
impregnated with 0.1% gelatin (w/v) and 10% polyacrylamide (w/v) as
described previously (14
, 16)
. Cells were treated with or
without PMA for 6–8 h, and then the culture medium was replaced with
fresh serum-free medium. The conditioned medium was collected 48 h
later, and medium containing equal amounts of protein (20 µg) was
mixed with sample buffer before electrophoresis. The gels were run at a
constant current and then washed twice for 30 min in 50 mM
Tris-HCl (pH 7.5) plus 2.5% Triton X-100; the gels were then incubated
overnight at 37°C in 50 mM Tris-HCl (pH 7.6), 10
mM CaCl2, 150 mM NaCl,
and 0.05% NaN3. The gels were stained with
Coomassie Brilliant Blue R-250 and then destained.
Western Blotting.
Western blotting for MMP-9 was performed by lysing the cells with
radioimmunoprecipitation assay buffer (1% NP40, 20 mM
Tris, 150 mM NaCl, 5 mM EDTA, 0.1 mg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
Twenty-µg aliquots of cell lysate from each treatment were then
loaded onto a 10% resolving SDS-PAGE gel with a 4.5% stacking gel.
Samples were electrophoresed and then electroblotted onto a
nitrocellulose membrane. Western blot analysis was performed with a
1:2000 dilution of anti-MMP-9 antibody (Oncogene Science, Cambridge,
MA). Immunoreactive bands were visualized for MMP-9 using a horseradish
peroxidase-conjugated antimouse IgG secondary antibody and enhanced
chemiluminescence reagents and exposed to X-ray film.
Matrigel Invasion Assay.
Invasiveness of parental SNB19 cells and vector-, sense MMP-9-, and
antisense MMP-9-transfected SNB19 clones was measured by the invasion
of cells through Matrigel-coated transwell inserts (Becton Dickinson,
Boston, MA). Briefly, transwell inserts with 8-µm pores were coated
with a final concentration of 1 mg/ml Matrigel, and 200 µl of cell
suspension (1 x 106 cells/ml)
were added in triplicate wells. After a 24-h incubation, the cells that
passed through the filter into the lower wells were quantified as
described elsewhere (19)
and expressed as a percentage of
the sum of the cells in the upper and lower wells. Cells on the lower
side of the membrane were fixed, stained with Hema-3, and photographed.
Three-dimensional Spheroid Invasion Assays.
Invasiveness of glioma spheroids in a three-dimensional invasiveness
model was measured according to a previously described procedure
(20)
in which glioma spheroids were cocultured with fetal
rat brain aggregates. Tumor spheroids were stained with the fluorescent
dye DiI and combined with fetal rat brain aggregates that had been
stained with DiO. At different time intervals, serial 1-µm-thick
sections were obtained from the surface through the center of the
cocultures with a confocal laser-scanning microscope. DiI and DiO
fluorescence was detected by using an argon laser at 488 nm with a
bandpass filter at 520–560 nm and a helium/neon laser at 543 nm with a
long-pass filter at 590 nm, respectively. The remaining volume of the
brain aggregate or tumor spheroid during cocultures at 24, 48, and
72 h was quantitated using the following formula: volume of
aggregate = (V1 + V2 + V3 + VM) x 2.
Intracranial Implantation.
For these experiments, parental cells and vector, sense, and antisense
stable transfectant cells were trypsinized and resuspended in
serum-free medium. Mice were anesthetized, and 10-µl aliquots of a
cell suspension containing 2 x 106 cells in serum-free medium were injected
using a stereotactic frame as described elsewhere (20)
.
Four weeks later, the mice were anesthetized and killed by intracardiac
perfusion with PBS followed by 4% paraformaldehyde in saline for
in situ fixation of the tumors. The brains were removed,
placed in 6% paraformaldehyde, and allowed to stand at 4°C for
4 h, and then they were transferred to a solution of 0.5
M sucrose in PBS and incubated overnight at
4°C. The following day, the brains were cut, embedded in microscopic
slides, and frozen at -20°C. The frozen sections were stained with
H&E to examine tumor growth. The sections were blindly reviewed by a
neuropathologist (G. N. F.) and scored for the size of the tumor in
each case semiquantitatively. The maximal cross-sectional diameter
measured in sections of each tumor was taken as a measurement of tumor
size and compared between controls and antisense transfectants.
The variation between the sections in each group was less than 10%.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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MMP-9 Activity and mRNA and Protein Levels in Parental and Stable
Transfectants by Gelatin Zymography, Northern Blotting, and Western
Blotting.
Vector- and sense MMP-9 construct-transfected clones expressed MMP-9 in
response to PMA, whereas in the absence of PMA, these cells expressed
only MMP-2 (Fig. 1A)
. In
contrast, the antisense clones in the presence of PMA expressed very
little or no MMP-9 activity and no significant change in MMP-2
activity. Occasionally, all of the antisense clones and parental cells
responded to PMA by expressing activated forms of MMP-2
(21)
. Quantitatively, MMP-9 enzymatic activity was 10–14
times higher in parental and vector controls and sense clones compared
with antisense clones. After gelatin zymography confirmed that MMP-9
activity was reduced in the antisense clones, we characterized mRNA
expression in parental clones, vector-transfected clones, sense MMP-9
construct-transfected clones, and antisense MMP-9 construct-transfected
clones. Of these clones, the parental clones, the vector-transfected
clones, and the sense-transfected clones all expressed MMP-9 mRNA in
the presence of PMA (Fig. 1B)
. In contrast, none of the
three antisense MMP-9 construct-transfected SNB19 cells expressed the
message for MMP-9 in the presence or absence of PMA. Probing the
stripped membranes with GAPDH showed that similar amounts of mRNA had
been loaded on the Northern blots. Densitometric quantification of
MMP-9 mRNA was significantly higher (10–12-fold) in parental,
vector-transfected, and sense-transfected clones compared with
antisense-transfected clones. After confirming by Northern
blotting that MMP-9 mRNA was reduced in antisense-transfected clones,
we determined MMP-9 protein levels in parental clones,
vector-transfected clones, sense MMP-9 construct-transfected clones,
and antisense MMP-9 construct-transfected clones by Western blotting
using MMP-9-specific antibodies. Use of an anti-MMP-9 antibody
revealed that MMP-9 protein was present in parental cells,
vector-transfected cells, and sense MMP-9 construct-transfected
cells treated with PMA (Fig. 1C)
, but MMP-9 protein
was not detected in antisense transfectants, regardless of PMA
treatment (Fig. 1C)
.
|
. In contrast, the
antisense MMP-9 construct-transfected clones were much less invasive,
and PMA treatment did not augment their invasiveness (Fig. 2A)
. The percentage of invasion of parental, vector, and
sense clones was much higher (45–48%) than that of antisense clones
(14–16%; Fig. 2B
). PMA treatment slightly increased
the invasive behavior of parental, vector, sense (from 48% to 58%),
and antisense clones (from 15% to 18%), and the effect was
slightly higher in the parental, vector, and antisense clones (Fig. 2B)
. In spheroid coculture assays, fetal rat brain cell
aggregates stained with a fluorescent dye, DiO, were confronted with
spheroids of parental, vector, sense, and antisense MMP-9 clones that
had been stained with DiI fluorescent dye. The tumor spheroids merged
with the rat brain aggregates within 24 h. At 48 and 72 h,
the tumor spheroids consisting of parental, vector, or sense clones
(red fluorescence) had progressively invaded the rat brain
aggregates (green fluorescence), producing a corresponding
decrease in brain aggregate volume. On the other hand, the antisense
MMP-9 spheroids failed to invade rat brain aggregates even at 72 h, although the two cell types did become attached to each other after
24 h (Fig. 3A)
.
Quantitative analysis of the remaining fetal brain aggregates were
10–15% in parental, vector, and sense clones compared with 92–95%
in antisense clones (Fig. 3B)
. The invasiveness of
fetal rat brain aggregates was significantly decreased in antisense
clones compared with parental, vector-, and sense construct-transfected
clones (P < 0.001).
|
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. In contrast, mice that received an injection of
antisense-transfected cells showed no tumor formation or very small
tumor formation at 4 weeks after the injection (Fig. 3C)
.
The sections analyzed using H&E staining and blindly reviewed by a
neuropathologist (G. N. F.) revealed that there was no significant
difference in tumor size in mice that received an injection of
parental, vector-, and sense-transfected clones. However, the tumor
size was significantly reduced (P < 0.001)
in antisense clones compared with parental, vector, and sense clones
(Fig. 3D)
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Glioblastomas express greater amounts of MMPs than do either low-grade gliomas or normal brain in vivo (13, 14, 15, 16, 17) . We found that MMP-9 activity in surgical specimens was proportional to the grade of the glioma (16) . Others have reported that overexpression of MMP-2 and MMP-9 associated with glioma malignancy was accompanied by overexpression of the TIMP-1 gene (24) . In contrast, we found glioma progression to be associated with down-regulation of TIMP-1 and TIMP-2 (25) , suggesting that an imbalance between expression and/or activity of MMPs and their specific inhibitors may be involved in tumor development. If the abundant expression of MMP-9 is responsible for the aggressive invasive behavior of gliomas, then down-regulation of MMP-9 could reduce the MMP-9-mediated invasiveness in glioblastomas.
Strategies such as expression of antisense RNA enable cell surface events to be bypassed such that the antisense sequence can directly influence the expression of a given gene of interest. Expression of cDNA constructs resulting in antisense RNA provides a direct and unambiguous experimental approach for studying the involvement of MMP-9 in brain tumor progression. In the present study, we stably transfected the glioblastoma cell line SNB19 with an expression vector containing a 528-bp MMP-9 cDNA in the sense and antisense orientations. Our results showed that successful transfection of the SNB19 cells with MMP-9 antisense construct led to decreases in MMP-9 enzymatic activity, protein contents, and mRNA transcripts, suggesting that this antisense construct has profound biological consequences. Parental, vector-, and sense-transfected clones showed no changes in these variables, which indirectly confirms that the antisense construct was responsible for the observed decrease in transcripts and protein. The mechanism by which antisense RNA affects the expression of MMP-9 in antisense-transfected cells is unclear, but it may interfere with mRNA transport or hybridization with cytoplasmic MMP-9 mRNA, which presumably would interfere with translation.
Antisense-transfected MMP-9 clones showed reduced invasiveness in Matrigel and spheroid invasion assays, indicating that MMP-9 is required for invasiveness. Antisense-transfected MMP-9 clones were also unable to form tumors in nude mice, unlike parental cells or vector-transfected or sense-transfected clones. In a rat sarcoma model, MMP-9 expression was shown with ribozyme directed against MMP-9 mRNA to be involved in metastasis but not in tumor growth (26) .The importance of MMP gene expression is underlined by the studies that demonstrated that synthetic MMP inhibitors limit tumor growth in animal models. Synthetic MMP inhibitors batimastat and marimastat effectively reduced glioma invasion in Matrigel-coated transwell assays and cocultures of tumor spheroids with fetal rat brain aggregates, although higher concentrations were required in coculture systems (27 , 28) . AG3340, another novel synthetic MMP inhibitor, inhibits the growth of the U87 glioma cell line s.c. implanted in SCID-NOD mice (29) .
In summary, our stable transfection of an established human glioblastoma cell line with antisense MMP-9 cDNA produced stable antisense transfectants that were less invasive than their parental cells because of the inhibition of MMP-9 expression. Our results suggest that MMP-9 would be a promising target for gene therapy to reduce the invasiveness of human gliomas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grant CA56792 (to
J. S. R.). 
2 S. K. and S. M. contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Department of Neurosurgery, Box 64, The University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
TX 77030. Phone: (713) 792-3266; Fax (713) 745-4687; E-mail: jrao{at}mdanderson.org
4 The abbreviations used are: ECM, extracellular
matrix; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; TIMP,
tissue inhibitors of metalloproteinase; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; PMA, phorbol myristate acetate.
Received 7/20/00. Accepted 10/23/00.
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