A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

Elevated levels of M(r) 92,000 type IV collagenase in human brain tumors.

Local invasive growth is one of the key features of primary malignant brain tumors accompanied by remodeling of the vasculature and destruction of normal brain tissue. Tissue invasiveness is an essential biological function used by a tumor to overcome the various barriers to its progression. The expression of metalloproteases has been shown to play a critical role in the invasive process in a number of tumors; however, their expression in human brain tumors has not been previously reported. In this study we showed metalloprotease activities at M(r) 240,000, 123,000, 92,000, 72,000, and 67,000 in brain tumor extracts. These enzyme activities were inhibited by EDTA, an inhibitor of metalloproteases. Significant increases in levels of protease bands at M(r) 92,000, 123,000, and 240,000 were observed in glioblastoma and metastatic lung tumors. Enzymatic inhibition and Western blotting with M(r) 92,000 type IV collagenase antibody confirmed the presence of M(r) 92,000 type IV collagenase in all samples. Quantitative analysis by densitometry showed 8-10-fold and 6-8-fold increases in M(r) 92,000 type IV collagenase activity in glioblastoma and metastatic lung carcinoma samples, respectively, when compared with normal brain, meningioma, astrocytoma, metastatic colon, and breast carcinoma samples. These findings provide evidence for elevated levels of metalloproteases in glioblastomas and suggest a therapeutic target for minimizing the invasive propensity of gliomas using protease inhibitors.

Modulation of in vitro invasion of human glioblastoma cells by urokinase-type plasminogen activator receptor antibody.

Four human glioblastoma cell lines (U251, UWR1, UWR2, and UWR3) were tested for the expression of the cell surface receptor for urokinase-type plasminogen activator (uPA). To our knowledge there have been no previous reports about the uPA receptors (uPARs) in glioblastoma cell lines. All four glioblastoma cell lines we tested were found to bind recombinant Pro-uPA saturably and reversibly. Scatchard analysis of radioligand binding with acid-pretreated cells showed the presence of a single population of high-affinity uPARs on glioblastoma cells. Northern blot analysis confirmed that glioblastoma cells like other human cell lines express a 1.4-kilobase uPAR mRNA and 2.4-kilobase uPA mRNA. The significance of the uPAR in the invasive potential of the cells was examined by incubating uPAR antibody in an in vitro invasion assay. The anti-uPAR monoclonal antibody blocked the invasion effectively in a Matrigel assay, in which inhibition of invasion ranged between 20 and 57% for the cells studied. These data suggest that the uPARs contribute significantly to the invasive capacity of the cells, possibly by facilitating uPA activity.

Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro.

In this study, we investigated the expression of activated gelatinase A and membrane-type metalloproteinase (MT-MMP) induced by concanavalin A (ConA) in four highly invasive glioma cell lines (UWR2, UWR3, U251MG, and SNB-19). We also examined gelatinase A and MT-MMP expression in human brain tumor tissues in vivo. Gelatin zymography showed that all four cell lines expressed latent progelatinase A (M(r) 66,000). Activated gelatinase A (M(r) 62,000) was induced by ConA in only UWR2 or UWR3 cells. MT-MMP mRNA was present in all four cell lines prior to ConA treatment, and the relative hybridization signals were 1, 0.80, 0.25, and 0.15 in UWR2, UWR3, U251MG, and SNB-19 cells, respectively. These mRNA signals were dramatically increased (2,8-, 5.4-, and 2.2-fold in UWR2, UWR3, and U251MG cells, respectively) following ConA treatment; however, MT-MMP mRNA expression was unchanged in SNB-19 cells. MT-MMP protein was detected in various amounts in the four cell lines, but only after ConA pretreatment. The amount of MT-MMP mRNA was unchanged in SNB-19 after ConA treatment, and the MT-MMP mRNA level in ConA-treated U251MG was lower than in UWR2 and UWR3 without ConA treatment. MT-MMP protein was detected in SNB-19 and U251 cell lines only after ConA treatment. Gelatin zymography of human brain tumor tissues revealed that almost all samples examined contained a latent form of gelatinase A, whereas the activated form of gelatinase A was only seen in metastatic lung adenocarcinomas and malignant astrocytomas, and especially in glioblastomas. MT-MMP mRNA levels were significantly higher in malignant astrocytomas than in low-grade gliomas and normal brain tissues. These results were confirmed by PCR analysis, which showed that MT-MMP mRNA was absent or barely detectable in normal brain white matter but was easily detectable in malignant astrocytomas. Immunohistochemistry of MT-MMP in frozen sections showed that MT-MMP was localized in neoplastic astrocytes of malignant astrocytomas but was undetectable in normal white brain matter. The data indicate that MT-MMP is present in malignant human glial tumors and that MT-MMP expression correlates with expression and activation of gelatinase A during malignant progression in vivo. A direct correlation between the levels of MT-MMP protein and its transcripts was not found in vitro, suggesting that MT-MMP expression in glioma cell lines might be regulated either at the level of transcription message stability or at posttranscription. Altered MT-MMP expression might contribute, in part, to gelatinase A activation, which in turn facilitates invasion of these tumors.

Expression and localization of urokinase-type plasminogen activator receptor in human gliomas.

Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. In the present study, we examined the presence and distribution of uPARs in human gliomas in vivo. The amounts of uPARs were measured by radioreceptor assays and Northern blotting and were significantly higher in anaplastic astrocytomas and glioblastomas than they were in normal brain tissues and low-grade gliomas. In situ hybridization was performed to investigate the cellular source of uPAR mRNA in various types of astrocytomas and normal brain tissues. uPAR mRNA was localized in astrocytoma cells and endothelial cells within tumor tissue, especially near sites of vascular proliferation and at the leading edges of tumors. uPAR mRNA was also expressed in tumor cells near necrotic areas. Expression was barely detectable in low-grade astrocytomas and normal brain tissues. These results suggest that expression of uPAR in the invading astrocytoma cells may play a significant role in the invasive behaviors of glioblastomas.

Expression and cellular localization of messenger RNA for plasminogen activator inhibitor type 1 in human astrocytomas in vivo.

We investigated the expression and cellular localization of plasminogen activator inhibitor type 1 (PAI-1) in human astrocytoma in vivo. Northern blot and densitometric quantitation of PAI-1 mRNA indicated that PAI-1 transcripts were significantly higher in human malignant astrocytomas and especially in glioblastomas than in low-grade gliomas and normal brain tissues in vivo. Using in situ hybridization with paraffin-embedded surgical specimens of human gliomas and normal brain tissues, PAI-1 mRNA was abundantly expressed in glioblastomas. PAI-1 mRNA was localized mainly in tumor cells and endothelial cells. The distribution of PAI-1 mRNA expression was particularly abundant around areas of vascular proliferation and in remnant tumor cells surrounding necrotic foci. PAI-1 mRNA was also expressed in both the tumor and endothelial cells of anaplastic astrocytomas, whereas it was not expressed or only weakly expressed in low-grade astrocytomas or normal brain tissues. These results suggest that high expression of PAI-1 is associated with the malignant progression of astrocytic tumors and that excessive PAI-1 expression might be associated with intratumoral necrosis in glioblastomas.

Expression and localization of urokinase-type plasminogen activator in human astrocytomas in vivo.

Plasminogen activators regulate a variety of processes involved in tissue morphogenesis, as well as cell differentiation, migration, and invasion. We examined the relative amounts of mRNA and protein and localization of urokinase-type plasminogen activator (uPA) in human astrocytomas in vivo. Using fibrin zymography and densitometric quantitation, we found that uPA activity was significantly higher in malignant astrocytomas, especially in glioblastomas, than it was in normal brain tissues or low-grade gliomas. The amounts of uPA mRNA, as determined by Northern blot analysis, were higher in anaplastic astrocytomas and glioblastomas than in normal brain tissues and low-grade gliomas, consistent with the amount of uPA activity. To investigate the cellular source of uPA in various tissues, we performed immunocytochemical localization of uPA protein and in situ hybridization of uPA mRNA with astrocytomas and normal brain tissues. Immunocytochemical staining for uPA showed strong immunoreactivity in the tumor cells and vasculature of glioblastomas and anaplastic astrocytomas but undetectable or very low immunoreactivity for uPA in low-grade gliomas and normal brain tissues. uPA mRNA was located in astrocytoma and endothelial cells and was heterogeneously distributed within glioblastoma, with preferential localization near vascular proliferation and at the leading edge of the tumor. uPA expression was dramatically higher in highly malignant astrocytomas, especially glioblastomas, and was correlated with malignant progression of astrocytomas.

Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion.

Increased expression of matrix metalloproteinases (MMPs) has been associated with human glioblastoma tumor progression. In this study, we sought to down-regulate MMP-9 expression by stably transfecting a high-grade glioblastoma cell line with a plasmid vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Stable transfectants were obtained through selection with G418. Of the clones transfected with vector, sense, and antisense constructs, Northern blotting, Western blotting, and gelatin zymography showed that MMP-9 expression was significantly reduced only in the antisense-transfected cells. A Matrigel invasion assay revealed marked reductions in invasiveness for the antisense clones relative to the parental, vector, and sense clones. Cocultures of tumor spheroids and fetal rat brain aggregates showed that the antisense-transfected stable clones showed no invasion of the rat brain aggregates; in contrast, 90% of the parental, vector, and sense clones invaded the rat brain aggregates. Intracerebral injection of antisense stable transfectants in nude mice produced no tumors or very small tumors, but intracerebral injection of parental or vector clones did produce tumors. These results suggest that MMP-9 expression is essential for the invasiveness of glioblastoma cells.

Adenovirus-mediated delivery of antisense gene to urokinase-type plasminogen activator receptor suppresses glioma invasion and tumor growth.

The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.

In vitro inhibition of human glioblastoma cell line invasiveness by antisense uPA receptor.

The cell surface urokinase-type plasminogen activator receptor (uPAR) has been shown to be a key molecule in regulating plasminogen-mediated extracellular proteolysis. To investigate the role of uPAR in invasion of brain tumors, human glioblastoma cell line SNB19 was stably transfected with a vector capable of expressing an antisense transcript complementary to the 300 base pair of the 5' end of the uPAR mRNA. Parental and stably transfected (vector, sense, and antisense) cell lines were analysed for uPAR mRNA transcript by Northern blot analysis, and receptor protein levels were measured by radioreceptor assays and Western blotting. Significant reduction of uPAR sites was observed in the antisense transfected cell lines. The levels of uPAR mRNA were significantly decreased in antisense clones compared to control, vector and sense clones. The invasive potential of the cell lines in vitro was measured by Matrigel invasion assay and migration of cells from spheroids to monolayers. The antisense transfected cells showed a markedly lower level of invasion and migration than the controls. The antisense clones were more adhesive to the ECM components compared to parental, vector and sense clones. All transfected (vector, sense and antisense) clones and parental cells produced similar levels of uPA activity without any significant difference however, MMP-2 activity was decreased in antisense clones compared to controls. These results demonstrate that uPAR expression is critical for the invasiveness of human gliomas and down regulation of uPAR expression may be a feasible approach to decrease invasiveness.

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells.

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Regulation of the urokinase-type plasminogen activator receptor gene in different grades of human glioma cell lines.

We reported previously that the production of urokinase-type plasminogen activator receptor (uPAR) protein is greater in high-grade glioblastomas than in low-grade gliomas. Transcriptional activation of the uPAR gene or increased stability of the uPAR mRNA that encodes this protein could cause the increased production of this protein in cell lines of different grades of gliomas. We found similar half-life of uPAR mRNA of 10-12 h in glioblastoma multiforme (UWR3) and anaplastic astrocytoma (SW1783) cells. However, the human uPAR promoter was up-regulated 6-8-fold in SW1783 cells and 11-13-fold in UWR3 cells as compared with its activity in low-grade gliomas, a finding that correlates well with previous findings of increases in uPAR mRNA and protein levels in higher-grade gliomas. uPAR mRNA level was increased 11-fold over a 24-h period in low-grade glioma cell lines after treatment with phorbol myristate acetate. The region spanning -144 to -123 bp of the human uPAR promoter that contains the Sp-1 site and a PEA-3 element and an AP-1 site at -184 plays major roles in uPAR promoter activity in glioblastoma cells. Specific antibodies used in an electrophoretic mobility shift assay identified fra-1, fra-2, Jun D, and c-Jun proteins in the nuclear protein complex that bind a 51-mer containing the AP-1 consensus sequence at -184 and its flanking sequences in the uPAR promoter. We further studied the inhibition of uPAR promoter by coexpression of a transactivation domain lacking C-Jun; a dominant-negative ERK1 and ERK2 mutant and a dominant-negative C-raf in glioblastoma cell lines showed the repressed uPAR promoter activity compared with the effect of the empty expression vector. We conclude from our findings that increased transcription is the more likely mechanism underlying the increase in uPAR production in high-grade gliomas.

Expression of tissue factor pathway inhibitor 2 inversely correlates during the progression of human gliomas.

Protease inhibitors regulate a variety of physiological and pathological processes including angiogenesis, embryo implantation, intravascular fibrinolysis, wound healing, and tumor invasion. Tissue factor pathway inhibitor (TFPI) 2 is a Mr 32,000 Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. In this study, we determined the relative amounts of TFPI-2 in low-, intermediate-, and high-grade human glioma cell lines and tumor tissue samples. TFPI-2 protein and mRNA levels (measured by Western and Northern blotting) were highest in low-grade glioma cells (Hs683), lower in anaplastic astrocytoma cells (SW1088 and SW1783), and undetectable in high-grade glioma cells (SNB19). Analysis of TFPI-2 protein in human normal brain and in glioma tumor tissues for TFPI-2 revealed the highest levels in normal brain, lesser amounts in low-grade gliomas and anaplastic astrocytomas, and undetectable amounts in glioblastomas. In situ hybridization of TFPI-2 mRNA with normal brain tissues revealed the greatest positivity in neurons, with moderate positivity in both glial and endothelial cells and moderate, little, or no TFPI-2 mRNA in low-grade glioma, anaplastic astrocytoma, and glioblastoma tumor tissue samples, respectively. We also found that recombinant TFPI-2 inhibited the invasiveness of SNB19 glioblastoma cells in a Matrigel assay in a dose-dependent manner. Collectively, these results suggest that TFPI-2 has a regulatory role in the invasiveness of gliomas in vitro and in vivo.

Stable transfection of urokinase-type plasminogen activator antisense construct modulates invasion of human glioblastoma cells.

The diffuse and extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modifications of the proteolysis of extracellular matrix components. A key molecule in regulating plasminogen-mediated extracellular proteolysis is the urokinase-type plasminogen activator (uPA). To investigate the role of uPA in the invasive process of brain tumors, we stably transfected a human glioblastoma cell line SNB19 with a vector capable of expressing an antisense transcript complementary to the 1020 bases at the 3' end of the uPA cDNA. Parental, vector-, and antisense construct-stably transfected cell lines were analyzed for uPA mRNA transcript by Northern blot analysis, for uPA enzyme activity by zymography, and for uPA protein levels by Western blotting. The levels of uPA mRNA, protein, and enzyme activities were significantly lower in antisense clones than in parental and vector controls. Radioreceptor binding studies demonstrated that uPA receptor levels remained the same in parental, vector-, and antisense-transfected cells. The antisense-transfected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Green fluorescent protein (GFP) expressing parental and antisense transfectants was generated for detection in mouse brain tissue without any posttreatment. Intracerebral injection of antisense stable transfectants significantly reduced tumor formation compared with that in controls. Our results suggested that down-regulation of uPA expression may be a feasible approach to reducing the malignancy and invasiveness of glial tumors.

In vitro modulation of human lung cancer cell line invasiveness by antisense cDNA of tissue factor pathway inhibitor-2.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase (uPA) or tissue-type plasminogen activator and thrombin. Earlier studies from our and other laboratories have shown that the production of TFPI-2 is downregulated during the progression of various cancers. To investigate the role of TFPI-2 in the invasion and metastasis of lung tumors, the human lung cancer cell line A549, which produces high levels of TFPI-2, was stably transfected with a vector capable of expressing an antisense transcript complementary to the full-length TFPI-2 mRNA. Northern blot analysis was used to quantify the TFPI-2 mRNA transcript, and western blot analysis was used to measure TFPI-2 protein levels in parental cells and stably transfected (vector and antisense) clones. The levels of TFPI-2 mRNA and protein were significantly less in antisense clones than in the parental and vector controls. The invasive potential of the parental cells and stably transfected vector clones in vitro, as measured by the Matrigel invasion assay, was also markedly less than that of antisense clones. Further characterization of these clones showed that more cells migrated from antisense clones than from parental and vector clones. These data suggest that TFPI-2 is critical for the invasion and metastasis of lung cancer and that the downregulation of TFPI-2 production may be a feasible approach to increase invasiveness and metastasis.

Downregulation of urokinase-type plasminogen activator receptor (uPAR) induces caspase-mediated cell death in human glioblastoma cells.

Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. Our previous studies with human glioblastoma cell line SNB19 expressing AS-uPAR stable tranfectant lose their invasive properties when injected in vivo. The aim of the present study is to investigate whether the inhibition of tumor formation is due to apoptosis. Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Key to the apoptotic program is a family of cystein proteases termed caspases, which are important for execution of apoptosis by cleavage of essential cellular proteins. We found loss of mitochondrial transmembrane potential, release of cytochrome C from mitochondria and subsequent activation of Caspase-9 in SNB 19 AS-uPAR cells compared to parental and vector controls. Our results indicate that suppression of uPAR results in apoptosis and suggest that Caspase-9 dependent apoptosis plays an important role in SNB19 AS-uPAR-induced apoptosis.

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors.

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5' end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.

Downregulation of the urokinase-type plasminogen activator receptor through inhibition of translation by antisense oligonucleotide suppresses invasion of human glioblastoma cells.

We previously showed that downregulation of the urokinase-type plasminogen activator receptor (uPAR) in the SNB19 human glioblastoma cell line by the stable transfection of a plasmid expressing a 300 bp antisense sequence to the 5' end of the uPAR gene produced a decrease in the amount of target mRNA. In a more recent study, we found that adenovirus-mediated transduction (Ad-uPAR) of the same uPAR antisense gene construct in SNB19 cells also downregulated uPAR protein levels. We report here that Ad-uPAR-transfected SNB19 cells produced the same amounts of target uPAR mRNA but significantly less protein by in vitro translation and by in situ [35S] labeling compared to Ad-CMV vector-transfected and mock-transfected cells. This antisense construct also inhibited glioblastoma cell invasion confirming previous results. We conclude that downregulation of uPAR by this antisense gene construct results from inhibition of protein translation.

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions.

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro.