Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock-or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.

Transduction of the cytosine deaminase (CD) gene into tumor cells followed by administration of 5-fluorocytosine (5-FC), called 5-FC/CD gene therapy, was created as suicide gene therapy for various cancers. The uracil phosphoribosyltransferase (UPRT) gene, which is absent from mammalian cells, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine 5'-monophosphate. We evaluated whether the coexpression of CD and UPRT genes could generate a synergistic antitumor effect on experimental brain tumors. In vitro study showed that 9L cells, transduced with the UPRT gene by an adenovirus, were 16 times more sensitive to 5-FU, and CD + UPRT-transduced cells were 6,000 times more sensitive to 5-FC than parent cells, indicating that the acquisition of CD and UPRT further increased the 5-FC sensitivity of 9L cells compared with cells transduced with CD alone. In a rat brain tumor model, decreased amounts of CD and UPRT vectors were inoculated into the tumors to detect any additional effect of UPRT. CD and UPRT coexpression followed by 5-FC administration showed an antitumor effect as detected by sequential magnetic resonance imaging. This therapy significantly prolonged animal survival. These results suggest that 5-FC/CD + UPRT gene therapy can enhance the antitumor effect of 5-FC/CD gene therapy. Consequently, this approach might be a more feasible modality for the treatment of malignant brain tumors.

We evaluated the therapeutic efficacy and neurotoxicity of adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for experimental malignant brain tumors. The 5-FC sensitivity in 9 L cells infected by an adenovirus vector expressing CD (AdexCACD) was increased 1700-fold compared with control cells. Rats bearing 9 L brain tumors were treated with an intratumoral injection of AdexCACD followed by intraperitoneal administration of 5-FC. The rats demonstrated remarkable inhibition of tumor growth by magnetic resonance imaging, and 7 of 10 rats survived for Ͼ90 days. To evaluate the potential side-effects of the 5-FC/CD gene therapy, rats were treated with an intracerebral injection of AdexCACD into the right basal ganglia and with 5-FC. The magnetic resonance imaging showed a highly enhanced area on the gadollinium-enhanced T1-weighted image at 18 days postinjection. Pathologically, this corresponded to an area of necrosis with surrounding apoptotic cells. In addition, there was demyelination and gliosis with enlargement of the lateral ventricles. These results suggest that the 5-FC/CD gene therapy may provide an anticancer effect for malignant brain tumors in humans, but also show that there are neurotoxic effects on normal brain tissue. Cancer Gene Therapy (2000) 7, 74 -82

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor ␣ v ␤ 3 integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of ␣ v ␤ 3 integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the ␣ v ␤ 3 integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of ␣ v ␤ 3 integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/ p16 in the presence of vitronectin resulted in decreased ␣ v ␤ 3 integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36delta5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36delta5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.