Despite many therapeutic strategies for glioblastoma multiforme, the survival rate in patients with this aggressive cerebral malignancy remains poor. These gliomas are highly resistant even to combinations of different therapies such as surgery, radiotherapy, and chemotherapy. Recent compelling evidence from cellular and molecular studies of the mechanisms underlying the invasiveness of human gliomas has implicated serine proteases and their receptors in the invasion process. We propose here to identify the mechanisms that lead to the inhibition of one of these molecules by using an adenoviral construct carrying antisense messages for uPA and its receptor, uPAR, and by using specific inhibitors of signaling pathway molecules.
Our specific aims are to: (1) Construct adenovirus carrying a truncated 1020 bp uPA gene and a truncated 300 bp uPAR gene that express antisense messages that downregulate a key step in the proteolytic cascade on glioma cell growth, adhesion, migration, invasion and tumor formation in both in vivo and in vitro models. (la) Construct a bicistronic adenovirus vector (Ad-uPAR-uPA) that is driven by the independent promoter elements CMV, bovine growth hormone, and SV40 polyadenylation signals and investigate the effect of this bicistronic construct on the invasion, adhesion and migration of human glioma cells. (ib) Determine the efficiency of the bicistronic construct in inhibiting the invasion and growth of human glioma cells in vivo in nude mice; and evaluate the toxicity of the intracerebrally injected constructs in Fischer/Wistar rats. (ic) Determine the effect of the bicistronic construct on the levels of integrins, MMP-2, and other signaling pathway molecules in glioma cell cultures. (2) Determine how the c-raf ERK, MEKK-JNK, and FAK-MAPK signaling pathways participate in regulating uPA and uPAR in human glioma cell lines. (2a) Determine whether uPA and uPAR gene expression is downregulated in glioblastoma cells transfected with expression vectors encoding dominant-negative ERK- 1 and EKR-2 or kinase-deficient c-raf constructs, (2b) Assess the ability of a kinase-inactive JNK and a kinase-inactive MEKK to downregulate uPA and uPAR expression. (2c) Assess the ability of focal adhesion kinase (FAK) to downregulate uPA and uPAR expression. (2d) Identify inhibitors of the signaling pathway(s) that reduce the expression of uPA and uPAR and the invasiveness of glioblastoma cell lines in vitro. We believe that identifying the molecular mechanisms that regulate the overexpression of uPA or uPAR could lead to the development of novel anti-invasive therapeutic agents whose mode of action depends on the antagonism of uPA or uPAR overexpression.
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