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Stable Transfection of Urokinase-Type Plasminogen Activator Antisense Construct Modulates Invasion of Human Glioblastoma Cells¹

Division of Cancer Biology, Department of Biomedical and Therapeutic Sciences [S. M., N. C., S. S. L., J. S. R.] and Departments of Neurosurgery [D. H. D., W. C. O., J. S. R.] and Pathology [M. G.], University of Illinois College of Medicine at Peoria, Peoria, Illinois 61656, and Departments of Neurosurgery [S. R. K., Y. K.], Neuro-Oncology [S. L. J., A. P. K., W. K. A. Y.], and Neuropathology [G. N. F.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

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.

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