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 surrou nding normal brain is believed to rely on modifications of the proteolysis of extracellular matrix components. A key molecule in regulating plasminoge n-mediated extracellular proteolysis is the urokinase-type plasminogen acti vator (uPA). To investigate the role of uPA in the invasive process of brai n 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 transcr ipt by Northern blot analysis, for uPA enzyme activity by zymography, and f or 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 t hat uPA receptor levels remained the same in parental, vector-, and antisen se-transfected cells. The antisense-transfected cells showed a markedly low er level of invasion in the Matrigel invasion assays, and their spheroids f ailed to invade the fetal rat brain aggregates in the coculture system. Gre en fluorescent protein (GFP) expressing parental and antisense transfectant s was generated for detection in mouse brain tissue without any posttreatme nt. Intracerebral injection of antisense stable transfectants significantly reduced tumor formation compared with that in controls. Our results sugges ted that down-regulation of uPA expression may be a feasible approach to re ducing the malignancy and invasiveness of glial tumors.