Stable antisense RNA expression neutralizes the activity of low-density lipoprotein receptor-related protein and promotes urokinase accumulation in the medium of an astrocytic tumor cell line

Low-density lipoprotein receptor-related protein (LRP) binds and internaliz es multiple ligands that are structurally and functionally diverse. However , the effects of LRP on cellular phenotype remain unclear. To study LRP in human astrocytic tumor cells, we designed LRP antisense RNA expression cons tructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter, U-1242 MG astrocytic tumor cells we re transfected with the antisense constructs and cloned from single cells t o yield multiple cell lines with decreased LRP expression. Further studies were performed with two cell lines in which LRP antigen was completely elim inated (L alpha 42) or substantially decreased (L alpha 47), as determined by Western blot analysis. Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated alpha(2)-ma croglobulin (alpha(2)M) in a specific and saturable manner, The B-max was a bout 5000 receptors/cell, L alpha 42 cells did not bind alpha(2)M, and bind ing was decreased by >60% in L alpha 47 cells, L alpha 42 and L alpha 47 ce lls also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plas minogen activator (uPA) in conditioned medium, The accumulation of uPA demo nstrates a major role for LRP in the catabolism of this protein in astrocyt ic tumor cells, The LRP-deficient cell lines, developed using antisense tec hnology, represent a new model system for studying LRP function in astrocyt es.