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

Abstract

Low-density lipoprotein receptor-related protein (LRP) binds and internalizes 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 constructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter. U-1242 MG astrocytic tumor cells were transfected with the antisense constructs and cloned from single cells to yield multiple cell lines with decreased LRP expression. Further studies were performed with two cell lines in which LRP antigen was completely eliminated (Lα42) or substantially decreased (Lα47), as determined by Western blot analysis. Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated α₂-macroglobulin (α₂M) in a specific and saturable manner. The B_max was about 5000 receptors/cell. Lα42 cells did not bind α₂M, and binding was decreased by >60% in Lα47 cells. Lα42 and Lα47 cells also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plasminogen activator (uPA) in conditioned medium. The accumulation of uPA demonstrates a major role for LRP in the catabolism of this protein in astrocytic tumor cells. The LRP-deficient cell lines, developed using antisense technology, represent a new model system for studying LRP function in astrocytes.