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
Im. Hussaini et al., 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, ANTISENSE N, 9(2), 1999, pp. 183-190
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.