Insulin-like growth factor II modulates the routing of cathepsin D in MCF-7 breast cancer cells.
A previous observation that insulin-like growth factor II (IGF-II) inhibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, in a cell producing IGF-II, the routing of lysosomal enzymes might be altered. To test this hypothesis MCF-7 breast cancer cells were transfected with pRc/CMV vector only (CMV) or vector containing IGF-II complementary DNA encoding either mature (M-II) or precursor (P-II) IGF-II, and the routing of cathepsin D, a predominant lysosomal enzyme in this cell line, was examined. The concentration of IGF-II in media conditioned by P-II clones (11.2 +/- 4.3 micrograms/ml) was much higher than in media conditioned by M-II clones (1.3 +/- 1.5 micrograms/ml). Metabolic labeling experiments were performed with 10 mM mannose 6-phosphate present in the medium to block reuptake of lysosomal enzymes. Cell extracts (C) and media (M) were immuno-precipitated with a cathepsin D antiserum, and immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M ratio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was significantly lower than for the six CMV clones (3.47 +/- 0.48). Similar results were obtained when conditioned M and C were examined by immunoblotting after a 48-h incubation. The mean of the C/M ratio for the seven P-II clones (11.4 +/- 1.6) was significantly lower than for the six CMV clones (24.9 +/- 5.2). There was also a strong negative correlation between the ratio of intracellular cathepsin D to extracellular cathepsin D and relative cathepsin D synthesis (r = 0.843), consistent with increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsin D in MCF-7 cells.
De Leon
D D
DD
Loma Linda School of Medicine, California 92350, USA.
Terry
C
C
Asmerom
Y
Y
Nissley
P
P
D001943:Breast Neoplasms / Q000378:metabolism*; D002402:Cathepsin D / Q000096:biosynthesis / Q000378:metabolism*; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007163:Immunosorbent Techniques; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008247:Lysosomes / Q000201:enzymology; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D017527:Receptor, IGF Type 2; D007335:Insulin-Like Growth Factor II; D002402:Cathepsin D