INSULIN-LIKE GROWTH-FACTOR-II MODULATES THE ROUTING OF CATHEPSIN-D INMCF-7 BREAST-CANCER CELLS

A previous observation that insulin-like growth factor II (IGF-II) inh ibits the cellular uptake of a lysosomal enzyme by inhibiting binding to the IGF-II/mannose 6-phosphate receptor led to the proposal that, i n a cell producing IGF-II, the routing of lysosomal enzymes might be a ltered. To test this hypothesis MCF-7 breast cancer cells were transfe cted with pRc/CMV vector only (CMV) or vector containing IGF-II comple mentary 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 conditi oned by P-II clones (11.2 +/- 4.3 mu g/ml) was much higher than in med ia conditioned by M-II clones (1.3 +/- 1.5 mu g/ml). Metabolic labelin g 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, a nd immunoprecipitates were analyzed by SDS-PAGE. The mean of the C/M r atio of cathepsin D for the seven P-II clones (1.60 +/- 0.13) was sign ificantly lower than for the six CMV clones (3.47 +/- 0.48). Similar r esults were obtained when conditioned M and C were examined by immunob lotting after a 48-h incubation. The mean of the C/M ratio for the sev en 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 correlati on between the ratio of intracellular cathepsin D to extracellular cat hepsin D and relative cathepsin D synthesis (r = 0.843), consistent wi th increased cathepsin D production in cells overexpressing IGF-II. It is concluded that endogenous IGF-II modulates the routing of cathepsi n D in MCF-7 cells.