Fig. 4. Heparanase processing is inhibited by lysosomal proteinase inhibitors. (A-D) Chloroquine treatment. (A) Heparanase-transfected 293 (upper panel), MDA-435 breast carcinoma (second panel), C6 rat glioma (third panel) and NMU rat mammary adenocarcinoma (fourth panel) cells were left untreated (0) or incubated for 20 hours with the indicated concentrations (µM) of chloroquine. Total cell lysates were immunoblotted with anti-heparanase antibody 1453. Note a dose-response inhibition of heparanase processing and accumulation of the unprocessed 65 kDa heparanase precursor. (B) Chloroquine treatment is reversible. Heparanase-transfected C6 glioma cells were left untreated (Con) or treated with 50 µM chloroquine for 20 hours. Cells were then lysed (Chl) or washed and chased for an additional 24 hours with chloroquine-free medium (Chase). Total cell lysates were then analyzed for heparanase processing by immunoblotting as above. Note re-appearance of the processed 50 kDa heparanase form upon chloroquine removal. (C,D) Uptake studies. (C) U87 glioma cells were left untreated (Control) or pre-treated with 100 µM chloroquine (Chloroquine) for 2 hours. The latent 65 kDa heparanase protein was then added for the indicated time points and total cell lysates were analyzed for heparanase processing by immunoblotting as above. Note complete inhibition of exogenously added heparanase processing upon chloroquine pre-treatment. (D) U87 glioma cells were left untreated (Control) or incubated with chloroquine (100 µM) for 2 hours, followed by the addition of the 65 kDa heparanase protein for additional 2 hours. Cells were then fixed and immunostained with anti-heparanase monoclonal antibody (BD) and 733 anti-heparanase (733) antibodies. Merged images are shown in the third row. Negative control, in which the primary antibody was omitted, is shown in the bottom row. Note the complete absence of heparanase processing as evident by the lack of reactivity with antibody 733 upon chloroquine treatment (second panel, right), and the accumulation of the latent heparanase form in large vesicles (upper and 3rd panels, right). Original magnifications x100. (E) Heparanase-transfected C6 glioma (upper panel) and NMU (lower panel) cells were left untreated (0) or incubated with the indicated concentrations (µM) of bafilomycin A1 or chloroquine (Chl, 50 µM) for 20 hours. Total cell lysates were analyzed by immunoblotting as above. Note complete inhibition of heparanase processing upon bafilomycin treatment.