MM cells proteolytically convert HGF into its active form and express the serine protease HGFA. (A) MM cells convert HGF into its active form. MM cell lines NCI-H929, XG-1, and OMP-1 were incubated with scHGF for 6 hours in the presence or absence of thrombin, the serine protease inhibitor leupeptin, or both, as indicated. HGF conversion was determined by immunoblotting with anti-HGF. As positive control, HGF conversion by recombinant HGFA is shown (left panel). The right panel shows the time kinetics of scHGF conversion by MM cells (in the presence of thrombin). As positive and negative controls, scHGF conversion by COS-7 cells transfected with either a plasmid containing HGFA or empty vector are shown. (B) Expression of HGFA in MM cell lines. Cell lysates were immunoblotted by using a monoclonal anti-HGFA antibody (A-1). COS-7 cells transfected with HGFA and the colorectal carcinoma cell lines DLD-1 and SW480 were used as positive controls. COS-7 cells transfected with empty vector were used as negative controls. β-Actin was used as loading control (bottom panel). (C) Expression of HGFA in MM cell lines and primary myeloma cells. MM cell line NCI-H929, primary myeloma (PM) cells, or COS-7 cells transfected with either empty vector or a plasmid containing HGFA were immunocytochemically stained with mAb A-1 against HGFA or isotype control. (D) HGFA expression in MM cells is intracellular. The indicated MM cells, either permeabilized (right panel) or not (left panel), were stained with anti-HGFA mAb PI-4 (bold line) or isotype control antibody (gray line). Expression was measured by FACS analysis.