INDUCTION OF APOPTOSIS IN PLASMACYTOMA CELLS BY A CYTOTOXIC FACTOR SECRETED BY P388D(1) MACROPHAGE-LIKE CELL-LINE

Tumoricidal activity is one of the major effector functions of activat ed macrophages. A previous study of ours demonstrated that the culture supernatant of P388D(1) murine macrophage-like cells show a cytotoxic effect on plasmacytoma MOPC-315, MPG-11 and myeloma FO but have no ef fect on J558 myeloma cells. In this study, the plasmacytoma cytotoxic factor in P388D(1) culture supernatant was partially purified by a DEA E-Sephacel ionic-exchanger chromatography and a panel of monoclonal an tibodies against plasmacytoma cytotoxic factor was prepared. All monoc lonal antibodies partially blocked the P388D(1)-mediated tumoricidal a ctivity. A large-scale purification was performed by ammonium sulfate fractionation (40-60% saturation), followed by an immunoaffinity chrom atography using one of the antiplasmacytoma cytotoxic factor monoclona l antibodies, CB7-C2. The affinity-purified plasmacytoma cytotoxic fac tor had IC50 at 3.11 mu g/ml for 2 x 10(4) MOPC-315 cells and showed a major band with an estimated molecular weight of 62 kDa on SDS-PAGE g el. However, CB7.C2 recognized a single band with an estimated molecul ar weight of 120-130 kDa on Western blotting, suggesting that the nati ve form of plasmacytoma cytotoxic factor could be a dimer. Plasmacytom a cytotoxic factor-mediated cytotoxicity involved apoptosis. Data from both agarose gel electrophoresis and terminal deoxynucleotidyl transf erase-mediated deoxyuridine 5-triphosphate (dUTP) nick-end-labeling me thod indicated that a significant amount of DNA fragmentation was indu ced in plasmacytoma cytotoxic factor-treated MOPC-315 cells. Using an Annexin V staining technique, the plasmacytoma cytotoxic factor-induce d apoptosis was confirmed further by observing the phosphatidylserine redistribution on the plasma membrane of plasmacytoma cytotoxic factor -treated cells. The plasmacytoma cytotoxic factor-induced apoptosis wa s dose-dependent and time-dependent and could be neutralized by CB7.C2 antiplasmacytoma cytotoxic factor antibody. Taken together, our studi es demonstrate that a 62 kDa protein secreted by P388D(1) macrophage-l ike cells shows its cytotoxic effect on MOPC-315 plasmacytoma cells vi a induction of apoptosis.