CHARACTERIZATION OF NON PIGMENTED B16 MELANOMA CELL-DERIVED CYTOTOXICFACTORS

We analyzed and tried to characterize substance(s) responsible for cyt otoxic activities detected in culture media conditioned by non pigment ed B16 melanoma cells (NPB16). The different cytological tests used sh owed that ultrafiltrated conditioned media (CM U1 fraction) contained several cytotoxic factors with a M(w) lower than 1000 Da. These factor s seemed to act either directly or indirectly on cell membranes, mitoc hondria, on the cell cycle and on protein and DNA synthesis. A cytotox ic activity could be found even after high dilution of CM U1. These cy totoxic factors were rapidly released by B16 cells in culture, indepen dently of cell confluence. Their activities in the treated cells were also very fast and the cytotoxic effects were irreversible after only a few hours of treatment. These factors were not intermediate products during melanogenesis, neither polyamines, nor proteases. At least one of them seemed to be a small acidic and basic stable peptide without disulfide bounds but not heat stable. The synthesis of at least one of these cytotoxic factors was inhibited by cycloheximide and the cytoto xic activity was partially destroyed by pronase and trypsin, but not b y pepsin. The cytotoxicity was not modified by copper complexants or f ree radical inhibitors (bovine serum albumin (BSA), tyrosine, superoxy de dismutase (SOD), catalase, vitamin E). Furthermore the levels of gl utathione peroxydase activity and reduced glutathione did not change a fter treatment by CM U1 as compared to controls. (C) 1997 Elsevier Sci ence Ireland Ltd.