B-CELL LYMPHOMA-2 TRANSFECTED P815 CELLS RESIST REACTIVE NITROGEN INTERMEDIATE-MEDIATED MACROPHAGE-DEPENDENT CYTOTOXICITY

Activated murine peritoneal macrophage cytotoxicity against P815 tumor cells has been shown to be mediated by the reactive nitrogen intermed iates (RNI) produced by macrophages from L-arginine through nitric oxi de (NO) synthase. Previous results from this laboratory indicated that NO-dependent killing of P815 fulfilled the criteria for apoptotic dea th. Work by others, in turn, demonstrated that the product of the bcl- 2 gene confers protection against various inducers of apoptosis, inclu ding reactive oxygen intermediates. Experiments were performed to dete rmine whether Bcl-2 could equally protect sensitive cells from RNI-dep endent apoptosis within the context of a relevant biologic system such as the delivery of such RNI by activated macrophages. Results demonst rated that transfection of P815 cells with the human bcl-2 gene confer s immunity from RNI-dependent, macrophage-mediated cytotoxicity. In co ntrast with wild-type or mock-transfected P815 cells, which do not con tain detectable Bcl-2, bcl-2-transfected cells showed minimal DNA frag mentation and cell membrane failure when cocultured with activated mac rophages. Additional findings indicate that Bcl-2 affords the transfec ted cells almost complete resistance to the DNA-fragmenting effects of chemically generated NO or H2O2, and partial protection from their cy tolytic effects. These findings are consistent with the hypothesis tha t tumor cells expressing bcl-2 may escape destruction by macrophage-de pendent immune surveillance mechanisms.