APOPTOTIC DEATH OF HUMAN LEUKEMIC-CELLS INDUCED BY VASCULAR CELLS EXPRESSING NITRIC-OXIDE SYNTHASE IN RESPONSE TO GAMMA-INTERFERON AND TUMOR-NECROSIS-FACTOR-ALPHA

The host defense against tumor cells is in part based on the productio n of nitric oxide (NO) by activated macrophages. However, cells of the blood vessels can also participate in antitumor defense responses. Th ey produce NO either constitutively [endothelial cells (ECs)] or after stimulation by proinflammatory cytokines (ECs and vascular smooth mus cle tells). We have used a tumor cell-vascular cell coculture system t o evaluate whether vascular cells ran mediate cytotoxic effects on tum or cells. Treatment with IFN-gamma and tumor necrosis factor-alpha ind uced death of human erythroleukemic K562 cells cocultured with rodent vascular smooth muscle cells or ECs. The synergistic antitumor activit y of the two cytokines depended on de novo gene expression of the indu cible isoform of NO synthase and on synthesis of reactive nitrogen int ermediates (RNIs) in the vascular cells. K562 cells did not produce an y appreciable levels of NO, but they were targeted by RNIs released fr om the cytokine-stimulated vascular cells, as demonstrated by electron paramagnetic resonance spectrometry, which showed formation of nonhem e iron-nitrosyl complexes in the tumor cells. Assays for mitochondrial respiration demonstrated that the tumor cells suffered from a block o f the complexes I and II of the mitochondrial respiratory chain. Furth er analysis of the cytotoxic mechanism by fluorescent microscopy, now cytometry, and DNA electrophoresis revealed that K562 cells attacked b y NO-producing vascular cells underwent apoptosis with plasma membrane blebbing, cell volume reduction, condensation of cytoplasm and chroma tin, and fragmentation of genomic DNA at internucleosomal sites. In co ntrast, only a few vascular cells exhibited these apoptotic changes, s uggesting that these cells resist the RNI attack. Inhibition of NO pro duction in vascular cells by N-G-monomethyl-L-arginine, an inhibitor o f NO synthases, significantly reduced the death of the K562 cells. The se observations suggest that vascular cells induce apoptotic death of tumor cells by producing RNIs in response to cytokine stimulation.