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 production of nitric oxide (NO) by activated macrophages. However, cells of the blood vessels can also participate in antitumor defense responses. They produce NO either constitutively [endothelial cells (ECs)] or after stimulation by proinflammatory cytokines (ECs and vascular smooth muscle cells). We have used a tumor cell-vascular cell coculture system to evaluate whether vascular cells can mediate cytotoxic effects on tumor cells. Treatment with IFN-gamma and tumor necrosis factor-alpha induced death of human erythroleukemic K562 cells cocultured with rodent vascular smooth muscle cells or ECs. The synergistic antitumor activity of the two cytokines depended on de novo gene expression of the inducible isoform of NO synthase and on synthesis of reactive nitrogen intermediates (RNIs) in the vascular cells. K562 cells did not produce any appreciable levels of NO, but they were targeted by RNIs released from the cytokine-stimulated vascular cells, as demonstrated by electron paramagnetic resonance spectrometry, which showed formation of nonheme iron-nitrosyl complexes in the tumor cells. Assays for mitochondrial respiration demonstrated that the tumor cells suffered from a block of the complexes I and II of the mitochondrial respiratory chain. Further analysis of the cytotoxic mechanism by fluorescent microscopy, flow cytometry, and DNA electrophoresis revealed that K562 cells attacked by NO-producing vascular cells underwent apoptosis with plasma membrane blebbing, cell volume reduction, condensation of cytoplasm and chromatin, and fragmentation of genomic DNA at internucleosomal sites. In contrast, only a few vascular cells exhibited these apoptotic changes, suggesting that these cells resist the RNI attack. Inhibition of NO production in vascular cells by NG-monomethyl-L-arginine, an inhibitor of NO synthases, significantly reduced the death of the K562 cells. These observations suggest that vascular cells induce apoptotic death of tumor cells by producing RNIs in response to cytokine stimulation.