INTERFERON-GAMMA AND TUMOR-NECROSIS-FACTOR-ALPHA INDUCE A SYNERGISTIC ANTIPROLIFERATIVE RESPONSE IN HUMAN EWING SARCOMA-CELLS IN-VITRO

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon gamma (rhIFNgamma) and tumour necrosis factor alpha (rhTNFalpha) on cell proliferation and survival and to characterize IFNgamma and TNFalpha receptor expression. Incorporation of [H-3]thymidine into cells was inhibited by rhIFNgamma after 24 h of incubation. Half-maximal inhibition was observed with 10-80 U/ml rhIFNgamma. A maximal effect (50%-70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFNgamma. The influence of rhTNFalpha on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [H-3]thymidine incorporation was not affected by rhTNFalpha up to 2000 U/ml after 96 h of incubation, whereas in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNFalpha. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFNgamma and rhTNFalpha after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1alpha IL-1beta, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophage-colony-stimulating factor, tested alone and in combination with rhIFNgamma and rhTNFalpha, had no influence on cell proliferation Binding studies in the cell lines with I-125-rhIFNgamma revealed a dissociation constant (K(d)) of 160-306 pM and approximately 8000-13 500 receptors/cell. Binding experiments with I-125-rhTNFalpha indicated 430-1250 receptors/cell with K(d) ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNFalpha are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNFalpha may be useful in the design of novel strategies in Ewing's sarcoma therapy.