Interferon γ and tumour necrosis factor α induce a synergistic antiproliferative response in human Ewing's 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 γ (rhIFNγ) and tumour necrosis factor α (rhTNFα) on cell proliferation and survival and to characterize IFNγ and TNFα receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFNγ after 24 h of incubation. Half-maximal inhibition was observed with 10-80 U/ml rhIFNγ. A maximal effect (50%-70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFNγ. The influence of rhTNFα 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 [³H]thymidine incorporation was not affected by rhTNFα up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNFα. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFNγ and rhTNFα after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFNγ and rhTNFα, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFNγ revealed a dissociation constant (K_d) of 160-306 p M and approximately 8000-13500 receptors/cell. Binding experiments with¹²⁵I-rhTNFα indicated 430-1250 receptors/cell with K_d ranging from 13 p M to 162 p M. These data indicate that, among various cytokines, only IFN and TNFα are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNFα may be useful in the design of novel strategies in Ewing's sarcoma therapy.