RECEPTOR-BINDING AND BIOLOGICAL-ACTIVITY OF IL-1-ALPHA, IL-1-BETA, IL-1-BETA ANALOGS AND AN IL-1 ANTAGONIST IN A375 HUMAN-MELANOMA CELLS

A receptor binding assay for IL-1 peptides on human melanoma cells of the A 375 cell line is reported. Strains differing in their sensitivity to the cytotoxic effects of 1L-1beta were compared. In both strains, binding equilibrium at temperatures between 0-degrees and 37-degrees-C was reached after 4 to 8 hours. At 37-degrees-C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0-degrees-C revealed a single class of high affinity receptors with a similar K(D) in both IL-1 resistant (0.18+/-0.07 nM) and sensitive strains (0.14+/-0.06 nM) but a 10-fold difference in the number of binding sites. Whereas > 1000 binding sites per cell were regularly observed in all resistant strains, only 100-200 sites could be detected on the IL-1 sensitive cells. In displacement assays, IL-1beta was found to be slightly more potent than IL-1alpha in both strain .In an attempt to further characterize the IL-1 binding site in these cells, the binding characteristics and biological activity of 20 point mutations of IL-1beta were examined. EC50 values similar to those of the wild type peptide were found in all these analogues with the exception R11S and E128K: their EC50 was increased by a factor of 10 but the biological activity was reduced 1000-fold as compared to IL-1beta. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1beta in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1beta. These results show that A375 human melanoma cells are useful for screening the binding and biological properties of analogues of the IL-1 family of peptides.