Receptor Binding and Biological Activity of IL-α, IL-1β, IL-1β Analogues and an IL-1 Antagonist in A375 Human Melanoma Cells

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Abstract
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 IL-1β were compared. In both strains, binding equilibrium at temperatures between 0° and 37°C was reached after 4 to 8 hours. At 37°C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0°C revealed a single class of high affinity receptors with a similar KD 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-1β was found to be slightly more potent than IL-1α in both strains. In an attempt to further characteristics the IL-1 binding site in these cells, the binding characteristics and biological activity of 20 point mutations of IL-1β 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-1p. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1p in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1s. 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.