title: Receptor binding and biological activity of IL-1 alpha, IL-1 beta, IL-1 beta analogues and an IL-1 antagonist in A375 human melanoma cells.
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-1 beta 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 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-1 beta was found to be slightly more potent than IL-1 alpha in both strains. 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-1 beta 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-1 beta. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1 beta in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1 beta. 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.
authors.affiliation: Department of Research (ZLF), University Hospital, Basel, Switzerland.
authors.forename: J B
authors.initials: JB
authors.lastname: Baumann
authors.forename: E
authors.initials: E
authors.lastname: Christen
authors.forename: G
authors.initials: G
authors.lastname: Gamboni
authors.forename: U
authors.initials: U
authors.lastname: Joss
authors.forename: J
authors.initials: J
authors.lastname: van Oostrum
authors.forename: J
authors.initials: J
authors.lastname: Girard
authors.forename: A N
authors.initials: AN
authors.lastname: Eberle
mesh_terms: D001667:Binding, Competitive; D006801:Humans; D007375:Interleukin-1 / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism*; D017354:Point Mutation; D011869:Radioligand Assay; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D007375:Interleukin-1; D017472:Receptors, Interleukin-1