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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.
Journal article

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.

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  • 1993-01-01
Published in:
  • Journal of receptor research. - 1993
Binding, Competitive
Humans
Interleukin-1
Melanoma
Point Mutation
Radioligand Assay
Receptors, Interleukin-1
Sensitivity and Specificity
Tumor Cells, Cultured
English 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.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/164741
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