Molecular analysis of constitutive IL-1α gene expression in human melanoma cells:: Autocrine stimulation through NF-κB activation by endogenous IL-1α

Constitutive production of/and acquired resistance to anti-proliferative cytokines are implicated in pathogenesis and progression of human melanoma cells. Human melanoma cells A375-C6 are sensitive to interleukin 1 (IL-1) anti-proliferative effect and do not produce IL-1, After long period of culture we have obtained cells which acquired resistance to IL-I, The resistant cells exhibited constitutive production of IL-1 alpha, TO analyse the mechanisms that lead to the expression of IL-1 alpha in the cells, we transfected of the resistant clone A375-R8 with CAT (chloramphenicol acetyltransferase) expression plasmids linked to a 5'-flanking deletion mutants of the human IL-1 alpha gene. Two nucleotide regions (-103 to -70 bp) and (-70 to -47 bp) from the start of the first exon appeared to contain a positive regulatory element(s), while the one -421 to -103 bp contained a negative regulatory element(s), The -103 to -70 bp region contained the consensus NF-kappa B (nuclear factor-kappa B) binding motif, Immunofluorescence analysis revealed that NF-kappa B is activated in A375-R8 cells. IL-1 receptor antagonist (IL-1Ra) decreased the level of IL-1 alpha mRNA and production of IL-1 alpha. IL-1Ra also inhibited the localization of p65 in the nuclei and CAT activity in transfectants with the plasmids containing NF-kappa B binding motif, These results indicate that endogenous IL-1 alpha stimulates the gene expression and production of IL-1 alpha in an autocrine manner through activation of NF-kappa B, (C) 1998 Academic Press.