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

Constitutive production of/and acquired resistance to anti-proliferative cy tokines are implicated in pathogenesis and progression of human melanoma ce lls. Human melanoma cells A375-C6 are sensitive to interleukin 1 (IL-1) ant i-proliferative effect and do not produce IL-1, After long period of cultur e we have obtained cells which acquired resistance to IL-I, The resistant c ells exhibited constitutive production of IL-1 alpha, TO analyse the mechan isms 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 b p) from the start of the first exon appeared to contain a positive regulato ry element(s), while the one -421 to -103 bp contained a negative regulator y element(s), The -103 to -70 bp region contained the consensus NF-kappa B (nuclear factor-kappa B) binding motif, Immunofluorescence analysis reveale d 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 activ ity in transfectants with the plasmids containing NF-kappa B binding motif, These results indicate that endogenous IL-1 alpha stimulates the gene expr ession and production of IL-1 alpha in an autocrine manner through activati on of NF-kappa B, (C) 1998 Academic Press.