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-1. The resistant cells exhibited constitutive production of IL-1α. To analyse the mechanisms that lead to the expression of IL-1α 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-1α 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(-k)B (nuclear factor-kB) binding motif.(Immunofluorescence analysis revealed that NF-kB is activated in A375-R8 cells. IL-1 receptor antagonist (IL-1Ra) decreased the level of IL-1α mRNA and production of IL-1α. IL-1Ra also inhibited the localization of p65 in the nuclei and CAT activity in transfectants with the plasmids containing NF-kB binding motif. These results indicate that endogenous IL-1α stimulates the gene expression and production of IL-1α in an autocrine manner through activation of NF-kB.
Copyright 1998 Academic Press