EXPRESSION OF INTERLEUKIN-1-ALPHA, INTERLEUKIN-6, AND TUMOR-NECROSIS-FACTOR-ALPHA GENES IN HUMAN-MELANOMA CLONES IS ASSOCIATED WITH THAT OFMUTATED N-RAS ONCOGENE

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma m RNA has been analyzed by reverse transcriptase-polymerase chain reacti on in 19 melanoma clones derived from the metastatic cell line 665/2 a nd previously characterized for RAS mutation and expression. Five of t hese clones and the parental cell line shelved a mutation at codon 61 of N-RAS that resulted in Gln--> Arg substitution (N-RAS/61(+)), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61(-)). With the exception of interferon gamma and IL-4, all th e cytokines tested were expressed by the parental 665/2 cell line, whe reas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed onl y in the subset of the clones bearing the mutated N-RAS gene. The othe r cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not corr elated to the N-RAS phenotype of the clones. The association between N -RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS gene s simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-a lpha expression. Direct evidence that N-RAS oncogene could influence t he pattern of cytokine expression was provided by the coordinate induc tion of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RA S/61(+) transfectants of the N-RAS wild-type melanoma clone 2/21. Furt hermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-l inked immunosorbent assay in the culture medium obtained from N-RAS/61 (+) melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS onco gene lead to a secreted protein. In an N-RAS/61(+) melanoma clone, by adding specific antibodies against each cytokine, it was found that so luble LL-1 alpha exerted a positive control on IL-6 mRNA and a negativ e one on its own expression. In addition, IL-1 alpha and IL-6 were neg atively regulated by soluble IL-6 and TNF-alpha.