Expression of Interleukin 1α, Interleukin 6, and Tumor Necrosis Factor α Genes in Human Melanoma Clones Is Associated with That of Mutated N-RAS Oncogene

Abstract

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1α, 1β, 4, 6, 7, and 8, tumor necrosis factor (TNF) α and interferon γ mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed 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 γ and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1α, IL-6, and TNF-α were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1β, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1α, IL-6, and TNF-α expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of Il-1α, IL-6, and TNF-α expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1α, IL-6, and TNF-α messenger RNA achieved in N-RAS/61⁺ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1α, IL-6, and TNF-α could be detected by enzyme-linked 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 oncogene lead to a secreted protein. In an N-RAS/61⁺ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1α exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1α and IL-6 were negatively regulated by soluble IL-6 and TNF-α.