Expression of cytokine genes, including IL-6, in human malignant melanoma cell lines


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Abstract

As a preliminary to transducing human melanoma cells with lymphokine genes, we sought for constitutive gene expression and production of eight interleukins, tumour necrosis factors and granulocytecolony stimulating factor in 19 human melanoma cell lines. Conversion of RNA into cDNA by reverse transcriptase and polymerase chain reaction (RT-PCR) were employed to evaluate gene expression while enzyme-linked immunosorbent assays (ELISA) or biological assays were used to assess the presence of proteins. No expression of interleukins (IL) 3, 4, and 5 or interferon-γ RNA was found, while the other cytokines were variably expressed in melanoma lines, with IL-1α, IL-1β, IL-6, IL-8, being detectable in most of the lines. At protein level, 10 melanoma cells were tested with ELISA and all were found to produce IL-8, five produced IL-6, two tumour necrosis factor (TNF)-α, one IL-1α and two TNFβ. The levels of TNFβ were at the limit of test sensitivity. The amount of various cytokines released by the different lines varied widely. Biological assay with the D10-G4 clone confirmed the presence of IL-1α in the supernatant of melanoma (ME) 10221 and revealed an IL-1 activity in the supernatant of Me 4024/1. The proliferating activity of melanoma supernatants on D10-G4 was inhibited by treatment with polyclonal antibodies against IL-1α but not with antibodies against IL-1β. TNF biological activity was tested against the TNF-susceptible fibrosarcoma WEHI 164 clone 13. Supernatant from the two melanomas positive in ELISA for TNFα (Me 4405 and Me 665/1) and from two others not tested in ELISA (Me 665/2 and Me 14932), were cytotoxic against WEHI cells. Among the TNF-producing melanomas, Me 4405 and Me 665/1 were resistant, Me 1492 moderately susceptible and Me 665/2 susceptible to recombinant TNFα cytotoxic activity. No correlation was found between cytokine expression and clinical stage of patients at the time when the tumours were obtained. These results show the variability of cytokine expression in melanoma cells that could explain their heterogeneity in the ability to stimulate host immune responses and to progress and form metastases.

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