Cytokines play an important role in the growth regulation of melanoma cells. Whereas the growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, melanoma cells of advanced tumor stages have often been reported to be “multi-cytokine resistant”. Thus, the aim of this study was to investigate the molecular mechanisms underlying cytokine resistance of melanoma cells. Transcription factors of the STAT family are crucial in signal transduction of cytokines. Previously, it has been reported that methylation of STAT1 on a conserved arginine residue (Arg31) is important for STAT1 function and loss of this modification was proposed to be involved in interferon resistance of cancer cells. Since STAT3 is involved in growth inhibition of melanoma cells, in the first part of this thesis I thus investigated the arginine methylation of STAT3 and STAT1. Here I provide several independent lines of evidence that did not support the occurrence of arginine methylation of STAT1 or STAT3. First, it was shown that the anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Second, it was shown that the methylation inhibitors, MTA and MDA, had profound and rapid effects on phosphorylation of STAT1 and STAT3, but MDA additionally also affected the p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Third, it was shown that mutation of Arg31 to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg31. Finally, the in vitro methylation assay using purified catalytically active protein arginine methyltransferases (PRMT1, -2, -3, -4, and -6) demonstrated that STAT proteins are not methylated, and cotransfection of PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, the data presented in this thesis suggest the absence of arginine methylation of STAT1 and STAT3. The Jak/STAT signaling pathway is subject to feedback inhibition by members of the suppressors of cytokine signaling (SOCS) family. In the second part of this thesis, I analysed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of IL-6 and OSM, to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to activation of Jak1, STAT3 and STAT1. I found a high level constitutive expression of SOCS3 that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the anti-proliferative action of IL-6 and OSM, but not of IFN-alpha. Interestingly, SOCS3-siRNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes while at protein level SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors.
info:eu-repo/classification/ddc/610, Medizin, Zytokine, Melanom, Jak/STAT, SOCS3, Argininmethylierung, cytokine, melanoma, arginine methylation
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