Interleukin-6-resistant melanoma cells exhibit reduced activation of STAT3and lack of inhibition of cyclin E-associated kinase activity

Development of cytokine resistance is an important feature of melanoma cell s during tumor progression. To study the mechanisms of interleukin-6 resist ance, we examined an interleukin-6 sensitive (WM35) and an interleukin-6 un responsive cell line (WM9), Interleukin-6 treatment resulted in rapid inhib ition of cyclin-dependent kinase 2/cyclin E activity and accumulation of th e hypophosphorylated retinoblastoma protein in WM35 but not in WM9 cells. I n contrast to previous reports, no differences in the expression of the cyc lin-dependent kinase 2 inhibitor p21(Cip1/WAF1) upon interleukin-6 treatmen t were found in both cell lines. Interleukin-6-induced inhibition of cyclin -dependent kinase 2 was also not due to changes in protein expression of cy clin-dependent kinase 2, cyclin E, p27(Kip1) and cdc25A, a phosphatase posi tively regulating cyclin-dependent kinase 2 activity. As it is established that interleukin-6 resistance of WM9 cells is not caused by differential in terleukin-6 receptor expression, we studied whether this is due to defectiv e interleukin-6 signaling in which activation of signal transducer and acti vator of transcription 3 is a critical step. WM9 cells showed reduced tyros ine phosphorylation, DNA binding, and delayed nuclear translocation of sign al transducer and activator of transcription 3 as compared with WM35 cells. The kinase upstream of signal transducer and activator of transcription 3, Janus kinase 1, was constitutively tyrosine-phosphorylated ill WM9 cells a nd did not respond to interleukin-6 with increased phosphorylation, As comp ared with WM35 cells, interleukin-6 treatment of WM9 cells was not parallel ed by reduced activity of the mitogen-activated protein kinase kinase-1, wh ich suppresses activation of signal transducer and activator of transcripti on 3, Our data suggest that resistance of advanced melanoma cells to interl eukin-6 is associated with reduced inhibition of cyclin-dependent kinase 2, which appears to be a consequence of a complex alteration in interleukin-6 signal transduction.