The melanoma cell line Colo 857 was completely resistant to IFN

The melanoma cell line Colo 857 was completely resistant to IFN,treatment, lacking IFN,mediated upregulation of both HLA class I and class II surface antigens as well as responsiveness to the antiproliferative effect of IFN, The resistance of Colo 857 cells was selective for IFN,because HLA class I surface expression was induced in these cells in a dose and time dependent manner by IFN,as well as by TNF, although the degree of upregulation varied between both cytokines. Because the IFN,receptor was expressed in the IFN, resistant Colo 857 cells to levels comparable with the IFN,sensitive control cell line Colo 794, the IFN,resistance seemed to be due to defects in the IFN,signal transduction pathway rather than at the receptor level. To investigate whether the loss of IFN,inducibility of HLA class I surface antigens was associated with altered expression levels of HLA class I APM components, constitutive and IFN,inducible LMP10, TAP2, tapasin, HLA class I HC, LMP2, TAP1, and B2 m mRNA and protein expression levels were monitored by qRT PCR and Western blot analysis. With the exception of B2 m, the constitutive expression pattern of these molecules was lower and not inducible in IFN,resistant Colo 857 cells than that in IFN,sensitive Colo 794 melanoma cells. In contrast, IFN,treatment increased APM component transcription levels and protein expression in both Colo 857 and Colo 794 cells. Lack of IFN,sensitivity due to defects in the IFN,signal cascade To determine whether the resistance of Colo 857 cells to IFN,is due to an impaired IFN,signal transduction, constitutive and IFN,inducible transcription of the major signal transduction molecules including JAK1, JAK2, and STAT1 were investigated. In contrast to the IFN,sensitive cell line Colo 794, RT PCR revealed a lack of constitutive and IFN, inducible JAK2 mRNA expression in Colo 857 cells, whereas the mRNA of JAK1 and STAT1 was expressed in these cells. With the exception of JAK1, the signal transduction components were not upregulated by IFN, These data were further confirmed by Western blot analysis, which showed JAK2 protein expression in Colo 794 and all other melanoma cells analyzed but not in Colo 857 cells despite their constitutive expression of JAK1 and STAT1 proteins. The functionality of the IFN,signaling components was determined using antibodies specifically directed against the selected phosphorylated counterparts JAK1 and STAT1. In Colo 794 cells, an increased phosphorylation of JAK1 and STAT1 was observed after IFN,treatment. In contrast, phosphorylation of JAK1 and STAT1 was not detected in Colo 857 cells. This defect is selective for IFN, as IFN,did induce STAT1 phosphorylation. Thus, the impaired phosphorylation of signal cascade members by IFN,treatment reflects the loss of JAK2 expression. Molecular mechanisms underlying deficient JAK2 expression To define the molecular mechanisms involved in the lack of JAK2 mRNA and protein expression in Colo 857 cells, JAK2 specific genomic PCR was carried out.