The molecular mechanisms causing IFN

The molecular mechanisms causing IFN,resistance have been investigated only to a limited extent, although this information might have an important impact on the development of targeted therapies. So far, IFN,responsive genes have been shown to be frequently downregulated in tumor cells because of impaired IRF1 expression as well as defective transcriptional and posttranscriptional regulation of components of the IFN,signal transduction cascade. In addition, to the best of our knowledge, loss of the IFN,mediated upregulation of TAP in one renal cell carcinoma cell line has been found to be associated with the lack of IRF1 and STAT1 binding activities as well as of JAK1, JAK2, and STAT1 phosphorylation upon incubation with IFN, Although JAK1 andor JAK2 gene transfer could not restore the IFN,mediated phosphorylation in this RCC cell line, their overexpression increased constitutive LMP2 and TAP1 expression independent of IFN, Furthermore, an impaired STAT1 phosphorylation was accompanied by loss of IFN,mediated HLA class I upregulation in melanoma and colorectal carcinoma cell lines. The purpose of this study was to determine the mechanisms of IFN,unresponsiveness of melanoma cells regarding the HLA class I upregulation as well as the role of the IFN,signal cascade for HLA class I APM component expression. Our results show loss of JAK2 expression in 1 of 8 melanoma cell lines, which associated with a lack of IFN,inducibility of HLA class I surface antigens and with a low constitutive HLA class I APM component expression. These defects could be corrected by JAK2 transfection, vice versa, JAK2 specific short hairpin RNA and the pharmacologic inhibitor AG490 inversely impairs constitutive APM component expression in JAK2 positive cells, which is associated with reduced HLA class I surface expression. Results Impaired constitutive HLA class I APM component expression in IFN,resistant melanoma cells Flow cytometric analysis of 8 untreated or IFN,treated melanoma cell lines using the HLA class I antigen specific mAb B9. 12. 1 or the HLA class II antigen specific mAb Tü39 showed a marked variability in the IFNγ mediated modulation of both HLA antigen classes. The different melanoma cell lines heterogeneously responded in a dose and time dependent manner to IFN,treatment, ranging from lack of to low to strong IFN,mediated upregulation of HLA class I and class II surface antigens. The representative results shown in Supplementary Figure S1 show that 4 of 8 melanoma cell lines tested exhibited a 2 to 3 fold upregulation of both HLA class I and class II surface antigens, whereas the remaining 4 failed to upregulate HLA class II antigens.