Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines Rodríguez Ruiz, Teresa Méndez Vales, Rosa Campo, Ana del Jiménez Gámiz, Pilar Aptsiauri, Natalia Garrido Torres-Puchol, Federico Ruiz-Cabello, Francisco Antineoplastic agents Epigenesis Genes HLA antigents Melanoma Skin neoplasms IRF1 protein Interferon regulatory factor-1 Background The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-γ-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-γ treatment in human melanoma cell lines. Methods Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan® Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-γ-treated cells. Results Altered IFN-γ mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-α led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-γ treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-α treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-γ in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-γ signaling pathway. Conclusion We observed two distinct mechanisms of loss of IFN-γ inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-γ signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-γ-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation. 2013-11-20T09:26:38Z 2013-11-20T09:26:38Z 2007 info:eu-repo/semantics/article Rodríguez, T.; et al. Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines. BMC Cancer, 7: 34 (2007). [http://hdl.handle.net/10481/29167] 1471-2407 doi: 10.1186/1471-2407-7-34 PMCID: PMC1808467 http://hdl.handle.net/10481/29167 eng http://creativecommons.org/licenses/by-nc-nd/3.0/ info:eu-repo/semantics/openAccess Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Biomed Central