Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines.
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-gamma-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-gamma 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(R) 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-gamma-treated cells.


RESULTS
Altered IFN-gamma 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-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway.


CONCLUSIONS
We observed two distinct mechanisms of loss of IFN-gamma 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-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.
Rodríguez
Teresa
T
Servicio de Análisis Clínicos, Hospital Universitario Virgen de las Nieves, Granada, Spain. teresa1rr@hotmail.com <teresa1rr@hotmail.com>
Méndez
Rosa
R
Del Campo
Ana
A
Jiménez
Pilar
P
Aptsiauri
Natalia
N
Garrido
Federico
F
Ruiz-Cabello
Francisco
F
D000970:Antineoplastic Agents / Q000494:pharmacology*; D044127:Epigenesis, Genetic; D005805:Genes, MHC Class I; D006680:HLA Antigens / Q000096:biosynthesis*; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology*; D008545:Melanoma / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D050794:STAT1 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D006680:HLA Antigens; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D007371:Interferon-gamma