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Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines.

Rodríguez T, Méndez R, Del Campo A, Jiménez P, Aptsiauri N, Garrido F, Ruiz-Cabello F - BMC Cancer (2007)

Bottom Line: 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.We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Servicio de Análisis Clínicos, Hospital Universitario Virgen de las Nieves, Granada, Spain. teresa1rr@hotmail.com <teresa1rr@hotmail.com>

ABSTRACT

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.

Conclusion: 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.

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Time course effect of IFN-γ on the expression of IRF-1 and iNOS (A). Total RNAs were isolated from ESTDAB-159 cell line and form the control cell line ESTDAB-056 grown in the presence or absence of 800 U/ml IFN-γ for the indicated periods of time and analyzed for the relative levels of mRNA by RT-PCR.
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Figure 4: Time course effect of IFN-γ on the expression of IRF-1 and iNOS (A). Total RNAs were isolated from ESTDAB-159 cell line and form the control cell line ESTDAB-056 grown in the presence or absence of 800 U/ml IFN-γ for the indicated periods of time and analyzed for the relative levels of mRNA by RT-PCR.

Mentions: After tyrosine-phosphorylation, STATs form homo- or heterodimers and translocate from the cytoplasm to the nucleus, where they bind specific DNA sequences and activate transcription of many genes. IRF-1 is transactivated by binding of STAT-1 dimers to the GAS sequence of the IRF-1 promoter. IRF-1 is the principal factor mediating the IFN-γ-induced expression of MHC class I antigen. In order to analyze the Jak-STAT signalling cascade, we investigated the induction of IRF-1 mRNA expression in response to IFN-γ treatment. As shown in Figure 4A, IRF-1 mRNA was upregulated by IFN-γ treatment in ESTDAB-159 cells. We also investigated the expression of another IRF-1- regulated gene, iNOS, to determine whether MHC class I gene expression is selectively blocked. We measured iNOS mRNA level in IFN-γ-treated ESTDAB-159 cells. As shown in Fig 4B, IFN-γ did not induce expression of iNOS mRNA, indicating an inhibition of IRF-1 transactivated genes.


Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines.

Rodríguez T, Méndez R, Del Campo A, Jiménez P, Aptsiauri N, Garrido F, Ruiz-Cabello F - BMC Cancer (2007)

Time course effect of IFN-γ on the expression of IRF-1 and iNOS (A). Total RNAs were isolated from ESTDAB-159 cell line and form the control cell line ESTDAB-056 grown in the presence or absence of 800 U/ml IFN-γ for the indicated periods of time and analyzed for the relative levels of mRNA by RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808467&req=5

Figure 4: Time course effect of IFN-γ on the expression of IRF-1 and iNOS (A). Total RNAs were isolated from ESTDAB-159 cell line and form the control cell line ESTDAB-056 grown in the presence or absence of 800 U/ml IFN-γ for the indicated periods of time and analyzed for the relative levels of mRNA by RT-PCR.
Mentions: After tyrosine-phosphorylation, STATs form homo- or heterodimers and translocate from the cytoplasm to the nucleus, where they bind specific DNA sequences and activate transcription of many genes. IRF-1 is transactivated by binding of STAT-1 dimers to the GAS sequence of the IRF-1 promoter. IRF-1 is the principal factor mediating the IFN-γ-induced expression of MHC class I antigen. In order to analyze the Jak-STAT signalling cascade, we investigated the induction of IRF-1 mRNA expression in response to IFN-γ treatment. As shown in Figure 4A, IRF-1 mRNA was upregulated by IFN-γ treatment in ESTDAB-159 cells. We also investigated the expression of another IRF-1- regulated gene, iNOS, to determine whether MHC class I gene expression is selectively blocked. We measured iNOS mRNA level in IFN-γ-treated ESTDAB-159 cells. As shown in Fig 4B, IFN-γ did not induce expression of iNOS mRNA, indicating an inhibition of IRF-1 transactivated genes.

Bottom Line: 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.We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Servicio de Análisis Clínicos, Hospital Universitario Virgen de las Nieves, Granada, Spain. teresa1rr@hotmail.com <teresa1rr@hotmail.com>

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus