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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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EMSA showing the binding of IRF-1 to a 32P labeled probe containing the IRF-1 consensus sequence. EMSA was performed with nuclear extracts obtained from untreated ESTDAB-056 (3) and ESTDAB-159 (5) melanoma cells or treated with 800 U/ml IFN-γ for 4 h (lanes 1, 4 and 7 corresponding to ESTDAB-056; and lanes 2, 6 and 8 corresponding to ESTDAB-159). A 50-fold molar excess of unlabeled IRF-1 probe was added to the binding reaction (lane 1, 2) to compete out the formation of a detectable complex. Anti-IRF-1 antibody was used to block IRF-1 binding to test the specificity of the interaction (lanes 7 and 8). Results shown are representative of at least three independent experiments.
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Figure 5: EMSA showing the binding of IRF-1 to a 32P labeled probe containing the IRF-1 consensus sequence. EMSA was performed with nuclear extracts obtained from untreated ESTDAB-056 (3) and ESTDAB-159 (5) melanoma cells or treated with 800 U/ml IFN-γ for 4 h (lanes 1, 4 and 7 corresponding to ESTDAB-056; and lanes 2, 6 and 8 corresponding to ESTDAB-159). A 50-fold molar excess of unlabeled IRF-1 probe was added to the binding reaction (lane 1, 2) to compete out the formation of a detectable complex. Anti-IRF-1 antibody was used to block IRF-1 binding to test the specificity of the interaction (lanes 7 and 8). Results shown are representative of at least three independent experiments.

Mentions: IFN-γ-stimulated IRF-1 induction was assayed using electrophoretic mobility shift assay (EMSA). As shown in Fig. 5, IRF-1 binding to the consensus element was induced after IFN-γ stimulation of ESTDAB-159 and of control cell line ESTDAB-056. Protein complex formation between IRF-1 and DNA probes was increased in the nuclear extract of IFN-γ-treated melanoma cells (lanes 3, 4, 5 and 6). Formation of detectable complex was competed out with 50-fold excess of unlabeled cold probe (lanes 1, 2). Supershift experiments using an anti-IRF-1 antibody (lanes 7, 8) indicated that the complex was specific. These results strongly suggest that the nuclear protein complex that binds to the putative IRF-1 binding probe contains a functional IRF-1 protein.


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)

EMSA showing the binding of IRF-1 to a 32P labeled probe containing the IRF-1 consensus sequence. EMSA was performed with nuclear extracts obtained from untreated ESTDAB-056 (3) and ESTDAB-159 (5) melanoma cells or treated with 800 U/ml IFN-γ for 4 h (lanes 1, 4 and 7 corresponding to ESTDAB-056; and lanes 2, 6 and 8 corresponding to ESTDAB-159). A 50-fold molar excess of unlabeled IRF-1 probe was added to the binding reaction (lane 1, 2) to compete out the formation of a detectable complex. Anti-IRF-1 antibody was used to block IRF-1 binding to test the specificity of the interaction (lanes 7 and 8). Results shown are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: EMSA showing the binding of IRF-1 to a 32P labeled probe containing the IRF-1 consensus sequence. EMSA was performed with nuclear extracts obtained from untreated ESTDAB-056 (3) and ESTDAB-159 (5) melanoma cells or treated with 800 U/ml IFN-γ for 4 h (lanes 1, 4 and 7 corresponding to ESTDAB-056; and lanes 2, 6 and 8 corresponding to ESTDAB-159). A 50-fold molar excess of unlabeled IRF-1 probe was added to the binding reaction (lane 1, 2) to compete out the formation of a detectable complex. Anti-IRF-1 antibody was used to block IRF-1 binding to test the specificity of the interaction (lanes 7 and 8). Results shown are representative of at least three independent experiments.
Mentions: IFN-γ-stimulated IRF-1 induction was assayed using electrophoretic mobility shift assay (EMSA). As shown in Fig. 5, IRF-1 binding to the consensus element was induced after IFN-γ stimulation of ESTDAB-159 and of control cell line ESTDAB-056. Protein complex formation between IRF-1 and DNA probes was increased in the nuclear extract of IFN-γ-treated melanoma cells (lanes 3, 4, 5 and 6). Formation of detectable complex was competed out with 50-fold excess of unlabeled cold probe (lanes 1, 2). Supershift experiments using an anti-IRF-1 antibody (lanes 7, 8) indicated that the complex was specific. These results strongly suggest that the nuclear protein complex that binds to the putative IRF-1 binding probe contains a functional IRF-1 protein.

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