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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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5-Aza-2-deoxycytidine (5-dAzaC) recovers IFN-γ mediated HLA-class I inducibility in ESTDAB-159 melanoma cells. Cells were untreated or pretreated for 7 days with 3 μM 5-dAzaC and/then untreated or incubated with 800 U/ml IFN-γ for 48 hrs. HLA class I expression before and after treatment with 5-dAzaC was determined by flow cytometry analyzes of HLA-ABC surface expression using W6/32 monoclonal antibody (B), and by quantitative RT-PCR (A). Results of HLA-B expression are normalized against GUSB expression (HLAB/GUSB).
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Figure 6: 5-Aza-2-deoxycytidine (5-dAzaC) recovers IFN-γ mediated HLA-class I inducibility in ESTDAB-159 melanoma cells. Cells were untreated or pretreated for 7 days with 3 μM 5-dAzaC and/then untreated or incubated with 800 U/ml IFN-γ for 48 hrs. HLA class I expression before and after treatment with 5-dAzaC was determined by flow cytometry analyzes of HLA-ABC surface expression using W6/32 monoclonal antibody (B), and by quantitative RT-PCR (A). Results of HLA-B expression are normalized against GUSB expression (HLAB/GUSB).

Mentions: We demonstrated the role of methylation in the loss of the inducibility of HLA class I expression by incubating ESTDAB-159 cells with the demethylating substance 5-Aza-2'-deoxycytidine. After 7 days of culture, 5-dAzaC restored IFN-γ-induced HLA-class I expression. Treatment of cells with either 5-dAzaC or IFN-γ alone did not lead to increase (or only to a marginal increase) in HLA class I protein or mRNA expression, respectively (Figure 6A). Interestingly, administration of both 5-dAzaC and IFN-γ produced an increase in the level of transcripts and led to cell surface expression of HLA class I molecules, as revealed by quantitative RT-PCR and flow cytometry analysis (Figure 6B).


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)

5-Aza-2-deoxycytidine (5-dAzaC) recovers IFN-γ mediated HLA-class I inducibility in ESTDAB-159 melanoma cells. Cells were untreated or pretreated for 7 days with 3 μM 5-dAzaC and/then untreated or incubated with 800 U/ml IFN-γ for 48 hrs. HLA class I expression before and after treatment with 5-dAzaC was determined by flow cytometry analyzes of HLA-ABC surface expression using W6/32 monoclonal antibody (B), and by quantitative RT-PCR (A). Results of HLA-B expression are normalized against GUSB expression (HLAB/GUSB).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: 5-Aza-2-deoxycytidine (5-dAzaC) recovers IFN-γ mediated HLA-class I inducibility in ESTDAB-159 melanoma cells. Cells were untreated or pretreated for 7 days with 3 μM 5-dAzaC and/then untreated or incubated with 800 U/ml IFN-γ for 48 hrs. HLA class I expression before and after treatment with 5-dAzaC was determined by flow cytometry analyzes of HLA-ABC surface expression using W6/32 monoclonal antibody (B), and by quantitative RT-PCR (A). Results of HLA-B expression are normalized against GUSB expression (HLAB/GUSB).
Mentions: We demonstrated the role of methylation in the loss of the inducibility of HLA class I expression by incubating ESTDAB-159 cells with the demethylating substance 5-Aza-2'-deoxycytidine. After 7 days of culture, 5-dAzaC restored IFN-γ-induced HLA-class I expression. Treatment of cells with either 5-dAzaC or IFN-γ alone did not lead to increase (or only to a marginal increase) in HLA class I protein or mRNA expression, respectively (Figure 6A). Interestingly, administration of both 5-dAzaC and IFN-γ produced an increase in the level of transcripts and led to cell surface expression of HLA class I molecules, as revealed by quantitative RT-PCR and flow cytometry analysis (Figure 6B).

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