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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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Transcription level (A) and cell surface expression (B) of HLA-ABC molecules in ESTDAB-004 and ESTDAB-159 melanoma cell lines [in (B) examined by indirect immunofluorescence using W6/32 and L-362 mAb]. Cells were treated with 800 U/ml of either IFN-γ or IFN-α for 48 h or with culture medium alone. The dark histogram corresponds to the isotypic control. Panel A – the results of real-time RT-PCR analysis. On the Y-axis on panel A – mRNA copy number of the gene of interest (HLA-A or HLA-B) normalized against the mRNA copy number of the reference gene GUSB.
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Figure 1: Transcription level (A) and cell surface expression (B) of HLA-ABC molecules in ESTDAB-004 and ESTDAB-159 melanoma cell lines [in (B) examined by indirect immunofluorescence using W6/32 and L-362 mAb]. Cells were treated with 800 U/ml of either IFN-γ or IFN-α for 48 h or with culture medium alone. The dark histogram corresponds to the isotypic control. Panel A – the results of real-time RT-PCR analysis. On the Y-axis on panel A – mRNA copy number of the gene of interest (HLA-A or HLA-B) normalized against the mRNA copy number of the reference gene GUSB.

Mentions: Cell surface expression of HLA class I molecules was analysed in a panel of 57 melanoma cell lines by flow cytometry. The response of MHC class I expression to IFN-treatment varied. Almost all of the melanoma cell lines showed increased surface expression of HLA class I antigens after 48 h of culture. Only four melanoma cell lines did not increase MHC class I expression after incubation with IFN-γ. Since two of them had β2 microglobulin mutation [14], they were not included in this study. However, the other two lines (ESTDAB-004 and ESTDAB-159) (Fig. 1) with absence of IFN-γ mediated increase in expression of the HLA class I molecules demonstrated HLA class I induction after treatment with IFN-α. Loss of IFN-γ mediated inducibility was also observed in MHC class II molecules (not shown). MHC class I expression in these two cell lines was also analysed by quantitative RT-PCR, and no upregulation of MHC class I mRNA was detected (Fig 1A). Treatment of both cell lines with 800 U/ml IFN-γ for 48 h produced no significant increase in the expression of MHC class I mRNA in comparison with a control cell line (ESTDAB-052, which responded with HLA-class I induction) (Fig. 1A). This finding was consistent with the protein expression as demonstrated by FACS analysis (Fig. 1B). Loss of IFN-γ mediated class I inducibility was not caused by a post-transcriptional defect. Because the IFN-γ signal transduction pathway shares two intracellular signalling molecules with the IFN-α pathway, these cells were also tested for their ability to up-regulate MHC class I in response to IFN-α. Both melanoma cell lines responded to IFN-α treatment with an increased MHC class I cell surface expression, indicating the possibility of a specific and selective alteration only in the IFN-γ signalling pathway.


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)

Transcription level (A) and cell surface expression (B) of HLA-ABC molecules in ESTDAB-004 and ESTDAB-159 melanoma cell lines [in (B) examined by indirect immunofluorescence using W6/32 and L-362 mAb]. Cells were treated with 800 U/ml of either IFN-γ or IFN-α for 48 h or with culture medium alone. The dark histogram corresponds to the isotypic control. Panel A – the results of real-time RT-PCR analysis. On the Y-axis on panel A – mRNA copy number of the gene of interest (HLA-A or HLA-B) normalized against the mRNA copy number of the reference gene GUSB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Transcription level (A) and cell surface expression (B) of HLA-ABC molecules in ESTDAB-004 and ESTDAB-159 melanoma cell lines [in (B) examined by indirect immunofluorescence using W6/32 and L-362 mAb]. Cells were treated with 800 U/ml of either IFN-γ or IFN-α for 48 h or with culture medium alone. The dark histogram corresponds to the isotypic control. Panel A – the results of real-time RT-PCR analysis. On the Y-axis on panel A – mRNA copy number of the gene of interest (HLA-A or HLA-B) normalized against the mRNA copy number of the reference gene GUSB.
Mentions: Cell surface expression of HLA class I molecules was analysed in a panel of 57 melanoma cell lines by flow cytometry. The response of MHC class I expression to IFN-treatment varied. Almost all of the melanoma cell lines showed increased surface expression of HLA class I antigens after 48 h of culture. Only four melanoma cell lines did not increase MHC class I expression after incubation with IFN-γ. Since two of them had β2 microglobulin mutation [14], they were not included in this study. However, the other two lines (ESTDAB-004 and ESTDAB-159) (Fig. 1) with absence of IFN-γ mediated increase in expression of the HLA class I molecules demonstrated HLA class I induction after treatment with IFN-α. Loss of IFN-γ mediated inducibility was also observed in MHC class II molecules (not shown). MHC class I expression in these two cell lines was also analysed by quantitative RT-PCR, and no upregulation of MHC class I mRNA was detected (Fig 1A). Treatment of both cell lines with 800 U/ml IFN-γ for 48 h produced no significant increase in the expression of MHC class I mRNA in comparison with a control cell line (ESTDAB-052, which responded with HLA-class I induction) (Fig. 1A). This finding was consistent with the protein expression as demonstrated by FACS analysis (Fig. 1B). Loss of IFN-γ mediated class I inducibility was not caused by a post-transcriptional defect. Because the IFN-γ signal transduction pathway shares two intracellular signalling molecules with the IFN-α pathway, these cells were also tested for their ability to up-regulate MHC class I in response to IFN-α. Both melanoma cell lines responded to IFN-α treatment with an increased MHC class I cell surface expression, indicating the possibility of a specific and selective alteration only in the IFN-γ signalling pathway.

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