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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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The expression and the activation of STAT proteins. Cells were treated with IFN-γ or IFN-α at 800 IU/ml. Proteins from cellular protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was performed as described in Materials and Methods. Anti-STAT1 and anti-phospho-STAT1 antibodies were used to assess STAT activation. Anti-γ-tubulin antibody was used to normalize the amounts of protein loaded in each well of the gel. Positive control (C+) of phosphorylated proteins from cellular extract A549 was obtained from Cell Signaling Technology.
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Figure 2: The expression and the activation of STAT proteins. Cells were treated with IFN-γ or IFN-α at 800 IU/ml. Proteins from cellular protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was performed as described in Materials and Methods. Anti-STAT1 and anti-phospho-STAT1 antibodies were used to assess STAT activation. Anti-γ-tubulin antibody was used to normalize the amounts of protein loaded in each well of the gel. Positive control (C+) of phosphorylated proteins from cellular extract A549 was obtained from Cell Signaling Technology.

Mentions: STAT-1 phosphorylation was examined by western blot using anti-STAT-1 or anti -phospho-STAT-1 antibodies that recognize unphosphorylated or phosphorylated STAT-1, respectively. Cells were incubated with 800 U/ml IFN-γ or IFN-α before western blot analysis. STAT-1 induction and activation were analyzed in both cell lines to yield further information on the possible mechanism of altered response to IFN-γ treatment. As shown in Figure 2, STAT-1 tyrosine phosphorylation occurred only in the ESTDAB-159 cells, whereas phosphorylation was undetectable in ESTDAB-004 cells. STAT-1 phosphorylation was upregulated and rapidly decreased after 60 min of incubation with IFN-γ. However, both cell lines responded with STAT-1 phosphorylation to IFN-α treatment (Figure 2). The tyrosine phosphorylation of Jak-2 in response to IFN-γ stimulation was also investigated. Absence of phosphorylation in response to IFN-γ was observed in ESTDAB-004 cells, which was correlated with decreased IFN-γ signaling (Figure 3). Suppressor of cytokine signaling-1 (SOCS1) is a critical negative regulator of IFN-γ responses. Its expression is induced in response to stimulation of a variety of cytokines, and over-expression of this protein results in inhibition of cytokine signaling [15]. SOCS1 protein expression was detected in ESTDAB-004 melanoma cells without treatment with IFN-γ and did not change after this treatment (Figure 3).


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)

The expression and the activation of STAT proteins. Cells were treated with IFN-γ or IFN-α at 800 IU/ml. Proteins from cellular protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was performed as described in Materials and Methods. Anti-STAT1 and anti-phospho-STAT1 antibodies were used to assess STAT activation. Anti-γ-tubulin antibody was used to normalize the amounts of protein loaded in each well of the gel. Positive control (C+) of phosphorylated proteins from cellular extract A549 was obtained from Cell Signaling Technology.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The expression and the activation of STAT proteins. Cells were treated with IFN-γ or IFN-α at 800 IU/ml. Proteins from cellular protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was performed as described in Materials and Methods. Anti-STAT1 and anti-phospho-STAT1 antibodies were used to assess STAT activation. Anti-γ-tubulin antibody was used to normalize the amounts of protein loaded in each well of the gel. Positive control (C+) of phosphorylated proteins from cellular extract A549 was obtained from Cell Signaling Technology.
Mentions: STAT-1 phosphorylation was examined by western blot using anti-STAT-1 or anti -phospho-STAT-1 antibodies that recognize unphosphorylated or phosphorylated STAT-1, respectively. Cells were incubated with 800 U/ml IFN-γ or IFN-α before western blot analysis. STAT-1 induction and activation were analyzed in both cell lines to yield further information on the possible mechanism of altered response to IFN-γ treatment. As shown in Figure 2, STAT-1 tyrosine phosphorylation occurred only in the ESTDAB-159 cells, whereas phosphorylation was undetectable in ESTDAB-004 cells. STAT-1 phosphorylation was upregulated and rapidly decreased after 60 min of incubation with IFN-γ. However, both cell lines responded with STAT-1 phosphorylation to IFN-α treatment (Figure 2). The tyrosine phosphorylation of Jak-2 in response to IFN-γ stimulation was also investigated. Absence of phosphorylation in response to IFN-γ was observed in ESTDAB-004 cells, which was correlated with decreased IFN-γ signaling (Figure 3). Suppressor of cytokine signaling-1 (SOCS1) is a critical negative regulator of IFN-γ responses. Its expression is induced in response to stimulation of a variety of cytokines, and over-expression of this protein results in inhibition of cytokine signaling [15]. SOCS1 protein expression was detected in ESTDAB-004 melanoma cells without treatment with IFN-γ and did not change after this treatment (Figure 3).

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