Association of IFN-gamma signal transduction defects with impaired HLA class I antigen processing in melanoma cell lines - PubMed (original) (raw)

Association of IFN-gamma signal transduction defects with impaired HLA class I antigen processing in melanoma cell lines

Annedore Respa et al. Clin Cancer Res. 2011.

Abstract

Purpose: Abnormalities in the constitutive and IFN-γ-inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance.

Experimental design: The basal and IFN-γ-inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2(-) cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay.

Results: The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2(-) cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2(-) Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity.

Conclusions: Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities.

©2011 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors have no conflicting financial interests.

Figures

Figure 1

Figure 1

Constitutive and cytokine-mediated regulation of HLA class I and class II surface expression. A, constitutive and cytokine-mediated dose-dependent upregulation of HLA class I surface antigen expression. B, constitutive and cytokine-mediated time-dependent upregulation of HLA class I and HLA class II antigen surface expression. The cells were either left untreated or treated with 200 units of IFN-γ for the time points indicated. Flow cytometry was carried out using FITC-conjugated HLA class I- and class II-specific mAb. Results are expressed as MFI − SD. The experiments were carried out at least 3 times. C, antiproliferative effect of IFN-γ in Colo 794. Cells were treated with the indicated concentrations of IFN-γ for 48 hours and the number of viable cells was quantified by XTT measurements. Results are expressed as relative growth to untreated cells. D, presence of IFN-γR on the cell surface expression. The IFN-γR expression was determined by flow cytometry by using IFN-γR chain-specific mAbs. The results are presented as histograms. The dotted line represents the IgG1 isotype control, the thin line the IFN-γR1, and the thick line the IFN-γR2.

Figure 2

Figure 2

IFN-γ resistance of melanoma cells associated with low levels of HLA class I APM component expression. mRNA and protein expression patterns of HLA class I APM components in melanoma cells either left untreated or treated for 24 to 48 hours with IFN-γ were determined by qRT-PCR and Western blot analysis as described in Materials and Methods by using APM-specific primers and antibodies, respectively. A, LMP10; B, TAP2; C, HLA class I HC; D, tapasin. At least 3 independent experiments were carried out. The results are either expressed as mean of the values obtained in 3 independent experiments (qRT-PCR) or shown by a representative Western blot.

Figure 3

Figure 3

Association of impaired JAK2 expression in Colo 857 cells with impaired STAT1 phosphorylation. Melanoma cells were either left untreated or treated with IFN-γ for various time points before being harvested for protein extraction. Western blot analysis was carried out using unphosphorylated and phosphorylated JAK/STAT pathway component-specific antibodies. Staining of the Western blot with an anti-GAPDH- or β-actin–specific antibody served as controls.

Figure 4

Figure 4

Genomic deletion of JAK2 in Colo 857 cells. A, genomic PCR using JAK2-specific primers showed no amplification product in Colo 857 but in Colo 794. B, locus-specific gene amplification (end point PCR) was carried out as described in Materials and Methods. The RCC cell line MZ 2733RC known to have no observed abnormalities in IFN-γ signaling pathway served as a control (C) CGH. The deletion of JAK2 was determined by 3 independent technologies as described in detail in Materials and Methods.

Figure 5

Figure 5

Reconstitution of HLA class I APM component expression by Colo 857 cells following JAK2 gene transfer. A, JAK2 and pSTAT1 expression were determined in untreated and IFN-γ–treated Colo 857 cells transfected with the JAK2 expression vector as described in Materials and Methods. The results showed restoration of JAK2 expression and pSTAT1 upregulation by JAK2 gene transfer. B, constitutive and IFN-γ–inducible upregulation of APM component expression in mock-transfected and JAK2-transfected Colo 857 cells was carried out as described in Materials and Methods by using APM-specific mAbs. C, restoration of constitutive HLA class I surface expression in Colo 857 cells. Flow cytometry of untransfected, mock, and JAK2-overexpressing cells was carried out as described using an anti-HLA class I–specific mAb. The results are expressed as MFI with subtraction of the isotype control.

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