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. 2007 Feb 23:7:34.
doi: 10.1186/1471-2407-7-34.

"VSports app下载" Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

Teresa Rodríguez  1 , Rosa Méndez, Ana Del Campo, Pilar Jiménez, Natalia Aptsiauri, Federico Garrido, Francisco Ruiz-Cabello
Affiliations

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

Teresa Rodríguez et al. BMC Cancer. .

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 VSports手机版. 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 V体育安卓版. 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 V体育ios版. 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 VSports最新版本. .

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Figures

Figure 1
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.
Figure 2
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.
Figure 3
Figure 3
Western blot analysis of the expression level and phosphorylation status of Jak-2 (A), and of the expression of SOCS-1 (B) in ESTDAB-004 cell line. We observed that Jak-2 was present but its phosphorylation was very low and was accompanied by a high SOCS-1 expression (negative regulator of IFN-γ response). Arrows indicate the specific proteins. Nuclear extracts as a positive control (C+) for Jak-2 were obtained from Cell Signaling.
Figure 4
Figure 4
Time course effect of IFN-γ on the expression of IRF-1 and iNOS (A). Total RNAs were isolated from ESTDAB-159 cell line and form the control cell line ESTDAB-056 grown in the presence or absence of 800 U/ml IFN-γ for the indicated periods of time and analyzed for the relative levels of mRNA by RT-PCR.
Figure 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.
Figure 6
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).
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"VSports app下载" References

    1. Darnell JE, Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–1421. doi: 10.1126/science.8197455. - "V体育平台登录" DOI - PubMed
    1. Hibino Y, Kumar CS, Mariano TM, Lai DH, Pestka S. Chimeric interferon-gamma receptors demonstrate that an accessory factor required for activity interacts with the extracellular domain. J Biol Chem. 1992;267:3741–9. - PubMed
    1. Kaplan DH, Shankaran V, Dighe AS, Stockert E, Aguet M, Old LJ, Schreiber RD. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc Natl Acad Sci USA. 1998;95:7556–7561. doi: 10.1073/pnas.95.13.7556. - DOI - PMC - PubMed
    1. Garrido F, Ruiz-Cabello F, Cabrera T, Perez-Villar JJ, Lopez-Botet M, Duggan-Keen M, Stern PL. Implications for immunosurveillance of altered HLA class I phenotypes in human tumours. Immunol Today. 1997;18:89–95. doi: 10.1016/S0167-5699(96)10075-X. - V体育官网 - DOI - PubMed
    1. Baillat G, Garrouste F, Remacle-Bonnet M, Marvaldi J, Pommier G. Bcl-xL/Bax ratio is altered by IFNgamma in TNFalpha- but not in TRAIL-induced apoptosis in colon cancer cell line. Biochim Biophys Acta. 2005;1745:101–110. doi: 10.1016/j.bbamcr.2004.12.005. - DOI - PubMed

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