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Variable Resistance of RMS to Interferon γ Signaling.

Simon-Keller K, Mößinger K, Bohlender AL, Ströbel P, Marx A - ISRN Oncol (2012)

Bottom Line: Conclusions.IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells.Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Medical Centre Mannheim, University of Heidelberg, 68135 Mannheim, Germany.

ABSTRACT
Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.

No MeSH data available.


Related in: MedlinePlus

IFNγ treatment does not alter protein expression of AChR. Induction of AChRγ in RMS cell lines 48 h after IFNγ treatment (100 ng/mL); filled histograms represent expression levels after incubation with IFNγ; open histograms represent expression levels without IFNγ incubation.
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fig4: IFNγ treatment does not alter protein expression of AChR. Induction of AChRγ in RMS cell lines 48 h after IFNγ treatment (100 ng/mL); filled histograms represent expression levels after incubation with IFNγ; open histograms represent expression levels without IFNγ incubation.

Mentions: To check whether resistance of most RMS cell lines against IFNγ-mediated killing reflects a facet of a broader block of IFNγ-driven gene expression, we analyzed AChR and MHC expression on RMS cell lines after incubation with IFNγ for up to 72 h. In contrast to a previous report about IFNγ-driven AChR induction in RMS-like transformed myoid cells [24], AChR expression on RMS cell was not altered either by IFNγ treatment alone (Figure 4) or when combined with TNFα (data not shown). As to bona fide IFNγ targets, expression of MHC class II and its upstream regulator, CIITA, was not inducible in any RMS cell line (Figures 5(a) and 5(c)), while MHC class I expression was slightly inducible in RH41, RD6, and TE671 but only marginally in CRL2061, RH30, and FLOH1 cells (Figure 5(b)). Of note, IFNγ-susceptible, apoptosis-prone HT29 cells exhibited strong induction of MHCI, MHCII (Figure 5(d)), and CIITA (Figure 5(c)) expression on IFNγ treatment.


Variable Resistance of RMS to Interferon γ Signaling.

Simon-Keller K, Mößinger K, Bohlender AL, Ströbel P, Marx A - ISRN Oncol (2012)

IFNγ treatment does not alter protein expression of AChR. Induction of AChRγ in RMS cell lines 48 h after IFNγ treatment (100 ng/mL); filled histograms represent expression levels after incubation with IFNγ; open histograms represent expression levels without IFNγ incubation.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3420146&req=5

fig4: IFNγ treatment does not alter protein expression of AChR. Induction of AChRγ in RMS cell lines 48 h after IFNγ treatment (100 ng/mL); filled histograms represent expression levels after incubation with IFNγ; open histograms represent expression levels without IFNγ incubation.
Mentions: To check whether resistance of most RMS cell lines against IFNγ-mediated killing reflects a facet of a broader block of IFNγ-driven gene expression, we analyzed AChR and MHC expression on RMS cell lines after incubation with IFNγ for up to 72 h. In contrast to a previous report about IFNγ-driven AChR induction in RMS-like transformed myoid cells [24], AChR expression on RMS cell was not altered either by IFNγ treatment alone (Figure 4) or when combined with TNFα (data not shown). As to bona fide IFNγ targets, expression of MHC class II and its upstream regulator, CIITA, was not inducible in any RMS cell line (Figures 5(a) and 5(c)), while MHC class I expression was slightly inducible in RH41, RD6, and TE671 but only marginally in CRL2061, RH30, and FLOH1 cells (Figure 5(b)). Of note, IFNγ-susceptible, apoptosis-prone HT29 cells exhibited strong induction of MHCI, MHCII (Figure 5(d)), and CIITA (Figure 5(c)) expression on IFNγ treatment.

Bottom Line: Conclusions.IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells.Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Medical Centre Mannheim, University of Heidelberg, 68135 Mannheim, Germany.

ABSTRACT
Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines.

No MeSH data available.


Related in: MedlinePlus