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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

Detection of IFNγ induced cell death by flow cytometry and western blot. (a and b) Apoptotic cell detection via flow cytometry of FLOH1, RH30, TE671, and HT29 cell lines after IFNγ treatment; cells were incubated for different periods of time with 100 ng/mL IFNγ in starvation media with 1% FCS and stained with propidium iodide and annexin V; (a) shows proportion of propidium iodide positive cells after IFNγ treatment at different time points; (b) reflects the complete flow cytometry data for HT29 control cell lines; (c) western blot analysis of caspase 8 cleavage 24 h after IFNγ treatment; β-actin serves as loading control.
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fig2: Detection of IFNγ induced cell death by flow cytometry and western blot. (a and b) Apoptotic cell detection via flow cytometry of FLOH1, RH30, TE671, and HT29 cell lines after IFNγ treatment; cells were incubated for different periods of time with 100 ng/mL IFNγ in starvation media with 1% FCS and stained with propidium iodide and annexin V; (a) shows proportion of propidium iodide positive cells after IFNγ treatment at different time points; (b) reflects the complete flow cytometry data for HT29 control cell lines; (c) western blot analysis of caspase 8 cleavage 24 h after IFNγ treatment; β-actin serves as loading control.

Mentions: Apoptosis was checked in RH30, FLOH1, TE671, and HT29 cells by Annexin V/Propidium iodide (PI) double staining and caspase 8 cleavage assay. Percentage of PI positive cells after 96 h of treatment approached 100% in HT29 cells, 60% in RH30 cells and <20% in the other, IFNγ-resistant cell lines (Figures 2(a) and 2(b)). Surprisingly, caspase 8 cleavage after 24 h (Figure 2(c)), 48 h, and 96 h (not shown) was only observed in HT29 cells but not in any RMS cell line tested, including apoptosis-prone RH30 cells (Figure 2(c) and data not shown).


Variable Resistance of RMS to Interferon γ Signaling.

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

Detection of IFNγ induced cell death by flow cytometry and western blot. (a and b) Apoptotic cell detection via flow cytometry of FLOH1, RH30, TE671, and HT29 cell lines after IFNγ treatment; cells were incubated for different periods of time with 100 ng/mL IFNγ in starvation media with 1% FCS and stained with propidium iodide and annexin V; (a) shows proportion of propidium iodide positive cells after IFNγ treatment at different time points; (b) reflects the complete flow cytometry data for HT29 control cell lines; (c) western blot analysis of caspase 8 cleavage 24 h after IFNγ treatment; β-actin serves as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Detection of IFNγ induced cell death by flow cytometry and western blot. (a and b) Apoptotic cell detection via flow cytometry of FLOH1, RH30, TE671, and HT29 cell lines after IFNγ treatment; cells were incubated for different periods of time with 100 ng/mL IFNγ in starvation media with 1% FCS and stained with propidium iodide and annexin V; (a) shows proportion of propidium iodide positive cells after IFNγ treatment at different time points; (b) reflects the complete flow cytometry data for HT29 control cell lines; (c) western blot analysis of caspase 8 cleavage 24 h after IFNγ treatment; β-actin serves as loading control.
Mentions: Apoptosis was checked in RH30, FLOH1, TE671, and HT29 cells by Annexin V/Propidium iodide (PI) double staining and caspase 8 cleavage assay. Percentage of PI positive cells after 96 h of treatment approached 100% in HT29 cells, 60% in RH30 cells and <20% in the other, IFNγ-resistant cell lines (Figures 2(a) and 2(b)). Surprisingly, caspase 8 cleavage after 24 h (Figure 2(c)), 48 h, and 96 h (not shown) was only observed in HT29 cells but not in any RMS cell line tested, including apoptosis-prone RH30 cells (Figure 2(c) and data not shown).

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