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Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line

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Abstract

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

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Abbreviations

IFN:

Interferon

cFLIP:

Cellular FLICE-like inhibitory protein

TNF-α:

Tumour necrosis factor-alpha

IFNres:

Interferon resistant cells

ISGs:

Interferon stimulated genes

JAK:

Janus-kinase

STAT:

Signal transducers and activators of transcription

qRT-PCR:

Quantitative real-time PCR

shRNA:

Short hairpin RNA

α-Fas:

Anti-Fas agonistic antibody

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Acknowledgments

We would like to thank Ingrid Dacklin for providing us with the IFN resistant cells. We are also grateful to Sofia Kapsi for valuable technical assistance. This work was supported by grants from The Swedish Cancer Society (Young Investigator Award and project grant to J.A.N), The Association of International Cancer Research (J.A.N), The Swedish Research Council (J.A.N.), The Kempe foundation (J.A.N) and a Career grant of Umeå University, Viranative Ltd, Umeå Sweden (E.L.) and The Medical Faculty. Leukocyte IFN was a kind gift from Viranative Ltd.

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Authors and Affiliations

  1. Department of Molecular Biology, Umeå University, 90187, Umeå, Sweden

    Andreas Höglund, Kristina Ruuth, Maria Jacobsson, Erik Lundgren & Jonas A. Nilsson

  2. Department of Immunology, Umeå University, 90185, Umeå, Sweden

    David Eriksson

  3. Department of Medical Chemistry and Biophysics, Umeå University, 90187, Umeå, Sweden

    Jeanette Blomberg

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  1. Jeanette Blomberg
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  2. Andreas Höglund
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Correspondence to Jeanette Blomberg.

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10495_2011_606_MOESM1_ESM.tif

Supplementary Figure 1. Reduced expression of IFN-receptor signalling molecules does not affect STAT1 activation. (a) Expression of STAT1, STAT2, JAK1 and TYK2 was investigated in un-stimulated parental and IFN resistant cells by immunoblot. (b) Cells were either untreated or stimulated with 1,000 U/ml IFN-α for 48 h and STAT1 was detected by immunoblot. (c) Parental cells and cells of clone I1 were treated with 1,000 U/ml IFN-α for the indicated time points in the figure. Cytoplasmic and nuclear extracts were separated and STAT1 localization was investigated by immunoblot. Actin was used as an equal loading control in all experiments. (TIFF 6952 kb)

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Blomberg, J., Höglund, A., Eriksson, D. et al. Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α and death receptor stimulation in resistant variants of the human U937 cell line. Apoptosis 16, 783–794 (2011). https://doi.org/10.1007/s10495-011-0606-0

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  • DOI: https://doi.org/10.1007/s10495-011-0606-0

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