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Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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2011 (English)In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, no 8, 783-794 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2011. Vol. 16, no 8, 783-794 p.
Keyword [en]
Apoptosis, Death receptor, Resistance, Fas, Interferon, cFLIP
National Category
Biophysics Cell and Molecular Biology Medical and Health Sciences Immunology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-3478DOI: 10.1007/s10495-011-0606-0OAI: oai:DiVA.org:umu-3478DiVA: diva2:142197
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Regulation of apoptosis during treatment and resistance development in tumour cells
Open this publication in new window or tab >>Regulation of apoptosis during treatment and resistance development in tumour cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Induction of apoptosis is the most studied cell death process and it is a tightly regulated physiological event that enables elimination of damaged and unwanted cells. Apoptosis can be induced via activation of either the intrinsic or the extrinsic signalling pathway. The intrinsic pathway involves activation of the mitochondria by stress stimuli, whereas the extrinsic pathway is triggered by ligand induced activation of death receptors such as Fas. Apoptosis induction via Fas activation plays an important role in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis.

Several studies have shown that anticancer therapies require functional cell death signalling pathways. Irradiation based therapy has been successful in treatment of several malignancies but the usage of high doses has been associated with side effects. Therefore, low dose therapies, that either is optimized for specific delivery or administrated in combination with other treatments, are promising modalities. However, in order to achieve high-quality effects of such treatments, the death effector mechanisms involved in tumour eradication needs to be further explored. Importantly, tumour cells frequently acquire resistance to apoptosis, which consequently allows tumour cells to escape from elimination by the immune system and/or treatment.

Interferons constitute a large family of pleotrophic cytokines that are important for the immune response against viruses and other microorganisms. The interferon signalling pathway mediates transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. Interferon has successfully been used in therapy against some tumours. However, several drawbacks have been reported, such as reduced sensitivity to interferon during treatment.

The aim of this thesis was to elucidate mechanisms that mediate resistance to death receptor or interferon induced apoptosis in human tumour cell models, as well as investigate what molecular events that underlie cell death following radiation therapy of tumour cells.

In order to elucidate mechanisms involved in acquired resistance to Fas- or interferon-induced apoptosis, a Fas- and interferon-sensitive human cell line, U937, was subjected to conditions where resistance to either Fas- or interferon induced apoptosis was acquired. Characterization of the Fas resistant cells showed that multiple resistant mechanisms had been acquired. Reduced Fas expression and increased cFLIP expression, which is an inhibitor of death receptor signalling, were two important changes found. To further examine the importance of these two alterations, clones from the Fas resistant population were established. The reduced Fas expression was determined to account for the resistant phenotype in approximately 70% of the clones. In the Fas resistant clones with normal Fas expression, the importance of an increased amount of the cFLIP protein was confirmed with shRNA interference. A cross-resistance to death receptor induced apoptosis was detected in the interferon resistant variant, which illustrates that a connection between death receptor and interferon induced apoptosis exists. Notably, interferon resistant cells also contained increased cFLIP expression, which were determined to mediate resistance to both interferon and death receptor mediated apoptosis. Finally, when cell death induced by irradiation treatment was investigated in HeLa Hep2 cells we could demonstrate that cell death was mediated by centrosome hyperamplification and mitotic aberrations, which forced the cells into mitotic catastrophes and delayed apoptosis.

In conclusion, we have described model systems where selection for resistance to Fas or interferon induced apoptosis generated a heterogeneous population, where several signalling molecules were altered. Furthermore, we have shown that a complex cell death network was activated by irradiation based therapy.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Medicinska fakulteten), 2008. 65 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1199
Keyword
Tumörbiologi
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1851 (URN)978-917264-627-8 (ISBN)
Public defence
2008-10-03, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
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Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2010-04-06Bibliographically approved

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Blomberg, JeanetteHöglund, AndreasEriksson, DavidRuuth, KristinaJacobsson, MariaNilsson, JonasLundgren, Erik
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Department of Medical Biochemistry and BiophysicsDepartment of Molecular Biology (Faculty of Medicine)Immunology/Immunchemistry
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Apoptosis (London)
BiophysicsCell and Molecular BiologyMedical and Health SciencesImmunology in the medical area

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