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Regulation of apoptosis during treatment and resistance development in tumour cells
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
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 [sv]
Tumörbiologi
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1851ISBN: 978-917264-627-8 OAI: oai:DiVA.org:umu-1851DiVA: diva2:142199
Public defence
2008-10-03, Major Groove, 6L, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2010-04-06Bibliographically approved
List of papers
1. Acquired resistance to Fas/CD95 ligation in U937 cells is associated with multiple molecular mechanisms
Open this publication in new window or tab >>Acquired resistance to Fas/CD95 ligation in U937 cells is associated with multiple molecular mechanisms
2008 In: Anticancer research, ISSN 0250-7005, Vol. 28, no 2A, 593-600 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-3476 (URN)
Available from: 2008-09-22 Created: 2008-09-22Bibliographically approved
2. Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis
Open this publication in new window or tab >>Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis
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2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 2, 497-508 p.Article in journal (Refereed) Published
Abstract [en]

Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.

Keyword
CpG methylation, ERK activation by PD98059, Fas, Fas expression, TNF-a Fas expression, TNF-a
National Category
Medical and Health Sciences Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-32859 (URN)10.1111/j.1742-4658.2008.06790.x (DOI)19076218 (PubMedID)
Available from: 2010-03-29 Created: 2010-03-29 Last updated: 2010-04-07Bibliographically approved
3. Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line
Open this publication in new window or tab >>Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line
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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.

Keyword
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:nbn:se:umu:diva-3478 (URN)10.1007/s10495-011-0606-0 (DOI)
Available from: 2008-09-22 Created: 2008-09-22 Last updated: 2012-02-17Bibliographically approved
4. Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells
Open this publication in new window or tab >>Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells
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2008 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 23, no 5, 541-549 p.Article in journal (Refereed) Published
Abstract [en]

Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.

Keyword
iodine-131, mitotic catastrophe, apoptosis, caspases, centrosomes, radioimmunotherapy
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-21104 (URN)10.1089/cbr.2007.0471 (DOI)18986216 (PubMedID)
Available from: 2009-04-06 Created: 2009-04-02 Last updated: 2013-09-13Bibliographically approved

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