Regulation of apoptosis during treatment and resistance development in tumour cells

Abstract: 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.