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dc.contributor.advisorSamali, Afshin
dc.contributor.authorDeegan, Shane
dc.description.abstractAutophagy is tightly regulated by the unfolded protein response (UPR). It plays an important role in the removal of unfolded proteins, damaged mitochondria and expanded endoplasmic reticulum (ER), to help relieve ER stress and reinstate homeostasis. However, when persistent, ER stress responses can switch the cytoprotective functions of UPR and autophagy into cell death promoting mechanisms. Depending on the cellular context, autophagy can either serve as a cell survival pathway, suppressing apoptosis, or it can lead to death itself. Although most cells primarily use apoptosis as a mode of cell death it is often not an option in many types of cancers and thus it is important to explore how the cell regulates other forms of cell death. The objective of this project was to determine the mode of ER stress-induced cell death in cells where the mitochondrial apoptotic pathway is compromised, and investigate how autophagy influences cell death in these conditions. For this we used caspase-9-/- mouse embryonic fibroblasts (MEFs), Bax/Bak-/- MEFs and Bax-/- HCT116 colon carcinoma cell line. Here we show that ER stress induces two phases of cell death, the first of which is rapid, caspase-9-dependent apoptosis and the second is a slow caspase-9-independent cell death. Further we show that the caspase-9-independent cell death is executed through a caspase-8 mediated activation of the executioner caspases. Inhibition of caspase-8 further delayed cell death however did not completely rescue the cells. Combination of caspase-8 knockdown and addition of the RIP1 kinase inhibitor, necrostatin-1, showed almost a complete rescue from cell death, further these cells were able to proliferate following removal of the drug. Interestingly knockdown of ATG5 in caspase-9-/- MEFs inhibited the activation of the executioner caspases and reduced the levels of cell death, implicating ATG5 as an essential component for caspase-8 activation in this model. To this end we carried out immunoprecipitation of endogenous ATG5 and showed its interaction with caspase-8, RIP1 kinase and FADD. For the first time we have identified a RIP1-containing death inducing protein complex assembled in response ER stress which executes cell death in conditions where the intrinsic pathway is compromised; furthermore we show that the formation of this complex is dependent on the autophagy protein, ATG5. In addition to this, we studied the interplay between the UPR and autophagy during ER stress. We carried out a microarray analysis in HCT116 cells and identified the transcriptional upregulation of an array of autophagy related genes. These genes included the autophagy receptors NIX, NBR1 and p62 and were confirmed by real-time RT-PCR. We further demonstrated that these genes were transcriptionaly upregulated by the PERK arm of the UPR, instigating ER stress-induced autophagy as a selective process. The data presented in this thesis show that in conditions where the intrinsic mitochondrial-mediated apoptotic pathway is compromised, exposure of cells to multiple stress stimuli will execute death via alternative means. We show that this caspase-9 independent cell death requires RIP1 kinase, FADD, ATG5 and caspase-8. Further we show that depending on the cellular context, cell death can be executed through apoptosis or necroptosis. Inhibiting components of the apoptotic pathway or the necroptotic pathway independently does not completely inhibit cell death; however, coperativly inhibiting these two processes results in a significant resistance from cell death. In addition to these findings, a microarray analysis showed that the autophagy process in reponse to ER stress is highly regulated by the UPR. We focused our study on the autophagy receptor proteins and showed that these proteins are transcriptionally upregulated in response to ER stress. Further we show the PERK arm of the UPR is responsible for their transcriptional upregulation.en_US
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 Ireland
dc.subjectER stressen_US
dc.subjectCell deathen_US
dc.titleThe molecular characterization of ER stress-induced autophagy and cell deathen_US
dc.contributor.funderScience Foundation Irelanden_US
dc.local.noteIn this thesis we identified a novel complex assembled under stress conditions that can sensitize cancer cells to death.en_US

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