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McComb, Scott. "The Paradoxical Roles of Cell Death Pathways in Immune Cells." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24331.
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Cell death plays a vital role throughout the immune response, from the onset of inflammation to the elimination of primed T cells. Understanding the regulation of cell death within immune cells is of vital importance to understanding the immune system and developing therapies against various immune-disorders. In this thesis I have investigated the regulation of cell death and its functional role in of the innate and adaptive arms of the immune system.
The mechanisms that govern expansion and contraction of antigen stimulated CD8+ T cells are not well understood. In the first section of this thesis, I show that caspase-3 becomes activated in proliferating CD8+ proliferation, yet this does not result in cell death. I used both in vivo and in vitro models to demonstrate that caspase-3 activation is specifically driven by antigen presentation and not inflammation, and that it likely plays a role in promoting T cell proliferation.
Next, I present novel data regarding the regulation of a newly identified form of programmed cell death via necrosis, known as necroptosis. I show that the cellular inhibitor of apoptosis (cIAP) proteins act to limit activation of key necroptosis proteins in macrophage cells. Furthermore, I show that necroptosis can be exploited by intracellular bacterial pathogens to escape removal by the immune system. I also demonstrate that necroptosis is highly intertwined with the pathway of inflammation, and the autocrine production of type-I interferon constitutes a vital positive feedback loop in the induction of inflammatory cell death. In the final section of my thesis work, I delve into
the specific regulation of Rip1 kinase and demonstrate that in addition to previously demonstrated regulation by caspase-8, cathepsins are also able to cleave Rip1 kinase and limit necroptosis.
This thesis presents a wide variety of novel data regarding the regulation of cell death within immune cells. In total, the results reveal a picture of two divergent forms of programmed cell death, apoptosis and necroptosis. Through improving the understanding of the cross-regulation of these two key cell death pathways this work aims to improve the understanding of the immune function.
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Yung, Hong Wa. "Regulation of astrocyte cell death by kinase signalling pathways." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620576.
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Mässenhausen, Anne von, Wulf Tonnus, and Andreas Linkermann. "Cell Death Pathways Drive Necroinflammation during Acute Kidney Injury." Karger, 2018. https://tud.qucosa.de/id/qucosa%3A71624.
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Renal tubules represent an intercellular unit and function as a syncytium. When acute tubular necrosis was first visualized to occur through a process of synchronized regulated necrosis (SRN) in handpicked primary renal tubules, it became obvious that SRN actually promotes nephron loss. This realization adds to our current understanding of acute kidney injury (AKI)-chronic kidney disease (CKD) transition and argues for the prevention of AKI episodes to prevent CKD progression. Because SRN is triggered by necroptosis and executed by ferroptosis, 2 recently identified signaling pathways of regulated necrosis, a combination therapy employing necrostatins and ferrostatins may be beneficial for protection against nephron loss. Clinical trials in AKI and during the process of kidney transplantation are now required to prevent SRN. Additionally, necrotic cell death drives autoimmunity and necroinflammation and therefore represents a therapeutic target even for the prevention of antibody-mediated rejection of allografts years after the transplantation process.
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Guo, Jing. "Studying the signaling pathways in ROS-induced neuronal cell death /." View abstract or full-text, 2005. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202005%20GUO.
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Benford, Helena L. "Molecular pathways of bisphosphonate-induced apoptosis." Thesis, University of Aberdeen, 2000. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602025.
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Recent studies have proposed that non-nitrogen-containing and nitrogen- containing bisphosphonate drugs inhibit osteoclastic bone resorption by different molecular mechanisms. The aim of this thesis was to investigate the molecular mechanisms of action of bisphosphonates in macrophages and osteoclasts and, in particular, the activation of caspase proteases and their role in apoptotic cell death. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates was found to involve the activation of caspase-3. By contrast, non-nitrogen- containing bisphosphonates did not cause caspase activation or J774 apoptosis, indicating that these bisphosphonates have different cellular effects. Further studies demonstrated that nitrogen-containing bisphosphonates induced J774 macrophage apoptosis by inhibiting the mevalonate pathway and preventing protein farnesylation and/or geranylgeranylation, since these compounds inhibited incorporation of [14 C] mevalonate into isoprenylated proteins, and addition of cell-permeable intermediates of the mevalonate pathway (FPP and GGPP) prevented bisphosphonate-induced apoptosis. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates or mevastatin (another inhibitor of the mevalonate pathway) was dependent on protein synthesis, since cycloheximide effectively prevented the activation of caspase-3 and prevented J774 cell apoptosis. Both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates caused caspase-3 activation and apoptosis of rabbit and human osteoclasts in vitro. The active form of caspase-3 was detected in apoptotic osteoclasts by immunofluorescence staining, whilst caspase-3 activity was visualised in osteoclasts using a cell-permeable, fluorogenic substrate and detected in cell lysates using caspase-specific substrates. Bisphosphonate-induced osteoclast apoptosis involved loss of mitochondrial membrane potential and could be prevented by a specific inhibitor of caspase-3/-7. The ability of bisphosphonates to activate caspase-3 and cause apoptosis was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, suggesting that caspase activation and apoptosis in osteoclasts induced by bisphosphonates is the consequence of loss of geranylgeranylated proteins. Bisphosphonate-induced osteoclast apoptosis and inhibition of bone resorption in vitro was suppressed by RANK ligand. This did notappear to involve changes in Akt phosphorylation or increased expression of cIAP-1 or cIAP-2. These studies have helped to identify the molecular mechanisms of action of bisphosphonate drugs and have provided new insights into the involvement of caspases in osteoclast apoptosis.
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Zhang, Tongli. "Mathematical Models of Some Signaling Pathways Regulating Cell Survival and Death." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/29443.
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In a multi-cellular organism, cells constantly receive signals on their internal condition and surrounding environment. In response to various signals, cells proliferate, move around or even undergo suicide. The signal-response is controlled by complex molecular machinery, understanding of which is an important goal of basic molecular biological research. Such understanding is also valuable for clinical application, since lethal diseases like cancer show maladaptive responses to growth-regulating signals. Because the multiple feedbacks in the molecular regulatory machinery obscure cause-effect relations, it is hard to understand these control systems by intuition alone. Here we translate the molecular interactions into differential equations and recapture the cellular physiological properties with the help of numerical simulations and non-linear dynamical tools. The models address the physiological features of programmed cell death, the cell fate decision by p53 and the dynamics of the NF-?B control system. These models identify key molecular interactions responsible for the observed physiological properties, and they generate experimentally testable predictions to validate the assumptions made in the models.<br>Ph. D.
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Yatim, Nader. "Coordinated activation of cell death and inflammatory pathways in dying cells regulate adaptive immunity." Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC233.
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Les Cellules mourantes initient l'immunité adaptative en fournissant des stimuli inflammatoires et des antigènes pour les cellules dendritiques (CD), qui à leurs tour activent les cellules T CD8 + par un processus appelé la présentation-croisée. Pour définir comment les différentes formes programmées de la mort cellulaire influencent l'immunité, nous avions établi des modèles de nécroptose et apoptose, par l'activation spécifique de RIPK3 ou CASP8, que nous avons utilisé pour évaluer in vitro et in vivo la réponse immunitaire. Nous avons alors trouvé que les cellules nécroptotiques exprimant l'antigène ovalbumin (OVA) induisent une forte réponse T CD8+ anti-OVA. Elles étaient plus immunogènes que les cellules apoptotiques ou nécrotiques. Étonnamment, l'activation simultanée de RIPK1 lors de la nécroptose etait responsable de l'immunogencité. En effet, l'abolition du recrutement de RIPK1 aux oligomères RIPK3 diminue le cross-priming et l'immunité anti-tumorale, en dépit d'un relargage équivalent de DAMPs (ATP et HMGB1), d'activation similaires des CDs et de l'inflammation in vivo. Nous avons aussi démontré que RIPK1 active la voie NF-kB et l'expression d'un programme transcriptionnel inflammatoire au sein des cellules nécroptotiques, nécessaire au cross-priming. De même, l'axe RIPK1-NF-kB était requis dans un modèle d'apoptose immunogenique. Nos résultats montrent que RIPK1, par sa capacité à coordonner la mort cellulaire et l'inflammation, orchestre la réponse T CD8 + et fournissent de nouveaux éclairages sur les interconnexions complexes entre la mort cellulaire, l'immunité innée et l'immunité adaptative<br>Dying cells initiate adaptive immunity by providing both antigens and inflammatory stimuli for dendritic cells (DCs), which in turn activate CD8' T cells through a process called antigen cross-priming. To define how different forms of programmed cell death influence immunity, we established models of necroptosis and apoptosis, where dying cells are generated by RIPK3 and CASP8 dimerization, respectively. We found that release of inflammatory mediators such as damage-associated molecular patterns (DAMPs) by dying cells was not sufficient for CD8+ T cell cross-priming. Instead, robust cross-priming required RIPK1 signaling and NF-KB-induced transcription within dying cells. Decoupling NF-1(13 signaling from necroptosis or inflammatory apoptosis reduced priming efficiency and tumor immunity. Our results reveal that coordinated inflammatory and cell death signaling pathways within dying cells orchestrate adaptive immunity
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Janson, Veronica. "Cisplatin-resistance and cell death in malignant pleural mesothelioma cells." Doctoral thesis, Umeå universitet, Medicinsk biovetenskap, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1680.
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Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant tumour. Cisplatin (cis-diamminedichloroplatinum (II)) is the best single-agent chemotherapy for MPM, but platinum-based combination therapies give the best overall response rates. However, cisplatin use is limited by resistance and severe side effects. This thesis has increased the knowledge concerning cisplatin-induced cell death in MPM by describing a novel potential therapeutic target, and three novel phenotypes of cisplatin-resistance in a human MPM cell line (P31) and its cisplatin-resistant sub-line (P31res1.2). The novel potential therapeutic target, and one of the novel phenotypes, was cisplatin-resistant pro-apoptotic BH3-only proteins. In the P31 cells, cisplatin transiently increased pro-apoptotic BH3-only proteins during 6 h of exposure. This response was almost completely abrogated in the P31res1.2 cells. De-regulated caspase activity and activation was the second novel phenotype identified. The P31res1.2 cells had earlier, possibly mitochondria-independent, caspase-3 activation, increased basal caspase-3 activity and increased basal cleavage of caspase-8 and -9. Despite these differences, 6-h equitoxic cisplatin exposures rendered 50-60% of the cells apoptotic in both cell lines. The third novel phenotype was abrogated Na+K+2Cl--cotransporter (NKCC1) activity. Although NKCC1 activity was dispensable for cisplatin-induced apoptosis, balanced potassium transport activity was essential for P31 cell survival. Finally, the survival signalling protein Protein Kinase B (PKB or Akt) isoforms α and γ were constitutively activated in a PI3K-independent manner in P31 cells. In the P31res1.2 cells, PKBα and γ activities were increased, and there was PI3K-dependent activation of PKBβ. However, this increase in PKB isoform activity was not strongly associated to the cisplatin-resistance of the P31res1.2 cells.
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Davies, Carwyn Children's Cancer Institute Australia for Medical Research UNSW. "The influence of p21WAF1 on cell death pathways in acute lymphoblastic leukaemia." Publisher:University of New South Wales. Children's Cancer Institute Australia for Medical Research, 2009. http://handle.unsw.edu.au/1959.4/44416.
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The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.
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Zhang, Tejia. "Discovery of bioactive lipids and lipid pathways in cell death and disease." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11483.
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Apoptosis is an intricately regulated cellular process required for the health and homeostasis of living systems. The mitochondrial apoptotic pathway depends on the BCL-2 family of pro- and anti-apoptotic members whose interactions regulate cell fate. BAX and BAK are key pro-apoptotic proteins required for mitochondrial permeabilization during apoptosis. While the mitochondrial death program relies heavily on its protein components, evidences support equally crucial roles for lipids and lipid metabolism in promoting or hindering apoptosis at the mitochondria. To gain insight into the interplay between lipids and BCL-2 proteins we used a liquid chromatography (LC)-mass spectrometry (MS)-based comparative lipidomics approach to uncover lipid changes in the absence of BAX and/or BAK. Our analysis revealed novel functions for BAX and BAK in inflammation and ceramide metabolism. A targeted LC-MS workflow was also developed for characterization of a novel lipid class involved in type 2 diabetes. Targeted LC-MS revealed altered oxysterol metabolism following perturbation of the Sonic hedgehog pathway. Taken together, our findings demonstrate interesting connections among lipids, cell death and disease.<br>Chemistry and Chemical Biology
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Chakraborty, Sayantan [Verfasser], and Barbara [Akademischer Betreuer] Conradt. "Novel roles of the central cell death pathway and cell corpse engulfment pathways in C. elegans / Sayantan Chakraborty. Betreuer: Barbara Conradt." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1100396004/34.
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Thakker, Parva. "T Cell Intrinsic and Extrinsic Role of XIAP, During CD8 T Cell Response Against Intracellular Pathogens." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42419.
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The magnitude and effectiveness of CD8 response against intracellular pathogens is
directed by survival and apoptotic signals that govern the fate of T cells. XIAP is a bona fide endogenous inhibitor of apoptotic signals. In this thesis, I have investigated the role of XIAP at various stages of CD8 T cell response. I used both in vivo and in vitro models to show that XIAP acts in a CD8 T cell extrinsic and intrinsic manner to regulate the expansion and contraction phases of the CD8 T cell response, respectively. During the expansion phase, XIAP prevents the cell death of APCs to promote APC-T cell interaction and cytokine release, which facilitates the proliferation and survival of activated T cells. During the contraction phase, XIAP functions in a cell-intrinsic fashion to inhibit the proapoptotic signals in the activated CD8 T cells to prolong the immune response. Finally, I also demonstrate that the expression of XIAP in T cells is critical for their differentiation in to memory subsets. Overall, I present that XIAP plays a critical role in generating an effective CD8 T cell immune response.
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Haseroth, Elke [Verfasser]. "Cell death pathways in the photodynamic therapy of hepatocellular carcinoma cells with tetrasulfonated aluminum phthalocyanine / Elke Haseroth." Ulm : Universität Ulm. Medizinische Fakultät, 2016. http://d-nb.info/1081456663/34.
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Popescu, Bogdan O. "Cell death and signal transduction pathways in Alzheimer's disease: the role of presenilin 1 /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-786-X/.
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Alotaibi, Moureq. "CELL DEATH AND GROWTH ARREST PATHWAYS MEDIATING THE ACTIONS OF STILBENE 5C IN HCT-116 COLON CANCER CELLS." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2851.
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Abstract The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells. The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. However, our studies indicated that stilbene 5c works in a p53-independent manner. Exposure of P53-null HCT116 cells to stilbene resulted in a similar sensitivity as in p53-wild type HCT116 cells. We found that autophagic vacuoles were formed in response to stilbene 5c in p53-null HCT116 cells as well. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death.
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Senaratne, Siddhika Gaurie. "Action and interaction of various drugs with signal transduction pathways, leading to cancer cell death." Thesis, St George's, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398083.
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Heywood, Darren J. "Investigating the involvement of the JNK and PKC signalling pathways in mediating neuronal cell death." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288333.
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Glover, Colin P. J. "Optimization of gene transfer systems to facilitate studies investigating transcriptional pathways mediating neuronal cell death." Thesis, University of Bristol, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274633.
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Venables, Luanne. "In vitro induction of cell death pathways by artemisia afra extract and isolation of an active compound, isoalantolactone." Thesis, Nelson Mandela Metropolitan University, 2014. http://hdl.handle.net/10948/d1021087.
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Artemisia afra is one of the oldest, most well known and widely used traditional medicinal plants in South Africa. It is used to treat many different medical conditions, particularly respiratory and inflammatory ailments. There is no reported evidence of its use for the treatment of cancer but due to its reported cytotoxicity, an investigation of the mode of cell death induced by an ethanol A. afra extract using two cancer cell lines was done. IC50 values of 18.21 and 31.88 μg/mL of ethanol extracts were determined against U937 and HeLa cancer cells, respectively. An IC50 value of the aqueous extract was greater than 250 μg/mL. The ethanol extract was not cytotoxic against confluent control cell lines, Chang Liver and Vero cells. The effect of the cytotoxic ethanol A. afra extract on U937 and HeLa cells and their progression through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. After 12 hours of treatment with A. afra a delay in G2/M phase of the cell cycle was evident. Apoptosis was confirmed using the TUNEL assay for DNA fragmentation, as well as fluorescent staining with annexin V-FITC. Apoptosis was evident with the positive control and A. afra treatment at 24 and 48 hours. JC-1 staining showed a decrease in mitochondrial membrane potential at 24 hours. It was deduced that A. afra ethanol extract induces caspase-dependent apoptosis in a mitochondrial dependent manner. Plants harbour many compounds that are not only useful to the plants but also to mankind. Many metabolites have been isolated from A. afra and their biological activity characterised. Due to observed apoptosis induction, isolation of cytotoxic compounds was done and a new sesquiterpene lactone from A. afra was isolated. Structural elucidation of the compound was done by IR, 1D and 2D NMR, CD and mass spectrometry and it was identified as isoalantolactone. HeLa cancer cells were treated with isoalantolactone and cytotoxicity was exhibited in a dose-dependent manner. A low IC50 value of 8.15 ± 1.16 μM was achieved. This study showed that isoalantolactone is partly responsible for the observed A. afra cytotoxicity. Due to the evidence of G2/M arrest, the anti-mitotic potential and the possible onset of mitotic catastrophe by A. afra and isoalantolactone was investigated. It was evident from various flow cytometric analysis of cyclin B1 and phospho-H3 and confocal microscopy that A. afra does possess anti-mitotic activity by causing hyperpolymerisation of tubulin and cells progress into the mitotic phase where M arrest is experienced. The anti-inflammatory activity of sesquiterpene lactones is well documented; however, the anti-inflammatory activity of A. afra is not. Here, it is reported that the production of NO and COX-2 protein levels in RAW 264.7 cells decrease in the presence of A. afra and isoalantolactone after stimulation with LPS. The activated NF-κB subunit, p65 was also investigated. The results suggest that A. afra and isoalantolactone inhibit p65 activation as a decrease in the activated subunit was evident. Thus, the results indicate that exposure to A. afra and isoalantolactone induces an anti-inflammatory response. In conclusion, this study shows, for the first time, the mechanism of induced apoptosis, the anti-mitotic and anti-inflammatory activity of A. afra and its isolated compound, isoalantolactone. It also proves that although extensive research may have been done on a particular plant, as with A. afra, more can be discovered leading to the identification of new compounds and integration of signalling pathways that can be exploited for the treatment of various diseases and ailments.
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Sathasivam, Sivakumar. "Investigation of cell death pathways in a cell culture model of Cu/Zn superoxide dismutase-related familial motor neurone disease." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412435.
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Maya-Pineda, Héctor Rubén. "Sensitization of prostate cancer cells to cytotoxic drugs induced by the small adenoviral E1A12S protein through multiple cell death/signalling pathways." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8482.
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Replication-selective oncolytic adenoviruses represent a promising anticancer approach with proven efficacy in cancer cell lines and tumour xenografts in vivo. Anti-tumour efficacy, both in preclinical studies and clinical trials, was significantly improved in combination with chemotherapeutics in numerous cancers, including prostate cancer. It has been established that expression of the viral E1A gene is essential for the enhancement of cell killing in combination with cytotoxic drugs. The overall goal of this project is to identify specific E1A gene regions involved in the sensitization to the cytotoxic drugs mitoxantrone and docetaxel, the current standard of care for late stage prostate cancers, to enable the development of improved anti-cancer therapies. Specific regions in the E1A proteins bind to numerous cellular factors to regulate the host cell function and the viral life cycle, including the p300, p400 and pRb family proteins. This work was aimed at determining the mechanisms involved in the synergistic cell killing in prostate cancer cells in response to the combination of the replication-selective (oncolytic) mutant AdΔΔ with cytotoxic drugs. Previous findings suggested an enhancement of drug-induced apoptosis. I found that the small E1A12S protein, unable to induce viral replication, is sufficient to sensitize the prostate cancer cells, 22Rv-1 (AR+), and PC-3 and DU145 (AR-), to drugs. The non-replicating AdE1A12S-mutant AdE1A1104 (defective in p300-binding) could not sensitize the cells while mutants with intact E1A-p300 binding (AdE1A12S, AdE1A1102, AdE1A1108) and defective in p400- (AdE1A1102) or pRb-binding (AdE1A1108) potently sensitized all tested cell lines. In fact, all mutants except AdE1A1104 potently synergised with mitoxantrone and docetaxel to kill the prostate cancer cells. When comparing the non-replicating E1A12S mutants with the corresponding replicating E1A-deletion mutants (expressing E1A12S and 13S) synergy was demonstrated with all replicating mutants except dl1104, which caused an additive effect with mitoxantrone. We hypothesised that the synergistic cell killing is the result of pathway convergence through E1A-p300 and mitoxantrone-activated DNA-damage/apoptosis events. To address this I employed an extensive miRNA array screen to identify potential pathways. Several miRNAs were found to be differentially regulated in response to the combination of AdE1A12S with mitoxantrone compared to each single agent treatment. The majority of these miRNAs are reported to be part of cell death and survival pathways (e.g. apoptosis and autophagy) and to be differentially regulated in prostate cancer. To further investigate the role of these pathways, I determined changes in expression levels of key proteins that had previously been suggested to be targeted by the identified miRNAs, thereby preventing translation of the respective mRNAs. The greatest changes in protein levels in response to AdE1A12S and mitoxantrone were observed for Bcl-2, p-Akt, LC3BII and p62. Finally, I verified similar mechanisms of action when the oncolytic AdΔΔ was combined with mitoxantrone under synergistic conditions. These findings will direct future investigations aimed at dissecting the mechanisms of action for virus-induced sensitization to cytotoxic drugs and may aid in the development of improved therapies for prostate cancer by design of novel oncolytic mutants and combination strategies and/or identification of targets for small molecules inhibitors.
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Zimmermann, Angela K. "A novel mechanism underlying BCL-2 antioxidant function : its role in mitochondrial apoptotic pathways and virus-induced neuronal cell death /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007.<br>Typescript. Includes bibliographical references (leaves 140-162). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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DeMasters, Gerald Alan. "Influence of the Vitamin D3 Analog EB 1089 on Senescence and Cell Death Pathways in the Response of Breast Tumor Cells to Ionizing Radiation." Abstract, 24-page preview and downloadable full-text (PDF format) available to VCU users at:, 2006. http://hdl.handle.net/10156/1620.
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Lema, Asqui Saúl Stalin. "Pathogen-triggered programmed cell death in plants: Metacaspase 1-dependent pathways = Muerte celular programada desencadenada por patógenos en plantas: vías dependientes de Metacaspasa 1." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586256.
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The hypersensitive response (HR) is a paradigmatic type of programmed cell death, that occurs in response to pathogen recognition at the site of attempted invasion. Notwithstanding more than a century of research on HR, many are the aspect that are still unknown about how it is so tightly regulated and how it can be contained spatially to a few cells. The hypersensitive response in the Arabidopsis thaliana is controlled by type I metacaspase AtMC1 which is a positive regulator of pathogen-triggered PCD and autophagy, and that negative regulation of AtMC1 by AtLSD1 or AtMC2 can prevent runaway cell death. However, it remains unclear how the cell death signaling is activated after pathogen attack and whether additional HR negative regulators exist to control the AtMC1 activity.
In our lab was set upping an unbiased approach to identify new AtMC1 regulators based in an immunoaffinity purification of AtMC1-containing complexes coupled to mass spectrometry. The use of this approach has allowed us to identify new regulators of AtMC1 activity, in basal versus cell death inducing conditions.
In the context of the second objective we were able to revealed that in basal conditions AtSerpin1 acts in vivo as an inhibitor of AtMC1-mediated cell death and autocatalytic processing in plants, emerging as a new inhibitor of cell death proteases in plants. Indicating a conserved function of a protease inhibitor on cell death regulators across different kingdoms.
The third part of this work continued with the characterization of AtHIR2 as positive regulator under cell death inducing conditions. We set out to characterize AtHIR2, a putative AtMC1 interactor retrieved from our immunoaffinity purification screening. Our results show that AtMC1 co-immunoprecipitates in planta with AtHIR2, and this interaction is not dependent of an intact catalytic site. Subcellular fractionation demonstrates that this interaction exclusively occurs in the microsomal fraction, indicating an active recruitment of AtMC1 to the plasma membrane.
Taken together all these results, we expected to contribute into elucidation of the regulation of Hypersensitive Response and the complex machinery that allows to cells make fate vital decisions to defense against pathogens. Our results will contribute on future approaches to developed new strategies in the fight against plant pathogens diseases in crops.<br>La respuesta hipersensible (RH) es un tipo paradigmático de muerte celular programada, que ocurre en respuesta al reconocimiento de patógenos en el sitio del intento de invasión. A pesar de más de un siglo de investigación sobre RH, muchos son los aspectos que aún se desconocen acerca de cómo está tan fuertemente regulada y cómo puede ser contenida espacialmente a solo unas pocas células. La respuesta hipersensible en Arabidopsis thaliana está controlada por la metacaspasa tipo I (AtMC1), que es un regulador positivo de la muerte celular programada desencadenada por patógenos y la autofagia, donde la regulación negativa de AtMC1 por AtLSD1 o AtMC2 puede prevenir la muerte celular incontrolada. Sin embargo, aún no está claro cómo se activa la señalización de muerte celular después de un ataque de patógenos y si existen reguladores negativos de RH adicionales para controlar la actividad del AtMC1.
En nuestro laboratorio se estableció un enfoque imparcial para identificar nuevos reguladores AtMC1 basados en una purificación de inmunoafinidad de complejos que contienen AtMC1 acoplados a la espectrometría de masas. El uso de este enfoque nos ha permitido identificar nuevos reguladores de la actividad de AtMC1, en condiciones basales versus condiciones de inducción de muerte celular.
En el contexto del segundo objetivo pudimos revelar que en condiciones basales AtSerpin1 actúa in vivo como un inhibidor de la muerte celular mediada por AtMC1 y del procesamiento auto-catalítico en plantas, emergiendo como un nuevo inhibidor de proteasas de muerte celular en plantas. Indicando una función conservada de un inhibidor de proteasa en reguladores de muerte celular a través de diferentes reinos.
La tercera parte de este trabajo continuó con la caracterización de AtHIR2 como regulador positivo bajo condiciones inducidas de muerte celular. Nos propusimos caracterizar AtHIR2, un interactor putativo de AtMC1 recuperado de nuestro análisis de purificación de inmunoafinidad. Nuestros resultados muestran que AtMC1 co-inmunoprecipita en planta con AtHIR2, y esta interacción no depende de un sitio catalítico intacto. El fraccionamiento sub celular demuestra que esta interacción ocurre exclusivamente en la fracción microsomal, lo que indica un reclutamiento activo de AtMC1 a la membrana plasmática.
Tomados en conjunto todos estos resultados, esperamos contribuir en la elucidación de la regulación de la Respuesta Hipersensible y la compleja maquinaria que permite a las células tomar decisiones vitales para la defensa contra los patógenos. Nuestros resultados contribuirán a futuros enfoques para desarrollar nuevas estrategias en la lucha contra las enfermedades de los patógenos de las plantas en los cultivos.
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Abrantes, Julia Laura Fernandes 1984. "Expressão ectópica de miR-34a em células de melanoma metastático humano = efeitos sobre vias de sinalização relacionadas com sobrevivência, proliferação e morte celular = Ectopic expression of miR-34a in human metastatic melanoma cells: effects on signaling pathways related to survival, proliferation and cell death." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314040.
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Orientador: Carmen Veríssima Ferreira Halder<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-22T16:08:32Z (GMT). No. of bitstreams: 1
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Previous issue date: 2013<br>Resumo: O melanoma é o tipo mais agressivo de câncer de pele. Seu tratamento permanece como um grande desafio, já que em estágio avançado torna-se extremamente refratário aos tratamentos convencionais. miR-34a é um microRNA supressor de tumor com expressão normalmente reduzida em células cancerosas. A fim de investigar o papel de miR-34a como supressor do melanoma, o principal objetivo deste estudo foi identificar alvos moleculares modulados pela expressão ectópica de miR-34a na linhagem celular de melanoma metastático humano SK-mel-103. miR-34a reduziu significativamente a viabilidade das células de melanoma, o que deve estar relacionado, pelo menos em parte, com o aumento na expressão da proteína pró-apoptótica Bax, ativação da caspase-3 e clivagem da PARP-1. Estes dados sugerem que miR-34a foi capaz de induzir apoptose nas células de melanoma. Além disso, houve redução na expressão de CDK4, CDK6, E2F3 e pRb, proteínas relacionadas com a progressão do ciclo celular. Aumento na expressão de p21, um inibidor de CDKs, também foi observado nessas células. Algumas moléculaschave envolvidas com os processos de proliferação celular e apoptose, como proteínas oncogênicas (Axl, AKT, ERK 1/2, ?-catenina e c-myc) e proteínas supressoras de tumor (p53 e PTEN), foram "down- e upreguladas" por miR-34a, respectivamente. Interessantemente, o fluxo autofágico foi aumentado por miR-34a, efeito que não foi correlacionado com alterações adicionais na viabilidade das células de melanoma. O aumento no fluxo autofágico ocorreu, provavelmente, como uma resposta celular ao estresse de retículo e a agregação de proteínas induzidos por miR-34a, fenômenos que também podem ter contribuído para a indução de apoptose nesse contexto. Os dados obtidos neste estudo trouxeram novos aspectos moleculares da ação de miR-34a como supressor tumoral, e permitem apontar este microRNA como um potencial alvo terapêutico contra o melanoma metastático humano<br>Abstract: Melanoma is the most aggressive form of skin cancer. Its treatment remains a big challenge, since in advanced stage it is extremely refractory to conventional treatments. miR-34a has emerged as an important tumor suppressor, and its expression is normally reduced in cancer cells. To provide more information about the role of miR-34a as a melanoma suppressor, the main goal of this study was to identify key molecular players modulated by ectopic expression of this microRNA in the metastatic melanoma cell line SK-mel-103. miR-34a caused a reduction of melanoma cells viability, what may be related, at least in part, with the increased expression of pro-apoptotic marker, Bax, activation of caspase 3 and PARP-1 cleavage, which indicates that miR-34a triggered apoptosis in melanoma cells. In addition, the expression of CDK4, CDK6, E2F3 and pRb, proteins related to the cell cycle progression, was reduced. An increase in p21 expression, a CDK inhibitor, was also detected in these cells. Some key molecules involved with proliferation and apoptosis processes, such as oncogenic proteins (Axl, AKT, ERK 1/2 kinases, ?- catenin and c-myc) and tumor suppressor proteins (p53 and PTEN), were down- and upregulated by miR-34a, respectively. Interestingly, the autophagic flux was stimulated by miR-34a, but this effect was not correlated with further alterations in cell viability. The increased autophagy occurred probably as a cellular response against the reticulum stress and the protein aggregation induced by miR-34a in melanoma cells, which can also be contributing to the cell death by apoptosis in this context. Our findings brought up novel molecular aspects about the role of miR-34a as melanoma suppressor. The broad action of this microRNA on key molecular players of melanoma aggressiveness was crucial for reprogramming these cells in favor of apoptosis. Altogether, this study pointed out miR-34a as a potential therapeutic agent against advanced melanoma<br>Doutorado<br>Bioquimica<br>Doutora em Biologia Funcional e Molecular
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Palmer, Daniel Harrison. "A study of the novel VDEPT cancer gene therapy combination nitroreductase / CB1954 : mechanisms of cell death, modulation of cellular signalling pathways and early phase clinical trials." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403904.
Full textAbstract:
A major limitation to the success of cancer gene therapy is the low efficiency of gene delivery achieved by currently available vectors. Virus-directed enzyme prodrug therapy aims to overcome this limitation by delivery of an enzyme to the tumour site, which catalyses the conversion of an inert prodrug to a potent cytotoxic. Activation of the prodrug at the tumour site maximises local cell kill whilst minimising systemic toxicity. Importantly, the activated species should pass to neighbouring nontransduced cells to mediate 'bystander' killing. In this way, significant anti-tumour effects may be observed even when only a fraction of cancer cells express the enzyme. A novel VDEPT system utilises bacterial nitroreductase (NR) to convert the monofunctional alkylating agent CB 1954 to a highly potent bifunctional agent. This is the first clinical experience of this combination, using a replication defective adenovirus to deliver NR. We report safety and kinetic data for prodrug and vector; host immune responses to vector; and extent of transgene expression. To facilitate the further development of this approach, we have investigated the mechanism of cell death induced by activated CB 1954, its interaction with conventional chemotherapeutic agents, and the effects of adenovirus vectors on key cellular pathways regulating cell survival and inflammatory/immune responses. We have identified synergistic cell killing with NRiCB 1954 and 5-fluorouracil, providing a rational framework for future clinical trial design. Adenovirus vectors (replicationdefective and conditionally-replicating) activate several signalling pathways, including PI3-kinase/Akt and NF-KB, mediating anti-apoptotic and inflammatory responses and significantly affecting therapeutic efficacy. Manipulation of signalling events presents a potential therapeutic target for the enhancement of adenovirus-based cancer gene therapy strategies.
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Dallimore, Elizabeth Jane. "Molecular and cellular characteristics of early vs late born retinal ganglion cells." University of Western Australia. School of Anatomy and Human Biology, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0138.
Full textAbstract:
[Truncated abstract] Developmentally, the rodent retinocollicular projection is often thought of as a homogenous projection of retinal ganglion cell (RGC) axons, however the extensive period of RGC neurogenesis and sequential arrival of their axons into central targets such as the superior colliulus (SC) suggests otherwise. RGC axons are already present in the developing SC at embryonic (E) day 16.5-17. RGCs born on E15 have innervated the SC by birth, whereas axons derived from RGCs that are born last (E19) do not grow into the SC until postnatal (P) days 4-6 (Dallimore et al., 2002). These observations may go someway to explaining why, after SC lesions in rats at P2, there is greater growth distal to the lesion site compared to lesions made at P6 (Tan and Harvey, 1997b). It may be that the post lesion growth is simply de novo growth of axons from late-born RGCs rather than regeneration of pre-existing, injured axons. Early and late cohorts of growing RGC axons presumably encounter different developmental terrains as they grow from retina to central targets, possibly resulting in differences in developmental milestones and growth potentials. There may also be differences in guidance cues, further suggesting that gene expression in early vs late born RGCs may differ. To examine differences between early (E15) and late (E19) born RGCs during development, the time-course and extent of programmed RGC death in normal rat pups, and RGC death following the removal of target-derived trophic factors, was assessed. ... On the other hand, LCM captured GCL analysed for gene expression at P0 and P7 revealed decreases in AKT, Math5, Notch1, c-jun, DCC, Arginase-1 mRNA levels and a considerable decrease in GAP-43 expression. It is not surprising to see differences in gene expression between whole eye and the more specific GCL samples, as the cells in all layers of the retina have very different functions and different developmental profiles. It is important to note decreases in mRNA expression in the GCL for a number of the genes analysed at P0 and P7, reflecting cessation of RGC death and completion of axonal growth into central visual targets. I also examined at the protein level expression of DCC, Arginase1, c-Jun and Bcl-2 at birth (P0) in BrdU labeled RGCs born on E15 or E19. When comparing the percentage of double labelled cells compared to the total number of cells expressing each protein, Bcl-2, c-Jun and Arg1 were expressed more in E15 RGCs (22.90%, 72.71%, and 16.44% respectively in E15 RGCs, compared with 0.52%, 13.17% and 3.59% in E19 RGCs). In contrast, DCC was expressed more at birth in E19 RGCs (18.05% in E19 RGCs compared with 9.23% in E15 RGCs). This shows there is clearly a difference in the expression of proteins in the two cohorts of RGCs, which is consistent with PCR data and with their growth state as their axons encounter the changes in the newborn brain. The overall findings of this research suggest that seemingly homogenous populations of neurons are quite different in their developmental profile and in their response to injury. This work may provide new ways of determining better strategies for CNS repair and the most effective way of targeting cells for regeneration and survival.
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Akizuki, Mayumi. "Optineurin suppression causes neuronal cell death via NF-κB pathway". Kyoto University, 2014. http://hdl.handle.net/2433/188648.
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Abdo, Michael A. "Tumour necrosis factor : alpha signal transduction in rat corpus luteum apoptosis." University of Western Australia. School of Anatomy and Human Biology, 2002. http://theses.library.uwa.edu.au/adt-WU2003.0024.
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[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Apoptosis is a morphologically distinct form of cell death that is involved in the regulation of normal and aberrant cell systems. The complexities of the apoptotic cell death pathway arise from variation in both the cellular specialisation and initial stimulus. The corpus luteum (CL) is an endocrine gland that whilst critical to the maintenance of pregnancy in the rat, regresses at the completion of each oestrous cycle and pregnancy. This regression is facilitated through apoptosis; though, the stimulus and factors involved in the apoptotic pathway are poorly understood. Previous studies suggest that CL regression is not initiated through failure of luteotrophic support, but rather the active production of a luteolytic factor, of which tumour necrosis factor -alpha (TNFα) is one possible candidate. Several publications have reported the participation of the immune system in ovarian events. There is evidence that TNFα expression within the ovary is coordinated between cells of the immune system and the hormonal regulation of the CL. This study has focussed on the role of TNFα in CL apoptosis and the factors involved in this apoptotic pathway. TNFα-induced cell death is governed by the presence of the two TNFα receptors (TNFR) and several second messenger systems that include; the sphingolipids, mitogen-activated protein (MAP) kinases, nitric oxide (NO), nuclear factor-kappaB (NF-κB) and the caspases. These factors and their interactions were assessed in the rat CL during pregnancy and post-partum, and in vitro. Apoptosis was measured through the analysis of DNA fragmentation using DNA 3’ end labelling and single cell electrophoresis (COMET assay). Assessment of mRNA and protein expression was through Real-time RT-PCR and Western blot analysis; proteins were localised within the CL by immunocytochemistry. In addition, specific measurement of sphingolipid expression and nitric oxide (NO) production was by high performance liquid chromatography (HPLC) and NO assay respectively. Following parturition, TNFα mRNA and protein expression increased corresponding to the onset of CL apoptosis and increased expression of the chemotactic factor monocyte chemoattractant protein -1 (MCP-1). Furthermore, CL apoptosis was induced by treatment with recombinant TNFα in a time- and dose-dependent manner. A similar effect was observed in isolated luteal cells. Simultaneously, the functional regression of the CL was assessed by measurement of both progesterone synthesis and steroidogenic acute regulatory (StAR) protein expression. StAR mRNA and protein expression declined toward parturition in vivo. Immunocytochemical studies revealed the presence of TNFα receptors 1 (TNFR1) and 2 (TNFR2) in luteal cells. Furthermore, TNFR mRNA was isolated from CL throughout pregnancy and post-partum. Subsequently, the role of the sphingolipids ceramide and sphingosine was examined during CL apoptosis in vitro. Ceramide and sphingosine were found to be potent apoptotic agents when administered in vitro (50µM). The downstream signal transduction of TNFα and ceramide was assessed through MAP kinase expression. Both TNFα and ceramide increased expression of the pro-apoptotic p38 MAP kinase with no change to the non-apoptotic extracellular signal-related kinase (ERK1&2). Despite previous reports of c-Jun NH2 terminal kinase (JNK) involvement in the cell death pathway, JNK expression was not evident in the rat CL. The caspases are a family of cysteine proteases central to the regulation and execution of apoptosis. General inhibition of the caspase cascade in vitro was effective in preventing apoptosis regardless of the apoptotic stimulus (TNFα, ceramide and sphingosine), suggesting that this pathway is central to CL apoptosis. Specific inhibition of several caspases produced a varying effect; inhibition of caspases 3, 6 and 8 significantly reduced the level of TNFα-induced apoptosis, thus supporting their classification as either regulatory or effector caspases. NO is endowed with the unique ability to initiate and to block apoptosis and this dichotomy extends to the cytotoxic actions of TNFα. Inhibition of NO production by treating CL with L-NAME prevented the onset of apoptosis, whilst NO production increased in response to increasing levels of apoptosis following trophic withdrawal. However, this effect was not seen during TNFα-induced apoptosis, suggesting that the actions of NO are independent of TNFα. The data presented within this study examine multiple elements of the TNFα cell death pathway in a single system. The results suggest that these elements are involved in TNFα signal transduction and furthermore, in rat CL apoptosis. It can be said that TNFα plays an active role in CL regression through the activation of the caspases, the sphingolipids and the MAP kinases.
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Rajamani, Uthra. "Hyperglycemia-induced activation of the hexosamine biosynthetic pathway causes myocardial cell death." Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/1142.
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Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2009.<br>ENGLISH ABSTRACT: OBJECTIVE – Oxidative stress increases flux through the hexosamine biosynthetic pathway
(HBP) resulting in greater O-GlcNAcylation of target proteins. Since increased oxidative stress
and HBP flux are associated with insulin resistance, we hypothesized that its activation leads to
greater O-GlcNAcylation of BAD (pro-apoptotic) and increased myocardial apoptosis.
RESEARCH DESIGN AND METHODS – To investigate our hypothesis, we employed two
experimental models: 1) H9c2 cardiomyoblasts exposed to high glucose (33 mM glucose) ± HBP
modulators ± antioxidant treatment vs. matched controls (5.5 mM glucose); and 2) a rat model of
high fat diet-induced insulin resistance and hyperglycemia. We evaluated apoptosis in vitro by
Hoechst nuclear staining, Annexin-V staining, caspase activity measurements and
immunoblotting while in vivo apoptosis was assessed by immunoblotting. In vitro reactive
oxygen species (ROS) levels were quantified by H2DCFDA staining (fluorescence microscopy,
flow cytometry). We determined overall and BAD O-GlcNAcylation, both by immunoblotting
and immunofluorescence microscopy. As BAD-Bcl-2 dimer formation enhances apoptosis, we
performed immunoprecipitation analysis and immunofluorescence microscopy (co-localization)
to determine BAD-cl-2 dimerization. In vivo overall O-GlcNAcylation, BAD O-GlcNAcylation
and BAD-Bcl-2 dimerization was determined by immunoprecipitation and immunoblotting.
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RESULTS – High glucose treatment of cells significantly increased the degree of apoptosis as
revealed by Hoechst nuclear staining (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V staining (43 ±
5%), caspase activity assay (26 ± 2%) and immunoblotting. In parallel, overall OGlcNAcylation
(p<0.001 vs. 5.5 mM), BAD O-GlcNAcylation (p<0.05 vs. 5.5 mM) and ROS
levels were increased (fluorescence microscopy – p<0.05 vs. 5.5 mM; flow cytometry – p<0.001
vs. 5.5 mM). HBP inhibition using DON and antioxidant treatment (α-OHCA) attenuated these
effects while HBP activation by PUGNAc exacerbated it. Likewise, insulin resistant rat hearts
exhibited significantly higher caspase-3 (p<0.05 vs. controls), overall O-GlcNAcylation (p<0.05
vs. controls) and BAD O-GlcNAcylation levels (p<0.05 vs. 5.5 mM). BAD-Bcl-2 dimer
formation was increased in cells exposed to hyperglycemia [immunoprecipitation analysis and
co-localization] and in insulin resistant hearts.
CONCLUSIONS - Our study identified a novel pathway whereby hyperglycemia results in
greater oxidative stress, resulting in increased HBP activation and increased BAD OGlcNAcylation.
We also found greater BAD-Bcl-2 dimerization increasing myocardial
apoptosis, suggesting that this pathway may play a crucial role in the onset of the diabetic
cardiomyopathy.<br>AFRIKAANSE OPSOMMING: DOELWIT – Oksidatiewe stres verhoog fluks deur die heksosamien biosintetiese weg (HBW)
wat in „n groter O-GlcNAsetilering van teiken proteïene resulteer. Weens die feit dat verhoogde
oksidatiewe stres en HBW fluks verband hou met insulienweerstandigheid, hipotetiseer ons dat
die aktivering hiervan tot groter O-GlcNAsetilering van BAD (pro-aptoptoties) en verhoogde
miokardiale apoptose lei.
NAVORSINGS ONTWERP EN METODES – Om die hipotese te ondersoek het ons twee modelle
ontplooi: 1) H9c2 kardiomioblaste is blootgestel aan hoë glukose konsentrasie (33mM glucose) ±
HBW moduleerders ± antioksidant behandeling vs. gepaarde kontrole (5.5mM glucose); en 2) „n
hoë vet dieetgeïnduseerde insulienweerstandige rotmodel en hiperglukemie. Ons het apoptose in
vitro deur middel van Hoescht nukleuskleuring geëvalueer, kasapase aktiwiteit bepalings en
immunoblotting terwyl apoptose in vivo getoets is deur immunoblotting. Reaktiewe
suurstofspesie (RSS) vlakke is deur middel van H2DCFDA verkleuring (fluoresensie
mikroskopie, vloeisitometrie) bepaal. Algehele en BAD O-GlcNAsetilering is beide deur
immunoblotting en immunofluoresensie mikroskopie bepaal. BAD-Bcl-2 dimeervorming
bevorder apoptose, om BAD-cl-2 dimerisasie te bepaal is daar van immunopresipitering analise
en immunofluoresensie mikroskopie (ko-lokalisasie) gebruik gemaak. In vivo is algehele OGlcNAsetiliering,
BAD O-GlcNAsetiliering en BAD-Bcl-2 dimerisasie deur immunopresipitasie
en immunoblotting bepaal.
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RESULTE – Hoë glukose behandeling van selle het die graad van apotpose betekenisvol verhoog
soos blootgelê deur Hoechst nukleuskleuring (54 ± 9%, p<0.01 vs. 5.5 mM), Annexin-V kleuring
(43 ± 5%), kaspase aktiviteit assay (26 ± 2%) en immunoblotting. In parallel, algehele OGlcNAsetilering
(p<0.001 vs. 5.5 mM), BAD O-GlcNAsetilering (p<0.05 vs. 5.5 mM) en RSS
vlakke is verhoog (fluoresensie mikroskopie– p<0.05 vs. 5.5 mM; vloeisitometrie– p<0.001 vs.
5.5 mM). HBW inhibering deur van DON en van antioksidant behandeling gebruik te maak (α-
OHCA) het hierdie effekte verlaag terwyl HBW aktivering deur PUGNAc dit verhoog het.
Netso, het insulienweerstandige rotharte betekenisvolle hoë kaspase -3 (p<0.05 vs. kontrole),
algeheel O-GlcNAsetilering (p<0.05 vs. kontrole) en BAD O-GlcNAsetiliering vlakke (p<0.05
vs. 5.5 mM) getoon. BAD-Bcl-2 dimeervorming is verhoog in hiperglukemies blootgestelde selle
[immunopresipitering analise en ko-lokalisering] en in insulienweerstandige harte.
GEVOLGTREKKINGS – Ons studie het „n nuwe weg geïdenifiseer waar hiperglukemie in groter
oksidatiewe stres resulteer wat weer HBW aktivering verhoog en BAD O-GlcNAsetilering
verhoog het. Ons het verder bevind dat groter BAD-Bcl-2 dimerisasie miokardiale apoptose
verhoog wat voorstel dat hierdie weg „n belangrike rol in diabetiese kardiomiopatie speel.
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O'Kane, H. F. "The FAS death receptor pathway in transitional cell carcinoma of the bladder." Thesis, Queen's University Belfast, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426738.
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Wardle, Fiona Claire. "Regulation of the BMP signalling pathway by BMP-1 related metalloproteases." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287477.
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Andrusiak, Matthew. "Differential Roles for the Retinoblastoma Protein in Cycling and Quiescent Neural Populations." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/24037.
Full textAbstract:
While the genetics of retinoblastoma and the implications of the retinoblastoma susceptibility gene, RB1, are well described, there is still scarce evidence to suggest why RB1 acts in such a cell-type specific manner. Using the murine cortex as a model, we examined the effects of RB1 deletion of cycling neural progenitors and post-mitotic neurons, in order to ascertain cell-type specific functions in the central nervous system. Using the previously identified cell-cycle independent role for Rb in tangential migration, we validated Rb/E2f regulation of neogenin and implicated it in this process. In quiescent cortical neurons, we identified a pivotal role for Rb in neuronal survival. Unlike in cycling progenitors, in post-mitotic neurons Rb specifically represses the expression of cell-cycle associated genes in an E2f-dependent manner. Finally, in cortical neurons in the absence of Rb, we observe an activation of chromatin at E2f associated promoters. To determine the role of direct interaction between Rb and chromatin modifying enzymes, we utilized an acute LXCXE-binding deficient mutant paradigm. We report that the LXCXE binding motif is dispensable in establishment and maintenance of cortical neuron quiescence and survival. The activation state of E2f-responsive promoters appears to be dependent on E2f-activity and not simply Rb-mediated repression. Taken as a whole, this thesis serves to support the hypothesis that Rb plays a diverse role in different cell-types by regulation of unique gene targets and regulatory mechanisms. Characterizing specific cancer-initiating populations and understanding the specific function of Rb will help in the treatment of many cancers resulting from RB1 mutation or mutation within the Rb/E2f pathway.
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Mbah, Nneka Elizabeth. "Defining the Mechanism of Methuosis, a Non-apoptotic Cell Death Pathway, Induced by Indolyl Chalcone Compounds in Glioblastoma Cells." University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1481303173869378.
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Darling, Nicola Jane. "Regulation of ER stress-induced cell death by the ERK1/2 signalling pathway." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708709.
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Park, Yoo Jin. "The role of Fas-mediated apoptotic pathway in amyloid-induced beta-cell death." Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/52420.
Full textAbstract:
In type 2 diabetes (T2D), progressive dysfunction and loss of beta-cells in pancreatic islets eventually leads to hyperglycemia. The formation of toxic protein aggregates known as islet amyloid contributes to beta-cell dysfunction and death in T2D. Islet amyloid is formed mainly by aggregation of the beta-cell hormone named islet amyloid polypeptide (IAPP), which is produced and released along with insulin. Amyloid also forms in cultured and transplanted human islets, indicating that in addition to its role in pathogenesis of T2D, amyloid formation contributes to islet graft failure in type 1 diabetes (T1D). The mechanism(s) underlying amyloid-induced beta-cell death are still unclear. Fas is a cell death receptor that has been implicated in the pathogenesis of both T1D and T2D. We hypothesised that amyloid-induced beta-cell death is mediated, at least partially, by activation of the Fas apoptotic pathway and that blocking the key steps in this pathway may protect beta-cells from amyloid toxicity in T2D and islet grafts in T1D.
Our studies showed that endogenously formed human IAPP aggregates induce beta-cell Fas upregulation leading to interaction of Fas and its ligand FasL, thereby promoting activation of the Fas apoptotic pathway initiated by caspase-8. Blocking Fas/FasL interaction reduced amyloid-induced beta-cell apoptosis, and beta-cell specific deletion of Fas or caspase-8 in hIAPP-expressing mouse islets improved beta-cell survival and function.
We further demonstrated that amyloid-induced beta-cell Fas upregulation is mediated by interleukin-1beta (IL-1beta). Amyloid formation in cultured islets closely correlated with elevated IL-1beta production and blocking IL-1beta signalling reduced amyloid-induced beta-cell Fas upregulation, caspase-8 activation and apoptosis. Moreover, IL-1beta-induced beta-cell dysfunction caused impaired prohIAPP processing and potentiated amyloid formation, which was restored by blocking IL-1 receptor. These findings support a dual role for IL-1beta in amyloid formation and its beta-cell toxicity. Similarly, enhancing islet function by the GLP-1 receptor agonist exenatide improved impaired prohIAPP processing and reduced amyloid formation.
In summary, our data show that the Fas apoptotic pathway plays a major role in amyloid-induced beta-cell death and that blocking the key mediators of this pathway may provide a new therapeutic strategy to preserve beta-cells in T2D and prolong islet graft survival in T1D.<br>Medicine, Faculty of<br>Medicine, Department of<br>Experimental Medicine, Division of<br>Graduate
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Wong, Helen Yunshan. "The role of mitochondrial (intrinsic) apoptotic pathway in hIAPP mediated β-cell death". Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58846.
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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.<br>Medicine, Faculty of<br>Experimental Medicine, Division of<br>Medicine, Department of<br>Graduate
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Lam, Wun. "A screen for genetic modifiers of polyglutamine pathology in Drosophila implicates a Dronc-related pathway in polyglutamine-induced cell death." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611432.
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Crichton, Jennifer E. "The Role of the E3-ubiquitin Ligase Trim17 in the Mitochondrial Cell Death Pathway." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23715.
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The upregulation of apoptosis is a hallmark of several neurodegenerative disorders including ischemic stroke. In neurons, as in other cell types, Bax and tBid are critical regulators of the intrinsic pathway upstream of mitochondrial outer membrane permeabilization (MOMP) and caspase activation. The characterization of the molecular events that occur during the early stages is therefore extremely important from a therapeutic standpoint. Here I show that two independent genetic pilot screens looking for novel regulators of Bax activation identified a common hit in the E3 ubiquitin ligase Trim17. Knockdown of Trim17 was found to protect against tBid-induced death in primary cortical neurons and allowed for the maintenance of mitochondrial function and oxidative phosphorylation under this apoptotic stress. The RING-domain of Trim17 was found to interact with Opa1 in mouse brain extracts. Furthermore, Opa1 co-immunoprecipitated with exogenously expressed full-length Trim17 from HEK293 cells. Knockdown of Trim17 in neurons increased Opa1 protein levels under steady-state conditions. These results suggest that Trim17 regulates Bax-dependent apoptosis in neurons via the modulation of Opa1 levels.
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Yu-Sheng, Yeh. "Studies on the role of cholesterol biosynthesis pathway on differentiation, cell death, and metabolism in adipocytes." Kyoto University, 2019. http://hdl.handle.net/2433/242687.
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Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第21810号<br>農博第2323号<br>新制||農||1066(附属図書館)<br>学位論文||H31||N5182(農学部図書室)<br>京都大学大学院農学研究科食品生物科学専攻<br>(主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛<br>学位規則第4条第1項該当
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Britt, Erin L. "Targeting BCL-2 Family Members in the Cell Death Pathway to Treat Head and Neck Cancer." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5352.
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Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.
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Wise, Randi. "The role of the secretory pathway and cell surface proteolysis in the regulation of the aggressiveness of breast cancer cells." Diss., Kansas State University, 2017. http://hdl.handle.net/2097/38199.
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Doctor of Philosophy<br>Biochemistry and Molecular Biophysics Interdepartmental Program<br>Anna Zolkiewska<br>Cancer cells exploit key signaling pathways in order to survive, proliferate, and metastasize. Understanding the intricacies of the aberrant signaling in cancer may provide new insight into how to therapeutically target tumor cells. The goal of my research was to explore the role of two modulators of transmembrane signaling, the secretory pathway and cell surface proteolysis, in the aggressiveness of breast cancer cells. To study the role of the secretory pathway, I focused on the family of endoplasmic reticulum (ER) chaperones. I found that several ER chaperones were upregulated in breast cancer cells grown under anchorage-independent conditions as mammospheres versus those grown under adherent conditions. Furthermore, certain members of the protein disulfide isomerase (PDI) family were consistently upregulated in two different cell lines at both the mRNA and protein levels. Knocking down these PDIs decreased the ability of the cells to form mammospheres. I demonstrated that the requirement for PDI chaperones in mammosphere growth is likely due to an increased flux of extracellular matrix (ECM) components through the ER. Next, I examined the role of cell surface proteolysis in modulating the aggressiveness of breast cancer cells. Cell-surface metalloproteases release soluble growth factors from cells and activate the corresponding growth factor receptors. I determined that specific metalloproteases (ADAM9 or ADAM12), modulate the activation of Epidermal Growth Factor Receptor (EGFR). I demonstrated that EGFR activation enhances the CD44⁺/CD24⁻ cell surface marker profile, which is a measure of cancer cell aggressiveness. I found that the MEK/ERK pathway, which is a downstream effector of EGFR activation, modulates the CD44⁺/CD24⁻ phenotype. When DUSP4, a negative regulator of the MEK/ERK pathway, is lost, activation of EGFR by metalloproteases no longer plays a significant role in cancer cell aggressiveness. This indicates that the ligand dependent activation of the EGFR/MEK/ERK pathway is a critical step in DUSP4-positive aggressive breast cancer. Finally, I examined the importance of metalloproteases in the regulation of Programmed-death ligand 1 (PD-L1), a transmembrane protein expressed by some cancer cells that plays a major role in suppressing the immune system. I demonstrated that cell-surface metalloproteases have the ability to cleave PD-L1 and release its receptor-binding domain to the extracellular environment. Collectively, these data indicate that (a) ER chaperones support anchorage-independent cell growth, (b) metalloproteases are important in regulation of an aggressive phenotype through the EGFR/MEK/ERK pathway, and (c) metalloproteases cleave PD-L1, a key component of immunosuppression in cancer.
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Matsumoto, Yoshihide. "Epithelial EP4 plays an essential role in maintaining homeostasis in colon." Kyoto University, 2020. http://hdl.handle.net/2433/253165.
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Kwon, Jungeun Sarah, Nicholas J. Everetts, Xia Wang, et al. "Controlling Depth of Cellular Quiescence by an Rb-E2F Network Switch." CELL PRESS, 2017. http://hdl.handle.net/10150/625987.
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Quiescence is a non-proliferative cellular state that is critical to tissue repair and regeneration. Although often described as the G0 phase, quiescence is not a single homogeneous state. As cells remain quiescent for longer durations, they move progressively deeper and display a reduced sensitivity to growth signals. Deep quiescent cells, unlike senescent cells, can still re-enter the cell cycle under physiological conditions. Mechanisms controlling quiescence depth are poorly understood, representing a currently underappreciated layer of complexity in growth control. Here, we show that the activation threshold of a Retinoblastoma (Rb)-E2F network switch controls quiescence depth. Particularly, deeper quiescent cells feature a higher E2F-switching threshold and exhibit a delayed traverse through the restriction point (R-point). We further show that different components of the Rb-E2F network can be experimentally perturbed, following computer model predictions, to coarse-or fine-tune the E2F-switching threshold and drive cells into varying quiescence depths.
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Evans, A. K. C. "The role of the programmed cell death (PD-1) pathway in the immunopathogenesis of chronic hepatitis B infection." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1138759/.
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Chronic hepatitis B (CHB) results from a complex interaction between a replicating non-cytopathic virus and an impaired antiviral host immune response. The Programmed Cell Death (PD-1) pathway is an immunoinhibitory T-cell pathway implicated in virus-specific T-cell dysfunction in several chronic viral infections. The role of the PD-1 pathway in the immunopathogenesis of chronic hepatitis B was investigated through several different approaches. Firstly, longitudinal changes in PD-1 expression in patients with CHB undergoing oral antiviral therapy was investigated. A direct correlation between viral load and PD-1 expression on virus-specific CD8+T-cells was observed in this patient cohort, and treatment induced suppression of viraemia resulted in a significant decrease in PD-1 expression on virus-specific CD8+ T-cells with a decrease in HBV-DNA and improvement in virus specific T-cell reactivity. Secondly, through the employment of a purposely-designed in vitro cell co-culture model of Hepatitis B virus infection the interactions between HBV-producing hepatoma cells (target cells) and HBV-specific CD8+ T-cells (effector cells) was investigated. This model provided evidence that both cytolytic and non-cytolytic CD8+ T-cell effector functions are important in effective control of viral replication, and blockade of the PD-1 pathway distorts the balance between these differential effector functions in vitro. Finally through the transfection of a human hepatoma cell line with hepatitis B virus (HBV) and the analysis of hepatoma cell lines that differentially express HBV these studies demonstrate that the Hepatitis B virus itself upregulates PDL1 expression on infected hepatocytes in vitro and in doing so, are able to alter the balance between cytolytic and non-cytolytic CD8+ T-cell effector functions favouring chronicity of infection. Manipulation of the PD-1 pathway may be a possible mechanism to improve virus-specific host immune responses and allow control of HBV infection. However, these immunotherapeutic strategies require careful application as there is a potential risk of immune-mediated host tissue damage.
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Sharma, Deepa [Verfasser], and Erich [Akademischer Betreuer] Gulbins. "Regulation and function of acid sphingomyelinase (ASM)/ceramide pathway in irradiation-induced cell death / Deepa Sharma. Betreuer: Erich Gulbins." Duisburg, 2015. http://d-nb.info/1079182489/34.
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Chang, Samantha J. "p53, CLIC, AND THE JAK/STAT PATHWAY: INVESTIGATING THE LINK BETWEEN CANCER STRESSES AND CELL DEATH IN DROSOPHILA MELANOGASTER." Ohio University Honors Tutorial College / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1399820564.
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Fraser, Alison. "An investigation of the cellular responses to the unsymmetrically substituted polyamine analogues and identification of the pathway to cell death." Thesis, University of Aberdeen, 2003. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU602324.
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HL-60 human promyelogenous leukaemic cells were used as a model to determine the cytotoxic potential of the unsymmetrically substituted polyamine analogues CHENSpm, CPENSpm and IPENSpm with a view to their use as chemotherapeutic agents. The cytotoxicity was compared with etoposide, an established cytotoxic drug. The analogues CHENSpm and IPENSpm induced cytotoxicity over 48 h, with decreases in cell number and protein content, with CPENSpm showing a growth inhibitory effect after 96 h. The cellular content of all 3 polyamines was decreased in all treatments, and this resulted from increases in the catabolic enzyme SS AT, and polyamine export, determined as active export by the presence of acetyl-polyamines and putrescine in the culture medium. All 3 polyamine analogues were detected intracellularly, and their internalisation was essential for their toxicity, as their co-incubation with transport inhibitors provided protection against analogue toxicity. Use of the transport inhibitors also confirmed the polyamine transporter as the route of analogue uptake. Analysis of the type of cell death induced by the unsymmetrically substituted polyamine analogues confirmed it as apoptosis, through morphological determination using DAPI staining, cell cycle analysis showing a pre G0/G1 peak, increased DNA fragmentation and induction of caspase-3-like enzymes. HL-60+ cells were used in conjunction with wild-type HL-60+ cells in an attempt to determine the pathway to apoptosis induced by these analogues. HL-60+ cells contained a stable vector that overexpressed bcl-2, an anti-apoptotic protein that prevents the mitochondrial release of apoptogenic factors including cytochrome c. These cells had shown virtually no toxicity to the analogues over all exposure periods, despite the intracellular accumulation of the analogues. Western blotting was used to probe for the presence of cytochrome c, an activator of apoptosis through the mitochondrial pathway, in the cytosol of both cell lines in response to both analogue and etoposide treatment. The detection of cytochrome c in the cytosol of wild-type HL-60 cells, but not the bcl-2 overexpressors, suggested that the mitochondrial route to apoptosis was being activated, and this was further confirmed by the increased activation of caspase-9, the initiator caspase in this route, with only basal levels of caspase-8, the initiator caspase in the death effector route, detected. These data strongly suggest that the exposure of HL-60 cells to unsymmetrically substituted analogues, CHENSpm and IPENSpm, results in the activation of apoptosis through the mitochondrial release of cytochrome c. Consideration of these analogues as future chemotherapeutic agents requires that their effects are decreased in normal cells. Two normal cell lines and primary human cell cultures were exposed to these analogues and results confirmed that the analogues were selectively cytotoxic towards malignant cells. Overall, these analogues were effective cytotoxic agents in the HL-60 human leukaemic cell model, and show promise as chemotherapeutic agents through a low dose and short exposure, which is directly comparable with etoposide. The identification of the mitochondrial pathway to apoptosis as the mechanism of action of these analogues means they would not be useful chemotherapeutic agents in tumours where anti-apoptotic proteins in the mitochondrial apoptotic pathway, such as bcl-2, are overexpressed. However, these analogues could be effective cytotoxic agents in other types of cancer, particularly those where the death effector pathway is suppressed due to a similar anti-apoptotic protein overexpression, and could be used either alone, or in conjunction with existing cytotoxic drugs.
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Turnbull, Stuart. "The central role of a metal-protein aggregation-dependent oxidative stress pathway in the pathogenesis of cell death in neurodegenerative diseases." Thesis, Lancaster University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413793.
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Chen, Szu-ying, and 陳思穎. "Signaling pathways of caspase inhibitor induced cell death." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/70347502503671890314.
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碩士<br>國立臺灣大學<br>藥理學研究所<br>96<br>Benzyloxycarbonyl-Val-Ala-Asp (ZVAD), a pancaspase inhibitor has been widely used to abolish apoptotic cell death. Interestingly, previous reports showed that ZVAD alone induces necrosis accompanying with autophagosome formation, which termed as autophagic cell death, in L929 fibrosarcoma cells. They found that ZVAD-induced autophagic cell necrosis relies on caspase 8 inhibition, RIP1, JNK activity, and ROS accumulation. Until now the connection of these molecules and signaling mechanisms in details, however, are unclear. Therefore the aim of this study is to elucidate the molecular mechanisms of ZVAD-induced autophagic cell death, and find out the sequential roles of JNK, ROS, poly (ADP-ribose) polymerase (PARP) , calcium and Src in the signaling pathways triggered upon caspase inhibition and their action levels either upstream or downstream of autophagosome formation.
First, we confirm ZVAD indeed can stimulate LC3 cleavage, beclin 1 gene expression, autophagosome formation, and cytosolic ROS accumulation in L929 cells. Further study with mitochondria specific fluorescence dye (MitoSox) and inhibitors (rotenone, FCCP and BHA), we suggest that the ROS production by ZVAD is generated from mitochondria. Antioxidants, beclin 1 silence, class III PI3K inhibitor (3-MA) all effectively block ROS production and cell death, implying ROS accumulation downstream of autophagy contributes to cell necrosis. Moreover, our results reveal that ZVAD can stimulate PARP activation and PARP inhibitor DPQ significantly reduces ZVAD-induced cell death, but does not affect ROS production, suggesting ROS increase leading to PARP activation, and in turn causing cell death. Besides, we find that ZVAD stimulates intracellular calcium elevation, and ZVAD-induced ROS production and cell death are abolished by either BAPTA/AM or Ca-free medium. When using specific kinase inhibitors to analyze their involvement in ZVAD-elicited events, we find that JNK, ERK and Src are involved in ZVAD-induced cell death and ROS increase. Biochemical data evidence ZVAD rapidly induces JNK and ERK phosphorylation, and both signaling activations are sensitive to Src inhibitor PP2 and its siRNA treatment. All these results prompt us to propose that ZVAD-induced autophagic cell necrosis is mediated sequentially through caspase 8 inhibition/Src activation/JNK and ERK activation/autophagy formation/ROS generation from mitochondria/PARP activation, and eventually leads to cell death.
Theses finding provide more information to understand the apoptosis- independent role played by caspase 8 in cell death. In addition to initiate classical caspase cascade leading to cell apoptosis, caspase 8 inhibition in contrast trigger a novel signaling pathway leading to cell autophagy, and in turn necrosis cell death.