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No description available.

Apoptosis is the process by which organisms eliminate excess, damaged or hazardous cells in a controlled manner. This process is controlled by the Bcl-2 family of proteins. Bcl-2 and Bcl-XL are anti-apoptotic paralogues that can replace CED-9, the sole homologue in C. elegans. It has therefore been assumed that Bcl-2 and Bcl-XL are replaceable and functionally identical. However, evidence in some mammalian cells indicates that this may not be the case. The purpose of this project was to exhaustively compare the anti-apoptotic activities of Bcl-2 and Bcl-XL in one cell type. As Bcl-2 and Bcl-XL have been found to localize to the ER and the outer mitochondrial membrane, we also determined whether subcellular location affects the function of these proteins differently. MCF-7 breast cancer cells were stably transfected with Bcl-2 and Bcl-XL alternatively targeted to the ER or mitochondria, and exposed to various doses of doxorubicin; PARP cleavage was measured using quantitative Western blotting as an indication of apoptosis to obtain EC₅₀ values in the different cell lines. The levels of both Bcl-2 and Bcl-XL affected anti-apoptotic activity; specific degradation of both proteins was noted at higher doses of doxorubicin. Nevertheless, the results indicated clearly that there was a difference between Bcl-2 and Bcl-XL. Using EC₅₀ values Bcl-XL mutants were at least 8 times more protective than Bcl-2 mutants. Furthermore, most of the cleavage products of PARP in Bcl-XL expressing clones were due to non-caspase-7 proteases, a pattern not seen with Bcl-2. Bcl-2 and Bcl-XL localized to mitochondria were most effective, while cytosolic and ER localized Bcl-XL were less effective, and Bcl-2 at these sites did not inhibit apoptosis. / Thesis / Master of Science (MSc)

Yes / Exposure to DNA-damaging agents produces a range of stress-related responses. These change the expression of genes leading to mutations that cause cell cycle arrest, induction of apoptosis and cancer. We have examined the contribution of haploid and diploid DNA damage and genes involved in the regulation of the apoptotic process associated with exposure, The Comet assay was used to detect DNA damage and quantitative RT-PCR analysis (qPCR) to detect gene expression changes in lymphocytes and sperm in response to methyl methanesulfonate. In the Comet assay, cells were administered 0–1.2 mM of MMS at 37 °C for 30 min for lymphocytes and 32 °C for 60 min for sperm to obtain optimal survival for both cell types. In the Comet assay a significant increase in Olive tail moment (OTM) and % tail DNA indicated DNA damage at increasing concentrations compared to the control group. In the qPCR study, cells were treated for 4 h, and RNA was isolated at the end of the treatment. qPCR analysis of genes associated with DNA stress responses showed that TP53 and CDKN1A are upregulated, while BCL2 is downregulated compared with the control. Thus, MMS caused DNA damage in lymphocytes at increasing concentrations, but appeared not to have the same effect in sperm at the low concentrations. These results indicate that exposure to MMS increased DNA damage and triggered the apoptotic response by activating TP53, CDKN1A and BCL2. These findings of the processing of DNA damage in human lymphocytes and sperm should be taken into account when genotoxic alterations in both cell types are produced when monitoring human exposure. / Libyan Government

Parkinson’s disease, the second most common neurodegenerative disease, is characterized by the loss of dopaminergic neurons within the substantia nigra. Currently, the treatments available for PD are symptomatic treatments that do not stop the progression of the disease. Trophic molecules, such as glial cell-line derived neurotrophic factor (GDNF), have been evaluated as potential therapeutic molecules that could stop the loss of neurons and potentially restore some of the neurons that have already been lost. However, these trophic molecules are large making them difficult to produce and delivery. Here we characterize three peptides (DNSP-5, DNSP-11, and DNSP-17) to determine it they are stable and offer protective effects similar to GNDF allowing them to be potential therapeutic molecules. The data presented here involves the evaluation of the molecular and cellular mechanism of DNSP-5, DNSP-11, and DNSP-17, which are derived from prosequence of GDNF. Initial studies were carried out to evaluate the physical characteristics of these three peptides to determine their viability as potential therapeutic molecules. The structure and stability of these peptides were evaluated. Based on the data it was determined that the three peptides do not interact in vitro, allowing for further individual evaluations of the peptides. It was also determined that the peptides were stable when stored at both -80°C and 37°C for one month, allowing them to both potentially be stored during treatment. Cell culture assays and proteomic profiling were utilized to determine binding partners and potential mechanisms through which DNSP-11 may be able to mediate apoptosis. It was determined that DNSP-11 was able to interact with a variety of binding partners that are involved in metabolism. These studies have aided in the understanding of neurotrophic factor prosequence function, but will also serve as a starting point for the development of novel trophic factors for PD treatment. Finally, the interaction between DNSP-11 and GAPDH was evaluated as a potential anti-apoptotic mechanism. GAPDH has previously shown to play a role in mediating apoptotic pathways. It was hypothesized that the observed interaction between DNSP-11 and GAPDH could mediate that role of GAPDH in apoptosis and afford DNSP-11 its observed anti-apoptotic effects. It was observed that while DNSP-11’s interaction with GAPDH may play a role in its anti-apoptotic effects, it does not appear to be the only mechanism involved. Based on this data, it is likely that the other metabolic binding partners play a role in DNSP-11’s anti-apoptotic mechanisms and therefore, these interactions should be further evaluated.

Novel strategies for the treatment of malignant gliomas are urgently needed. They are characterised by an inherent resistance to both chemo- and radiotherapeutics resulting in unrelenting tumour progression. While the exact mechanisms of treatment resistance remain undefined, it is now recognized that multiple components within the apoptotic pathway are heavily dysregulated in glioma cells and that the over-expression of anti-apoptotic proteins in patient samples correlates with inferior patient survival. The Inhibitor of Apoptosis Proteins (lAPs) represent the final molecular blockade preventing cellular apoptosis and have been identified as a potential rational therapeutic target in gliomas. The work described herein was focused on the development of novel therapeutic strategies that target the lAPs in malignant gliomas, that are readily translatable to the clinic, and that have the potential to improve patient outcomes. The first series of studies examined the hypothesis that targeting the lAPs in conjunction with other conventional and targeted therapies would overcome treatment resistance, and enhance anti-tumour activity. The novel, small molecule, lAP inhibitor LBW242 was shown to successfully target the lAPs in glioma cells and inhibit their ability to bind to and inactivate caspases. However when tested as a single agent in vitro, no stand alone anti-glioma activity of LBW242 was demonstrated. A screen of the activity of LBW242 in combination other pro-apoptotic compounds led to the discovery that lAP inhibition applied in combination with receptor tyrosine kinase (RTK) inhibition led to enhanced caspase activation and induction of apoptosis with a subsequent synergistic anti-glioma effect. The most profound effect was demonstrated with the specific combination of PDGFR and lAP inhibition both in vitro and in vivo as well as in primary patient derived glioma tumourspheres. While multiple RTKs have previously been validated as rational therapeutic targets, the clinical failure of RTK inhibitors in glioma patients has to date remained unexplained. The results in this thesis provide a novel explanation for the resistance of glioma cells to these targeted therapies, and more importantly offer a clinically tractable strategy of overcoming that resistance and improving patient outcomes. The second series of studies investigated the mechanism of synergy between lAP and RTK inhibition. The results showed that PDGFR inhibition does not stimulate apoptosis in glioma cells by previously described pathways. A screen of the entire apoptotic pathway revealed that treatment with imatinib modulates the expression of the anti-apoptotic protein NOL3/ARC. The results showed that imatinib treatment leads to down-regulation of NOL3 and that this effect is critical to the synergy between lAP and PDGFR inhibition. Further analysis suggested a critical role for NOL3 in gliomagenesis and treatment resistance NOL3 was found to be highly expressed in malignant gliomas and with expression levels that are inversely correlated with patient outcomes. A role for NOL3 has not previously been described in malignant gliomas. Finally, a series of studies were undertaken that tested the use of LBW242 in combination with the standard-of-care therapies of irradiation and temozolomide. In vitro assays demonstrated that LBW242 enhanced the pro-apoptotic activity of radiotherapy, and clonogenic assays showed that the combination therapy led to a synergistic anti-glioma effect in multiple glioma cell lines. Athymic mice bearing established human malignant glioma tumour xenografts treated with LBW242 plus radiation and temozolomide demonstrated a profound and synergistic suppression of tumour growth. Neurosphere assays revealed that the combination of radiation and LBW242 led to a pro-apoptotic effect in highly resistant glioma stem cells with a corresponding inhibition of tumour growth. The results indicate a potentially powerful strategy to enhance the therapeutic activity of standard-of-care therapies in glioma patients. Collectively, the findings of the studies in this thesis contribute to a better understanding of the mechanisms of treatment resistance in malignant gliomas, and demonstrate that the pro-apoptotic and anti-glioma effects of radiotherapy, chemotherapy and specific targeted therapies can be enhanced by the addition of a novel, small molecule lAP inhibitor. These results are readily translatable to clinical trial, and offer the potential for improved treatment outcomes for glioma patients.

Acute kidney injury (AKI) induced by renal ischaemia reperfusion injury (IRI) is characterised by renal failure, acute tubular necrosis (ATN), inflammation and microvascular congestion. Apoptotic cell administration reduces inflammation in experimental models of acute inflammation in the lung, joints and peritoneum. Preliminary data suggested that administration of 20x106 apoptotic thymocytes to mice 24-hours prior to renal IRI ameliorated renal function without affecting ATN 24-hours following IRI. This thesis attempted to validate these finding and explore underlying hypothetical mechanisms. These studies examined if functional protection was conferred by apoptotic cell modulation of (a) circulating IgM antibodies or (b) coagulation status leading to improved intrarenal microvascular blood flow. Pathogenic IgM antibodies bind ischaemic cardiac or skeletal muscle and the intestine leading to complement activation and worse injury. We examined IgM binding to human renal (HK-2) cells by flow cytometry and to ischaemic murine kidney tissue. H2O2 or Antimycin A treated HK-2 cells incubated with human serum (IgM source) exhibited no IgM binding. Medullary IgM deposition assessed by immunofluorescence was minimal following IRI. We also assessed IgM deposition by immunohistochemistry following hepatic IRI and discovered dramatic deposition. These data suggest that IgM antibodies exhibit differential binding to injured tissues and are not directly involved in renal IRI, but may have a role in hepatic IRI. To support our second hypothesis we studied apoptotic cell modulation of coagulation. A thrombin generation assay revealed that early apoptotic cell-treated mice exhibited delayed thrombin generation. Furthermore, in vitro studies confirmed direct apoptotic cell-platelet binding. To replicate apoptotic cell derived functional protection Balb/c mice underwent 20, 24 or 25-minutes of ischaemia to induce mild, moderate or severe kidney dysfunction. Renal function and injury was determined 24-hours following IRI by plasma creatinine measurement and ATN scoring. Unexpectedly, intravenous pretreatment of mice with apoptotic thymocytes conferred no protection. Indeed, apoptotic thymocytes further impaired renal function depending upon injury severity. Impairment of renal function was not secondary to increased microvascular congestion, inferred by fibrin and platelet deposition, neither increased ATN nor inflammation, assessed by neutrophil infiltration. These data indicate that apoptotic cell administration does not protect from subsequent renal IRI and that apoptotic cells are thus not inherently anti-inflammatory in all models of acute inflammation. Unable to replicate apoptotic cell derived functional protection we explored the binding of IgM antibodies to apoptotic cells which acts to facilitate dead cell clearance. We characterised IgM binding to non-apoptotic and apoptotic murine thymocytes and human Jurkat cells using flow cytometry, confocal and electron microscopy. We demonstrated specific IgM binding to a subset of late apoptotic cells. Electron microscopy indicated that IgM+ apoptotic cells exhibited marked plasma membrane disruption, suggesting that access to intracellular epitopes was required for IgM binding. Binding of IgM to permeabilised non-apoptotic and apoptotic cells suggested that IgM bound epitopes are ‘apoptosis independent’ such that IgM may bind any cell with profound plasma membrane disruption. Interestingly, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognise and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.

<p> Bcl-2 proteins play a critical role in the regulation of apoptosis, a form of programmed cell death. Apoptosis is important during development to facilitate the elimination of supernumerary, damaged or harmful cells in multicellular organisms. Altered regulation of apoptosis is associated with many diseases such as several forms of cancer as well as autoimmune and degenerative disorders. The way in which Bcl-2 proteins regulate apoptosis is unknown and much research is focused on elucidating the molecular mechanism of their function. Bcl-2, an anti-apoptotic member of this family, is localized to the mitochondria, endoplasmic reticulum and nuclear envelope. In healthy cells, Bcl-2 adopts a typical tail-anchored topology in which the carboxyl-terminal helix (a9) is inserted into the membrane, anchoring the protein, leaving the majority of the protein in the cytosol. Previous results from our lab have shown that after the induction of apoptosis, Bcl-2 undergoes a conformational change in which the endogenous cysteine residue, C158, in the a5 helix becomes protected from a membrane impermeant cysteine specific labelling reagent, IASD (4-acetamido-4' ((iodoacetyl)amino)-stilbene-2,2'disulfonate). Modification of cysteine residues results in a change in migration ofBcl-2 in an isoelectric focusing, IEF, gel system. To investigate the nature of this conformational change, cysteine scanning mutagenesis was used to determine the topology of Bcl-2 in the late stages of apoptosis. The results from the current study showed that in rat 1 myc ERTM fibroblasts, a discontinuous sequence of residues in the a5 and a6 helices of Bcl-2 become protected from IASD labelling after the induction of apoptosis by etoposide or serum starvation. The data support a model topology in which, during apoptosis, Bcl-2 undergoes a functionally significant conformational change, going from a single spanning transmembrane protein to a polytopic membrane protein in which three helices span the membrane, a5, a6 and a9. </p> / Thesis / Master of Science (MSc)

Το πολλαπλούν μυέλωμα είναι μια νεοπλασία η οποία, έως και σήμερα, παραμένει ανίατη. Στο ΠΜ, το ανοσοποιητικό σύστημα δε κατορθώνει να καταστρέψει τα κακοήθη πλασμοκύτταρα, ενώ υπάρχουν ενδείξεις ότι τα κύτταρα του όγκου παίζουν ενεργό ρόλο σε αυτό. Στην παρούσα διατριβή, ελέγξαμε ποικίλλες μυελωματικές σειρές σχετικά με την έκφραση των προ- και αντι-αποπτωτικών γονιδίων. Βρήκαμε ότι τα μυελωματικά κύτταρα εκφράζουν έναν μη φυσιολογικό φαινότυπο (fas high/bcl low). Μελετώντας τα κύτταρα του μυελού των οστών ενός ασθενούς με ΠΜ τύπου IgG/k σταδίου ΙΙΙΑ, διαπιστώσαμε ότι αυτά τα κύτταρα εξέφραζαν τα γονίδια granzyme B και perforin, τα οποία υπό κανονικές συνθήκες εκφράζονται μόνο από τα κυτταροτοξικά Τ λεμφοκύτταρα (CTLs) και τα φυσικά φονικά κύτταρα (ΝΚ). Προτείνουμε ότι πιθανόν το παραπάνω γεγονός αποτελεί έναν επιπλέον αμυντικό μηχανισμό των κυττάρων του όγκου εναντίον των CTLs και ΝΚ κυττάρων. Συγκεκριμένα υποθέτουμε ότι τα κύτταρα του όγκου προσελκύουν τα CTLs και τα ΝΚ κύτταρα και τα καταστρέφουν με τη διαδικασία της κυτταρόλυσης. Τα διφωσφονικά οξέα χρησιμοποιούνται ευρέως για τη θεραπεία του ΠΜ, ενώ η θαλιδομίδη χρησιμοποιείται σήμερα για τη θεραπεία πολλών τύπων ΠΜ. Μελετήσαμε την επίδραση της θαλιδομίδης, του ζολεδρονικού οξέος και το συνδυασμό τους στην επιβίωση των μυελωματικών κυττάρων. Βρήκαμε ένα συνεργιστικό αποτέλεσμα των δύο αυτών ουσιών. Όταν η θαλιδομίδη συγχοηγηθεί με το ζολεδρονικό οξύ σε ελάχιστους μη δραστικούς μηχανισμούς, μειώνει την τοξική επίδραση 50% του τελευταίου 3-4 φορές. / Multiple Myeloma (MM) is a plasma cell neoplasia which, to this day, remains incurable. In MM, the immune system fails to destroy the malignant plasmocytes, and evidence exists that the tumor plays an active part in this. In the present study, we tested various MM cell lines for the expression of pro- and anti-apoptotic genes. We found that MM cells express an abnormal phenotype, namely fas high/bcl low. By studying bone marrow cells from a patient suffering of MM IgG/k type stage IIIA, we found that those cells expressed granzyme B nad perforin, normally expressed by cytotoxic T cells (CTLs) and natural killer (NK) cells. We propose that this may constitute an additional acquired mechanism by the tumor cells to protect themselves against the host.