Deregulated MYC expression contributes to cellular transformation as well as progression and maintenance of human tumours. Interestingly, in the absence of additional genetic alterations, potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced apoptosis has long been recognised as a major barrier against cancer development. However, it is largely unknown how cells discriminate physiological from supraphysiological levels of MYC in order to execute an appropriate biological response. The experiments described in this thesis demonstrate that induction of apoptosis in mammary epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required to invade low-affinity binding sites and repress target genes of the serum response factor SRF. These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1- dependent gene repression and increases AKT phosphorylation and downstream function. Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive role for the MYC/MIZ1 complex in epithelial cells. / Eine Deregulation der MYC Expression trägt entscheidend zur malignen Transformation und Progression humaner Tumoren bei. In Abwesenheit von zusätzlichen genetischen Läsionen machen potentiell onkogene MYC Proteinmengen Zellen jedoch anfällig für eine Reihe Apoptoseauslösender Reize. Daher kann MYC-induzierte Apoptose als bedeutende tumorsuppressive Maßnahme und wichtige Barriere gegen die Entstehung von Krebs betrachtet werden. Mechanistisch unklar ist allerdings wie genau Zellen physiologische von supraphysiologischen MYC-Mengen unterscheiden um adäquat darauf reagieren zu können. Die Experimente in dieser Dissertation zeigen, dass die repressive Eigenschaft von MYC/MIZ1 Komplexen für die Induktion von Apoptose in Brustepithelzellen essentiell ist. Die Analyse von Genexpressions- und ChIP-Sequenzier-Experimenten verdeutlicht, dass hohe Level an MYC benötigt werden um niedrig-affine Bindestellen im Genom zu besetzen und Zielgene des SRF (serum response factor ) Transkriptionsfaktors zu reprimieren. Diese Gene haben eine wichtige Funktion in Prozessen wie Zytoskelettdynamik und Zelladhäsion und sind vermutlich daran beteiligt notwendige Überlebenssignale an die Kinase AKT weiterzuleiten. Eine Wiederherstellung der SRF Aktivität revertiert die MIZ1-abhängige Repression der Zielgene und führt zu einer vermehrten AKT Phosphorylierung und Funktion. Insgesamt deuten diese Resultate auf eine tumorsuppressive Rolle des MYC/MIZ1 Komplexes in epithelialen Zellen hin, da eine Veränderung der genregulatorischen Aktivität als Folge der Assoziation mit MIZ1 dazu beiträgen könnte onkogene Mengen an MYC zu erkennen.

ApoE is a polymorphic protein that has been found to play many different roles in biological processes including lipid transport, neurobiology and immunoregulation. ApoE occurs in the human population in three major isoforms; apoE2, apoE3 and apoE4. The apoE4 isoform has been identified as a major risk factor for several diseases including atherosclerosis and Alzheimer's disease, therefore a greater understanding of apoE biology is highly sought after. In my thesis, I have investigated several novel aspects of apoE biology. I have identified an association between increased apoE expression and apoptosis in a neuronal cell type and demonstrated that apoE becomes enriched within the neuronal apoptotic debris, consistent with a possible role for apoE in facilitating apoptotic debris clearance. A possible anti-apoptotic role of apoE in macrophages was assessed by reducing or eliminating apoE expression using siRNA and cells isolated from apoE knockout animals, respectively. The removal of apoE did not alter overall sensitivity to apoptosis, however, it did significantly increase staurosporine-induced caspase-3 activation. In other studies, the poorly understood accumulation of apoE within the nucleus was found to be enhanced during serum starvation and to localise in intra-nuclear structures that are distinct from inter-chromatin granule clusters. Analysis of apoE within the human brain revealed a correlation between fragmentation and the apoE3 isoform which was independent from AD status and brain region examined. Additionally, a portion of brain apoE3 was found to be present in the form of disulphide-linked dimers. Collectively, these studies have further expanded the current knowledge of apoE biology in terms of its association with apoptosis, nuclear localization and structural differences between the apoE3 and apoE4 isoforms in the human brain.

Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Apoptosis plays important roles in host defences against virus infection. It is therefore not surprising that viruses have developed a vast array of modulators that block this process at different stages within the apoptotic pathways. Intrestingly, Orf virus (ORFV), a member of the Parapoxvirus genus, did not reveal any of the known poxviral inhibitors of apoptosis, but was found to express a unique anti-apoptotic protein, ORFV125. The aim of this PhD project was to determine the subcellular localisation of this protein and to further characterise its anti-apoptotic activity. This included exploring its ability to inhibit early, intermediate and late events of apoptosis and identifying the mechanism by which this viral protein functions to prevent cell death. Experiments revealed that ORFV125 was localised to the mitochondria through a C-terminal mitochondrial-targeting motif, and this specific location was necessary for the protein�s anti-apoptotic function. Furthermore, the viral protein inhibited UV-induced apoptotic events at and downstream of the mitochondria such as cytochrome c release, caspase activation and DNA fragmentation. However, it was not able to prevent UV-induced activation of the c-Jun-NH₂ kinase (JNK), an event occurring upstream of the mitochondria, consistent with its localisation to this organelle. The ability to prevent apoptosis was comparable with that of the cellular anti-apoptotic protein Bcl-2, which belongs to a family of mitochondrial regulators of apoptosis. Although standard BLAST analysis failed to detect homology to anti-apoptotic members of the Bcl-2 family, a manual alignment of the primary sequence of ORFV125 with these proteins revealed characteristic residues of Bcl-2 homology (BH) domains within ORFV125. These motifs are conserved within the Bcl-2 proteins and important for their structure and function. In addition, mutating amino acids within the ORFV125 BH domains led to a loss of the anti-apoptotic function of the mutated proteins, indicating the functional importance of these residues for the viral protein. These observations suggest that ORFV125 might be classified as a viral Bcl-2-like protein. To provide evidence for this hypothesis, it was investigated if ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. The viral protein was able to entirely block the activation of the pro-apoptotic Bcl-2 family members Bak and Bax, although it did not directly bind to these proteins. Instead, ORFV125 interacted with a subset of the pro-apoptotic BH3-only proteins, which can trigger the activation of Bax and Bak. Furthermore, this study demonstrated that ORFV125 could inhibit apoptosis induced by BH3-only proteins to which the viral protein could bind. On the other hand, ORFV125 was not able to prevent the activity of pro-apoptotic proteins that it failed to interact with. This shows that ORFV125�s mechanism of action is to inhibit the activity of BH3-only proteins by binding and neutralising their function. Overall, these results provided evidence that ORFV125 is potent anti-apoptotic protein that can prevent UV-induced cell death without the participation of other ORFV proteins. Furthermore, the viral protein shared primary sequence and secondary structure similarities to Bcl-2 family members and acted in a Bcl-2-like manner to inhibit apoptosis.

In the biopharmaceutical industry, essentially, there are three components that play the main role in producing biopharmaceutical products, the host cell, the expression vector and the bioreactor and/or production environment. To produce the highly valued and desired products, the choice of a suitable host is one of the most important aspects. The host required is not only required to produce the desired product, but also needs to demonstrate robustness in a bioreactor system. Constantly facing challenges in a bioreactor, cells often undergo apoptosis, a well-known limiting factor in biopharmaceutical production, which ultimately leads to low yield of valuable protein(s). We have genetically engineered a CHO-K1 cell line to constitutively express human insulin-like growth factor-1 (IGF-1) and murine polyoma large T-antigen (PyLT-Ag) to generate Super-CHO and CHO-T respectively, two cell lines that can potentially serve different niches in the biopharmaceutical industry. In the first part of the project, we hypothesised that suspension-adapted Super-CHO and CHO-T cells are both resilient cell lines relative to the suspension-adapted CHO-K1 (designated as CHO-XL-99) when facing nutrient depletion, one of the most common problems in a bioreactor. Furthermore, in the second part of this project, the suspension-adapted CHO cell lines were also tested against a cytotoxic heavy metal, cadmium. Without the protection of the metal-resistance element, metallothionein, both Super-CHO and CHO-T cells were also challenged with cadmium to demonstrate their robustness over the parental cell line, CHO-XL-99. In the subsequent study, this project also focussed on the transfection efficiency of each parental and engineered CHO cell lines. Different strategies have been employed in the past in an attempt to improve productivity in the biopharmaceutical industry, from alterations in vector construction, improved culture condition, down to enhanced product recovery. However, the transfer and expression of the gene-of-interest (GOI) has still proven to be the limiting factor for achieving increased specific productivity. In an effort to improve transfection efficiency, strategies including cell cycle synchronisation and various transfection methods to deliver the GOI into the cells have been employed. Thus, the third part of this project has used synchronising agents in conjunction with commercially available lipid- and polymer-based reagents as delivery vehicle for the model protein, EGFP. The combination of cell synchronisation and transfection vehicle on transfection efficiency is studied here, in addition to their individual or collective effect on cell growth, apoptosis and viability. In summary, this project demonstrates the incidence of apoptosis in the cell culture induced by nutrient depletion and heavy metal, and that the use of transfection reagents solely, or in combination with synchronising agents also correlates with the increase of apoptotic indices in the cell culture. The use of the robust cell lines for transfection is an important aspect, and the balance between cell viability and the effort for augmenting transfection efficiency has to be met in order to achieve the maximum biopharmaceutical yields.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007. / Includes bibliographical references (p. 129-140).

Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanomas striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of cancer. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. My results demonstrate that the combination of ABT-737 and RNA silencing of Mcl-1 induces significant cell death in six different melanoma cell lines, representing a potential new therapeutic strategy. I show that the apoptotic response to the combination treatment involves both the intrinsic pathway and a death receptor-independent role for extrinsic pathway proteins. The combination treatment also induces a number of gene expression changes as assessed by cDNA microarray and follow-up analyses. Based on the results of the array, I investigated the effects of inhibition of MAPK proteins combined with ABT-737 and/or Mcl-1 knockdown. I found that the combination of a p38 MAPK inhibitor and ABT-737 strongly and synergistically induces apoptosis in melanoma cell lines, thus suggesting a second novel treatment combination with potential for melanoma therapy. Finally, I provide novel evidence that Bcl-2 family member PUMA is cleaved in a caspase-dependent fashion during apoptosis and may play a role in treatment response. Currently, there are no effective treatments for metastatic melanoma. My findings describe two potential combination therapies for melanoma as well as provide novel evidence as to the mechanisms involved in treatment response.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 14

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.