Organochlorine pesticides (OCs) are environmental toxicants with important links to human health. They have been found to activate signalling pathways within cells and thereby affect cell survival and proliferation. Receptor Activator of Nuclear Factor kB (RANK) ligand and its receptor RANK are crucial for mammary epithelial proliferation in pregnancy and have recently been linked to hormone induced breast cancers. The objectives of this study were to confirm the proliferative effects of an OC (endosulfan) on human MCF-7 breast cancer cells, identify activated intracellular signaling pathways and investigate changes in RANK and RANKL gene expression. This study showed that endosulfan has a stimulatory effect on human MCF-7 cell proliferation, which may be invoked through activated intracellular signaling pathways (JNK, ERK1/2 and p38). In addition, there was a down regulation of RANK and upregulation of RANKL gene expression suggesting endosulfan is capable of modulating both cellular behavior and gene expression. / UOIT

Cells of the cardiovascular system translate incoming extracellular signals from hormones and drugs through binding of cell surface receptors, and activation of intracellular signaling cascades allowing modulation of specific cellular function. cAMP and cGMP are ubiquitous second messengers that activate specific signaling machinery used to promote or inhibit cellular functions such as cell migration, cell adhesion and proliferation. Increases in intracellular cAMP or cGMP levels occurs through activation of adenylyl cyclase (cAMP) or guanylyl cyclase (cGMP) or by inhibition of the cAMP and cGMP hydrolyzing enzymes, cyclic nucleotide phosphodiesterases (PDEs). Cyclic nucleotides achieve signaling specificity through compartmentation, a mechanism allowing effective regulation of cAMP or cGMP signaling in discrete parts of the cell in a spatial and temporal manner. Cells of the cardiovascular system such as platelets, vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs) maintain cyclic nucleotide compartmentation through coordinating signaling complexes containing a cAMP or cGMP effector protein and PDEs. Studies reported in this thesis demonstrate that human platelets, VECs and VSMCs each contain distinct cyclic nucleotide signaling complexes, and that based on their composition and selective subcellular localization, regulate specific cellular functions. In platelets, subcellular localization of PDE5 results in differential regulation of PDE5 and selective regulation of Ca2+ release from endoplasmic reticulum stores, an initial step in platelet aggregation and provides a potential therapeutic target in preventing thrombosis. VECs utilize multiple signaling systems to regulate cellular function including cAMP signaling pathways and modification of phosphatidylinositols. These studies demonstrate that a PDE3B-based signaling complex allows integration of both cAMP and phosphatidylinositol-3-kinase-γ (PI3Kγ) signals resulting in increased cell adhesion and cell spreading. Finally, studies in VSMCs demonstrate that PDE5 localization in cells allows cAMP/cGMP cross talk through PDE5 and PDE3A. These results are discussed in the context of further understanding the role of PDEs in mediating cAMP and cGMP signaling and modulation of cell function in cells of the cardiovascular system. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-06-28 13:31:51.428

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

CC2D1A (coiled-coil and C2 domain containing protein 1A), also known as Freud-1, has been identified as a transcriptional repressor of the serotonin receptor 5-HT1A, a regulator of endosomal budding and an activator of NF-KB signaling. It also acts as a scaffold that promotes activity of the PI3K/Akt pathway upon stimulation by the epidermal growth factor (EGF). Moreover, several studies highlight naturally occurring mutations of CC2D1A in humans that produce varying degrees of intellectual disorder and autism. Use of the Cre-LoxP system to conditionally knockout CC2D1A in mice has provided promising results regarding its effect on 5-HT1A expression and behaviour. This thesis aims to extend the use of this knockout model by studying cell signaling activity in mouse embryonic fibroblasts (MEFs), derived from the CC2D1Aflx/flx transgenic line, that have been treated with a commercially available Cre recombinase to completely knock out CC2D1A. I hypothesize that CC2D1A directly regulates EGF receptor activity and that its Cre-mediated knock down in vitro will entirely block cell signaling pathways activated by the EGF receptor. Western blot analysis demonstrated that, after Cre-mediated CC2D1A knockout, Akt and Erk1/2 phosphorylation were still maintained upon EGF treatment. In addition, overexpressing Freud-1 via transfection had no effect on cell signaling compared to the wild-type control. Analysis of recombinant Freud-1 constructs reveal that a C-terminal truncation enhances its ability to bind to PIP2 and PIP3 – phospholipids essential to the Akt pathway. In addition, immunocytochemistry analysis demonstrates a responsiveness of CC2D1A to EGF treatment. Altogether, these data highlight a unique and effective way in carrying out gene knockout in vitro while also emphasizing the need to further investigate CC2D1A’s importance in regulating cell signaling pathways and functional compensation by other homologous proteins

<p>The neuropeptides orexin-A and -B act as endogenous ligands for G-protein-coupled receptors (GPCRs) called OX₁ and OX₂ receptors. Previous observations have established that orexin receptors have an ability to couple to different G-proteins and signaling pathways and induce Ca²⁺ elevations via both receptor-operated Ca²⁺ channels (ROCs) and store-operated Ca²⁺ channels (SOCs). This thesis further elucidates the intracellular signaling mechanisms of orexin receptors.</p><p>Orexin receptors were shown to activate ERK (extracellular signal-regulated kinase) via Ras, protein kinase C, phosphatidylinositol-3 kinase and Src. Ca²⁺ influx was shown to be obligatory for the activation of ERK and adenylyl cyclase, wherewith a hypothesis was formed that submembrane Ca²⁺ elevation is of central importance for the regulation of orexin receptors' coupling to different signaling pathways. This was further investigated with respect to OX₁R-mediated activation of phospholipase C (PLC) showing that ROC influx was of more central importance for the OX₁R signaling, but also SOCs amplified PLC activity. A technique to block OX₁R-induced IP₃increase and subsequent Ca²⁺ release was devised, leaving ROC influx as the only source of Ca²⁺ elevation upon OX₁R activation. This block had no effect on OX₁R-mediated activation of ERK, showing that ROC-dependent influx is the most central Ca²⁺ elevating process in OX₁R signaling. OX₁Rs' coupling to PLC was further investigated by measuring the metabolites generated, inositol phosphates and diacylglycerol (DAG). The results indicate involvement of two different PLC activities with different substrate specificities, which results in, at low orexin-A concentrations, DAG production without concomitant production of IP₃. At even lower orexin-A concentrations, OX₁Rs generate DAG by activating phospholipase D. In conclusion, the results strengthen the hypothesis that ROCs have a central role in orexin receptor signaling and DAG may be the signal of preference.</p>

Thesis advisor: Mary F. Roberts / Thesis advisor: Eranthie Weerapana / Small molecules are of interest both as metabolites in tumor cell biology and as potential therapeutics in the fight against cancer. In this work, small molecules in both roles have been examined. Modulation of tumor cell metabolism holds promise as a strategy to combat cancer, and both glucose and glutamine have been identified as critical fuels for tumor cell growth and proliferation. However, the reason for glutamine addiction is poorly understood. The differential metabolism of glutamine and glucose was therefore examined using ¹³C labeling and NMR-based metabolomics in the VM-M3 tumor cell line, which requires both glucose and glutamine for survival and proliferation. In the course of this study, a novel mammalian metabolite itaconic acid was identified. Itaconic acid was detected in extracts and tissue culture media from the murine macrophage-derived tumor cell lines VM-M3 and RAW 264.7 as well as in primary macrophages. Production and secretion of itaconic acid was increased upon stimulation. LC-MS and NMR based metabolomics studies show that this metabolite is synthesized by the decarboxylation of cis-aconitate from the TCA cycle, and provided evidence for a novel mammalian homologue of the enzyme cis-aconitic decarboxylase. D-3-deoxy diC₈PI is a small molecule of interest as a potential cancer therapeutic. This compound was designed to induce apoptosis in tumor cells by competitively binding to the Akt PH domain and preventing Akt translocation. However, high resolution ³¹P field-cycling studies show that both D-3-deoxy diC₈PI and an inactive analogue L-3,5-dideoxy diC₈PI bind to the same site on the PH domain, which is distinct from the binding site of the ligand diC₈PI(3,4,5)P₃. This makes the aforementioned mechanism of cytotoxicity unlikely. Aggregation of the PH domain in the presence of soluble headgroup IP₆ was also observed, which may be related to a physiological function of this protein and invalidates at least one other binding assay. Investigation into alterations in signaling pathways in the MCF-7 breast cancer cell line showed that D-3-deoxy diC₈PI activates the p38MAPK pathway which results in CREB hyperphosphorylation. However, activation of this pathway appears to be compensatory and unrelated to the mechanism of action. D-3-deoxy diC₈PI also decreases levels of cyclin D1 and cyclin D3, which regulate the progression of the cell cycle. These decreases appear to be occurring at the transcriptional level rather than due to increased proteasomal degradation. The loss of these two proteins does not cause apoptosis in MCF-7 cells, but siRNA knockdown of specifically cyclin D1 inhibits proliferation. This is consistent with the cell cycle arrest observed upon D-3-deoxy diC₈PI treatment in these cells. These findings do not conclusively elucidate the mechanism of cytotoxicity of D-3-deoxy diC₈PI, but provide a characterization of some of its effects in the MCF-7 cell line which may be useful for further studies. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

In recent years, dietary fat has been shown to be capable of activing taste receptor cells in the tongue. Fatty acids (FAs), which act as the chemical cue and are found in dietary fat, activate a cellular signaling pathway that results in a unique signal being sent to the brain that is then interpreted as the taste of fat. One important element in this pathway is the ion channel TrpM5. It is responsible for depolarizing the taste cells that are activated by fatty acids; depolarization is an essential step in cellular response, making TrpM5 essential in the functioning of the FA signaling pathway. To study the potential roles of the FA signaling pathway, a mouse model, in which mice lacked the TrpM5 gene (TrpM5-/-), was used. From this model, I show that TrpM5 is essential for detection of fatty acids in the oral cavity; without TrpM5, mice were not able to detect FAs in the mouth. I also show here that TrpM5-/- mice eat significantly less and gain significantly less weight on a high fat diet than wildtype mice, who have the TrpM5 gene, linking TrpM5 to both fat intake and weight gain. Interestingly, these responses are only seen in male mice. Females lacking TrpM5 show no deficit in calorie intake compared to the wildtype females. Despite taking in the same amount of calories as the wildtype females, TrpM5-/- females still gain significantly less weight than the wildtypes. This posits a sex-specific response in terms of calorie intake on a high fat diet. Additionally, I show that the TrpM5 pathway is specific for a subtype of fatty acids, primarily the long-chain polyunsaturated fatty acids (PUFAs) and does not contribute to saturated fatty acid taste transduction. Lastly, in this study I show that both male and female mice who do not have TrpM5 excrete significantly less lipids in their feces than the wildtype mice; surprisingly not implicating TrpM5 in fat malabsorption. We are currently looking for other roles of TrpM5 in fat metabolism.

<p>Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.</p><p>The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.</p><p>Finally, the structure of YegS, an <i>Escherichia coli</i> protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.</p>

Human T-cell lymphotropic virus type 1 (HTLV-1) infects about 20 million individuals worldwide. This retrovirus induces two major types of pathologies: adult T-cell leukemia (ATL) or a neurodegenerative disorder called HAM/TSP (HTLV-associated myelopathy/tropical spastic paraparesis). The HTLV-1 transactivator protein (Tax) plays a central role in the development of these pathologies. We hypothesized that Tax activities are modulated through complexation with cellular proteins. Using the yeast two-hybrid method, we isolated two cellular Tax binding proteins, Gβ2 and MCM3, involved in cell signaling and DNA replication, respectively. We demonstrate that the interplay between Tax and Gβ2 modulates migration of infected T-lymphocytes toward chemokines. On the other hand, by interacting with MCM3, Tax accelerates DNA replication during the synthesis phase of the cell cycle and generates DNA damages responsible for cell transformation. We have thus identified two novel Tax partners potentially participating to the mechanisms by which the viral protein ensures viral persistence and leads to the development of HTLV-1 pathologies.