Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies. To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway. In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.

As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

The clinical manifestations of hemolytic disease of the fetus and newborn mediated by anti-K, an antibody of the Kell blood group system, are distinguishable from the classical form of the disease. Affected fetuses have low numbers of circulating reticulocytes and antibody titers and bilirubin levels are not reliable predictors of anemia. These observations suggest that antibodies to Kell glycoprotein lead to anemia through suppression of erythropoiesis. This study established a liquid erythroid progenitor cell culture model in which to perform analyses on the mechanism of the suppressive growth effect of anti-Kell glycoprotein. Using this culture model, this study demonstrated the requirement for co-ligation of Kell glycoprotein by a bivalent antibody for growth suppression. The absence of markers of apoptosis in cell cultures treated with anti-Kell glycoprotein suggests that the mechanism of growth suppression is distinct from programmed cell death and necrosis. Furthermore, this growth suppression cannot be rescued by erythropoietin.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. Subscription resource available via Digital Dissertations / Amylin is a 37-amino acid peptide usually cosecreted with insulin from pancreatic islet β-cells. It is implicated in the regulation of normal glucose metabolism and thought to induce pathological features of non-insulin-dependent diabetes mellitus (NIDDM). In particular, human amylin (hA) deposits as islet amyloid, and is associated with the loss of insulin-producing islet β-cells in NIDDM. The biochemical mechanism of hA-evoked death in cultured RINm5F pancreatic islet β-cells has been investigated in this thesis. Synthetic hA but not rat amylin (rA) aggregated in aqueous solution, formed fibrils, and evoked β-cell death in a time- and concentration-dependent manner. The cell death exhibited apoptotic features, including inter-nucleosomal DNA fragmentation, mitochondrial dysfunction, delayed membrane lysis, aurintricarboxylic acid suppression and cell membrane blebbling. Cytotoxicity of hA was inhibited by Congo red (an amyloid-binding dye), 8-37hA fragment (fibril-forming but non-toxic), 1-40βA or 25-35βA (Alzheimer-associated peptide), but neither by sorbitol (inhibitory to hA fibril formation), rA nor its 8-37rA peptide (non-fibril-forming and non-toxic). Preformed large amyloid deposits of hA were less potent in causing β-cell death than small aggregates. These data suggest that hA induces β-cell apoptosis via small aggregates through a possible membrane receptor pathway. Inhibitors of protein and mRNA synthesis did not inhibit hA-evoked apoptosis, but rather enhanced or directly triggered β-cell death during prolonged exposure. Likewise, Ca2+ modulators, which alter intracellular free Ca2+ concentration ([Ca2+]i), failed to prevent hA cytotoxicity and were ultimately cytotoxic themselves. Fura-2 loading and 45Ca2+ uptake studies indicated that hA did not mobilise intracellular Ca2+ during its toxicity. These results indicate a protein synthesis- and Ca2+-independent process of hA toxicity RINm5F islet β-cells. The studies reported in this thesis have established a new in vitro model of hA-evoked apoptosis using cultured RINm5F pancreatic islet β-cells. A new model of NIDDM pathogenesis is presented and discussed.

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells. / Subscription resource available via Digital Dissertations only.