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Doctor of Philosophy(PhD) / Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

Methotrexate is a folic acid analogue widely used as a chemotherapeutic agent. It is known to be a potent inhibitor of the enzyme dihydrofolate reductase, therefore, perturbing intracellular pools of purine and pyrimidine bases for DNA synthesis, as well as pools of reduced folates used in a variety of metabolic reactions. It has been postulated, and subsequently widely accepted, that methotrexate kills cells by perturbing the intracellular ratio of dUTP:dTTP thereby leading to dUMP misincorporation into DNA. This would initiate an excision repair pathway designed to rid cellular DNA of this aberrant base. However, because of the imbalance of nucleotide pools, dUMP may well be re-incorporated during repair thus initiating a futile cycle of dUMP misincorporation and repair eventually leading to single-strand breaks in the DNA. From the results presented in this thesis, no evidence for dUMP misincorporation could be found in the two human cell lines studied (HeLa and CCRF-HSB2), despite the drug exhibiting dose-dependent cytotoxicity to both cell lines. This was true after a variety of methotrexate treatment times and at two different drug concentrations. Subsequent analysis of the drug treated cells, using the nucleoid sedimentation technique, for evidence of single-strand breaks in DNA yielded some anomalous results. Single-strand breaks, in the form of slower sedimenting nucleoids, were easily detectable after exposure of cells to low doses of methotrexate. However, treatment with higher doses resulted in the creation of faster sedimenting nucleoids. Subsequent analysis using other techniques showed that this faster sedimentation was occurring in the presence of DNA single-strand breaks. Collaborative work involving electron microscopy revealed methotrexate induced gross morphological changes in chromatin structure. Analogies with other unrelated anti-tumour agents interacting with topoisomerase enzymes are discussed.

This work reported is divided into two parts: the first part deals with quinoxaline derivatives and includes the preparation and characterisation of novel linear tricyclic quinones 1,4-diazanthracen-9,10-diones, (54) and (55). The reaction of diazanaphthoquinones and 1-acetyl-1,3-butadiene are used to produce these quinones through the Diels-Alder reaction. In addition hexaazapentacyclic 5,6,7,12,13, 14-hexaazapentacene was prepared by the reaction ofbis(2-chloroquinoxalin-3-yl)sulfide with thioxamide and the reaction of the sulfide with amines was investigated. Two different approaches to 6,13-dibutyl-5,6,7,12,13,14-hexaazapentacene are given. Derivatives of the pentacyclic, 6-thia- 5,7,12,13,14-pentaazapentacene and the unsubstituted 6,13 -dihydro compound are described. The novel N-(2,5-dimethoxy-6-nitrophenyl)guanidine is used to obtain 3-amino-5,6-dimethoxy-1 ,2,4-benzotriazine-1-oxide and 4,7 -dimethoxy-1 ,2,3 -benzotriazole is shown to be second product. Second part of the work is concerned with the development of a preparative route to 2,6-diphenylpyridines substituted with different groups on the phenyl nuclei. Several approaches were attempted. Finally, success was achieved and a series of compounds having basic chains of different length on the phenyl groups was prepared. One chain in each case had a terminal primary amine. The binding constants of the primary amines and their N-acetyl derivatives with DNA were determined using fluorescence spectroscopy.

Doctor of Philosophy(PhD) / Iron (Fe) is essential for cell growth and replication as many Fe-containing proteins catalyse key reactions involved in energy metabolism (cytochromes, mitochondrial aconitase and Fe-S proteins of the electron transport chain), respiration (hemoglobin and myoglobin) and DNA synthesis (ribonucleotide reductase). If not appropriately shielded, Fe could participate in one-electron transfer reactions that lead to the production of extremely toxic free radicals. The Fe storage protein, ferritin, is essential to protect cells against Fe-mediated oxidative stress by accommodating excess Fe into its protein shell (Xu et al., 2005). However, despite intensive research over the last few decades, many questions relating to intracellular Fe metabolism, e.g. Fe release from ferritin remain unanswered. Therefore, it is important to elucidate the molecular mechanisms of Fe trafficking in cells. At the beginning of my candidature, little was understood regarding the effect of anti-cancer agents, anthracyclines on the Fe-regulated genes, including transferrin receptor-1 (TfR1), N-myc downstream-regulated gene-1 (Ndrg1) and ferritin. Furthermore, the mechanisms of ferritin-Fe release and anthracycline-mediated ferritin-Fe accumulation are unclear. The work presented in Chapters 3 and 4 has addressed these issues. Apart from the studies examining the molecular interactions of anthracyclines with Fe, a mouse model with perturbed Fe metabolism was used and the marked alterations of protein expression in the heart of this knockout mouse model was discussed in Chapter 5. Chapter 3 Anthracyclines are effective anti-cancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron (Fe) and perturb Fe metabolism (Xu et al., 2005). These effects on Fe-trafficking remain poorly understood, but are important to decipher as treatment for anthracycline cardiotoxicity utilises the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the Fe-regulated genes, transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by Fe-depletion, as it was reversed by adding Fe and was prevented by saturating the anthracycline metal-binding site with Fe. However, DOX did not act like a typical chelator, as it did not induce cellular Fe mobilisation. In the presence of DOX and 59Fe-transferrin, Fe-trafficking studies demonstrated ferritin-59Fe accumulation and decreased cytosolic-59Fe incorporation. This could induce cytosolic Fe-deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via HIF-1α-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on Fe metabolism were complex due to its multiple effector mechanisms. Chapter 4 The Fe storage protein, ferritin, can accommodate up to 4500 atoms of Fe in its protein shell (Harrison and Arosio, 1996). However, the underlying mechanism of ferritin-Fe release remains unknown. Previous studies demonstrated that anti-cancer agents, anthracyclines, led to ferritin-59Fe accumulation (Kwok and Richardson, 2003). The increase in ferritin-59Fe was shown to be due to a decrease in the release of Fe from this protein. It could be speculated that DOX may impair the Fe release pathway by preventing the synthesis of essential ferritin partner proteins that induce Fe release. In this study, a native protein purification technique has been utilised to isolate ferritin-associated partners by combining ultra-centrifugation, anion-exchange chromatography, size exclusion chromatography and native gel electrophoresis. In addition to cells in culture (namely, SK-Mel-28 melanoma cells), liver taken from the mouse was used as a physiological in vivo model, as this organ is a major source of ferritin. Four potential partner proteins were identified along with ferritin, e.g. aldehyde dehydrogenase 1 family, member L1 (ALDH1L1). Future studies are required to clarify the relationship of these proteins with cellular Fe metabolism and ferritin-Fe release. Chapter 5 A frequent cause of death in Friedreich’s ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed two dimensional electrophoresis to characterise the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in the protein expression profile were observed in 9-week-old KO mice with severe cardiomyopathy. In contrast, only a few proteins showed altered expression in asymptomatic 4-week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilisation and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.

No description available.