Background: Thalassaemia is a genetic disorder disease, one of the most clinically relevant haemoglobinopathies in paediatric population. It interferes with the synthesis of haemoglobin chain. For the sake of maintaining the serum haemoglobin at a normal level, regular blood cell transfusion is required to the patients with thalassaemia. In general, patients with thalassaemia are often diagnosed at an early age and need to take a life-long iron chelating therapy to prevent the multi-organ failure caused by iron-overload. The safety issue is considered a very importance aspect in the treatment among paediatric population and young people. In the past decades, numerous randomised controlled trials (RCTs) and meta-analysis regarding the efficacy and safety of iron chelating agents including deferoxamine, deferiprone, deferasirox, in reducing iron accumulation among patients with thalassaemia had been published, yet limited meta-analysis reveal the same issue among paediatric population and young adults. Further evidence and understanding are therefore needed to confirm whether or not the iron chelating agents are safe among young patients with thalassaemia. Objective: To conduct a meta-analysis to evaluate the safety of iron chelating agents in paediatric population and young adults with thalassaemia. Methods: Literature search was carried out in PubMed, EMBASE, BIOSIS Previews, Science Citation Index Expanded and Cochrane Library databases. This meta-analysis of observational studies was conducted following the PRISMA and MOOSE statements. Selection Criteria All prospective uncontrolled cohort studies were eligible to include. Articles were assessed according to the age range of its participants and the quality of the reported adverse effects. All enrolled studies should record countable cases of adverse effects of paediatric population and young patients up to 25 years of age, diagnosed with alpha/beta thalassaemia or sickle cell disease and under the treatment of iron chelating agents. Data Collection and analysis Two reviewers independently retrieved the data and conducted the quality assessment for each of the included studies. Agency for healthcare research and quality assessment tool was used to evaluate the general risk of bias, whilst the quality of harms was assessed with the Mcharm question criterions. Meta-analysis was carried out for detecting the entire proportion value of six adverse effects including liver abnormality, renal abnormality, rash, abdominal pain/discomfort, nausea and neutropenia, and the forest plot was generated accordingly. The I2 was estimated to assess the methodological quality of each outcome. Random or fixed model was used for the analysis. The sensitivity analysis was conducted to assess the robust of the result. Results A total of 8199 articles were identified in the initial database search. After removal of duplications, update from other sources and exclusion based on the exclusion criteria, 25 full articles were retrieved and 14 uncontrolled cohort studies were included in this review. Eight hundred and fifty-four patients up to 25 years of age were included in the analysis. The general quality of the studies was moderate while the quality of adverse effects was low to moderate. Out of the 14 included studies, nine were under the deferasirox treatment; five for deferiprone therapy. No study included deferoxamine. Most adverse effects were observed among the paediatric patients under deferasirox treatment. The meta-analysis of pooled proportion under deferasirox were 17.23% (95%CI, 8.78-25.68%) for liver abnormality, 11.58% (95%CI, 5.91-17.25%) for renal abnormality, 5.41% (95%CI, 3.23-7.58%) for rash, 11.03% (95%CI, 1.83-20.22%) for abdominal pain/discomfort and 5.77% (95%CI, 1,50-10.03%) for nausea. Only one study reported case of neutropenia in the patients under deferasirox, whereas more cases were recorded within the paediatric patients with deferiprone, estimated proportion of 5.98% (95%CI, 2.79-9.16%). However, the meta-analysis of estimated proportion for liver abnormality in paediatric patients with deferiprone was 10.08% (95%CI, 2.67-17.49%), abdominal pain/discomfort was 4.31% (95%CI, 1.65-6.98%). Only one study reported case of renal abnormality, rash and nausea respectively in the patients with deferasirox, which a meta-analysis could not be conducted. Conclusions Among paediatric population and young patients with thalassaemia disease, most drug-related adverse effects were liver injury among patients under both deferasirox and deferiprone. For patients under deferasirox, the proportions of the risk of abdominal pain/discomfort and renal abnormality were in a secondary high-level, whereas the proportions of the risk of rash and nausea were comparatively very low. Few adverse effects were detected among young patients with deferiprone. In addition, the proportion of liver abnormality and abdominal pain/discomfort were lower for deferiprone than deferasirox. Further investigation is needed to assess the safety and efficacy between different dosage of iron chelating agents and the risk of other adverse effects among paediatric population, which are necessary to guide the clinical practice in the treatment of paediatric patients with thalassaemia. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 28-33). Also available via the Internet.

Thesis (Ph. D.) in Biological Sciences--University of Maine, 2001. / Includes vita. Includes bibliographical references (leaves 148-166).

Thesis (M.S.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains viii, 85 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

M.Sc. / Sub-Saharan Africa accounts for a large fraction of the world’s infectious diseases, particularly AIDS (Acquired Immunodeficiency Syndrome) and Tuberculosis (TB) and at the same time, iron (Fe) overload is common to several of its regions. Excess Fe aids in the replication of both Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (M.tuberculosis, Lounis, 2001; Georgiou, 2000) and also causes a malfunction of the host’s defense system and may ultimately lead to cell death. Not only does iron assist in pathogen survival but the pathogens themselves have a synergistic relationship where infection of one supports the replication of the other (Toosi, 2001; Bonecini-Almeida et al., 1998). Controlling the replication of these pathogens as well as the iron overload simultaneously becomes a huge task as many pathogenic and host factors needs to be considered at once. Desferrioxamine (DFO), a chelator commonly used to treat clinical conditions of iron overload has been reported to inhibit the multiplication of pathogens and at the same time extract the excess iron.

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2. In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclindependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p &lt 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15-21- fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21CIP1/WAF1 nuclear accumulation normally observed in response to these DNAdamaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation. In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58;4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63;1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands??? permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1?? and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1?? knockout (HIF-1??-KO) mouse demonstrated that the transcriptional upregulation of Ndrg1 following Fe chelation was mediated by a HIF-1??-dependent and - independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis. In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.