The Cdc25 phosphatases are highly conserved from yeast through humans and play pivotal roles in regulating the activities of cyclin-dependent kinases (Cdks). Cdc25A is one of three human Cdc25 family members, and has previously been shown to be overexpressed in numerous cancers and to transform rodent fibroblasts. Cdc25A therefore represents a rational target for chemotherapeutic development. Further, a thorough understanding of its biology and regulation in normal and transformed cells may facilitate the development of strategies to specifically interfere with the proliferation of cancerous cells. In this work I describe experiments which demonstrate that bisperoxoVanadium compounds, and specifically bpV(Me2Phen), inhibit Cdc25A phosphatase in vitro and in vivo. Further, these compounds cause cell-cycle arrest, are cytotoxic to cancer cells, and slow the growth of tumours in mouse models. With respect to the fundamental biology of Cdc25A, I have identified a sequence element (NLS) responsible for nuclear localization of Cdc25A phosphatase. An analysis of this sequence demonstrated high conservation of flanking phosphoacceptor sites, notably Serine 292. S292 was predicted to be a consensus PKA or CamKII substrate. Using site-directed mutagenesis I have shown that S292 is the sole site of PKA phosphorylation in vitro. The functional importance of S292 phosphorylation was investigated via transfections of phospho-mimetic mutants of S292 (S292E) expressed as GFP-fusion proteins; these studies indicate that S292 phosphorylation may promote nuclear localization. Studies by other groups have indicated that S292 is a phosphorylation site for inhibitory kinases, namely Chk1 and Chk2 (4). I generated a phospho-specific antibody to this site and demonstrate by immunofluorescence and western blotting an unexpected pattern of S292 phosphorylation associated with nuclear bodies and the mitotic apparatus. I provide evidence to suggest that these sites represent local fine-tuning of Cdc25A, allowing Cdk activity to be controlled at the level of specific subcellular structures. These studies highlight the complexity of Cdc25 regulation and indicate a previously unappreciated degree of control of their activity such that these enzymes exist in multiple discrete pools within a given cell.

The survival of breast cancer patients declines when tumors are invasive and have an increased possibility of metastasizing to distal sites. Transforming Growth Factor-beta (TGF-beta) suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells. However, at late stage of mammary carcinogenesis, due to genetic and epigenetic alterations, TGF-beta loses its cytostatic actions, and contributes to tumor invasion by promoting cell proliferation, Actin cytoskeletal reorganization, as well as Epithelial to Mesenchymal Transition (EMT). Despite the key role of TGF-beta1 in tumor suppression as well as tumor progression, the molecular mechanisms underlying the conversion of TGF-beta form an inhibitor of proliferation in mammary breast cancer cells to an inducer of their cell growth and EMT have not been fully elucidated. Thus, acquiring a basic knowledge on the mechanism of TGF-beta regulating its target genes and its contribution to cancer progression may highlight new avenues for cancer therapy development. This prompted us to further investigate and identify TGF-beta-inducible genes that may be involved in TGF-beta biological responses during tumorigenesis. / In this thesis, we identified Talin as a novel TGF-beta1 target gene that acts as an antagonist to inhibit TGF-beta-mediated cell growth arrest and transcriptional activity in mammary cancer cell line, MCF-7. Searching for new partners of activated Smads, we found that TGF-beta1 induces Talin translocation from cytosol to the plasma membrane where Talin physically interacts with the TGF-beta1 signaling components, the Smads and the receptors. Furthermore, we observed that TGF-beta1 stimulation leads to the formation of Actin stress fibers where Talin was detected at the end of these stress fibers. Taken all together, the obtained data show that TGF-beta1 positively induced expression of Talin and suggests a role for Talin, which acts as a negative feedback loop to control TGF-beta biological responses.

It has long been known that oxygen has the potential to be toxic to biologic systems and that this toxicity is not due to oxygen itself, but due to the production of oxygen radicals. One of these potentially toxic radicals, superoxide (O₂⁻) can be generated as a result of ionizing radiation, and if not adequately removed can proceed to cause cell damage. Superoxide dismutase (SOD) is one of the key enzymes involved in the defence against oxygen toxicity. SOD activity can be inhibited by diethyldithiocarbamate (DOC), a powerful copper chelator. If inhibition of SOD by DOC increases the lifetime and effectiveness of radiation induced O₂⁻, it follows that the potential exists for DOC to enhance the effect of radiation. DOC is however also a thiol compound, and thus may act as a radioprotector by modifying tissue oxygenation status or by free radical scavenging. This study has concerned itself primarily with the inhibition of superoxide dismutase by diethyldithiocarbamate in order to sensitize tumours to ionizing radiation. The use of DOC as an inhibitor of SOD has however meant that any sensitization resulting from SOD inhibition could be masked by a radioprotective effect by DOC. The inhibition of SOD by DDC was confirmed in a murine rhabdomyosarcoma, and it was shown that this inhibition can be maintained for up to twenty-four hours after DDC administration. It was hypothesised that there was a potential for the radioprotective effect of DDC to be overcome, if the levels of DDC were low enough at the time of irradiation. Indeed, if DDC was removed from the growth medium of B16 mouse melanoma cells in culture prior to irradiation, a significant sensitization was demonstrated. It was shown that DDC could act as both a radiosensitizer and as a radioprotector in the same experiment. The dominant action of DDC was found to be dependent on the time allowed between DDC administration and irradiation. If this time was approximately 4 hours, it was possible to show a radiosensitizing effect by means of a tumour growth delay assay. This time modulation effect of DOC was shown in larger tumours, rather than smaller tumours, which could indicate that tumour oxygenation is an important criterion in determining the response to radiation of DOC treated cells. It was shown that B16 mouse melanoma cells exposed to 43°C after DDC pre-treatment were sensitized to thermal damage. This work suggests that some caution should be exercised when DDC is put forward as either a radiosensitizer or a radioprotector in the clinic, but that DDC may have potential as a thermosensitizer.

Note:

by Lee Chi-Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 74-78). / Abstracts in English and Chinese. / Acknowledgment --- p.ii / Table of Contents --- p.iii / Abstract --- p.v / Abstract (Chinese Version) --- p.vi / Abbreviation --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General Background --- p.1 / Chapter 1.1.1 --- Valienamine --- p.2 / Chapter 1.1.2 --- Valienamine Derivatives --- p.4 / Chapter 1.2 --- Mechanistic Aspects of Glycosidase Inhibition --- p.6 / Chapter 1.2.1 --- General Background --- p.6 / Chapter 1.2.2 --- Mechanism of Enzyme Catalyzed Hydrolysis of Glycosides --- p.6 / Chapter 1.2.3 --- Types of Glucosidase Inhibitors --- p.7 / Chapter 1.2.4 --- Inhibition of Glycosidases by Valienamine Derivatives --- p.8 / Chapter 1.3 --- Previous Synthesis of Valienamine --- p.9 / Chapter 1.3.1 --- Enantiospecific Synthesis of Valienamine by Vasella and co-workers --- p.9 / Chapter 1.3.2 --- Enantiospecific Synthesis of Valienamine by Tatsuta and co-workers --- p.10 / Chapter 1.3.3 --- Synthesis of N-Alkyl Derivatives of Valienamine --- p.12 / Chapter 1.4 --- Previous Syntheses of Valienamine-containing Pseudodisaccharides and its Diastereomers --- p.12 / Chapter 1.4.1 --- Epoxide aminolysis --- p.12 / Chapter 1.4.2 --- Condensation of amine with ketone --- p.15 / Chapter 1.4.3 --- Synthesis of pseudoaminodisaccharide by Kapp and co-workers --- p.16 / Chapter 2. --- Results and Discussion --- p.18 / Chapter 2.1 --- General Strategy --- p.18 / Chapter 2.2 --- Syntheses of coupling precursors --- p.20 / Chapter 2.2.1 --- Syntheses of protected valienamine 65 and its 2-epimer80 --- p.20 / Chapter 2.2.1.1 --- Synthesis of Diol68 --- p.20 / Chapter 2.2.1.2 --- Synthesis of Diol67 --- p.21 / Chapter 2.2.1.3 --- Synthesis of protected valienamine 65 and its 2-epimer80 --- p.22 / Chapter 2.2.2 --- Syntheses of 6-deoxyaminosugars 63 and118 --- p.24 / Chapter 2.2.2.1 --- Synthesis of benzyl ether91 --- p.24 / Chapter 2.2.2.2 --- Synthesis of β-diol103 --- p.28 / Chapter 2.2.2.3 --- Synthesis of α-alcohol107 --- p.30 / Chapter 2.2.2.4 --- Synthesis of β-diol113 --- p.31 / Chapter 2.2.2.5 --- Syntheses of amines 63 and118 --- p.33 / Chapter 2.2.3 --- Syntheses of allylic chlorides --- p.34 / Chapter 2.3 --- Syntheses of pseudoaminodissaccharides --- p.36 / Chapter 3. --- Conclusion --- p.42 / Chapter 4. --- Experimental --- p.44 / Chapter 5. --- References --- p.74 / Chapter 6. --- Appendix --- p.79 / List of spectra --- p.79

Oligodendrocyte progenitors are highly sensitive to oxidative insults. Among the factors postulated to contribute to this susceptibility are high levels of intracellular iron and low antioxidant content. During ischemia, the neurotransmitter dopamine (DA) is released and may contribute to oxidative stress and oligodendrocyte injury in the hypomyelinating disorder, periventricular leucomalacia (PVL). In this thesis, I investigated the role of iron in DA-induced toxicity in primary cultures of oligodendrocyte progenitors, and assessed the contribution of the antioxidant defenses (glutathione (GSH), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) and other survival factors (heat shock proteins and the protein kinase Akt) in determining the response of the cells to DA. / Addition of iron to cultures increased DA-induced expression of the stress protein heme oxygenase-1 (HO-1), and toxicity as assessed by mitochondrial activity, cellular release of lactate dehydrogenase, nuclear condensation and caspase-3 activation. In contrast, an iron chelator reduced these events. Furthermore, DA induced accumulation of superoxide, which was also reduced by the iron chelator. Surprisingly, a mimetic of the superoxide detoxifying enzyme, SOD potentiated DA toxicity, suggesting that generation of hydrogen peroxide via superoxide dismutation may be contributing to toxicity. Both a mimetic of the peroxide-scavenging enzyme, GPx and a GSH analog blocked DA-induced superoxide accumulation, HO-1 expression and caspase-3 activation. In addition, the GPx mimetic blocked caspase-3 activation induced by the combination of DA with iron. In contrast, an inhibitor of glutathione synthesis potentiated DA-induced HO-1 expression and cell death. / Finally, in further examining the cellular defense mechanisms, I found that various heat shock proteins increased in expression levels during oligodendroglial differentiation, however only heat shock protein-90 (HSP-90) was detected in oligodendrocyte progenitors. An HSP-90 inhibitor decreased activated Akt levels, induced caspase-3 activation, increased nuclear condensation, reduced oligodendrocyte progenitor viability, and potentiated DA-induced apoptosis. In addition, an Akt inhibitor alone exacerbated DA toxicity and in combination with the HSP-90 inhibitor caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. / In conclusion, elevated levels of iron, superoxide, deficient detoxification of peroxides by glutathione peroxidase and inadequate defense by glutathione contribute to the susceptibility of oligodendrocyte progenitors to DA-induced toxicity. On the other hand, HSP-90 alone or in concert with Akt play important roles in oligodendrocyte progenitors survival following an insult that produces oxidative stress.

No description available.

Parkash Amarender. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-172). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.xi / 撮要 --- p.xiv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.2 --- General Properties of RIPs --- p.5 / Chapter 1.2.1 --- Structure --- p.5 / Chapter 1.2.1.1 --- Type I and Type II RIPs --- p.5 / Chapter 1.2.1.2 --- Small RIPs --- p.10 / Chapter 1.2.2 --- Distribution --- p.12 / Chapter 1.2.3 --- Physicochemical properties --- p.15 / Chapter 1.3 --- Enzymatic activities of RIPs --- p.17 / Chapter 1.3.1 --- N-glycosidase activity --- p.17 / Chapter 1.3.2 --- Polynucleotide:adenosine glycosidase activity --- p.21 / Chapter 1.3.3 --- Ribonuclease (RNase) activity --- p.24 / Chapter 1.3.4 --- Deoxyribonucleolytic (DNase) activity --- p.25 / Chapter 1.3.5 --- Multiple depurination --- p.26 / Chapter 1.3.6 --- Inhibition of protein synthesis --- p.27 / Chapter 1.4 --- Biological activities of RIPs --- p.29 / Chapter 1.4.1 --- Interaction of ribosome-inactivating proteins with cells --- p.29 / Chapter 1.4.1.1 --- Internalization of type 1 ribosome-inactivating proteins --- p.29 / Chapter 1.4.1.2 --- Internalization of type 2 ribosome-inactivating proteins --- p.32 / Chapter 1.4.2 --- Effects on laboratory animals --- p.33 / Chapter 1.4.3 --- Immunosuppressive activity --- p.33 / Chapter 1.4.4 --- Abortifacient activity --- p.34 / Chapter 1.4.5 --- Antiviral activity --- p.35 / Chapter 1.5 --- Physiological roles of RIPs --- p.37 / Chapter 1.6 --- Applications of RIPs --- p.39 / Chapter 1.6.1 --- Possible uses in experimental and clinical medicine --- p.39 / Chapter 1.6.1.1 --- Anti-tumor therapy --- p.40 / Chapter 1.6.1.2 --- Immune disorders --- p.42 / Chapter 1.6.1.3 --- Neuroscience research --- p.43 / Chapter 1.6.2 --- Applications in agriculture --- p.44 / Chapter 1.7 --- Arginine/Glutamate Rich Polypeptides (AGRPs) --- p.46 / Chapter 1.8 --- Objectives of the present study --- p.48 / Chapter 1.8.1 --- Rationale of the study --- p.48 / Chapter 1.8.2 --- Outline of the thesis --- p.50 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Introduction --- p.52 / Chapter 2.2 --- Materials and methods --- p.54 / Chapter 2.2.1 --- Materials --- p.54 / Chapter 2.2.2 --- Preparation of crude extract --- p.55 / Chapter 2.2.3 --- Purification of proteins --- p.55 / Chapter 2.2.4 --- Molecular weight determination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.61 / Chapter 2.2.5 --- Protein determination --- p.64 / Chapter 2.2.6 --- N-terminal amino acid sequence --- p.64 / Chapter 2.2.7 --- Preparation of rabbit reticulocyte lysate --- p.65 / Chapter 2.2.8 --- Assay for cell-free protein synthesis- inhibiting activity --- p.65 / Chapter 2.2.9 --- Assay for N-glycosidase activity --- p.66 / Chapter 2.2.10 --- Assay for ribonuclease activity --- p.70 / Chapter 2.2.11 --- Assay for antifungal activity --- p.71 / Chapter 2.2.12 --- Assay for dehydrogenase activity --- p.71 / Chapter Chapter 3 --- Purification and characterization of proteins from their respective sources. / Chapter 3.1. --- Purification and Characterization of Hispidin from Hairy melon (Benincasa hispida var. chieh-qua) / Chapter 3.1.1. --- Introduction --- p.73 / Chapter 3.1.2. --- Results --- p.76 / Chapter 3.1.2.1. --- Purification --- p.78 / Chapter 3.1.2.2. --- Molecular weight determination --- p.84 / Chapter 3.1.2.3. --- N-terminal amino acid sequence --- p.85 / Chapter 3.1.2.4. --- Assay for cell-free protein synthesis-inhibiting activity --- p.86 / Chapter 3.1.2.5. --- Assay for N-glycosidase activity --- p.87 / Chapter 3.1.2.6. --- Assay for ribonuclease activity --- p.88 / Chapter 3.1.2.7. --- Assay for dihydrodiol dehydrogenase activity --- p.88 / Chapter 3.1.2.8. --- Assay for antifungal activity --- p.89 / Chapter 3.1.2.9. --- "Assessment of purity, yield and activity" --- p.91 / Chapter 3.1.3. --- Discussion --- p.92 / Chapter 3.2. --- Purification and Characterization of Momorchin from Dried Bitter Gourd (Momordica charantia) Seeds / Chapter 3.2.1. --- Introduction --- p.95 / Chapter 3.2.2. --- Results --- p.99 / Chapter 3.2.2.1. --- Purification --- p.100 / Chapter 3.2.2.2. --- Molecular weight determination --- p.103 / Chapter 3.2.2.3. --- N-terminal amino acid sequence --- p.104 / Chapter 3.2.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.105 / Chapter 3.2.2.5. --- Assay for ribonuclease activity --- p.105 / Chapter 3.2.2.6. --- Assay for N-glycosidase activity --- p.106 / Chapter 3.2.2.7. --- "Assessment of purity, yield and activity" --- p.107 / Chapter 3.2.3. --- Discussion --- p.108 / Chapter 3.3.3. --- Purification and Characterization of Luffacylin from Sponge Gourd (Luffa cylindrica) / Chapter 3.3.1. --- Introduction --- p.110 / Chapter 3.3.2. --- Results --- p.113 / Chapter 3.3.2.1. --- Purification --- p.115 / Chapter 3.3.2.2. --- Molecular weight determination --- p.119 / Chapter 3.3.2.3. --- N-terminal amino acid sequencing --- p.120 / Chapter 3.3.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.121 / Chapter 3.3.2.5. --- Assay for ribonuclease activity --- p.121 / Chapter 3.3.2.6. --- Assay for N-glycosidase activity --- p.122 / Chapter 3.3.2.7. --- Assay for antifungal activity --- p.123 / Chapter 3.3.2.8. --- "Assessment of purity, activity and yield" --- p.124 / Chapter 3.3.3. --- Discussion --- p.125 / Chapter 3.4. --- Purification and Characterization of α and β Benincasin from fresh Winter Melon {Benincasa hispida var. dong-gua) Seeds / Chapter 3.4.1. --- Introduction --- p.127 / Chapter 3.4.2. --- Results --- p.129 / Chapter 3.4.2.1. --- Purification --- p.130 / Chapter 3.4.2.2. --- Molecular weight determination --- p.135 / Chapter 3.4.2.3. --- N-terminal amino acid sequence --- p.136 / Chapter 3.4.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.137 / Chapter 3.4.2.5. --- Assay for ribonuclease activity --- p.137 / Chapter 3.4.2.6. --- Assay for antifungal activity --- p.138 / Chapter 3.4.2.7. --- "Assessment of purity, activity and yield" --- p.140 / Chapter 3.4.3. --- Discussion --- p.141 / Chapter 3.5. --- Purification and characterization of Moschins from Pumpkin (Cucurbita moschata) Seeds / Chapter 3.5.1. --- Introduction --- p.143 / Chapter 3.5.2. --- Results --- p.145 / Chapter 3.5.2.1. --- Purification --- p.146 / Chapter 3.5.2.2. --- Molecular weight determination --- p.149 / Chapter 3.5.2.3. --- N-terminal amino acid sequence --- p.150 / Chapter 3.5.2.4. --- Assay for cell-free protein synthesis- inhibiting activity --- p.151 / Chapter 3.5.2.5. --- Assay for ribonuclease activity --- p.151 / Chapter 3.5.2.6. --- "Assessment of purity, activity and yield" --- p.152 / Chapter 3.5.3. --- Discussion --- p.153 / Chapter Chapter 4 --- General Discussion and Conclusion --- p.154 / References --- p.158

Recent population studies provide evidence that individuals with high circulating insulin levels have a poor prognosis and/or increased risk of cancer development; however, laboratory studies concerning the role of insulin in breast cancer biology are sparse. We compared the growth of 4T1 murine breast cancer allografts in control mice, alloxan-induced hypoinsulinemic mice, and mice treated with the insulin/insulin-like growth factor-1 receptor tyrosine kinase inhibitor BMS-536924. Both interventions significantly decreased tumor growth versus control and decreased pathway activation downstream of the insulin receptor as reflected by Aktser473 phosphorylation status in the neoplastic tissue. Alloxan-treated mice exhibited signs of insulin deficiency, while BMS-536924-treated animals showed only minor metabolic derangements. Skeletal muscle displayed reduced pAktser473 in alloxan-treated mice. In contrast, BMS-536924 treatment increased pAktser473 in muscle. This raises the possibility that the relative lack of metabolic toxicity of BMS-536924 involves varying tissue levels of the drug. These results support the view that host insulin physiology is a potentially modifiable determinant of breast cancer behaviour.

No description available.