Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 99-131).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 92-112). Also available in print.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 67-89).

Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate its expression. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-é is an in-vitro Cdk7 kinase and that neuroblastoma cells proliferate via a PKC-é/Cdk7/cdk2 cell signaling pathway. ICA-1 specifically inhibits the activity of PKC-é but not that of PKC-zeta (PKC-æ), the closely related atypical PKC family member. The IC50 for the kinase activity assay was approximately 0.1µM which is 1000 times less than that of aurothiomalate, a known PKC-é inhibitor. The phosphorylation of Cdk7 by PKC-é was potently inhibited by ICA-1. ICA-1 mediates its antiproliferative effects on neuroblastoma cells by inhibiting the PKC-é/Cdk7/cdk2 signaling pathway. ICA-1 (0.1µM) inhibited the in-vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-é inhibitor and chemotherapeutic agent for neuroblastoma. Bcr-Abl has been shown to regulate the activation of the transcription factor ELK-1 which in turn regulates the expression of PKC-é. Alternatively, we hypothesize that PKC-é can self regulate its expression by indirectly regulating the activity of Elk-1 in an ERK1 dependent manner. Our preliminary data shows that there was robust increase in the expression as well as association of PKC-é and Elk-1 in actively proliferating neuroblastoma cells suggesting a potential role of PKC-é in regulating the activity of Elk-1. Analysis of the subcellular fractions also presented a similar increase in the association between PKC-é and Elk-1 in the nuclear fraction of actively proliferating cells as compared to cytoplasm. Interestingly, the nuclear expression of PKC-é was also found to be higher in these cells, suggesting that PKC-é translocated to the nucleus in actively proliferating cells and regulated the transcriptional activity of Elk-1. However, our data from in-vitro kinase activity demonstrated that PKC-é was not an Elk-1 kinase but that it increased the phosphorylation of Elk-1 in the presence of ERK1, an upstream kinase of Elk-1 in the Bcr-Abl mediated regulatory pathway of PKC-é. This suggested that ERK1 was integral to the self-regulatory activity of PKC-é. In conclusion, we hypothesize that the self-regulatory mechanism of PKC-é is initiated by the translocation PKC-é into the nucleus where it activates ERK1. This promotes the activation of its downstream target Elk-1 which subsequently upregulates the expression of PKC-é

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Ovarian cancer is one of the most deadly female malignancies. Despite advances in the treatment of ovarian cancer for the past decade, the cure rate of this disease is moderately improved. Emerging evidence suggests the molecular personalized therapeutic approach become popular for human cancer treatment. The nuclear factor-kappa B (NF-κB) signaling pathway has been shown to play multiple roles in cancer progression such as anti-apoptosis, cell cycle, angiogenesis and metastasis. This study attempted to characterize the functional roles of transforming growth factor (TGF)-β-activating kinase 1 (TAK1) in the activation of NF-κB signaling. Importantly, this study provided evidence showing the significance of TAK1-NF-κB signaling axis in ovarian cancer aggressiveness during omental metastasis. Using quantitative RT-PCR and immunohistochemical analyses, TAK1 was frequently up-regulated and was significantly associated with high-grade (P=0.001), lymph node and distant metastasis (P=0.025), as well as a tendency toward advanced stage ovarian cancers (P=0.08). Functionally, enforced expression of TAK1 could augment cell proliferation, colony formation, anchorage-independent growth ability and migration/invasion in ovarian cancer cells. Conversely, repression of TAK1 expression by genetically or pharmaceutical approach abrogated these tumorigenic capacities including tumor growth in vivo. Furthermore, co-treatment of (5Z) -7-Oxozeaenol could sensitize ovarian cancer cells to cisplatin-induced cell apoptosis, indicating TAK1 is also involved in chemoresistance. Mechanistically, using Western blotting and NF-κB -reporter luciferase analyses, the elevation of TAK1 phosphorylation at Ser412 but not Thr184/187 was found to associate with the activation of NF-κB in ovarian cancer cells solely. A series of functional studies with genetic and pharmaceutical alterations revealed that the increased TAK1 Ser412 phosphorylation was required for exerting the ovarian cancer cell oncogenesis. Omental metastasis is the common phenomenon observed in most of advanced-stage ovarian cancer. Using omentum conditioned medium (OCM), the findings of this study showed that the omentum tissue was able to secrete numerous factors including chemokines such as GRO-α and IL8 in activating TAK1-NF-κB signaling cascade, which thereby induced increased oncogenic capacities in cell growth, migration and invasion. Taken together, this study suggests that TAK1-NF-κB signaling axis is indispensable for promoting oncogenesis of ovarian cancer and targeting this pathway may be a promising personalized cancer therapeutic approach in ovarian cancer. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Presenilin-1 (PS1) mutations caused by the PSEN1 gene mutations are the major cause of early onset familial Alzheimer’s disease (EOFAD). Two Chinesespecific EOFAD related PS1 mutations, V97L and A136G, have been found. Studies suggested that V97L mutation lead to the overexpression of Aβ42 and tau hyperphosphorylation, which are the major hallmarks of Alzheimer’s disease (AD), while properties of A136G were unclear. Since calcium dysregulation was suggested to play an important role in AD, the research project investigated if V97L and A136G mutations also lead to altered endoplasmic reticulum (ER) 〖Ca〗^(2+) regulation. SH-SY5Y cells transduced with retrovirus carrying V97L mutant or A136 mutant PSEN1 were used as the experiment models. In Western blotting, while the PS1 expression level was unaffected in V97L mutant, the expression level was significantly lower in A136G mutant. In carbachol (CCh) perfusion experiment, V97L mutant was found to exaggerate ER 〖Ca〗^(2+) release when stimulated by higher concentration (30, 100 and 300 μM) CCh, while A136G mutant exaggerated ER Ca2+ release when stimulated by 30 μM and 300 μM CCh, but not 100 μM CCh. In 5% fetal bovine serum (FBS) perfusion experiment, both V97L and A136G mutants were found to sensitize 〖Ca〗^(2+) oscillation, which the sensitization effect of V97L was 3 folds of A136G. The results suggested that V97L mutation exaggerates ER 〖Ca〗^(2+) release, possibly via interaction with IP3R. However the results of A136G were inconclusive and contradicting, therefore further investigation is needed. / published_or_final_version / Physiology / Master / Master of Medical Sciences