Man, Cheuk Him. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 219-263). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xvi / List of Publications --- p.xvii / Chapter Ch.l --- Introduction --- p.1 / Chapter 1.1 --- Aim of study --- p.1 / Chapter 1.2 --- Literature review --- p.3 / Chapter 1.2.1 --- Nasopharyngeal carcinoma (NPC) --- p.3 / Chapter 1.2.1.1 --- Structure and function of nasopharynx --- p.3 / Chapter 1.2.1.2 --- Histopathology of NPC --- p.3 / Chapter 1.2.1.3 --- Epidemiology of NPC --- p.4 / Chapter 1.2.2 --- Etiology of NPC --- p.6 / Chapter 1.2.2.1 --- Genetic factors --- p.6 / Chapter 1.2.2.2 --- Environment factors --- p.13 / Chapter 1.2.2.3 --- Epstein-Barr virus (EBV) infection --- p.14 / Chapter 1.2.3 --- Therapeutic treatment of NPC --- p.24 / Chapter 1.2.3.1 --- Radiotherapy (RT) --- p.24 / Chapter 1.2.3.2 --- Chemotherapy --- p.25 / Chapter 1.2.4 --- Notch signaling pathway --- p.26 / Chapter 1.2.4.1 --- Notch receptors and their ligands --- p.26 / Chapter 1.2.4.2 --- Activation of Notch signaling pathway --- p.29 / Chapter 1.2.4.3 --- Regulators of Notch signaling pathway --- p.32 / Chapter 1.2.4.4 --- Effectors of Notch signaling pathway --- p.32 / Chapter 1.2.5 --- Role of Notch signaling pathway in tumorigenesis --- p.33 / Chapter 1.2.5.1 --- Cell proliferation --- p.34 / Chapter 1.2.5.2 --- Cell survival --- p.35 / Chapter 1.2.5.3 --- Angiogenesis --- p.36 / Chapter 1.2.5.4 --- Cell invasion and metastasis --- p.36 / Chapter 1.2.6 --- Notch and oncogenic virus --- p.37 / Chapter 1.2.7 --- Crosstalk between Notch and other signaling pathways --- p.38 / Chapter 1.2.7.1 --- NFkB signaling pathway --- p.38 / Chapter 1.2.7.2 --- Ras signaling pathway --- p.39 / Chapter 1.2.7.3 --- Wnt signaling pathway --- p.40 / Chapter 1.2.7.4 --- Akt signaling pathway --- p.40 / Chapter 1.2.7.5 --- ErbB2 signaling pathway --- p.41 / Chapter 1.2.8 --- Notch as therapeutic target for cancer --- p.41 / Chapter Ch.2 --- Materials and Methods --- p.45 / Chapter 2.1 --- "Cell lines, xenografts and primary tumors" --- p.45 / Chapter 2.1.1 --- Cell lines --- p.45 / Chapter 2.1.2 --- Xenografts --- p.46 / Chapter 2.1.3 --- Primary tumors --- p.48 / Chapter 2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.1 --- Sample preparation for RT-PCR --- p.50 / Chapter 2.2.1.1 --- RNA extraction --- p.50 / Chapter 2.2.1.2 --- Quantitation of total RNA --- p.50 / Chapter 2.2.2 --- Conventional RT-PCR --- p.51 / Chapter 2.2.3 --- Quantitative RT-PCR --- p.51 / Chapter 2.3 --- Western immunoblot --- p.55 / Chapter 2.3.1 --- Protein extraction --- p.55 / Chapter 2.3.2 --- SDS-PAGE and immunoblotting --- p.55 / Chapter 2.4 --- Immunohistochemistry --- p.59 / Chapter 2.5 --- Cloning and plasmid DNA preparation --- p.62 / Chapter 2.5.1 --- Polymerase chain reaction (PCR) and purification of PCR products --- p.62 / Chapter 2.5.2 --- Restriction enzyme double digestion --- p.65 / Chapter 2.5.3 --- Ligation of plasmid and insert sequence --- p.65 / Chapter 2.5.4 --- Bacterial transformation --- p.66 / Chapter 2.5.5 --- Plasmid DNA extraction --- p.66 / Chapter 2.5.6 --- DNA sequencing --- p.67 / Chapter 2.6 --- Transient transfection of NPC cell lines --- p.67 / Chapter 2.7 --- Drug treatment on NPC cell lines --- p.69 / Chapter 2.8 --- Cell proliferation assays --- p.71 / Chapter 2.8.1 --- WST-1 assay --- p.71 / Chapter 2.8.2 --- BrdU assay --- p.71 / Chapter 2.9 --- Flow cytometry analysis --- p.72 / Chapter 2.9.1 --- Sample preparation --- p.72 / Chapter 2.9.2 --- Cell cycle analysis by propidium iodide staining --- p.73 / Chapter 2.9.3 --- Apoptosis analysis by AnnexinV-PI staining --- p.73 / Chapter 2.10 --- Apoptosis analysis by Caspase-3 activity assay --- p.74 / Chapter 2.11 --- RBP-Jk reporter assay --- p.75 / Chapter 2.12 --- NFKB1 reporter assay --- p.77 / Chapter 2.13 --- Dual luciferase reporter assay --- p.77 / Chapter 2.14 --- Expression array --- p.78 / Chapter 2.15 --- Statistical analysis --- p.79 / Chapter Ch.3 --- Characterization of Notch Signaling Molecules in NPC --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- "Expression of Notch ligands, receptors, effectors and regulators in NPC cell lines and xenografts" --- p.81 / Chapter 3.2.2 --- "Expression of Notch ligands, receptors, regulators and effectors in NPC primary tumors" --- p.104 / Chapter 3.3 --- Discussion --- p.111 / Chapter 3.3.1 --- Overexpression of Jagl and D114 in NPC --- p.112 / Chapter 3.3.2 --- Overexpression of Notch receptors in NPC --- p.114 / Chapter 3.3.3 --- "Downregulation of Negative regulator, Numb, in NPC" --- p.116 / Chapter 3.3.4 --- Overexpression of Notch effectors in NPC --- p.117 / Chapter 3.4 --- Summary --- p.119 / Chapter Ch.4 --- Mechanisms of Activation of Notch Signaling Pathway in NPC --- p.120 / Chapter 4.1 --- Introduction --- p.120 / Chapter 4.2 --- Results --- p.122 / Chapter 4.2.1 --- EBV mediated Notch activation --- p.122 / Chapter 4.2.1.1 --- No effect of EBERs and EBNA1 on the expression of Notch Components --- p.122 / Chapter 4.2.1.2 --- LMP1 induces expression of Notch components --- p.129 / Chapter 4.2.1.3 --- LMP2A induces expression of Notch components --- p.133 / Chapter 4.2.2 --- Effect of CXCR4 on Notch signaling pathway in C666-1 --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter 4.3.1 --- EBV-mediated induction of Notch components --- p.139 / Chapter 4.3.2 --- Regulation of Notch expression by CXCR4 signaling pathway --- p.142 / Chapter 4.4 --- Summary --- p.145 / Chapter Ch.5 --- Investigation of the Oncogenic Role of Notch3 --- p.146 / Chapter 5.1 --- Introduction --- p.146 / Chapter 5.2 --- Results --- p.148 / Chapter 5.2.1 --- Effect of knockdown Notch 1 by siRNA on the growth of C666-1 --- p.148 / Chapter 5.2.2 --- Effect of knockdown Notch3 by siRNA on the growth of C666-1 --- p.151 / Chapter 5.2.2.1 --- Effect of knockdown Notch3 by siRNA on the RBP-Jk promoter activity of C666-1 --- p.153 / Chapter 5.2.2.2 --- Effect of knockdown Notch3 by siRNA on the proliferation of C666-1 --- p.155 / Chapter 5.2.2.3 --- Effect of knockdown Notch3 by siRNA on cell cycle progression of C666-1 --- p.158 / Chapter 5.2.2.4 --- Effect of knockdown Notch3 by siRNA on resistant to apoptosis in C666-1 --- p.160 / Chapter 5.2.3 --- Investigation of the anti-proliferation effect of therapeutic agents targeting Notch signaling pathway in NPC cells --- p.168 / Chapter 5.2.3.1 --- "Effect of DAPT on the proliferation of HEK293T, C666-1 and HK-1" --- p.168 / Chapter 5.2.3.2 --- Effect of AMD3100 on Notch signaling pathway and proliferation of NPC cells --- p.172 / Chapter 5.2.4 --- Study of downstream targets of Notch3 in NPC cells --- p.178 / Chapter 5.3 --- Discussion --- p.200 / Chapter 5.3.1 --- Oncogenic role of Notch3 in C666-1 --- p.200 / Chapter 5.3.2 --- Potential therapeutic approach in treating NPC via Notch inhibition --- p.206 / Chapter 5.3.2.1 --- "Gamma secretase inhibitor, DAPT" --- p.206 / Chapter 5.3.2.2 --- "CXCR4 antagonist, AMD3100" --- p.207 / Chapter 5.4 --- Summary --- p.209 / Chapter Ch.6 --- General Discussion --- p.210 / Chapter Ch.7 --- Conclusion --- p.217 / Reference --- p.219 / Appendices --- p.263 / Appendix 1 Summary of immunohistochemical staining results on 23 primary NPC samples --- p.264 / Appendix 2 Summary of 581 selected genes from the expression array --- p.265