Meng Jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Abbreviation --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Black Mustard and Sinigrin --- p.2 / Chapter 1.2 --- Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Different Stages of HCC --- p.6 / Chapter 1.2.2 --- Risk Factors --- p.8 / Chapter 1.2.3 --- Treatments of HCC --- p.10 / Chapter 1.3 --- Biomarkers Used to Evaluate Effects of Sinigrin on HCC --- p.12 / Chapter 1.3.1 --- AST & ALT --- p.12 / Chapter 1.3.2 --- Glutathione S Transferase -p (GST-p) --- p.13 / Chapter 1.4 --- Tumor Suppressor Genes and Oncogenes --- p.14 / Chapter 1.4.1 --- "p53, the Tumor Suppressor Gene" --- p.15 / Chapter 1.4.2 --- p53-dependent pathway --- p.15 / Chapter 1.4.2.1 --- Mdm2 --- p.16 / Chapter 1.4.2.2 --- Bax and Bcl-2 --- p.17 / Chapter 1.4.2.3 --- PCNA and p21wAF1/CIP1 --- p.18 / Chapter 1.5 --- Aim of the Project --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- In vitro Studies --- p.21 / Chapter 2.1.1 --- Neutral Red Assay --- p.21 / Chapter 2.1.1.1 --- Chemicals and Reagents --- p.21 / Chapter 2.1.1.2 --- Liver Cells --- p.23 / Chapter 2.1.1.3 --- Neutral Red Assay --- p.24 / Chapter 2.1.2 --- Flow Cytometery --- p.24 / Chapter 2.1.2.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2.2 --- Flow Cytometery Analysis --- p.25 / Chapter 2.1.3 --- DNA Fragmentation --- p.26 / Chapter 2.1.3.1 --- Chemicals and Reagents --- p.26 / Chapter 2.1.3.2 --- DNA Extraction --- p.28 / Chapter 2.1.3.3 --- DNA Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.4 --- cDNA Microarray --- p.29 / Chapter 2.1.4.1 --- Chemicals and Reagents --- p.30 / Chapter 2.1.4.2 --- RNA Extraction --- p.33 / Chapter 2.1.4.3 --- RNA Quantity and Quality Control --- p.34 / Chapter 2.1.4.4 --- RT-PCR --- p.35 / Chapter 2.1.4.5 --- cRNA Convention and Purification --- p.36 / Chapter 2.1.4.6 --- Hybridization --- p.37 / Chapter 2.1.4.7 --- Washing and Detection --- p.37 / Chapter 2.1.4.8 --- Data Analysis --- p.38 / Chapter 2.2 --- In vivo Studies --- p.39 / Chapter 2.2.1 --- Animal Treatment --- p.39 / Chapter 2.2.1.1 --- Chemicals and Reagents --- p.39 / Chapter 2.2.1.2 --- Chemical Carcinogens --- p.40 / Chapter 2.2.1.3 --- Promotion Stage --- p.41 / Chapter 2.2.1.4 --- Progression Stage --- p.44 / Chapter 2.2.2 --- Measurement of Serum ALT and AST Activities --- p.46 / Chapter 2.2.2.1 --- Chemicals and Reagents --- p.46 / Chapter 2.2.2.2 --- Activity Assay --- p.46 / Chapter 2.2.3 --- Histological Analysis --- p.47 / Chapter 2.2.3.1 --- Chemicals and Reagents --- p.47 / Chapter 2.2.3.2 --- Preparation of Slides --- p.49 / Chapter 2.2.3.3 --- H&E Staining --- p.49 / Chapter 2.2.3.4 --- GST-p Immuno-staining --- p.50 / Chapter 2.2.4 --- Semi-Quantitative RT-PCR Analysis of mRNA Expression --- p.53 / Chapter 2.2.4.1 --- Chemicals and Reagents --- p.53 / Chapter 2.2.4.2 --- Extraction of total RNA from rat liver --- p.53 / Chapter 2.2.4.3 --- Quantity and Quality Control of RNA --- p.53 / Chapter 2.2.4.4 --- RT-PCR (Reverse Transcription) --- p.54 / Chapter 2.2.4.5 --- PCR --- p.54 / Chapter 2.2.4.6 --- DNA gel electrophoresis --- p.55 / Chapter 2.2.4.7 --- Data Analysis --- p.56 / Chapter 2.2.5 --- Western Blot Analysis for Biomarkers --- p.56 / Chapter 2.2.5.1 --- Chemicals and Reagents --- p.56 / Chapter 2.2.5.2 --- Extraction of the Cytosol Protein --- p.60 / Chapter 2.2.5.3 --- Extraction of the Nuclear protein --- p.61 / Chapter 2.2.5.4 --- SDS Gel Electrophoresis --- p.61 / Chapter 2.2.5.5 --- Western Blot --- p.62 / Chapter 2.2.5.6 --- Interaction with Antibodies --- p.63 / Chapter 2.2.5.7 --- ECL Detection --- p.63 / Chapter 2.2.5.8 --- Data Analysis --- p.64 / Chapter Chapter 3: --- Results --- p.65 / Chapter 3.1 --- In vitro Studies --- p.66 / Chapter 3.1.1 --- Cell Viability test and IC50 --- p.66 / Chapter 3.1.2 --- Cell Cycle Analysis --- p.68 / Chapter 3.1.3 --- DNA Fragmentation --- p.71 / Chapter 3.1.4 --- Effects of Sinigrin on Gene Expression --- p.73 / Chapter 3.2 --- In vivo Studies --- p.77 / Chapter 3.2.1 --- Effects of Sinigrin on HCC Development (Promotion stage) in Rats --- p.77 / Chapter 3.2.1.1 --- Direct Observation --- p.77 / Chapter 3.2.1.2 --- Relative Liver / Body Weight Ratio --- p.79 / Chapter 3.2.1.3 --- AST/ALT Assay --- p.81 / Chapter 3.2.1.4 --- Basic Structure of Hepatocytes --- p.83 / Chapter 3.2.1.5 --- GST-p Foci Area --- p.85 / Chapter 3.2.1.6 --- mRNA Expression of p53 and Mdm2 --- p.88 / Chapter 3.2.1.7 --- Protein Expression of Biomarkers --- p.90 / Chapter 3.2.2 --- Effects of Sinigrin on HCC Development (Progression stage) in Rats --- p.97 / Chapter 3.2.2.1 --- Direct Observation --- p.97 / Chapter 3.2.2.2 --- Relative Liver / Body Weight Ratio --- p.99 / Chapter 3.2.2.3 --- AST/ALT Assay --- p.101 / Chapter 3.2.2.4 --- Basic Structure of Hepatocytes --- p.103 / Chapter 3.2.2.5 --- GST-p Foci Area --- p.105 / Chapter 3.2.2.6 --- mRNA Expression of p53 and Mdm2 --- p.108 / Chapter 3.2.2.7 --- Protein Expression of Biomarkers --- p.110 / Chapter Chapter 4: --- Discussion --- p.116 / Chapter 4.1 --- Protective and Therapeutic Benefits of Sinigrin --- p.117 / Chapter 4.1.1 --- Effects of SIN on Cancer and Normal Cells --- p.117 / Chapter 4.1.2 --- Effective Tumor Induction by DEN-CC14 Treatment --- p.118 / Chapter 4.1.3 --- Protective Effect of SIN in the Promotion Stage of HCC --- p.118 / Chapter 4.1.4 --- Therapeutic Effect of SIN in the Progression Stage of HCC --- p.119 / Chapter 4.2 --- Biological Activities of SIN --- p.121 / Chapter 4.3 --- Summary --- p.134 / References --- p.xiv

Lau Ven Gie Vengie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-121). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 論文摘要 --- p.iv / TABLE OF CONTENTS --- p.vi / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.x / LIST OF ABBREVIATIONS --- p.xi / INTRODUCTION --- p.1 / Chapter I --- Hepatocellular Carcinoma --- p.1 / Risk factors --- p.1 / Stages in chemical carcinogenesis --- p.2 / Initiation --- p.2 / Promotion --- p.3 / Progression --- p.5 / Treatment of hepatocarcinoma --- p.6 / Chemotherapy ´ؤ hepatic arterial infusion (HAI) --- p.6 / Trans-arterial chemoembolization (TACE) --- p.7 / Radiofrequency ablation (RFA) --- p.8 / Percutaneous ethanol injection (PEI) --- p.9 / Liver resection --- p.9 / Liver transplantation --- p.10 / Chapter II --- Molecular Mechanisms: Oncogenes and Tumor-suppressor genes --- p.11 / Cell cycle control --- p.12 / p53 mutation in HCC --- p.13 / Normal functions of p53 and its target genes --- p.13 / p21(Wafl/Cipl/Sdil) --- p.13 / PCNA --- p.14 / Bcl-2 and Bax: the Bcl-2 family --- p.14 / Mdm2 --- p.17 / Chapter III --- Evaluation of the effects of hepatocarcinogenesis --- p.19 / GST-Pi --- p.19 / AST & ALT --- p.19 / Chapter IV --- Traditional Chinese Medicine (TCM) --- p.21 / Andrographis Paniculata --- p.21 / Pharmacological properties of andrographolide --- p.23 / Chapter V --- Aim of the project --- p.26 / MATERIALS AND METHODS --- p.27 / Chapter 1 --- Effects of andrographolide on cell viability and cell cycle --- p.27 / Chapter 1.1 --- Materials and solutions --- p.27 / Chapter 1.2 --- Preparation of solutions --- p.28 / Chapter 1.3 --- Procedures --- p.29 / Chapter 1.3.1 --- Seeding cells into culture flask --- p.29 / Chapter 1.3.2 --- Subculturing technique --- p.30 / Chapter 1.3.3 --- Neutral red assay --- p.30 / Chapter 1.3.4 --- DNA purification of HepG2 cells --- p.31 / Chapter 1.3.5 --- DNA gel electrophoresis --- p.32 / Chapter 1.3.6 --- Flow cytometry --- p.32 / Chapter 2 --- Effects of andrographolide on gene expressions --- p.33 / Chapter 2.1 --- Materials and solutions --- p.33 / Chapter 2.2 --- Preparation of solutions --- p.34 / Chapter 2.3 --- Procedures --- p.35 / Chapter 2.3.1 --- Cell treatments --- p.35 / Chapter 2.3.2 --- mRNA extraction from cell --- p.35 / Chapter 2.3.3 --- Determination of total RNA yield and quality yield --- p.36 / Chapter 2.3.4 --- RNA formaldehyde agarose gel electrophoresis --- p.36 / Chapter 2.3.5 --- cDNA synthesis --- p.37 / Chapter 2.3.6 --- "cRNA synthesis, labeling and amplification" --- p.39 / Chapter 2.3.7 --- cRNA purification --- p.40 / Chapter 2.3.8 --- Oligo GEArray hybridization --- p.41 / Chapter 2.3.9 --- Chemiluminescent detection --- p.43 / Chapter 2.3.10 --- Data analysis --- p.44 / Chapter 3 --- Effects of andrographolide on hepatocarcinogenesis in rats --- p.45 / Chapter 3.1 --- Materials and solutions --- p.45 / Chapter 3.2 --- Preparation of solutions --- p.46 / Chapter 3.3 --- Procedures --- p.47 / Chapter 3.3.1 --- Animal treatment --- p.47 / Chapter 3.3.2 --- Promotion (Experiment 1) --- p.48 / Chapter 3.3.3 --- Progression (Experiment 2) --- p.49 / Chapter 3.3.4 --- Extraction of blood serum --- p.52 / Chapter 3.3.5 --- Measurement of absorbance --- p.52 / Chapter 3.3.6 --- Tissue processing --- p.53 / Chapter 3.3.7 --- Hematoxylin and Eosin (H&E) Staining --- p.53 / Chapter 3.3.8 --- Immunohistochemical staining of GST-P --- p.54 / Chapter 3.3.9 --- Examination of liver sections --- p.55 / Chapter 4 --- "Effects of andrographolide on the expressions of Mdm2, p53, PCNA, Bax, Bcl-2 & p21" --- p.56 / Chapter 4.1 --- Materials and solutions --- p.56 / Chapter 4.2 --- Preparation of solutions --- p.57 / Chapter 4.3 --- Procedures --- p.59 / Chapter 4.3.1 --- Total mRNA extraction from liver --- p.59 / Chapter 4.3.2 --- Reverse transcription of mRNA to cDNA --- p.59 / Chapter 4.3.3 --- Protocol for polymerase chain reaction (PCR) --- p.60 / Chapter 4.3.4 --- DNA gel electrophoresis --- p.61 / Chapter 4.3.5 --- Nuclear protein extraction --- p.61 / Chapter 4.3.6 --- Cytosolic protein extraction --- p.62 / Chapter 4.3.7 --- Determination of protein concentration --- p.62 / Chapter 4.3.8 --- Immunoprecipitation of p53 from liver nuclear protein --- p.62 / Chapter 4.3.9 --- Protein gel electrophoresis by SDS-PAGE --- p.63 / Chapter 4.3.10 --- Western blotting --- p.64 / RESULTS --- p.66 / Chapter 1 --- Effects of andrographolide on cell viability and cell cycle --- p.66 / Chapter 2 --- Effects of andrographolide on gene expressions --- p.76 / Chapter 3 --- Effects of andrographolide on hepatocarcinogenesis in rats --- p.79 / Chapter 4 --- "Effects of andrographolide on the expressions of Mdm2, p53, PCNA, Bax, Bcl-2 & p21" --- p.91 / DISCUSSION --- p.102 / CONCLUSION --- p.111 / REFERENCES --- p.113

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

慢性乙型肝炎病毒（HBV）感染是肝癌發生的重要因素，其中乙肝病毒X蛋白（HBx）在這一過程起著關鍵作用。研究發現，一些HBV變體和HBx突變具有更高致癌風險，而且這些變體和突變存在地區差異。香港是HBV感染高發地帶，因此本研究目的是從這一地區120個肝癌組織標本中篩查出HBx突變位點。我們用巢式PCR從84.16% (101/120)的標本中提取和擴增了HBx，並進行基因測序。三種HBx突變被檢測出，包括點突變，遠端羧基端截斷和缺失突變。其中點突變位點有39個，特別的是在50%的標本中檢測出A1630G/G1721A 和 A1762T/G1764A雙突變。在31.68% (32/101)的標本中發現遠端羧基端截斷，以及在2.97% (3/101)的標本中檢測出缺失突變。總之，大多數突變集中在HBx轉錄啟動域，表明這些突變在肝癌發生中可能起著重要作用。 / 缺氧誘導因數-1α（HIF-1α）在肝癌的發生和發展中也起著重要作用。研究發現，野生型HBx可以啟動HIF-1α，但是變異型HBx和HIF-1α的關係還沒有研究清楚。我們研究表明HBx轉錄啟動域是必需而且足夠啟動HIF-1α的。在這個區域的突變中，雙突變K130M/V131Z增強HBx對HIF-1α的活性，但遠端羧基端截斷和缺失突變削弱其功能。進一步研究發現，羧基端特別是119-140氨基酸對HBx的穩定和功能非常重要。肝癌標本中，我們也發現HBx和HIF-1α的表達呈正相關。因此，雖然不同的突變對於HBx的功能有不同的影響，但總的來說這些突變可以促進HIF-1α的表達和啟動，進而導致肝癌患者的預後不良。 / 靶向治療在肝癌綜合治療中扮演重要角色。索拉菲尼（Sorafenib）是一種多激酶抑制劑，臨床實驗發現它對晚期肝癌治療有效，但其抑制腫瘤血管生成機制還不完全清楚。我們研究發現Sorafenib明顯而且劑量依賴性地降低HIF-1α的表達和活化，進而抑制血管內皮生長因數（VEGF）的表達。Sorafenib抑制mTOR, ERK, p70S6K, RP-S6, eIF4E和4E-BP1等翻譯起始因數的磷酸化，從而抑制HIF-1α的合成而不影響其降解。體外實驗進一步發現Sorafenib降低HIF-1α和VEGF的表達，從而抑制腫瘤的血管形成和生長。總之，我們的研究表明sorafenib可能通過阻斷mTOR/p70S6K/4E-BP1 和 ERK 信號通路來抑制HIF-1α的合成，從而發揮其抗腫瘤血管生成作用。 / Chronic HBV infection is the leading cause of hepatocellular carcinoma (HCC) and HBx plays a crucial role in the molecular pathogenesis of HBV-related HCC. Previous investigations have indicated that some variations of HBV or mutations of HBx are associated with higher risk of HCC development, whereas the mutations profiles may be disparate in different regions. In the present studies, we thus aim to screen and identify the HBx mutation hotspots in 120 HCC tissues from Hong Kong, a region with HBV hyper-endemic. HBV DNAs were successfully isolated and amplified in 84.16% (101/120) HCC specimens via nest-PCR, and then subjected to gene sequencing. Three types of HBx mutations, including point mutations, distal carboxyl-terminal truncations and deletion mutations, were discovered. Among the point mutations, 39 mutation hotspots were indentified, with two double mutations (A1630G/G1721A and A1762T/G1764A) occurring in approximate 50% of 101 HCC cases. Distal C-terminal truncated mutations were discovered in 31.68% (32/101) of HCC cases, whereas deletion mutations were detected in 2.97% (3/101) of them. Overall, majority of identified mutations were located at the transactivation domain of HBx, suggesting the crucial roles of these mutations in HCC development. / Hypoxia-inducible factor-1α (HIF-1α) also closely involves in the development and progression of HCC. Wild-type HBx has been shown to activate HIF-1α. But the relationship between HBx mutants and activation of HIF-1α has not been fully elucidated. We here revealed that the transactivaiton domain of HBx was necessary and sufficient to activate HIF-1α. Double mutations K130M/V131Z in this domain enhanced the functionality of HBx in upregulating the expression and the activation of HIF-1α, whereas C-terminal truncations and deletion mutations weakened this prosperity of HBx. We further uncovered that the C-terminus, especially the region of amino acids 119-140, was essential for the stability and transactivation of HBx. The positive association between the HBx mutants and HIF-1α was found in the HCC tissue samples. Therefore, although mutations exerted different effects on the functionality of HBx, the overall activity of HBx mutants was suggested to upregulate HIF-1α, whose level is related to poor prognosis of HCC patients. / The therapy targeting a critical molecule in the development of HCC such as HIF-1α may be a potential and effective treatment regimen for HCC patients. Sorafenib, a multikinase inhibitor, has demonstrated promising results for the treatment of advanced HCC in clinical trials, but the mechanism that accounts for the anti-angiogenic efficiency of this agent has not been fully elucidated. We here revealed that sorafenib remarkably and dose-dependently decreased the expression and the transcriptional activity of HIF-1α, and its target gene, vascular endothelial grow factor (VEGF). Further analysis revealed that this reduction of HIF-1α by sorafenib was caused by the inhibition of HIF-1α protein synthesis rather than by the promotion of HIF-1α protein degradation. Moreover, the phosphorylated levels of mTOR, ERK, p70S6K, RP-S6, eIF4E and 4E-BP1 were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease of tumor vascularisation and growth. Collectively, our data suggest that sorafenib may exhibit anti-angiogenic activity by inhibiting HIF-1α synthesis, which is likely to be achieved through suppressing the phosphorylation of mTOR/p70S6K/4E-BP1 and ERK. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liu, Liping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 133-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Publications --- p.VI / Acknowledgements --- p.VII / Abbreviations --- p.IX / List of Figures --- p.XI / List of Tables --- p.XIII / Table of Contents --- p.XIV / Chapter Chapter I --- General Introduction --- p.1 / Chapter 1.1 --- Overview of Hepatocellular Carcinoma --- p.1 / Chapter 1.2 --- HBV Infection and HCC Development --- p.6 / Chapter 1.3 --- Overview on Hepatitis B virus X Protein --- p.10 / Chapter 1.4 --- Roles of Hypoxia-inducible Factors in HCC --- p.17 / Chapter 1.5 --- Targeted Therapies and Sorafenib --- p.27 / Chapter Chapter II --- Identification of HBx Mutation Hotspots in HCC Tissues --- p.31 / Chapter 2.1 --- Abstract --- p.31 / Chapter 2.2 --- Introduction --- p.32 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.4 --- Results --- p.40 / Chapter 2.5 --- Discussion --- p.53 / Chapter Chapter III --- The Relationship between HBx Mutants and HIF-1α --- p.59 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.63 / Chapter 3.4 --- Results --- p.70 / Chapter 3.5 --- Discussion --- p.91 / Chapter Chapter IV --- The Effects of Sorafenib on Hypoxia-inducible Factor-1α --- p.96 / Chapter 4.1 --- Abstract --- p.96 / Chapter 4.2 --- Introduction --- p.98 / Chapter 4.3 --- Materials and Methods --- p.101 / Chapter 4.4 --- Results --- p.108 / Chapter 4.5 --- Discussion --- p.124 / Chapter Chapter V --- Conclusion and Future Plans --- p.129 / Chapter 5.1 --- Conclusion --- p.129 / Chapter 5.2 --- Future Plans --- p.131 / References --- p.133

Yau Mei-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 96-112). / Abstracts in English and Chinese. / Abstract / Abstract (Chinese Version) / Acknowledgements / List of Abbreviations / Table of Contents / List of Tables / List of Figures / Chapter Chapter 1 --- Introduction / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Hepatocellular Carcinoma / Chapter 2.2 --- Epidermal Growth Factor Receptor / Chapter 2.2.1 --- Activation of Epidermal Growth Factor Receptor / Chapter 2.2.2 --- Epidermal Growth Factor Receptor Signaling Pathways / Chapter 2.2.3 --- Expression Level and Patient Survival / Chapter 2.2.4 --- Epidermal Growth Factor Receptor Activity and Tumor Cell Growth / Chapter 2.2.5 --- Epidermal Growth Factor Receptor Activity and Radiation / Chapter 2.3 --- "Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, ZD1839" / Chapter 2.3.1 --- Tumor Cell Growth Control Activities of ZD1839 / Chapter 2.3.2 --- Factors Affecting the Tumor Cell Growth Control Activities of ZD1839 / Chapter 2.3.3 --- Radiosensitization Activities of ZD1839 / Chapter 2.3.4 --- Factors Affecting the Radiosensitization Activities of ZD1839 / Chapter 2.4 --- Study Objectives / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- ZD1839 / Chapter 3.2 --- Cell lines and Cell Culture / Chapter 3.3 --- Immunoblot Analysis / Chapter 3.3.1 --- Total Protein Extraction / Chapter 3.3.2 --- Protein Amount Determination / Chapter 3.3.3 --- Protein Separation / Chapter 3.3.4 --- Blotting / Chapter 3.3.5 --- Antibody Labeling / Chapter 3.3.6 --- Detection of Antibody Binding / Chapter 3.4 --- Cytotoxicity Assay / Chapter 3.5 --- Nucleotide sequence analysis / Chapter 3.5.1 --- Total RNA Extraction / Chapter 3.5.2 --- RNA Amount Determination / Chapter 3.5.3 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) / Chapter 3.5.3.1 --- Reverse Transcription / Chapter 3.5.3.2 --- High Fidelity Polymerase Chain Reaction / Chapter 3.5.4 --- Purification of PCR Product / Chapter 3.5.5 --- Cycle Sequencing Reaction / Chapter 3.5.6 --- DNA Precipitation and Sequencing / Chapter 3.6 --- Clonogenic Assay / Chapter 3.7 --- Immunohistochemical Analysis / Chapter Chapter 4 --- Results / Chapter 4.1 --- Immunoblot Analysis / Chapter 4.2 --- Cytotoxicity Assay / Chapter 4.2.1 --- Effect of ZD 1839 on cell morphology / Chapter 4.2.2 --- Effect of ZD 1839 on cell growth / Chapter 4.3 --- Nucleotide sequence analysis / Chapter 4.3.1 --- RNA Concentration of HCC cells / Chapter 4.3.2 --- Sequencing of TK domain within EGFR / Chapter 4.3.3 --- Sequencing of TK domain within HER2 / Chapter 4.4 --- Clonogenic assay / Chapter 4.4.1 --- Effects of ZD 1839 pre-treatment on radiation response / Chapter 4.4.2 --- Effects of ZD 1839 continuous treatment on radiation response / Chapter 4.5 --- Immunohistochemical Analysis / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Important Findings / Chapter 5.2 --- EGFR Expression of HCC Cells / Chapter 5.3 --- Cytotoxicity of ZD1839 on HCC Cell Lines / Chapter 5.4 --- Factors Affecting the Cytotoxicity of ZD1839 / Chapter 5.4.1 --- Effect of EGFR Expression on ZD1839 Cytotoxicity / Chapter 5.4.2 --- Effect of EGFR Mutations on ZD 1839 Cytotoxicity / Chapter 5.4.3 --- Effect of HER2 Expression on ZD1839 Cytotoxicity / Chapter 5.4.4 --- Effect of HER2 Mutations on ZD 1839 Cytotoxicity / Chapter 5.5 --- Radiation Response ofHCC Cell Lines upon ZD1839 Treatment / Chapter 5.6 --- Factors Affecting Radiation Response of ZD1839-treated HCC Cell Lines / Chapter 5.6.1 --- Effect of Growth Arrest on Radiation Response of HCC Cell Lines / Chapter 5.6.2 --- Other Factors Affecting Radiation Response of HCC Cell Lines / Chapter Chapter 6 --- Conclusion / References

Ng, Po Yan. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 109-120). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p.iv / List of Figures --- p.viii / List of Tables --- p.xi / List of Abbreviation --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- A General Introduction to the Development and Clinical Activities of Platinum Drugs --- p.1 / Chapter 1.1.1 --- Platinum Drugs used in a Clinical Setting --- p.4 / Chapter 1.1.2 --- Platinum Drugs under Clinical Trials --- p.5 / Chapter 1.1.3 --- Platinum Compounds with Dual Mechanisms --- p.7 / Chapter 1.2 --- Platinum Drug Antitumor Mechanism --- p.9 / Chapter 1.3 --- Limitations of Platinum Drugs --- p.12 / Chapter 1.3.1 --- Toxicity --- p.12 / Chapter 1.3.2 --- Drug Resistance or Cross Resistance --- p.15 / Chapter 1.3.2.1 --- Reduced Drug Accumulation or Increased Drug Efflux --- p.16 / Chapter 1.3.2.2 --- Drug Inactivation --- p.18 / Chapter 1.3.2.3 --- Enhanced DNA Repair --- p.19 / Chapter 1.4 --- Why Combinational Therapy? --- p.21 / Chapter 1.4.1 --- Antimetabolites --- p.20 / Chapter 1.4.2 --- Topoisomerase Inhibitors --- p.22 / Chapter 1.4.3 --- Tubulin-Active Antimitotic Agents --- p.24 / Chapter 1.4.4 --- Demethylcantharidin as a potential candidate for drug combination --- p.28 / Chapter 1.5 --- Study Objectives --- p.31 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell Lines --- p.33 / Chapter 2.2 --- Cancer Cell Preparation / Chapter 2.2.1 --- Chemicals and Reagents --- p.33 / Chapter 2.2.2 --- Cell Culture Practice --- p.34 / Chapter 2.2.2.1 --- Subcultures --- p.35 / Chapter 2.2.2.2 --- Cryopreservation --- p.37 / Chapter 2.2.2.3 --- Thawing Cryopreservated Cells --- p.38 / Chapter 2.2.3 --- Development of Drug-Resistant Cell Lines --- p.39 / Chapter 2.3 --- Growth Inhibition Assay / Chapter 2.3.1 --- Evaluation of Cytotoxicity in vitro --- p.40 / Chapter 2.3.2 --- Drug Pretreatment --- p.43 / Chapter 2.3.3 --- Drug Pre-sensitization with Concurrent Treatment --- p.44 / Chapter 2.4 --- Calculations for Drug Combinations --- p.46 / Chapter 2.5 --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Results and Discussions / Chapter 3.1 --- In vitro Cytotoxicity and Evaluation of Drug Resistance --- p.50 / Chapter 3.2 --- Role of Leaving Ligand in a Platinum Complex --- p.58 / Chapter 3.3 --- Priority in Selecting the Most Effective Drug Combination --- p.66 / Chapter 3.4 --- Drug Combination Studies / Chapter 3.4.1 --- Drug Combination Prescreening --- p.68 / Chapter 3.4.1.1 --- Comparison of the effectiveness of the three Drug Combinations --- p.72 / Chapter 3.4.1.2 --- Rationale for Drug Combination Studies presented in Section 3.4.2 & 3.4.3 --- p.73 / Chapter 3.4.2 --- Drug Pre-sensitization Studies in Colorectal Cancer Cell Lines --- p.74 / Chapter 3.4.2.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Colorectal Cancer Cell Lines --- p.84 / Chapter 3.4.2.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Oxaliplatin Resistant HCT116 Colorectal Cancer Cell Lines --- p.87 / Chapter 3.4.3 --- Drug Pre-sensitization Studies in Liver Cancer Cell Lines --- p.89 / Chapter 3.4.3.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Liver Cancer Cell Lines --- p.99 / Chapter 3.4.3.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Cisplatin Resistant SK-Hepl Liver Cancer Cell Line --- p.101 / Chapter 3.5 --- Possible Explanation to the Observed Drug Combination Effect --- p.103 / Chapter 3.6 --- General Protocols for Drug Combinations --- p.105 / Chapter Chapter 4 --- Conclusions / Reference --- p.109 / Appendices --- p.121 / Chapter I a. --- "Raw Data of Pre-screening for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.122 / Chapter I b. --- "Raw Data of Pre-screening for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.123 / Chapter II a. --- "Raw Data of Pre-screening for SK-Hepl (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.124 / Chapter II b. --- "Raw Data of Pre-screening for SK-Hepl ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.125 / Chapter III a. i) --- "Isobolograms for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.126 / Chapter III a. ii) --- "Raw Data for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.127 / Chapter III b. i) --- "Isobolograms for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.128 / Chapter III b. ii) --- "Raw Data for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.129 / Chapter IV a. i) --- "Isobolograms for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.130 / Chapter IV a. ii) --- "Raw Data for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.131 / Chapter IV b. i) --- "Isobolograms for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.132 / Chapter IV b. ii) --- "Raw Data for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.133 / Chapter V a. i) --- "Isobolograms for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.134 / Chapter V a. ii) --- "Raw Data for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.135 / Chapter V b. i) --- "Isobolograms for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.136 / Chapter V b. ii) --- "Raw Data for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.137 / Chapter VI a. i) --- Isobolograms for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.138 / Chapter VI a. ii) --- Raw Data for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.139 / Chapter VI b. i) --- "Isobolograms for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.140 / Chapter VI b. ii) --- "Raw Data for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.141 / Chapter VII a. i) --- "isobolograms for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.142 / Chapter VII a. ii) --- "Raw Data for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.143 / Chapter VII b.i) --- "Isobolograms for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.144 / Chapter VII b. ii) --- "Raw Data for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.145 / Chapter VIII a. i) --- "Isobolograms for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.146 / Chapter VIII a. ii) --- "Raw Data for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.147 / Chapter VIII b. i) --- "Isobolograms for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.148 / Chapter VIII b. ii) --- "Raw Data for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.149