by Chan Ka Kui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 104-113). / Abstracts in English and Chinese. / ABSTRACT --- p.1 / Chapter 1 --- INTRODUCTION --- p.5 / Chapter 1.1 --- Incidence rate of breast cancer in Hong Kong --- p.5 / Chapter 1.2 --- Estrogen and breast cancer --- p.6 / Chapter 1.3 --- The relation between glucose transporters and breast cancer --- p.7 / Chapter 1.4 --- Antisense oligonucleotide --- p.10 / Chapter 1.5 --- Action mechanisms of antisense oligonucleotide --- p.11 / Chapter 1.6 --- Modification of the oligonucleotide --- p.13 / Chapter 1.7 --- Length --- p.16 / Chapter 1.8 --- Sequence selection of the antisense oligonucleotide --- p.16 / Chapter 1.9 --- Delivery means in antisense oligonucleotide --- p.18 / Chapter 1.10 --- The therapeutic role of antisense oligonucleotide --- p.19 / Chapter 1.11 --- Objective of the project --- p.21 / Chapter 2 --- MATERIAL AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.2 --- Methods --- p.26 / Chapter 3 --- RESULTS --- p.37 / Chapter 3.1 --- The characteristics of MCF-7 and MDA-MB-231 cells --- p.37 / Chapter 3.2 --- Trend of uptake of antisense oligonucleotides in MCF-7 and MDA- MB-231 cells --- p.41 / Chapter 3.3 --- The integrity of the oligonucleotide in serum-free medium during transfection --- p.48 / Chapter 3.4 --- Detection of effects of Glut5 antisense oligonucleotides of breast tumor cells-MTT assay --- p.50 / Chapter 3.5 --- Detection of the antiproliferative effect by trypan blue exclusion assay and thymidine incorporation --- p.56 / Chapter 3.6 --- Cell cycle analysis and DNA extraction --- p.61 / Chapter 3.7 --- Suppression of Glut5 mRNA detected by RT-PCR --- p.66 / Chapter 3.8 --- Suppression of translation of Glut5 proteins as indicated by Western blotting --- p.73 / Chapter 3.9 --- Measurement of the fructose and glucose uptake in MCF-7 and MDA -MB-231 cells after antisense treatment --- p.76 / Chapter 3.10 --- Change of the phosphofructokinase-1 (PFK-1) activities in MDA- MB-231 cells --- p.82 / Chapter 3.11 --- Measurement of the change in the intracellular pH of the breast tumor cells --- p.84 / Chapter 4 --- DISCUSSION --- p.89 / Chapter 4.1 --- The insights of Glut5 antisense oligonucleotide into cancer therapy --- p.89 / Chapter 4.2 --- The uptake pattern of Glut5 antisense oligonucleotides in breast tumor cells --- p.90 / Chapter 4.3 --- Stability of antisense oligonucleotide during transfection --- p.92 / Chapter 4.4 --- Effects of Glut5 antisense oligonucleotide on MCF-7 and MDA-MB- 231cells --- p.93 / Chapter 4.5 --- Proofs of undergoing antisense action mechanism --- p.95 / Chapter 4.6 --- Physiological changes in breast tumor cells after antisense treatment --- p.97 / Chapter 5 --- CONCLUSION --- p.103 / Chapter 6 --- References --- p.104

Lam Mei Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgement --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- The facilitative glucose transporter family --- p.2 / Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5 / Chapter 1.3 --- Antisense strategy --- p.8 / Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9 / Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13 / Chapter 1.3.3 --- Factors influencing antisense activity --- p.16 / Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17 / Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2: --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Reagents and buffers --- p.25 / Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26 / Chapter 2.1.3 --- Culture medium --- p.28 / Chapter 2.1.4 --- Chemicals --- p.29 / Chapter 2.1.5 --- Culture of cells --- p.31 / Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31 / Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32 / Chapter 2.1.6 --- Animal Studies --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- In vitro studies --- p.34 / Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34 / Chapter 2.2.1.2 --- Transfection --- p.35 / Chapter 2.2.1.3 --- MTT assay --- p.36 / Chapter 2.2.1.4 --- Flow cytometry --- p.37 / Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45 / Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46 / Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47 / Chapter 2.2.1.8 --- Western blot analysis --- p.50 / Chapter 2.2.2 --- In vivo studies --- p.55 / Chapter 2.2.2.1 --- Animal studies --- p.55 / Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58 / Chapter (ii) --- Creatine kinase (CK) assay --- p.60 / Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62 / Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64 / Chapter Chapter 3: --- Results --- p.67 / Chapter 3.1 --- In vitro studies --- p.68 / Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68 / Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77 / Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82 / Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87 / Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96 / Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100 / Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108 / Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117 / Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125 / Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131 / Chapter 3.2 --- In vivo studies --- p.135 / Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135 / Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139 / Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139 / Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147 / Chapter Chapter 4: --- Discussion --- p.151 / Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152 / Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154 / Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155 / Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158 / Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166 / Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167 / Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168 / Chapter 4.3 --- Conclusion --- p.169 / Bibliography --- p.172