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by Ng Mei Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 173-183). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abbreviations --- p.3 / Abstracts --- p.4 / List of figures and tables --- p.7 / Contents Page No / General Introduction --- p.13 / Chapter CHAPTER ONE --- Biological Characterization of A431 Cells & SiHa Cells Under Different Microenvironments --- p.16 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.1.1 --- Microenvironment Surrounding Tumor Cells --- p.18 / Chapter 1.1.2 --- Hypoxic Environment --- p.20 / Chapter 1.1.2.1 --- The Hypoxic Chamber --- p.20 / Chapter 1.1.3 --- Reoxygenation --- p.22 / Chapter 1.1.4 --- Acidic Environment --- p.23 / Chapter 1.1.5 --- Glucose Depletion --- p.24 / Chapter 1.1.6 --- Irradiation --- p.25 / Chapter 1.2 --- Objectives --- p.26 / Chapter 1.3 --- Materials and Methods --- p.27 / Chapter 1.3.1 --- Materials --- p.27 / Chapter 1.3.2 --- Methods --- p.29 / Chapter 1.3.2.1 --- Cell Lines --- p.29 / Chapter 1.3.2.2 --- The Working Procedure for the Hypoxic Chamber --- p.30 / Chapter 1.3.2.3 --- "Aerobic, Hypoxic and Reoxygenated Conditions" --- p.33 / Chapter 1.3.2.4 --- Acidic Condition --- p.35 / Chapter 1.3.2.5 --- Glucose Depleted Condition --- p.36 / Chapter 1.3.2.6 --- Gamma-Irradiation --- p.37 / Chapter 1.3.2.7 --- Analysis of the Growth Pattern by MTT Assay and Cell Counting --- p.38 / Chapter 1.3.2.8 --- Cell Cycle Analysis --- p.39 / Chapter 1.3.2.9 --- Western Blot Analysis --- p.40 / Chapter 1.3.2.10 --- DNA Fragmentation Analysis --- p.42 / Chapter 1.4 --- Results --- p.43 / Chapter 1.4.1 --- Cell Proliferation Profile by MTT Assay --- p.43 / Chapter 1.4.1.1 --- Proliferation of cells under hypoxia --- p.43 / Chapter 1.4.1.2 --- Proliferation of cells under acidic pH environments --- p.49 / Chapter 1.4.1.3 --- Proliferation of cells under glucose depleted environment --- p.52 / Chapter 1.4.2 --- Distribution of cell cycles under different micro environments --- p.54 / Chapter 1.4.3 --- General Protein Expression Pattern by Western Blot Analysis --- p.57 / Chapter 1.4.4 --- Detection of Apoptosis by DNA Fragmentation Assay --- p.59 / Chapter 1.5 --- Discussion --- p.58 / Chapter CHAPTER TWO --- REACTION KINETICS OF A431 CELLS AND SiHa CELLS INDUCED BY EGF --- p.71 / Chapter 2.1 --- Introduction --- p.72 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.74 / Chapter 2.1.2 --- EGF Signaling Pathway --- p.76 / Chapter 2.2 --- Objectives --- p.79 / Chapter 2.3 --- Materials and Methods --- p.80 / Chapter 2.3.1 --- Materials --- p.80 / Chapter 2.3.2 --- Methods --- p.82 / Chapter 2.3.2.1 --- Cell Lines --- p.82 / Chapter 2.3.2.2 --- EGF Sensitivity Assay --- p.83 / Chapter 2.3.2.3 --- Combination Effect of Hypoxia and EGF --- p.83 / Chapter 2.3.2.4 --- Early Kinetics Analysis by Low EGF Concentration Treatment --- p.84 / Chapter 2.3.2.5 --- Late Kinetics Analysis by High EGF Concentration Treatment --- p.85 / Chapter 2.4 --- Results --- p.86 / Chapter 2.4.1 --- Sensitivity of A431 cells and SiHa cells to EGF by MTT Assay --- p.86 / Chapter 2.4.2 --- Early/Late Kinetics of EGF induced protein tyrosine phosphorylation Pattern --- p.90 / Chapter 2.4.3 --- Raf protein expression --- p.96 / Chapter 2.4.4 --- EGFR expression level --- p.100 / Chapter 2.5 --- Discussions --- p.104 / Chapter CHAPTER THREE --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN A431 CELLS BY DIFFERENTIAL DISPLAY UNDER DIFFERENT TUMOR MICROENVIRONMENTS --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and Methods --- p.112 / Chapter 3.2.1 --- Materials --- p.112 / Chapter 3.2.2 --- Methods --- p.114 / Chapter 3.2.2.1 --- Spheroid Cells --- p.114 / Chapter 3.2.2.2 --- Identification of Differentally Expressed Genes by RT-PCR --- p.117 / Chapter 3.2.2.3 --- Ligation and Cloning of Differentially Expressed cDNA --- p.120 / Chapter 3.2.2.4 --- Screening and Sequencing of the cDNA Inserts --- p.121 / Chapter 3.2.2.5 --- Northern Blot Analysis --- p.123 / Chapter 3.3 --- Results --- p.124 / Chapter 3.4 --- Discussions --- p.161 / GENERAL CONCLUSION --- p.165 / REFERENCES --- p.167

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 144-157). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 144-157). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 73-126).

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 140-172).

This study completed the analysis of mutational frequencies and clinicopathological patterns of six EGFR pathway-related genes (EGFR, HER2, HER4, KRAS, BRAF and MET) in 212 resected lung adenocarcinomas (AD) from 98 male and 114 female Chinese patients without prior chemotherapy or tyrosine kinase inhibitor (TKI) therapy. Genomic DNA and cDNA sequencing, quantitative PCR and fluorescence in-situ hybridization (FISH) were employed to investigate mutation and amplification status of the relevant genes. Overall, more than 75% of tumours were detected to harbour mutations or amplification in one of these six genes. The commonest mutation was found to involve EGFR, comprising 60.38% of cases, followed by KRAS (9.43%), HER2 (2.36%), MET (2.36%), BRAF (1.42%) and HER4 (0.47%). Four somatic mutations in MET exon 14 splicing region were found, leading to alternative splicing and a transcript lacking exon 14. Two of the MET mutant tumours and one MET wild-type tumour showed MET amplification of more than 3.5 fold increase in copy number. Mutations of EGFR were significantly more frequent in female (p = 0.0196), non-smokers (p < 0.001) and well differentiated tumours (p = 0.0209). KRAS mutations showed significant association with male (p = 0.0099) and smoking history (p = 0.0011). A novel HER2 D769Y mutation was found and HER2 mutations were associated with smokers (p = 0.0013) and poorly differentiated tumours (p = 0.0147). BRAF, MET mutations and MET amplification were not associated with clinicopathological factors. Mutations were mutually exclusive except for two cases with KRAS and HER4/BRAF. MET amplification was co-existent with MET mutations in two cases. MET amplification was found to negatively correlate with disease-free and cancer-specific survivals. The results suggested that MET amplification may contribute to disease progression and could be a therapeutic target in primary lung AD in Hong Kong Chinese patients. / published_or_final_version / Pathology / Master / Master of Medical Sciences