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Molecular analysis of BRAF and microsatellite analysis of chromosome 14q in astrocytic tumors.January 2004 (has links)
Chan Ching Yin. / Thesis submitted in: October 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 197-221). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.vi / List of abbreviations --- p.ix / List of tables --- p.xv / List of figures --- p.xvi / Contents --- p.xviii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1. --- What are astrocytic tumors? --- p.1 / Chapter 1.1.1. --- Histological characteristics and classification --- p.2 / Chapter 1.1.2. --- Epidemiology --- p.2 / Chapter 1.1.3. --- Treatment and patient survival --- p.4 / Chapter 1.2. --- "Cytogenetics, molecular genetics and epigenetics of astrocytic tumors" --- p.6 / Chapter 1.2.1. --- Cytogenetics --- p.6 / Chapter 1.2.2. --- Genetic imbalances --- p.7 / Chapter 1.2.3. --- Tumor suppressor genes --- p.13 / Chapter 1.2.4. --- Oncogenes --- p.22 / Chapter 1.2.5. --- Primary and secondary GBMs --- p.26 / Chapter 1.3. --- Major pathways involved in astrocytic tumorigenesis --- p.30 / Chapter 1.3.1. --- Cell cycle dysregulation and suppression of apoptosis --- p.30 / Chapter 1.3.2. --- Promotion of proliferation and survival --- p.33 / Chapter 1.4. --- BRAF mutation in human cancers --- p.38 / Chapter 1.5. --- Other CNS tumors included in the current study --- p.52 / Chapter 2. --- Aims of study --- p.61 / Chapter 3. --- Materials and methods --- p.64 / Chapter 3.1. --- Clinical materials --- p.64 / Chapter 3.2. --- Cell lines --- p.75 / Chapter 3.3. --- Cell culture --- p.77 / Chapter 3.4. --- DNA extraction --- p.78 / Chapter 3.4.1. --- Pre-treatment of samples --- p.78 / Chapter 3.4.2. --- Cell lysis and protein removal --- p.80 / Chapter 3.4.3. --- Precipitation of DNA --- p.81 / Chapter 3.4.4. --- Determination of DNA concentration --- p.81 / Chapter 3.5. --- Mutation analysis of BRAF by cycle sequencing --- p.83 / Chapter 3.5.1. --- Amplification of BRAF exons --- p.83 / Chapter 3.5.2. --- Cycle sequencing and automated gel electrophoresis --- p.84 / Chapter 3.6. --- Immunohistochemistry of B-Raf and GFAP --- p.87 / Chapter 3.6.1. --- Pre-treatment of samples --- p.87 / Chapter 3.6.2. --- Detection of B-Raf and GFAP antigens by ABC method --- p.88 / Chapter 3.6.3. --- Controls --- p.90 / Chapter 3.7. --- Quantification of EGFR gene dosage by TaqMan based real-time PCR --- p.91 / Chapter 3.7.1. --- Preparation of gene constructs --- p.92 / Chapter 3.7.2. --- Primers and TaqMan probes --- p.93 / Chapter 3.7.3. --- Experimental condition and PCR program --- p.95 / Chapter 3.7.4. --- DNA standards --- p.95 / Chapter 3.7.5. --- Controls --- p.96 / Chapter 3.7.6. --- Experimental layout --- p.96 / Chapter 3.8. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.97 / Chapter 4. --- Results --- p.101 / Chapter 4.1. --- Mutation analysis of BRAF --- p.101 / Chapter 4.2. --- Immunohistochemistry of B-Raf protein --- p.107 / Chapter 4.3. --- Quantification of EGFR gene dosage --- p.117 / Chapter 4.4. --- Correlation between EGFR dosage and BRAF mutation --- p.128 / Chapter 4.5. --- Correlation between EGFR dosage and B-Raf expression --- p.129 / Chapter 4.6. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.131 / Chapter 5. --- Discussions --- p.149 / Chapter 5.1. --- BRAF mutations as common events in human cancers --- p.149 / Chapter 5.2. --- BRAF mutation in CNS tumor specimens --- p.150 / Chapter 5.2.1. --- Tumorigenic effect of the V599E substitution --- p.153 / Chapter 5.2.2. --- V599E B-Raf mutant activation independent of Ras activation --- p.155 / Chapter 5.2.3. --- Autocrine stimulation of Ras signaling in V599E B-Raf mutant --- p.156 / Chapter 5.3. --- BRAF expression in astrocytic tumors --- p.159 / Chapter 5.4. --- Mutually exclusive pattern between EGFR amplification and BRAF expression --- p.161 / Chapter 5.4.1. --- Similar effect of EGFR activation and B-Raf activation --- p.163 / Chapter 5.4.2. --- Mutual effects between Ras/Raf/Mek/Erk and Akt signaling --- p.164 / Chapter 5.5. --- Microsatellite analysis of chromosome 14q in human cancers --- p.167 / Chapter 5.6. --- Microsatellite analysis of chromosome 14q in astrocytic tumors --- p.170 / Chapter 5.6.1. --- Finer mapping of common regions of deletion --- p.170 / Chapter 5.6.2. --- Genes within the common regions of deletion --- p.173 / Chapter 5.6.3. --- Overlapping deletion regions in astrocytic and non-CNS tumors --- p.186 / Chapter 6. --- Further studies --- p.190 / Chapter 6.1. --- Role of BRAF alterations in astrocytic tumors --- p.190 / Chapter 6.2. --- B-Raf expression in astrocytic tumors and correlation with EGFR overexpression --- p.193 / Chapter 6.3. --- Microsatellite analysis of 14q in astrocytic tumors --- p.194 / Chapter 7. --- Conclusions --- p.195 / Chapter 8. --- References --- p.198
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Investigation of putative tumor suppressors on chromosome 16q in nasopharyngeal carcinoma.January 2003 (has links)
Hui Wai Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 158-189). / Abstracts in English and Chinese. / Abstract / Acknowledgements / List of Tables / List of Figures / Table of Contents / Table of Contents / Chapter Chapter I: --- Introduction --- p.1 / Chapter I. --- Aim of Study --- p.1 / Chapter II. --- Literature Review --- p.3 / Chapter 1. --- Background --- p.3 / Chapter A. --- Epidemiology --- p.3 / Chapter B. --- Histopathology --- p.3 / Chapter C. --- Etiology --- p.4 / Chapter i. --- Environmental Factors --- p.5 / Chapter ii. --- Epstein-Barr Virus (EBV) Infection --- p.6 / Chapter iii. --- Genetic Factors --- p.9 / Chapter 2. --- Molecular Genetics of NPC --- p.11 / Chapter A. --- Genome-Wide Studies --- p.11 / Chapter i. --- Comparative Genomic Hybridization (CGH) --- p.11 / Chapter ii. --- Loss of Heterozygosity (LOH) Studies --- p.12 / Chapter iii. --- Homozygous Deletion Study --- p.12 / Chapter B. --- NPC-related Oncogenes and Tumor Suppressor Genes --- p.13 / Chapter i. --- Oncogenes --- p.13 / Chapter ii. --- Tumor Suppressor Genes --- p.14 / Chapter 3. --- Chromosome 14q and NPC --- p.19 / Chapter A. --- Tumor Suppressor Loci and Cancer-Related Genes on Chromosome14q --- p.20 / Chapter i. --- Tumor Suppressor Loci on Chromosome 14q --- p.20 / Chapter ii. --- Cancer-Related Genes on Chromosome 14q --- p.26 / Chapter 4. --- Chromosome 16q and NPC --- p.28 / Chapter A. --- Tumor Suppressor Loci and Candidate Tumor Suppressor genes on Chromosome16q --- p.28 / Chapter i. --- Tumor Suppressor Loci on Chromosome 16q --- p.28 / Chapter ii. --- Metastasis Suppressor Loci on Chromosome 16q --- p.34 / Chapter iii. --- Candidate Tumor Suppressor Genes on Chromosome 16q --- p.34 / Chapter Chapter II: --- Materials and Methods --- p.40 / Chapter I. --- Cell Lines and Xenografts --- p.40 / Chapter 1. --- Cell Lines --- p.40 / Chapter 2. --- Xenografts --- p.41 / Chapter 3. --- DNA Extraction --- p.42 / Chapter II. --- Patients and Biopsy Specimens --- p.44 / Chapter 1. --- Manual Microdissection --- p.44 / Chapter 2. --- Laser Captured Microdissection (LCM) --- p.46 / Chapter 3. --- DNA Extraction --- p.46 / Chapter III. --- Comprehensive Screening for Homozygous Deletion Regions on Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancers --- p.48 / Chapter 1. --- DNA of Human Cancer Cell Lines --- p.48 / Chapter 2. --- Sequence-Tagged Sites (STS) Markers --- p.48 / Chapter 3. --- Polymerase Chain Reaction (PCR) --- p.49 / Chapter IV --- . Investigation of Inactivation of Potential Tumor Suppressor Genes on Chromosome 14q32.12-32.33 and 16q23.1-24.3 --- p.58 / Chapter 1. --- Detection of Homozygous Deletion --- p.58 / Chapter 2. --- Expression Analysis --- p.58 / Chapter A. --- RNA Extraction --- p.58 / Chapter B. --- Reverse-Transcription (RT) PCR --- p.61 / Chapter i. --- DNase I Digestion --- p.62 / Chapter ii. --- First-strand cDNA Synthesis and RNase Digestion --- p.62 / Chapter iii. --- Reverse-Transcription (RT)-PCR --- p.63 / Chapter C. --- Real-Time RT PCR --- p.63 / Chapter 3. --- Methylation Analysis --- p.68 / Chapter A. --- Sodium Bisulfite Modification --- p.68 / Chapter B. --- Methylation-Specific PCR (MSP) --- p.69 / Chapter C. --- Bisulfite Sequencing --- p.70 / Chapter D. --- Combined Bisulfite Restriction Analysis (COBRA) --- p.75 / Chapter E. --- 5 -aza-2' -deoxycytidine Treatment --- p.76 / Chapter ChapterIII: --- Results --- p.78 / Chapter I. --- Comprehensive Screening for Homozygous Deletion Regions in Human Cancers --- p.78 / Chapter 1. --- Chromosome 14q32.12-3233 --- p.78 / Chapter 2. --- Chromosome 16q23.1-243 --- p.79 / Chapter II. --- Investigation of Inactivation of Potential Tumor Suppressor Genes in NPC --- p.86 / Chapter 1. --- Chromosome 14q --- p.86 / Chapter A. --- "WW Domain-Containing Protein, 45-kD (WW45)" --- p.86 / Chapter B. --- Apoptosis Stimulating Protein of p53(ASPP1) --- p.88 / Chapter 2. --- Chromosome 16q --- p.92 / Chapter A. --- WW Domain-Containing Oxidoreductase (WWOX) --- p.92 / Chapter i. --- Homozygous Deletion Screening of WWOX --- p.92 / Chapter ii. --- Expression of Aberrant Splicing Transcripts of WWOX in NPC --- p.94 / Chapter iii. --- Sequencing of WWOX Aberrant Transcripts --- p.95 / Chapter iv. --- Quantitative Analysis of WWOX Transcripts in NPC --- p.95 / Chapter v. --- Methylation Analysis --- p.99 / Chapter B. --- H-Cadherin (CDH13) --- p.102 / Chapter i. --- Analysis of H-cadherin Deletion on Cancer Cell Lines and Xenografts --- p.102 / Chapter ii. --- Expression Analysis of H-Cadherin by RT-PCR and Real-Time RT-PCR --- p.102 / Chapter iii. --- Analysis of Promoter Hypermethylation by Methylation-Specific PCR (MSP) and Bisulfite Sequencing in NPC Cell Lines and Xenografts --- p.104 / Chapter iv. --- Demethylation Study of H-Cadherin in C666-1 Cell Line --- p.105 / Chapter v. --- Methylation Analysis of H-Cadherin in Primary Tumors --- p.105 / Chapter vi. --- Methylation Analysis of H-Cadherin in Human Cancer Cell Lines --- p.106 / Chapter C. --- Myeloid Translocation Gene on Chromosome 16 (MTG16) --- p.113 / Chapter i. --- Deletion Analysis of MTG16 in Cancer Cell Lines and Xenografts --- p.113 / Chapter ii. --- Differential Expression of MTG16a and MTG16b Transcripts in NPC Cell Lines and Xenografts --- p.113 / Chapter iii. --- Methylation Analysis of MTG16b in NPC Cell Lines and Xenografts --- p.118 / Chapter iv. --- Sequencing of MTGl 6b RT-PCR Products --- p.119 / Chapter v. --- Demethylation Study of MTG16b in HK-1 Cell Line --- p.119 / Chapter vi. --- Promoter Methylation Analysis of MTG16b by MSP in Primary NPC and Cancer Cell Lines --- p.120 / Chapter Chapter IV: --- Discussion --- p.124 / Chapter I. --- Comprehensive Homozygous Deletion Screening of Chromosomes 14q32.12-32.33 and 16q23.1-24.3 in Human Cancer Cell Lines and Xenografts --- p.124 / Chapter II. --- Investigation of Candidate Tumor Suppressor Genes on Chromosome 14q in NPC --- p.128 / Chapter III. --- Alterations of Candidate Tumor Suppressor Genes on Chromosome 16q in NPC --- p.133 / Chapter 1. --- Expression of Aberrant Transcripts of WWOX in NPC --- p.133 / Chapter 2. --- Methylation-Associated Silencing of H-Cadherin and MTG16b in NPC --- p.140 / Chapter Chapter V: --- Conclusion --- p.154 / Chapter Chapter VI: --- References --- p.158
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