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The effect of 5-fluorouracil on the mRNA and proteins expression in a human colon cancer cell line SW480.

Wong, Wai Ki Vicky. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 105-131). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of contents --- p.vii / List of tables --- p.xii / List of figures --- p.xiii / List of abbreviations --- p.xiv / Chapter Chapter 1: --- Introduction --- p.1 / Chapter Chapter 2: --- Colorectal cancer / Chapter 2.1 --- Literature Review / Chapter 2.1.1 --- Colorectal cancer --- p.8 / Chapter 2.1.2 --- Incident rate of colorectal cancer --- p.8 / Chapter 2.1.3 --- Hereditary colorectal cancer --- p.9 / Chapter 2.1.4 --- Sporadic colorectal cancer and Wnt signaling pathway --- p.10 / Chapter 2.1.5 --- Chemotherapy treatment of colorectal cancer --- p.11 / Chapter 2.1.5.1 --- 5-Fluorouracil --- p.12 / Chapter 2.1.5.2 --- Oxaliplatin --- p.14 / Chapter 2.1.5.3 --- Irinotecan --- p.14 / Chapter 2.1.6 --- Biomarkers for colorectal cancer --- p.15 / Chapter 2.1.6.1 --- Thymidylate synthase --- p.15 / Chapter 2.1.6.2 --- Dihydropyrimidine dehydrogenase --- p.16 / Chapter 2.1.6.3 --- Thymidine phosphorylase --- p.16 / Chapter 2.1.6.4 --- Microsatellite-instability status --- p.16 / Chapter 2.1.6.5 --- Clinical uses of biomarkers for colorectal cancer --- p.17 / Chapter 2.1.7 --- Choice of cell line as colorectal cancer model --- p.17 / Chapter 2.1.8 --- Aims of study --- p.17 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Verification of SW480 as a nuclear β-catenin positive cell line / Chapter 2.2.1.1 --- Maintenance of cell lines --- p.21 / Chapter 2.2.1.2 --- Antibody --- p.21 / Chapter 2.2.1.3 --- Agar block preparation for SW480 and CCD-18C0 cells --- p.22 / Chapter 2.2.1.4 --- Immunocytochemical staining --- p.22 / Chapter 2.2.2 --- Effect of anti-cancer drugs on cell viability / Chapter 2.2.2.1 --- Maintenance of cell lines --- p.22 / Chapter 2.2.2.2 --- MTT cell viability assay --- p.23 / Chapter 2.3 --- Results / Chapter 2.3.1 --- SW480 is a β-catenin positive cell line --- p.24 / Chapter 2.3.2 --- Antiproliferative effects of cytotoxic drugs in SW480 cells / Chapter 2.3.2.1 --- 5-Fluorouracil --- p.26 / Chapter 2.3.2.2 --- Oxaliplatin --- p.29 / Chapter 2.3.2.3 --- Irinotecan --- p.31 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- SW480 as a nuclear β-catenin positive cell line --- p.33 / Chapter 2.4.2 --- Antiproliferative effects of 5-fluorouracil in SW480 cells --- p.33 / Chapter 2.4.3 --- Summary --- p.34 / Chapter Chapter 3: --- Effect of 5-fluorouracil on mRNA expression in SW480 cells / Chapter 3.1 --- Literature Review / Chapter 3.1.1 --- Application of quantitative real-time polymerase chain reaction in cancer research / Chapter 3.1.1.1 --- Principles of quantitative real-time polymerase chain reaction --- p.36 / Chapter 3.1.1.2 --- Advantages of quantitative real-time polymerase chain reaction over conventional polymerase chain reaction --- p.39 / Chapter 3.1.1.3 --- Determination of colorectal cancer biomarkers by quantitative real-time polymerase chain reaction --- p.39 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Determination of the effect of 5-fluorouracil on mRNA expression in SW480 cells / Chapter 3.2.1.1 --- Treatment of cells --- p.40 / Chapter 3.2.1.2 --- Extraction of total RNA from SW480 cells --- p.40 / Chapter 3.2.1.3 --- Removal of genomic DNA --- p.41 / Chapter 3.2.1.4 --- Determination of the efficiency of genomic DNA removal --- p.42 / Chapter 3.2.1.5 --- Determination of the purity and concentration of RNA --- p.42 / Chapter 3.2.1.6 --- Determination of the integrity of RNA --- p.43 / Chapter 3.2.1.7 --- First strand cDNA synthesis --- p.44 / Chapter 3.2.1.8 --- Real-time polymerase chain reaction using human Wnt signaling pathway RT2 ProfileŕёØ PCR array --- p.44 / Chapter 3.2.1.9 --- Calculation of the fold-change in genes expression between the 5-FU treated and control SW480 cells --- p.45 / Chapter 3.3 --- Results / Chapter 3.3.1 --- The quality and quantity of RNA --- p.46 / Chapter 3.3.2 --- Effects of 5-fluorouracil on genes expression in SW480 cells --- p.48 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Alterations in mRNA expression in 5-fluorouracil treated SW480 cells --- p.55 / Chapter 3.4.1.1 --- Extracellular signaling molecules --- p.55 / Chapter 3.4.1.2 --- Canonical Wnt signaling pathway --- p.56 / Chapter 3.4.1.3 --- Regulators of cell cycle --- p.57 / Chapter 3.4.1.4 --- Regulators of growth and proliferation --- p.58 / Chapter 3.4.1.5 --- Regulators of transcription --- p.58 / Chapter 3.4.1.6 --- Regulators of Wnt receptor signaling pathway --- p.60 / Chapter 3.4.1.7 --- Other genes involved in Wnt signaling --- p.61 / Chapter 3.4.2 --- Limitations of Q-RT-PCR --- p.61 / Chapter 3.4.3 --- Summary --- p.62 / Chapter Chapter 4: --- Effect of 5-fluorouracil on proteins expression in SW480 cells / Chapter 4.1 --- Literature Review / Chapter 4.1.1 --- From mRNA to proteins --- p.63 / Chapter 4.1.2 --- Application of proteomics in cancer research --- p.63 / Chapter 4.1.3 --- Two-dimensional gel electrophoresis --- p.64 / Chapter 4.1.4 --- Principles of MALDI TOF mass spectrometry --- p.64 / Chapter 4.1.5 --- Peptide mass fingerprinting --- p.65 / Chapter 4.1.6 --- Drug response proteins detected by proteomics in colorectal cancer cell lines --- p.65 / Chapter 4.1.7 --- Detection of biomarker in colorectal cancer formation using proteomics --- p.66 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Determination of the effect of 5-fluorouracil on proteins expression in SW480 cells / Chapter 4.2.1.1 --- Treatment of cells --- p.67 / Chapter 4.2.1.2 --- Cell lysis --- p.67 / Chapter 4.2.1.3 --- Protein quantitation of cell lysate --- p.67 / Chapter 4.2.1.4 --- Sample preparation for two-dimensional electrophoresis --- p.68 / Chapter 4.2.1.5 --- Two-dimensional electrophoresis --- p.69 / Chapter 4.2.1.6 --- Silver staining --- p.69 / Chapter 4.2.1.7 --- Image analysis --- p.70 / Chapter 4.2.1.8 --- In-gel protein digestion --- p.70 / Chapter 4.2.1.9 --- Peptide mass fingerprinting using mass spectrometry --- p.71 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Protein expression patterns of 5-fluorouracil treated and untreated SW480 cells by 2-dimensional electrophoresis --- p.72 / Chapter 4.3.2 --- Identification of the differentially expressed proteins after 5-fluorouracil treatment in SW480 cells --- p.75 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Effects of 5-fluorouracil on protein expression in SW480 cells --- p.82 / Chapter 4.4.1.1 --- Identified upregulated proteins after 5-fluorouracil treatment in SW480 cells / Chapter 4.4.1.1.1 --- Cyclophilin A --- p.83 / Chapter 4.4.1.1.2 --- Cytokeratin 19 --- p.83 / Chapter 4.4.1.1.3 --- Cytokeratin 8 --- p.84 / Chapter 4.4.1.1.4 --- RAN --- p.84 / Chapter 4.4.1.1.5 --- Heat shock protein 27 --- p.84 / Chapter 4.4.1.1.6 --- Peroxiredoxin 6 --- p.85 / Chapter 4.4.1.2 --- Identified dowiiregulated proteins after 5-fluorouracil treatment in SW480 cells / Chapter 4.4.1.2.1 --- Heat shock protein 60 --- p.86 / Chapter 4.4.1.2.2 --- Cytokeratin 18 --- p.86 / Chapter 4.4.1.2.3 --- Cytokeratin 9 --- p.86 / Chapter 4.4.1.2.4 --- Carbamoylphosphate synthetase I --- p.87 / Chapter 4.4.1.2.5 --- a-Enolase --- p.87 / Chapter 4.4.1.2.6 --- Heat shock protein 70 --- p.87 / Chapter 4.4.1.2.7 --- nm23 --- p.88 / Chapter 4.4.1.2.8 --- β-actin --- p.88 / Chapter 4.4.2 --- Limitations of proteomics profiling --- p.89 / Chapter 4.4.3 --- Summary --- p.90 / Chapter Chapter 5: --- Verification of proteinśة identities by immunocytochemical staining / Chapter 5.1 --- Materials and Methods / Chapter 5.1.1 --- Antibodies --- p.91 / Chapter 5.1.2 --- Treatment of cells --- p.91 / Chapter 5.1.3 --- Agar block preparation of SW480 cells --- p.92 / Chapter 5.1.4 --- Immunocytochemical staining and evaluation --- p.92 / Chapter 5.1.5 --- Polymer-based immunohistochemical detection system --- p.93 / Chapter 5.1.6 --- Statistical analyses --- p.93 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Confirmation of proteomic findings using immunocytochemical stainings in paraffin-embedded sections of 5-fluorouracil treated and untreated SW480 cells --- p.94 / Chapter 5.3 --- Discussion / Chapter 5.3.1 --- Immunocytochemical staining to verify proteomics findings of 5-fluorouracil treated and untreated SW480 cells --- p.99 / Chapter 5.3.2 --- Limitations of ICC staining --- p.100 / Chapter 5.3.3 --- Summary --- p.100 / Chapter Chapter 6: --- Conclusions and future perspectives / Chapter 6.1 --- Significance of study --- p.101 / Chapter 6.2 --- Future perspectives --- p.102 / References --- p.105

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326008
Date January 2007
ContributorsWong, Wai Ki Vicky., Chinese University of Hong Kong Graduate School. Division of Medical Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xv, 131 leaves : ill. (chiefly col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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