In vitro studies on the mechanisms of hyperthermia- and TNF-α-induced apoptosis.

by Yuen Wai Fan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.Includes bibliographical references (leaves 211-232).Abstracts in English and Chinese.Acknowledgements --- p.iList of Publications and Abstracts --- p.iiAbbreviations --- p.ivAbstract --- p.xiAbstract in Chinese --- p.xivList of Figures --- p.xviiList of Tables --- p.xxiiiContents --- p.xxivChapter Chapter 1. --- General Introduction --- p.1Chapter 1.1 --- Hyperthermia --- p.2Chapter 1.1.1 --- History of Hyperthermia --- p.2Chapter 1.1.2 --- Biological Functions of Hyperthermia --- p.3Chapter 1.1.3 --- Clinical Application of Hyperthermia --- p.4Chapter 1.1.3.1 --- Whole-body Hyperthermia --- p.4Chapter 1.1.3.2 --- Regional Hyperthermia --- p.4Chapter 1.1.3.3 --- Local Hyperthermia --- p.5Chapter 1.1.4 --- Combination Therapy --- p.5Chapter 1.1.4.1 --- Combined treatment with Hyperthermia and Radiotherapy --- p.6Chapter 1.1.4.2 --- Combined treatment with Hyperthermia and Chemotherapy --- p.6Chapter 1.2 --- Tumour Necrosis Factor --- p.9Chapter 1.2.1 --- History of Tumour Necrosis Factor --- p.9Chapter 1.2.2 --- Sources of TNF-α and TNF-β --- p.9Chapter 1.2.3 --- Biological Roles of TNF --- p.10Chapter 1.2.3.1 --- Receptors of TNF-α --- p.11Chapter 1.2.4 --- Signaling Pathway of TNF --- p.12Chapter 1.2.4.1 --- Activation of Death Domain --- p.12Chapter 1.2.4.2 --- Activation of Sphingomyelin Pathway --- p.13Chapter 1.2.4.3 --- Activation of NF-kB pathway --- p.13Chapter 1.3 --- Types of Cell Death: Necrosis and Apoptosis --- p.16Chapter 1.3.1 --- Necrosis --- p.16Chapter 1.3.2 --- Apoptosis --- p.16Chapter 1.4 --- Signaling Pathway in Apoptosis --- p.19Chapter 1.4.1 --- Factors Involved in Apoptotic Pathway --- p.19Chapter 1.4.1.1 --- Caspases --- p.19Chapter 1.4.1.2 --- Death Substrates --- p.20Chapter 1.4.1.3 --- Bcl-2 Protein Family --- p.21Chapter 1.4.1.4 --- Role of Mitochondria --- p.23Chapter 1.5 --- Objectives of the Project --- p.26Chapter Chapter 2. --- Materials and Methods --- p.28Chapter 2.1 --- Materials --- p.29Chapter 2.1.1 --- Culture of Cells --- p.34Chapter 2.1.1.1 --- "TNF-α Sensitive Cell Line, L929" --- p.34Chapter 2.1.1.2 --- "TNF-α Resistance Cell Line, L929-11E" --- p.34Chapter 2.1.1.3 --- Preservation of Cells --- p.35Chapter 2.1.2 --- Culture Media --- p.36Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-Free Medium) --- p.36Chapter 2.1.3 --- Buffers and Reagents --- p.37Chapter 2.1.3.1 --- Preparation of Buffers --- p.37Chapter 2.1.3.2 --- Buffer for Common Use --- p.37Chapter 2.1.3.3 --- Reagents for Annexin-V-FITC/PI assay --- p.37Chapter 2.1.3.4 --- Reagents for Cytotoxicity Assay --- p.37Chapter 2.1.3.5 --- Reagents for Molecular Biology Work --- p.38Chapter 2.1.3.6 --- Reagents for Western Blotting Analysis --- p.38Chapter 2.1.4 --- Chemicals --- p.40Chapter 2.1.4.1 --- Recombinant Murine TNF-α --- p.40Chapter 2.1.4.2 --- Dye for Cytotoxicity Assay --- p.41Chapter 2.1.4.3 --- Fluorescence Dyes --- p.41Chapter 2.1.4.4 --- Chemicals Related to Mitochondrial Studies --- p.41Chapter 2.1.4.5 --- Inhibitors of Caspases --- p.42Chapter 2.1.4.6 --- Antibodies for Western Blotting --- p.42Chapter 2.1.4.7 --- Other Chemicals --- p.43Chapter 2.2 --- Methods --- p.44Chapter 2.2.1 --- Treatment with TNF-α --- p.44Chapter 2.2.2 --- Treatment with Hyperthermia --- p.44Chapter 2.2.3 --- In vitro Cell Cytotoxicity Assay --- p.45Chapter 2.2.4 --- Flow Cytometry --- p.46Chapter 2.2.4.1 --- Introduction --- p.46Chapter 2.2.4.2 --- Analysis by FCM --- p.48Chapter 2.2.4.3 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50Chapter 2.2.4.4 --- Determination of Mitochondrial Membrane Potential (ΔΨm) --- p.51Chapter 2.2.4.5 --- Determination of Hydrogen Peroxide (H202) Release --- p.52Chapter 2.2.4.6 --- Determination of Intracellular Free Calcium ([Ca2+]i) Level --- p.52Chapter 2.2.4.7 --- Determination of the Relationship of ΔΨm and [Ca2+]i Level --- p.53Chapter 2.2.5 --- Western Blotting Analysis --- p.53Chapter 2.2.5.1 --- Preparation of Proteins from Cells --- p.53Chapter 2.2.5.2 --- SDS Polyacrylamide Gel Electophoresis (SDS- PAGE) --- p.56Chapter 2.2.5.3 --- Electroblotting of Proteins --- p.57Chapter 2.2.5.4 --- Probing Antibodies for Proteins --- p.57Chapter 2.2.5.5 --- Enhanced Chemiluminescence (ECL) assay --- p.58Chapter 2.2.6 --- Reverse Transcriptase Polymerase Chain Reaction --- p.58Chapter 2.2.6.1 --- Extraction of RNA by Trizol Reagent --- p.59Chapter 2.2.6.2 --- Determination of the Amount of RNA --- p.60Chapter 2.2.6.3 --- Agarose Gel Electrophoresis --- p.60Chapter 2.2.6.4 --- Reverse Transcription --- p.63Chapter 2.2.6.5 --- Polymerase Chain Reaction (PCR) --- p.63Chapter 2.2.6.6 --- Design of Primers for Different Genes --- p.64Chapter 2.2.6.7 --- Determination of the Number of Cycles in PCR for Different Genes --- p.67Chapter 2.2.7 --- Caspase Fluorescent Assay --- p.67Chapter 2.2.7.1 --- Caspase-3 or ´ؤ8 Assay --- p.67Chapter Chapter 3. --- Results --- p.59Chapter 3.1 --- Studies of the Characteristics of L929 and L929-11E cells --- p.70Chapter 3.1.1 --- Determination of the Growth Curve of L929 and L929-11E Cells --- p.70Chapter 3.2 --- Studies on the Effect of TNF-α on L929 and L929-11E Cells --- p.73Chapter 3.2.1 --- TNF-α Induced Cell Death in L929 Cells but not in L929- 11E Cells --- p.73Chapter 3.2.2 --- TNF-α Induced Apoptosis in a Time-dependent Manner in L929Cells but not in L929-11E Cells --- p.80Chapter 3.2.3 --- TNF-α Induced Mitochondrial Membrane Depolarization in a Time-dependent Manner in L929 Cells but notin L929-11E Cells --- p.87Chapter 3.2.4 --- TNF-α Induced Cytochrome c Release in a Time- dependent Manner in L929 Cells but not in L929-11E Cells --- p.92Chapter 3.3 --- Effect of Hyperthermia on L929 and L929-11E Cells --- p.96Chapter 3.3.1 --- Introduction --- p.95Chapter 3.3.2 --- Hyperthermia Induced Apoptosis in L929 and L929-11E Cells --- p.96Chapter 3.3.3 --- Effect of Hyperthermia on Mitochondrial Membrane Depolarization --- p.100Chapter 3.3.4 --- Hyperthermia Induced Cyto c Release in a Time-dependent Manner in L929 and L929-11E Cells --- p.105Chapter 3.4 --- Relationship of Hyperthermia and TNF-α with PTP in L929 Cells --- p.107Chapter 3.5 --- Effect of TNF-α and Hyperthermia on the Level of Hydrogen Peroxide (H202) in L929 and L929-11E Cells --- p.114Chapter 3.5.1 --- Introduction --- p.114Chapter 3.5.2 --- TNF-α Enhanced the Level of H202 in L929 cells but not in L929-11E Cells --- p.115Chapter 3.5.3 --- Hyperthermia Enhanced the Level of H202 in L929 and L929-11E cells --- p.117Chapter 3.6 --- Effect of TNF-α and Hyperthermia on the Level of Intracellular Calcium in L929 and L929-11E Cells --- p.122Chapter 3.6.1 --- Increase in the Intracellular Calcium Level Induced by TNF-α Was Related to the Mitochondrial Membrane Depolarization in L929 Cells but not in L929-11E Cells --- p.122Chapter 3.6.2 --- Hyperthermia Increased the Level of [Ca2+]i in L929 and L929-11E Cells in a Time-dependent Manner --- p.124Chapter 3.7 --- Effect of Combined Hyperthermia and TNF-α Treatment on the Induction of Apoptosis in L929 and L929-1 1E Cells --- p.129Chapter 3.7.1 --- Combined Treatment with Hyperthermia and TNF- α Induced Apoptosis in Both L929 and L929-11E cells --- p.129Chapter 3.7.2 --- Hyperthermia and Its Combined Treatment with TNF-α Induced Mitochondrial Membrane Depolarization in L929 and L929-11E Cells --- p.135Chapter 3.8 --- Investigation of the Downstream Apoptotic Pathway in L929 and L929-11E Cells Upon Hyperthermia and TNF-a treatment --- p.142Chapter 3.8.1 --- Introduction --- p.142Chapter 3.8.2 --- Effect ofTNF-α and Hyperthermia on p53 Expression --- p.142Chapter 3.8.3 --- Effect of Hyperthermia and TNF-α on PARP --- p.146Chapter 3.8.4 --- Effect of Hyperthermia and TNF-α on Caspase-3 Activity --- p.149Chapter 3.8.5 --- Effect of Hyperthermia and TNF-α on Bid protein --- p.158Chapter 3.8.6 --- Effect of Hyperthermia and TNF-α on Caspase-8 Activity --- p.165Chapter 3.8.7 --- Effect ofTNF-α on TNFR1 Expression --- p.169Chapter Chapter 4. --- DiscussionChapter 4.1 --- TNF-α Induced Apoptosis and Changed the Mitochondrial Activities in L929 Cells --- p.176Chapter 4.2 --- L929-11E cells Possessed Resistance Towards TNF-α --- p.187Chapter 4.3 --- Hyperthermia Triggered Apoptosis and Changed Mitochondrial Activities in L929 and L929-11E cells --- p.190Chapter 4.4 --- Combined hyperthermia and TNF-α treatment induced cell death and changed mitochondria activities in L929 and L929-11E cells --- p.195Chapter 4.5 --- Reversal of the TNF-α resistance and Enhancement of Sensitivity Towards Hyperthermia in L929-11E cells --- p.197Chapter 4.6 --- Proposed Pathway in the TNF-α- and Hyperthermia-mediated Apoptosis --- p.200Chapter 4.7 --- Application of TNF-α and Hyperthermia on Clinical Cancer Treatment --- p.203Chapter Chapter 5. --- Future Perspective of the Project --- p.206References --- p.21