Apoptosis : a practical approach by George P Studzinski

1. Overview of apoptosis George P. Studzinski 1. General introduction and overview of contents 2. Historical perspective 3. Distinction of apoptosis from other forms of cell death 4. Apoptotic cascades 5. Time course of apoptotic cascades 6. Selection of methods Procedure for determination of increased mitochondrial to nuclear DNA ratio, for the detection and quantitation of apoptosis 7. Pitfalls References 2. Morphological recognition of apoptotic cells James W. Wilson and Christopher S. Potten 1. Introduction Key morphological features of apoptotic cells 2. Light and fluorescent microscopy techniques for the assessment of apoptosis Preparation of cell or tissue samples Nuclear counterstains 3. Electron microscopic techniques 4. Quantitation of apoptotic events Methods Problems in scoring apoptotic events 5. In situ detection of DNA strand breaks 6. Other techniques 7. Conclusions References xv xix 1 1 5 5 8 8 11 12 14 16 19 19 19 20 20 23 27 29 29 31 33 37 38 38 Contents 3. Assessment of DNA damage in apoptosis Akira Yoshida, Rong-Guang Shao, and Yves Pommier 1. Introduction 2. Types of DNA fragmentation 3. Measurement of high molecular weight DNA fragmentation by pulse-field gel electrophoresis Pulse-field gel electrophoresis procedures Strategies for electrophoretic separations 4. Detection of internucleosomal DNA fragmentation by standard agarose gel electrophoresis 5. Filter elution assay to measure apoptotic DNA fragmentation Equipment Filters and solutions DNA labelling and preparation of experimental cell cultures Protocols Counting samples and computations References 4. Analysis of cell death by flow and laser-scanning cytometry Zbigniew Darzynkiewicz, Elzbieta Bedner, and Xun Li \. Introduction 2. Preparation of cells for analysis by LSC. Detection of apoptotic cells based on changes in nuclear chromatin 3. Light-scattering properties of apoptotic and necrotic cells 4. Analysis of mitochondrial transmembrane potential (Avm) 5. Detection of apoptotic cells exposing phosphatidylserine on plasma membrane 6. Detection of apoptotic cells based on their fractional DNA content 7. Identification of apoptotic cells based on the presence of DNA strand breaks 8. Identification of apoptotic cells based on the increased DNA sensitivity to denaturation 9. General comments References 41 41 41 43 44 46 47 48 49 49 50 51 52 53 57 57 59 61 63 67 68 71 75 77 78 x Contents 5. Cell-mediated cytotoxicity and cell death receptors Javier Naval and Alberto Anel 1. Overview of pathways for cell-mediated cytotoxicity Fas-based cytotoxicity Perforin/granzyme-based cytotoxicity 2. Standard methods for the evaluation of cell-mediated cytotoxicity Chromium release assay [125I]Iododeoxyuridine (125IUdR) release assay The JAM test BLT-esterase release assay Estimation of target cell nuclear fragmentation using fluorescent dyes Activation-induced cell death (AICD) 3. Separate studies of Fas- and perforin/granzyme-based cytotoxicity Fas-based cytotoxicity in the absence of perforin/granzyme contribution Perforin/granzyme-based cytotoxicity in the absence of FasL contribution 4. Use of caspase and granzyme inhibitors in cell-mediated cytotoxicity assays Caspase inhibitors Granzyme inhibitors References 6. Sphingolipids as messengers of cell death Gary M. Jenkins and Yusuf A. Hannun 1. Introduction 2. Sphingolipids and their role in apoptosis Overview of Sphingolipids Role of Sphingolipids in apoptosis Strategies and considerations in evaluating Sphingolipids 3. Extraction of Sphingolipids and normalization by lipid phosphate Extraction of sphingosines, ceramides, and sphingomyelins Alkaline hydrolysis of Sphingolipids Lipid phosphate measurement for normalization of experimental samples 4. Analysis of sphingoid backbones HPLC on sphingoid backbones 81 81 82 83 84 84 87 89 91 92 94 95 95 96 97 97 99 100 105 105 105 105 106 107 108 108 111 112 113 113 xi Contents 5. Analysis of ceramides 6. Analysis of sphingomyelins 7. Measurement of SMase and SM synthase activities References 7. Cytochemical detection of cytoskeletal and nucleoskeletal changes during apoptosis Manon van Engeland, Bert Schutte, Anton H. N. Hopman, Frans C. S. Ramaekers, and Chris P. M. Reutelingsperger 1. Introduction 2. Cyto- and nucleoskeletal changes during apoptosis General Microfilaments and microfilament-associated proteins Microtubules Intermediate filaments Nucleoskeletal components Generation of neo-epitopes in cytoskeletal proteins during apoptosis 3. Apoptosis detection systems Overview The annexin V affinity assay Detection of apoptosis using the TUNEL assay References 8. Metabolic alterations associated with apoptosis Ana P. Costa-Pereira and Thomas G. Cotter 1. Introduction 2. Detection of changes in the mitochondrial transmembrane potential 3. Detection of intracellular reactive oxygen intermediates (ROI) 4. Determination of glutathione levels and its oxidative state 5. Measurement of catalase in cells undergoing apoptosis References 115 118 120 123 125 125 126 126 126 126 127 128 128 129 129 129 135 138 141 141 142 144 146 153 155 xii Contents 9. Methods of measuring Bcl-2 family proteins and their functions John C. Reed, Zhihua Xie, Shinichi Kitada, Juan M. Zapata, Qunli Xu, Sharon Schendel, Maryla Krajewska, and Stanislaw Krajewski 1. Introduction 2. Immunoblot analysis Cell lysis and tissue processing procedures Immunoblotting procedure 3. Detection of Bcl-2 family proteins by flow cytometry (FACS) Indirect immunofluorescence procedure Antibodies 4. Northern blot analysis of BCL-2 m RNA General guidelines for RNA blotting Pre-hybridization and hybridization procedures Solutions 5. Reverse transcriptase-polymerase chain reaction (RT-PCR) cDNA synthesis PCR amplication Southern blot analysis of PCR products Primers and probes for PCR 6. Immunohistochemical detection of Bcl-2 family proteins in tissues Procedures for tissue preparation, embedding, and sectioning Pre-staining sample preparation Immunostaining Counterstaining and mounting Solutions 7. Immunohistochemical detection of Bcl-2 protein in cultured cells Procedures Immunostaining Solutions 8. Production of recombinant Bcl-2 family proteins in bacteria Human Bcl-2 Human Bcl-XL protein Mouse Bax protein Human Bid protein 9. Measurements of pore formation by Bcl-2 family proteins Liposome preparation Channel activity measurements 157 157 157 158 161 165 166 167 167 168 169 170 171 172 172 173 174 175 177 180 183 185 186 187 188 189 191 191 192 194 197 199 201 202 204 xiii Contents 10. Methods of assaying Bcl-2 and Bax family protein function in yeast Plasmid considerations Saccharomyces cerevisiae strains Media and growth conditions Transformation of S. cerevisiae by the LiOAc method Assay for Bax-induced lethality in S. cerevisiae 11. Summary References 10. Methods for detecting proteolysis during apoptosis in intact cells April L. Blajeski and Scott H. Kaufmann 1. Introduction 2. Involvement of proteases in apoptosis 3. Caspase nomenclature and classification 4. Detection of procaspases and their cleavage products by immunoblotting Theoretical considerations Practical considerations in immunoblotting for caspases Sample preparation and immunoblotting 5. Cleavage of caspase targets Establishing that proteases are activated in situ Establishing that a polypeptide is cleaved by caspases 6. Assays of caspase activity Theoretical considerations of caspase activity assays Measuring caspase activity in cell lysates 7. Detection of active caspases by affinity labelling Theoretical considerations in affinity labelling Practical considerations in the use of affinity labelling reagents Labelling and detecting enzymatically active caspases 8. Studying the biological effects of caspase cleavages A molecular approach for analysis of the effects of substrate cleavage Use of caspase inhibitors: promises and pitfalls References List of Suppliers Index 205 205 207 207 209 210 212 212 215 215 215 217 218 218 222 222 224 224 224 226 227 227 230 230 232 232 234 235 235 236 239 243 xiv