Programmed Cell Death Part B (eBook)

Cover 1
Copyright Page 5
TOC$Contents 6
Contributors 14
Preface 22
Volume in Series 22
CH$Chapter 1: Analysis of Autophagic and Necrotic Cell Death in Dictyostelium 50
1. Introduction 51
2. Growing Dictyostelium Cells in Monolayer Cultures 52
3. Inducing Autophagic Vacuolar Cell Death 53
4. Further Exploration of Autophagic Cell Death 55
5. Inducing Necrotic Cell Death 59
6. Further Exploration of Necrotic Cell Death 59
References 62
CH$Chapter 2: Methods and Protocols for Studying Cell Death in Drosophila 66
1. Introduction 67
2. Cell Death Analysis During Development 71
3. Biochemical Analysis 76
4. Genetic Analysis 80
References 84
CH$Chapter 3: In Vivo and In Vitro Methods for Studying Apoptotic Cell Engulfment in Drosophila 88
1. Introduction 89
2. Culture Conditions 90
3. Labeling of Apoptotic Cells 92
4. Visualizing the Macrophages 95
5. Quantification and Statistics 99
6. Engulfment in Cell Culture 101
Acknowledgments 106
References 106
CH$Chapter 4: A Mouse Mammary Epithelial Cell Model to Identify Molecular Mechanisms Regulating Breast Cancer Progression 110
1. Introduction 111
2. A Novel Mouse Mammary Epithelial Cell Model 112
3. Protocols 114
4. Concluding Remarks 123
References 123
CH$Chapter 5: Immortalized Mouse Epithelial Cell Models to Study the Role of Apoptosis in Cancer 126
1. Introduction 127
2. Immortalization of Baby Mouse Kidney, Mammary, Prostate, and Ovarian Surface Epithelial Cells 130
3. Development of iBMK Cell Lines for Identifying the Role of Apoptosis in Cancer 143
4. Aposcreen: An iBMK Cell-Based Screen for the Identification of Apoptosis-Inducing Compounds as Potential Anti-Cancer Agents 144
5. Adaptation of the iBMK Cell Model System for the Evaluation of Tissue-Specific Tumor-Promoting Functions 146
6. Tumorigenicity Assay 149
7. Noninvasive In Vivo Monitoring of Tumor Growth by Expression of Fluorescence and Chemiluminescence Markers 151
8. Tumor-Derived Cell Lines (TDCLs) as a Model System for Epithelial Tumor-Promoting Functions 152
9. Concluding Remarks and Future Perspectives 153
Acknowledgments 154
References 154
CH$Chapter 6: DNA Damage Response and Apoptosis 156
1. Introduction 157
2. Methods 159
Notes 168
Acknowledgments 169
References 169
CH$Chapter 7: Phorbol Ester-Induced Apoptosis and Senescence in Cancer Cell Models 172
1. Introduction 173
2. PKCdelta as a Pro-Apoptotic Kinase 173
3. PKCdelta as a Mediator of Apoptotic Responses in Prostate Cancer Cells: The Emergence of an Autocrine Paradigm 174
4. Roles of PKCalpha and PKCepsilon LNCAP Cell Death and Survival 176
5. Regulation of Cell Cycle and Senescence by PKC 177
6. Determination of Phorbol Ester-Induced Apoptosis in Prostate Cancer Cells 178
7. Adenoviral Expression of PKC Isozymes 180
8. RNA Interference of PKC Isozymes 181
9. Collection and Storage of CM from LNCaP Cells 182
10. Determination of Senescence in Response to Phorbol Esters 183
References 185
CH$Chapter 8: Manipulation of PKC Isozymes by RNA Interference and Inducible Expression of PKC Constructs 190
1. Introduction 190
2. Methods 192
Acknowledgments 204
References 204
CH$Chapter 9: Studying p53-Dependent Cell Death In Vitro and In Vivo 208
1. Introduction 208
2. Studying p53-Dependent Cell Death In Vitro 209
3. Overexpression of p53 by Adenovirus (Ad) 209
4. Silencing the p53 Gene by Retroviral Vector 213
5. Studying the Apoptotic Response 214
6. Studying p53-Dependent Apoptosis In Vivo 214
References 221
CH$Chapter 10: NF-kappaB as a Determinant of Distinct Cell Death Pathways 224
1. Introduction 225
2. Methods 227
3. DNA 229
4. Proteins 232
5. Metabolic Health 233
6. Senescence Detection 234
References 235
CH$Chapter 11: Purification and Bioassay of Hedgehog Ligands for the Study of Cell Death and Survival 238
1. Introduction 239
2. Hedgehog and the Regulation of Cell Death 242
3. Generation of Recombinant Hedgehog Ligands 244
References 250
CH$Chapter 12: Experimental Approaches to Investigate the Proteasomal Degradation Pathways Involved in Regulation of Apoptosis 254
1. Introduction 255
2. General Instructions 256
3. Protocols 256
References 271
CH$Chapter 13: Ubiquitination Mediated by Inhibitor of Apoptosis Proteins 274
1. Introduction 274
2. In Vitro Ubiquitination Assay 275
3. In Vivo Ubiquitination Assay 281
Acknowledgments 283
References 283
CH$Chapter 14: Phosphatases and Regulation of Cell Death 286
1. Introduction 287
2. Identification of the Putative Phosphatase in Cells 289
3. In Vitro Phosphatase Assays 297
4. Interaction of the Phosphatase and Apoptotic Protein 298
5. Linking Phosphatase Activity to Cell Death Protein Function 302
6. Conclusion 304
Acknowledgments 304
References 304
CH$Chapter 15: Analysis of Neuronal Cell Death in Mammals 308
1. Introduction 309
2. Apoptosis Detection In Vitro 309
3. Assessment of Cell Death 311
4. Apoptosis Detection in Brain Development 318
5. Detection of Synaptic Apoptosis (Synaptosis) 321
6. Conclusions 324
Acknowledgments 324
References 324
CH$Chapter 16: Dissecting Apoptosis and Intrinsic Death Pathways in the Heart 326
1. Introduction 327
2. Protocols 328
Acknowledgments 332
References 333
CH$Chapter 17: Quantification of Vascular Endothelial Cell Apoptosis In Vivo 336
1. Introduction 336
2. Protocols 337
References 341
CH$Chapter 18: Recombinant TRAIL and TRAIL Receptor Analysis 342
1. Introduction 343
2. Generation of Recombinant TRAIL 344
3. Analysis of TRAIL Receptors 351
4. Concluding Remarks 360
Acknowledgments 360
References 361
CH$Chapter 19: Analysis of Tnf-Related Apoptosis-Inducing Ligand In Vivo Through Bone Marrow Transduction and Transplantation 364
1. Introduction 365
2. Generation of Retroviral Infection Medium 366
3. Bone Marrow Cell Preparation 367
4. Retroviral Transduction of Bone Marrow Cells 368
5. Bone Marrow Transplantation 370
6. Assessment of Bone Marrow Transplantation 371
7. Pathologic Evaluation of TRAIL-Transduced Mice 372
8. Methods for Immunohistochemical Staining for TRAIL Expression 375
9. Applications that Use the Bone Marrow Transplantation Approach 377
Acknowledgments 378
References 378
CH$Chapter 20: Overcoming Resistance to Trail-Induced Apoptosis in Prostate Cancer by Regulation of c-FLIP 382
1. Introduction 383
2. Structure and Molecular Mechanisms of c-FLIP in Apoptosis 383
3. Expression of c-FLIP 384
4. Regulation of c-FLIP Expression 386
5. Targeting c-FLIP to Enhance Apoptosis in Cancer Cells 390
6. Methods and Materials 391
Acknowledgments 395
References 395
CH$Chapter 21: Caspase Assays: Identifying Caspase Activity and Substrates In Vitro and In Vivo 400
1. Introduction 401
2. Caspase Methods 402
3. Conclusion 414
References 415
CH$Chapter 22: Synthesis and Biophysical Characterization of Stabilized alpha-Helices of BCL-2 Domains 418
1. Introduction 419
2. Synthesis of Non-Natural Amino Acids for Peptide Stapling 421
3. Design and Synthesis of Stabilized alpha-Helices of BCL-2 Domains (SAHBs) 422
4. Structural Assessment of SAHBs by Circular Dichroism (CD) 425
5. Protease Resistance Testing of SAHBs 427
6. Cell Permeability Screening of SAHBs 430
7. Summary 432
Acknowledgments 433
References 433
CH$Chapter 23: Dissection of the BCL-2 Family Signaling Network with Stabilized alpha-Helices of BCL-2 Domains 436
1. Introduction 437
2. BCL-2 Family Binding Measurements by Fluorescence Polarization Assay 437
3. In Vitro Release Assays as a Measure of Pro-Apoptotic Activity 441
4. Measurement of Cellular Apoptosis Induction 449
5. Identification of In Situ Mechanistic Targets of SAHBs 452
6. Summary 454
Acknowledgments 454
References 454
CH$Chapter 24: ERM-Mediated Genetic Screens in Mammalian Cells 458
1. Introduction 458
2. Methods and Design 460
3. ERM-Mediated Genetic Screens and Re-PCA 464
4. Conclusions 466
Acknowledgments 467
References 467
Author Index 470
IDX$Subject Index 494
Color Plate Section 500