Hiyama Telomeres and Telomerase in Cancer

1. Auflage 2009
ISBN: 978-1-60327-879-9
Verlag: Humana Press
Format: PDF
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 380 Seiten

Telomerase, an enzyme that maintains telomeres and endows eukaryotic cells with immortality, was first discovered in tetrahymena in 1985. In 1990s, it was proven that this enzyme also plays a key role in the infinite proliferation of human cancer cells. Now telomere and telomerase are widely accepted as important factors involved in cancer biology, and as promising diagnostic tools and therapeutic targets. Recently, role of telomerase in 'cancer stem cells' has become another attractive story. Until now, there are several good books on telomere and telomerase focusing on biology in ciliates, yeasts, and mouse or basic sciences in human, providing basic scientists or students with updated knowledge.

Autoren/Hrsg.

Hiyama, Keiko

Inhaltsverzeichnis

1;Preface;5
2;Contents;6
3;Contributors;8
4;Part I: Basic Background;12
4.1;Chapter 1;14
4.1.1;Telomeres and Telomerase in Humans;14
4.1.1.1;1.1 Introduction;14
4.1.1.2;1.2 Telomere Structure;15
4.1.1.3;1.3 Why do Telomeres Gradually Shortened?;20
4.1.1.4;1.4 Functions of Telomeres and Telomerase;21
4.1.1.5;1.5 Two Mortality Stage Mechanisms and Telomere Hypothesis;22
4.1.1.6;1.6 Telomerase is a Conserved Reverse-Transcriptase;23
4.1.1.7;1.7 Telomerase Activity and Cellular Immortalization;24
4.1.1.8;1.8 Telomerase is Activated in >80% of Human Malignancies;24
4.1.1.9;1.9 Telomerase Activity in Normal Somatic Cells and Stem Cells;24
4.1.1.10;1.10 Telomere-Binding Proteins;25
4.1.1.11;1.11 Telomere Dysfunction and Human Diseases;26
4.1.1.12;1.12 Concluding Remarks;26
4.1.1.13;References;26
4.2;Chapter 2;33
4.2.1;Telomere-Binding Proteins in Humans;33
4.2.1.1;2.1 Introduction;33
4.2.1.2;2.2 Telomere-Binding Proteins in Human Cells;35
4.2.1.2.1;2.2.1 Shelterin;35
4.2.1.2.1.1;2.2.1.1 TRF1 and TRF2;35
4.2.1.2.1.2;2.2.1.2 TIN2;36
4.2.1.2.1.3;2.2.1.3 TPP1 and POT1;37
4.2.1.2.1.4;2.2.1.4 RAP1;37
4.2.1.2.1.5;2.2.1.5 Perspective;38
4.2.1.2.2;2.2.2 Telomere-Associated Proteins in Human Cells;38
4.2.1.3;2.3 Shelterin Shapes Telomeric DNA into a Protected Structure;39
4.2.1.3.1;2.3.1 The Role of Shelterin in Generation of the 30' Overhang;39
4.2.1.3.2;2.3.2 T-Loop Formation;41
4.2.1.3.3;2.3.3 Prevention of Inappropriate T-Loop Deletion;42
4.2.1.4;2.4 Suppression of the ATR and ATM DNA Damage Response Pathways;43
4.2.1.4.1;2.4.1 Suppression of ATR Pathway;43
4.2.1.4.2;2.4.2 Suppression of ATM Pathway;45
4.2.1.5;2.5 Prevention of Inappropriate NHEJ and HR Repair at Chromosome Ends;46
4.2.1.6;2.6 Telomere Length Regulation by the Shelterin Complex;47
4.2.1.6.1;2.6.1 Shelterin-Mediated Control of Telomerase;47
4.2.1.6.1.1;2.6.1.1 TRF1;48
4.2.1.6.1.2;2.6.1.2 POT1 and TPP1;48
4.2.1.6.1.3;2.6.1.3 TRF2 and RAP1;49
4.2.1.6.1.4;2.6.1.4 Tankylase 1;49
4.2.1.6.1.5;2.6.1.5 Perspective;50
4.2.1.6.2;2.6.2 Telomere Length Homeostasis in the Absence of Telomerase;50
4.2.1.7;2.7 Concluding Remarks;51
4.2.1.8;References;52
4.3;Chapter 3;57
4.3.1;Regulation of Telomerase Through Transcriptional and Posttranslational Mechanisms;57
4.3.1.1;3.1 Introduction;57
4.3.1.2;3.2 Transcriptional Regulators;58
4.3.1.2.1;3.2.1 Negative Transcriptional Regulators;58
4.3.1.2.1.1;3.2.1.1 Mad/Max;58
4.3.1.2.1.2;3.2.1.2 Other Repressors Targeting c-Myc-Dependent Telomerase Activation;60
4.3.1.2.1.3;3.2.1.3 BRCA1;60
4.3.1.2.1.4;3.2.1.4 p53;61
4.3.1.2.1.5;3.2.1.5 Rb;61
4.3.1.2.1.6;3.2.1.6 IRF1 and p27Kip1;62
4.3.1.2.1.7;3.2.1.7 TGFb, Smad3, and SIP1;62
4.3.1.2.1.8;3.2.1.8 AP1;62
4.3.1.2.1.9;3.2.1.9 WT1;63
4.3.1.2.1.10;3.2.1.10 Other Tumor Suppressors;63
4.3.1.2.1.11;3.2.1.11 Perspective;64
4.3.1.2.2;3.2.2 Positive Transcriptional Regulators;64
4.3.1.2.2.1;3.2.2.1 c-Myc;64
4.3.1.2.2.2;3.2.2.2 Survivin;65
4.3.1.2.2.3;3.2.2.3 hALP;65
4.3.1.2.2.4;3.2.2.4 TEIF;65
4.3.1.2.2.5;3.2.2.5 JNK Pathway;65
4.3.1.2.2.6;3.2.2.6 HPV 16 E6;66
4.3.1.2.2.7;3.2.2.7 STAT3;66
4.3.1.2.2.8;3.2.2.8 EWS-ETS;66
4.3.1.2.2.9;3.2.2.9 HIF1alpha;67
4.3.1.2.3;3.2.3 Both Positive and Negative Regulators;67
4.3.1.2.3.1;3.2.3.1 E2F;67
4.3.1.2.3.2;3.2.3.2 Sp1;68
4.3.1.2.3.3;3.2.3.3 USF1 and USF2;68
4.3.1.2.3.4;3.2.3.4 p73;69
4.3.1.2.3.5;3.2.3.5 Perspective;70
4.3.1.3;3.3 Posttranslational Regulators;70
4.3.1.3.1;3.3.1 Kinases;70
4.3.1.3.1.1;3.3.1.1 PP2A and Akt;70
4.3.1.3.1.2;3.3.1.2 PKC;71
4.3.1.3.1.3;3.3.1.3 c-Abl and KIP;72
4.3.1.3.1.4;3.3.1.4 MAPK;72
4.3.1.3.1.5;3.3.1.5 Perspective;72
4.3.1.3.2;3.3.2 Ligases;73
4.3.1.3.2.1;3.3.2.1 MKRN1 and Smurf2;73
4.3.1.3.2.2;3.3.2.2 E6/E6AP;73
4.3.1.3.2.3;3.3.2.3 Perspective;74
4.3.1.3.3;3.3.3 Polymerases;74
4.3.1.4;3.4 Regulators of Assembly;75
4.3.1.5;3.5 Additional Regulators;77
4.3.1.5.1;3.5.1 Telomere-Binding Proteins;77
4.3.1.5.1.1;3.5.1.1 Shelterin and Associated Proteins;77
4.3.1.5.1.2;3.5.1.2 PINX1;77
4.3.1.5.1.3;3.5.1.3 TopoIIa;77
4.3.1.5.1.4;3.5.1.4 HP1;78
4.3.1.5.1.5;3.5.1.5 Perspective;79
4.3.1.5.2;3.5.2 Localization;79
4.3.1.5.2.1;3.5.2.1 Nucleolin;79
4.3.1.5.2.2;3.5.2.2 TNFalpha;79
4.3.1.5.2.3;3.5.2.3 14-3-3 Family;80
4.3.1.5.2.4;3.5.2.4 PINX1;80
4.3.1.5.3;3.5.3 Viral Proteins;80
4.3.1.5.3.1;3.5.3.1 HPV E6 and E2;80
4.3.1.5.3.2;3.5.3.2 LMP2A;81
4.3.1.5.3.3;3.5.3.3 LANA and E1A;81
4.3.1.5.3.4;3.5.3.4 HBV;81
4.3.1.5.3.5;3.5.3.5 Perspective;82
4.3.1.5.4;3.5.4 Hormone Receptors;82
4.3.1.5.4.1;3.5.4.1 ER;82
4.3.1.5.4.2;3.5.4.2 PR;82
4.3.1.5.4.3;3.5.4.3 AR;83
4.3.1.5.5;3.5.5 hTR Binders;83
4.3.1.5.5.1;3.5.5.1 pRb and Small Nucleolar RNA-Binding Proteins;83
4.3.1.5.5.2;3.5.5.2 NF-Y and MDM2;83
4.3.1.5.5.3;3.5.5.3 hnRNPs;83
4.3.1.6;3.6 Concluding Remarks;84
4.3.1.7;References;86
4.4;Chapter 4;96
4.4.1;Telomere Dysfunction and the DNA Damage Response;96
4.4.1.1;4.1 Introduction;97
4.4.1.2;4.2 Telomeres;97
4.4.1.2.1;4.2.1 Structure, Function, and Telomere-Associated Proteins;97
4.4.1.2.2;4.2.2 Telomerase and Telomere Maintenance;100
4.4.1.2.3;4.2.3 Telomere Dysfunction and Genomic Instability;101
4.4.1.3;4.3 DNA Damage Response;102
4.4.1.3.1;4.3.1 Components of the Response and Signaling Pathways;103
4.4.1.3.2;4.3.2 Mechanisms of DNA Repair;105
4.4.1.3.2.1;4.3.2.1 Homologous Recombination;106
4.4.1.3.2.2;4.3.2.2 Nonhomologous End Joining;108
4.4.1.3.2.3;4.3.2.3 Perspective;108
4.4.1.4;4.4 Linking DNA Damage Response and Telomere Dysfunction;109
4.4.1.4.1;4.4.1 DNA Damage and Telomere-Binding Proteins: A Role in Telomere Maintenance;109
4.4.1.4.2;4.4.2 Resolution of the Paradox;111
4.4.1.4.3;4.4.3 Cellular Fates;112
4.4.1.5;4.5 Clinical Implications;114
4.4.1.5.1;4.5.1 Aging;115
4.4.1.5.2;4.5.2 Genome Instability and Premature Aging Syndromes;115
4.4.1.5.2.1;4.5.2.1 Ataxia Telangiectasia and Ataxia Telangiectasia-Like Disorder;115
4.4.1.5.2.2;4.5.2.2 Nijmegen Break Syndrome;116
4.4.1.5.2.3;4.5.2.3 Bloom and Werner Syndromes;116
4.4.1.5.2.4;4.5.2.4 Hutchinson–Guilford Progeria Syndrome;117
4.4.1.5.3;4.5.3 Dyskeratosis Congenita;118
4.4.1.5.4;4.5.4 Cancer;118
4.4.1.5.5;4.5.5 Neurodegenerative Diseases;119
4.4.1.6;4.6 Therapeutic Possibilities;120
4.4.1.6.1;4.6.1 Telomerase and Telomere-Based Therapies;120
4.4.1.6.2;4.6.2 Targeting DNA Damage Response and Repair;121
4.4.1.7;4.7 Concluding Remarks;122
4.4.1.8;References;123
4.5;Chapter 5;135
4.5.1;Alternative Lengthening of Telomeres in Human Cells;135
4.5.1.1;5.1 Introduction;135
4.5.1.2;5.2 Telomere Length Phenotype;136
4.5.1.3;5.3 ALT-Associated PML Bodies;138
4.5.1.4;5.4 Telomere Exchange Events in ALT Cells;141
4.5.1.5;5.5 Telomeric Recombination in ALT Cells;141
4.5.1.6;5.6 T-Loops and T-Circles;143
4.5.1.7;5.7 Minisatellite Instability Associated with ALT;145
4.5.1.8;5.8 Proteins Involved in the ALT Mechanism and its Repression;145
4.5.1.9;5.9 Does More than One ALT Mechanism Exist?;147
4.5.1.10;5.10 Telomere Maintenance in Normal Mammalian Biology;148
4.5.1.11;5.11 Repression of Telomerase in ALT-Positive Cell Lines;149
4.5.1.12;5.12 Coexistence of ALT and Telomerase in the Same Cells;149
4.5.1.13;5.13 ALT in Human Tumours;150
4.5.1.14;5.14 Concluding Remarks;151
4.5.1.15;References;152
4.6;Chapter 6;157
4.6.1;Mouse Model: Telomeres and Telomerase in Stem Cell and Cancer;157
4.6.1.1;6.1 Introduction;158
4.6.1.1.1;6.1.1 Telomeres and Telomerase;158
4.6.1.1.2;6.1.2 Telomeres and Telomerase in Stem Cell;158
4.6.1.1.3;6.1.3 Telomeres and Telomerase in Cancer Stem Cell;160
4.6.1.2;6.2 Mouse Model;160
4.6.1.2.1;6.2.1 Terc-Deficient Mouse Model;161
4.6.1.2.1.1;6.2.1.1 Terc–/– Mouse Model and Cancer;162
4.6.1.2.1.2;6.2.1.2 Terc–/–Mouse Model and Tissue Specific Stem Cells;163
4.6.1.2.2;6.2.2 Tert-Deficient Mouse Model;163
4.6.1.2.3;6.2.3 Super-Tert Mouse Model;164
4.6.1.2.4;6.2.4 Atm in Telomere Dysfunctional Mice;165
4.6.1.2.5;6.2.5 p53 and Telomere Dysfunction in Mice;165
4.6.1.2.6;6.2.6 Deletion of p21 in Terc–/–Mice;166
4.6.1.2.7;6.2.7 p16Ink4a/p19Arf in Terc–/–Mice;167
4.6.1.2.8;6.2.8 PKcs in Terc–/–Mice;168
4.6.1.2.9;6.2.9 Wrn and Blm in Terc–/–Mice;168
4.6.1.2.10;6.2.10 Exo-1 in Terc–/–Mice;169
4.6.1.2.11;6.2.11 Terc–/–and PMS2–/–;169
4.6.1.3;6.3 Concluding Remarks;170
4.6.1.4;References;170
5;Part II: Telomers and Telomerase in Human Cancers;176
5.1;Chapter 7;177
5.1.1;Role of Telomeres and Telomerase in Cancer;177
5.1.1.1;7.1 Introduction;177
5.1.1.2;7.2 Telomerase in Carcinogenesis and Cellular Immortalization;180
5.1.1.3;7.3 Additional Roles of Telomerase in Tumorigenesis;182
5.1.1.4;7.4 Telomeres and Telomerase in Cancer Stem Cells;183
5.1.1.5;7.5 Application to Anticancer Strategy;183
5.1.1.6;References;184
5.2;Chapter 8;187
5.2.1;Diagnostic Value I: Solid Tumors;187
5.2.1.1;8.1 Introduction;188
5.2.1.2;8.2 Detecting Human Telomerase/TERT in Clinical Materials of Human Malignant Tumors;191
5.2.1.3;8.3 Telomerase/TERT as Biomarkers for Human Malignant Tumors;193
5.2.1.3.1;8.3.1 Telomerase in the Screening for High-Risk Premalignant Lesions;193
5.2.1.3.2;8.3.2 Telomerase/TERT as a Diagnostic Marker;194
5.2.1.3.2.1;8.3.2.1 Head, Neck Tumors, and Esophageal Tumors;194
5.2.1.3.2.2;8.3.2.2 Thyroid and Breast Tumors;196
5.2.1.3.2.3;8.3.2.3 Lung;197
5.2.1.3.2.4;8.3.2.4 Digestive Organs;197
5.2.1.3.2.5;8.3.2.5 Pleural Effusion and Peritoneal Lavage;198
5.2.1.3.2.6;8.3.2.6 Genitourinary Organs;199
5.2.1.3.2.7;8.3.2.7 Skin;200
5.2.1.3.2.8;8.3.2.8 Detecting Circulating Cancer Cells;200
5.2.1.3.2.9;8.3.2.9 Sarcoma;200
5.2.1.3.3;8.3.3 Telomerase/TERT Expression in Prediction of Prognosis and Grading of Malignant Tumors;201
5.2.1.3.3.1;8.3.3.1 Brain Tumors;201
5.2.1.3.3.2;8.3.3.2 Childhood Malignant Tumors;201
5.2.1.3.3.3;8.3.3.3 Prognostic Marker for Other Cancers;203
5.2.1.4;8.4 Concluding Remarks;203
5.2.1.5;References;204
5.3;Chapter 9;217
5.3.1;Diagnostic Value II: Hematopoietic Malignancies;217
5.3.1.1;9.1 Introduction;217
5.3.1.2;9.2 Stem Cell, Niche, and Quiescence;218
5.3.1.3;9.3 Regulation on Telomere/Telomerase in Normal HSC;220
5.3.1.4;9.4 Telomere Biology in the Transplanted HSC Population;221
5.3.1.5;9.5 Telomeres and Telomerase in Human Leukemia Cells;221
5.3.1.6;9.6 Genomic Instability and Telomere Attrition in Leukemia and Bone Marrow Failure Syndromes;224
5.3.1.7;9.7 Concluding Remarks;225
5.3.1.8;References;226
5.4;Chapter 10;231
5.4.1;Therapeutic Targets and Drugs I: Telomerase and Telomerase Inhibitors;231
5.4.1.1;10.1 Introduction;231
5.4.1.2;10.2 Telomerase as a Cancer Therapeutic Target: Original Hypotheses and Proof-of-Principle Studies;232
5.4.1.3;10.3 Telomerase as a Cancer Therapeutic Target: Molecular Targets;234
5.4.1.3.1;10.3.1 Targeting hTERT;234
5.4.1.3.1.1;10.3.1.1 Nucleoside Analog;235
5.4.1.3.1.2;10.3.1.2 Nonnucleoside Agents;235
5.4.1.3.1.3;10.3.1.3 Other Direct and Indirect Methods;238
5.4.1.3.2;10.3.2 Targeting hTERC (hTR or hTER);239
5.4.1.3.2.1;10.3.2.1 Antisense Oligonucleotides;239
5.4.1.3.2.2;10.3.2.2 Chemically Modified Oligonucleotides;239
5.4.1.3.2.3;10.3.2.3 PS-, NP-, and NPS-ODNs;240
5.4.1.3.2.4;10.3.2.4 RNAi, Ribozymes, and Mutant hTERCs;241
5.4.1.3.2.5;10.3.2.5 Summary;242
5.4.1.3.3;10.3.3 Targeting Telomerase-Associated Proteins;242
5.4.1.3.4;10.3.4 Targeting Telomerase Activity and Accessibility to Telomeres;244
5.4.1.3.4.1;10.3.4.1 G-Quadruplex Interacting Agents;244
5.4.1.3.4.2;10.3.4.2 Tankyrase Inhibitors;245
5.4.1.4;10.4 Telomerase Inhibitors in Clinical Trials;246
5.4.1.4.1;10.4.1 GRN163L;246
5.4.1.4.2;10.4.2 Immunotherapy: TERT Vaccine;247
5.4.1.5;10.5 Summary and Perspectives;248
5.4.1.6;References;248
5.5;Chapter 11;256
5.5.1;Therapeutic Targets and Drugs II: G-Quadruplex and G-Quadruplex Inhibitors;256
5.5.1.1;11.1 Introduction;256
5.5.1.2;11.2 Biological Evidence of DNA G-Quadruplexes in Telomeres;259
5.5.1.3;11.3 Human Telomeric G-Quadruplex Structures and Polymorphism;261
5.5.1.3.1;11.3.1 The Hybrid-1 Type Intramolecular Human Telomere G-Quadruplex Structures Formed in K+ Solution by the Modified 26-nt Human Telomeric Sequence Tel26;264
5.5.1.3.2;11.3.2 The Hybrid-2 Type Intramolecular Human Telomere G-Quadruplex Structures Formed in K+ Solution by the Wild-Type 26-nt Human Telomeric Sequence wtTel26;264
5.5.1.3.3;11.3.3 The Hybrid-1 and Hybrid-2 Human Telomeric G-Quadruplexes are Closely Related Yet Distinct in Their Folding and Structures;266
5.5.1.3.4;11.3.4 Capping Structures Determine the Selective Formation of Hybrid-1 or Hybrid-2 Telomeric G-Quadruplex Structure;266
5.5.1.3.5;11.3.5 Human Telomeric Sequences Form a Mixture of Hybrid-1 and Hybrid-2 Structures with a Low Energy Barrier; the Population of Two Forms Largely Depends on the 3'-Flanking Sequences;267
5.5.1.3.6;11.3.6 Insights into the G-Quadruplex Loop Conformations;267
5.5.1.3.7;11.3.7 Hybrid-Type Structures are the Predominant Conformations for wtTel26 and Tel26 in K+ Solution, Even in the Copresence of a High Concentration of Na+;268
5.5.1.3.8;11.3.8 Structure Polymorphism of Human Telomeric G-Quadruplexes May be Related with the Highly Conserved Asymmetric Human Telomeric DNA Sequence;270
5.5.1.3.9;11.3.9 Potential of the Hybrid-Type Human Telomeric G-Quadruplexes to Form Higher-Order Multimers;271
5.5.1.4;11.4 Ligands Targeting the Telomeric G-Quadruplexes and Their Biological Effects;272
5.5.1.4.1;11.4.1 Telomestatin;273
5.5.1.4.2;11.4.2 BRACO19;274
5.5.1.4.3;11.4.3 RHPS4;275
5.5.1.4.4;11.4.4 TMPyP4;275
5.5.1.4.5;11.4.5 12459;276
5.5.1.4.6;11.4.6 307A;277
5.5.1.4.7;11.4.7 Other Compounds;277
5.5.1.4.8;References;277
5.6;Chapter 12;286
5.6.1;Therapeutic Targets and Drugs III: Tankyrase 1, Telomere-Binding Proteins, and Inhibitors;286
5.6.1.1;12.1 Introduction: Molecular Targeting Therapy of Cancer;286
5.6.1.2;12.2 Telomerase Inhibition as an Anticancer Strategy;287
5.6.1.3;12.3 Telomere Length Regulation by TRF1 and Tankyrase 1;288
5.6.1.4;12.4 Tankyrase 1 Inhibition Enhances the Anticancer Impact of Telomerase Inhibitors;290
5.6.1.5;12.5 Tankyrase 1 Expression and Nontelomeric Function;290
5.6.1.6;12.6 Telomere Integrity Maintenance by the 30-Overhang and the T-Loop Structure;291
5.6.1.7;12.7 Shelterin Components as Candidates for Anticancer Molecular Targets;292
5.6.1.8;12.8 Conclusion and Perspective;293
5.6.1.9;References;294
5.7;Chapter 13;297
5.7.1;Therapeutic Targets and Drugs IV: Telomerase-Specific Gene and Vector-Based Therapies for Human Cancer;297
5.7.1.1;13.1 Introduction;298
5.7.1.2;13.2 Regulation of hTERT Transcription;299
5.7.1.3;13.3 hTERT Promoter for Therapeutic Transgene Expression;300
5.7.1.4;13.4 hTERT Promoter for Oncolytic Virotherapy;301
5.7.1.4.1;13.4.1 Construction of Telomerase-Specific Oncolytic Adenovirus;301
5.7.1.4.2;13.4.2 Preclinical Studies of Telomelysin;302
5.7.1.4.3;13.4.3 Multidisciplinary Therapy with Telomelysin;304
5.7.1.4.4;13.4.4 Clinical Application of Telomelysin;306
5.7.1.5;13.5 hTERT Promoter for Cancer Diagnostics;307
5.7.1.5.1;13.5.1 Imaging of Tumor Cells Using Telomerase-Specific Oncolytic Adenovirus;307
5.7.1.5.2;13.5.2 In Vivo Imaging of Micrometastasis with TelomeScan;309
5.7.1.6;13.6 Conclusions and Perspectives;310
5.7.1.7;References;311
6;Part III: Experimental Protocols;317
6.1;Chapter 14;318
6.1.1;Protocol I: Telomerase Activity and Telomerase Expression;318
6.1.1.1;14.1 Introduction;318
6.1.1.2;14.2 Telomeric Repeat Amplification Protocol Assay;319
6.1.1.2.1;14.2.1 Original TRAP Assay;320
6.1.1.2.2;14.2.2 Modified TRAP Assay;324
6.1.1.2.3;14.2.3 Internal Standard and Quantification in TRAP Assay;325
6.1.1.2.4;14.2.4 Method for Removal of PCR Inhibitors;326
6.1.1.2.5;14.2.5 Advanced Techniques of TRAP Assay;327
6.1.1.2.5.1;14.2.5.1 Two-Primer-TRAP;327
6.1.1.2.5.2;14.2.5.2 Scintillation Proximity Assay;327
6.1.1.2.5.3;14.2.5.3 Real-Time Quantitative TRAP Assay;327
6.1.1.2.5.4;14.2.5.4 TRAP-Enzyme-Linked Immunosorbent Assay;328
6.1.1.2.5.5;14.2.5.5 Magnetic Bead Retrieval Assay and TRAP;329
6.1.1.2.5.6;14.2.5.6 Hybridization Protection Assay (HPA)-TRAP;329
6.1.1.2.5.7;14.2.5.7 Transcription-Mediated Amplification and Hybridization Protection Assay;329
6.1.1.2.5.8;14.2.5.8 In Situ TRAP Assay;329
6.1.1.3;14.3 Detection of Two Main Subunits of Human Telomerase: TERT (Human Telomerase Reverse Transcriptase) and TERC (Telomerase RNA Component);330
6.1.1.3.1;14.3.1 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR): Detection of TERT and TERC;330
6.1.1.3.2;14.3.2 In Situ Detection of TERT and TERC;331
6.1.1.3.2.1;14.3.2.1 In Situ Hybridization for TERC and TERT mRNA;331
6.1.1.3.2.2;14.3.2.2 In Situ Detection of the TERT Protein;333
6.1.1.4;14.4 Concluding Remarks;335
6.1.1.5;References;336
6.2;Chapter 15;339
6.2.1;Protocol II: Importance and Methods of Telomere G-Tail Length Quantification;339
6.2.1.1;15.1 Telomere G-Tail Generation and Telomere End-Protection;339
6.2.1.2;15.2 Telomere Length, G-Tail Length, and Diseases;341
6.2.1.3;15.3 Methods of Measuring Telomere G-Tail Length;343
6.2.1.3.1;15.3.1 T-OLA (Telomeric-Oligonucleotide Ligation Assay) (30);345
6.2.1.3.2;15.3.2 PENT (Primer Extension-Nick Translation) (4);346
6.2.1.3.3;15.3.3 30 Overhang Protection Assay (31);347
6.2.1.3.4;15.3.4 G-Tail Telomere HPA (Hybridization Protection Assay);348
6.2.1.4;15.4 Protocol for G-Tail Telomere HPA (9);349
6.2.1.4.1;15.4.1 Reagents;349
6.2.1.4.2;15.4.2 Protocol;349
6.2.1.4.3;15.4.3 Data Analysis;350
6.2.1.5;References;351
6.3;Chapter 16;353
6.3.1;Protocol III: Detection of Alternative Lengthening of Telomeres;353
6.3.1.1;16.1 Introduction;353
6.3.1.2;16.2 Southern-Based Terminal Restriction Fragment (TRF) Analysis;356
6.3.1.2.1;16.2.1 Buffers and Solutions;356
6.3.1.2.2;16.2.2 DNA Isolation;356
6.3.1.2.3;16.2.3 Southern Analysis of TRF Lengths;357
6.3.1.3;16.3 Detection of APBs by Immunostaining and Telomere Fluorescence in Situ Hybridisation (FISH);357
6.3.1.3.1;16.3.1 Buffers and Solutions;358
6.3.1.3.2;16.3.2 APBs in Cell Culture Monolayers;358
6.3.1.3.3;16.3.3 APBs in Tumour Specimens;359
6.3.1.3.3.1;16.3.3.1 Preparation of Tumour Sections;359
6.3.1.3.3.2;16.3.3.2 Staining Procedures for Tumour Sections;359
6.3.1.3.3.3;16.3.3.3 APB-Counting for Tumour Specimens;359
6.3.1.3.4;16.3.4 Identification of ALT Genes by APB-Screening Assay;360
6.3.1.3.4.1;16.3.4.1 Methionine Restriction;361
6.3.1.3.4.2;16.3.4.2 APB Screening by RNA Interference and Methionine Starvation;361
6.3.1.4;16.4 Telomere Length Fluctuation Analysis;361
6.3.1.4.1;16.4.1 Buffers and Solutions;361
6.3.1.4.2;16.4.2 Isolation of Metaphase Chromosomes;362
6.3.1.4.3;16.4.3 Telomere FISH;362
6.3.1.4.4;16.4.4 Telomere Fluorescence Histogram Analysis;362
6.3.1.5;16.5 Concluding Remarks;363
6.3.1.6;References;364
7;Index;367
7.1;A;367
7.2;B;367
7.3;C;368
7.4;D;368
7.5;E;368
7.6;F;368
7.7;G;369
7.8;H;369
7.9;I;370
7.10;J;370
7.11;K;370
7.12;L;370
7.13;M;370
7.14;N;370
7.15;O;371
7.16;P;371
7.17;Q;372
7.18;R;372
7.19;S;372
7.20;T;372
7.21;U;375
7.22;V;375
7.23;W;375
7.24;X;375
7.25;Y;375