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Cell-wide Metabolic Alterations Associated with Malignancy
1st Edition, Volume 543 - June 7, 2014
Editors: Lorenzo Galluzzi, Guido Kroemer
Language: English
Hardback ISBN:9780128013298
9 7 8 - 0 - 1 2 - 8 0 1 3 2 9 - 8
eBook ISBN:9780128014837
9 7 8 - 0 - 1 2 - 8 0 1 4 8 3 - 7
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods…Read more
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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods providing a a theoretical overview on metabolic alterations of cancer cells and a series of protocols that can be employed to study oncometabolism, in vitro, ex vivo and in vivo. Malignant cells exhibit metabolic changes when compared to their normal counterparts, owing to both genetic and epigenetic alterations. Although such a metabolic rewiring has recently been indicated as "yet another" general hallmark of cancer, accumulating evidence suggests that the metabolic alterations of each neoplasm rather represent a molecular signature that intimately accompanies, and hence cannot be severed from, all facets of malignant transformation.
Continues the legacy of this premier serial with quality chapters authored by leaders in the field
Covers research methods in biomineralization science
Contains sections on such topics providing a a theoretical overview on metabolic alterations of cancer cells and a series of protocols that can be employed to study oncometabolism, in vitro, ex vivo and in vivo.
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
Preface
Acknowledgments
Chapter One: Methods to Measure Cytoplasmic and Mitochondrial Ca2 + Concentration Using Ca2 +-Sensitive Dyes
Abstract
1 Introduction
2 Experimental Components and Considerations
3 Isolation and Differentiation of Naïve CD4+ CD25− T Cells from Spleen and Lymph Nodes of Wild-Type and Orai1−/− Mice
4 Mitochondrial Ca2 + Measurement
5 Data Handling and Processing
6 Notes
7 Conclusion
Acknowledgments
Chapter Two: Methods to Measure Intracellular Ca2 + Fluxes with Organelle-Targeted Aequorin-Based Probes
Abstract
1 Introduction
2 Experimental Components and Considerations
3 Application Examples
4 Aequorin Improvement by Fusion with GFP
5 Monitoring Ca2 + Fluxes in Living Organism by BRET
Acknowledgments
Chapter Three: Measuring Baseline Ca2 + Levels in Subcellular Compartments Using Genetically Engineered Fluorescent Indicators
Abstract
1 Introduction
2 Evolution of Recombinant Ca2 + Probes and Organelle Targeting
3 Enhancing the Fluorophores
4 Modulating Ca2 +-Binding Properties
5 Developing Organelle Targeting
6 Quantitative Considerations of Measurements of Organelle-Targeted Probes
7 Experimental Protocols
8 Semiquantitative Imaging of Basal Mitochondrial [Ca2 +] with a Novel Mitochondria-Targeted GCaMP6m (2mtGCaMP6m)
9 Semiquantitative Imaging of Agonist-Induced Mitochondrial Ca2 + Signals in Primary Neurons Using the Low-Affinity 2mtD4cpv Probe
10 Semiquantitative Imaging of Basal, Steady-State [Ca2 +] in the ER
11 Concluding Remarks
Chapter Four: Autophagy and Autophagic Flux in Tumor Cells
Abstract
1 Introduction
2 Autophagy Initiation and Autophagic Flux
3 Autophagic Flux and Degradation
4 Conclusion
Acknowledgments
Chapter Five: Methods to Assess Autophagy In Situ—Transmission Electron Microscopy Versus Immunohistochemistry
Abstract
1 Introduction
2 Transmission Electron Microscopy
3 Immunohistochemistry
4 Concluding Remarks
Acknowledgments
Chapter Six: Methods to Measure the Enzymatic Activity of PI3Ks
Abstract
1 Introduction
2 Analysis of PI3K Lipid Kinase Activity in Immunoprecipitate
3 Analysis of the Activity of Small Molecule PI3K Inhibitors
4 Analysis of the Localized Synthesis of PI3K Lipid Products
5 Materials
Acknowledgments
Chapter Seven: Luciferase-Based Reporter to Monitor the Transcriptional Activity of the SIRT3 Promoter
Abstract
1 Introduction
2 Protocol for Construction of Reporter Plasmid
3 Assaying SIRT3 Promoter Activity with the Construct
4 Verifying Reporter Results with Complementary Methods
5 Summary
Acknowledgments
Chapter Eight: Metabolomic Profiling of Cultured Cancer Cells
Abstract
1 Introduction
2 Cell Culture, Treatments, and Sample Collection
3 Chromatography, Mass Spectrometry, and Data Analysis
4 Concluding Remarks
5 Notes
Acknowledgments
Chapter Nine: Pulsed Stable Isotope-Resolved Metabolomic Studies of Cancer Cells
Abstract
1 Introduction
2 Experimental Procedures
3 Data Analysis
4 Outlook
Chapter Ten: Single-Cell Imaging for the Study of Oncometabolism
Abstract
1 Introduction
2 Methods
3 MAMS Arrays
Chapter Eleven: Study of Cellular Oncometabolism via Multidimensional Protein Identification Technology
Abstract
1 Introduction
2 Sample Protocol
3 Data Processing
4 Example Applications of MuDPIT
5 Summary
Chapter Twelve: In Vivo Quantitative Proteomics for the Study of Oncometabolism
Abstract
1 Introduction
2 Sample Preparation
3 MS Sample Preparation
4 Instruments
5 MS Data Analysis
6 Additional Considerations
7 Conclusions
Chapter Thirteen: Metabolomic Profiling of Neoplastic Lesions in Mice
Abstract
1 Introduction
2 Metabolic Profiling and Cancer Biology
3 Techniques Applied in Metabolomic Profiling of Neoplastic Lesions in Mice
4 Conclusion
Chapter Fourteen: Metabolomic Profiling of Tumor-Bearing Mice
Abstract
1 Introduction
2 Mouse Models
3 Sample Preparation and Mass Spectrometry Analysis (Fig. 14.3)
4 Statistics
5 Biomarker Validation
6 Summary
Chapter Fifteen: Metabolomic Studies of Patient Material by High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
Abstract
1 MAS-NMR Spectroscopy as a Tool for Metabolic Profiling
2 MAS-NMR Versus Solution-State NMR Spectroscopy of Tumors
3 MAS-NMR Tumor Profiling in Clinical Studies
4 Metabolic Perturbations in Tumors Detected by MAS-NMR Spectroscopy
5 Protocol for the Analysis of Tissue by 1H MAS-NMR Spectroscopy
Chapter Sixteen: Analysis of Metabolomic Profiling Data Acquired on GC–MS
Abstract
1 Introduction
2 Spectrum Deconvolution
3 Metabolite Identification
4 Metabolite Quantification
5 Association Network Analysis
6 Metabolic Pathway Analysis
Acknowledgments
Author Index
Subject Index
No. of pages: 384
Language: English
Edition: 1
Volume: 543
Published: June 7, 2014
Imprint: Academic Press
Hardback ISBN: 9780128013298
eBook ISBN: 9780128014837
LG
Lorenzo Galluzzi
Lorenzo Galluzzi is Assistant Professor of Cell Biology in Radiation Oncology at the Department of Radiation Oncology of the Weill Cornell Medical College, Honorary Assistant Professor Adjunct with the Department of Dermatology of the Yale School of Medicine, Honorary Associate Professor with the Faculty of Medicine of the University of Paris, and Faculty Member with the Graduate School of Biomedical Sciences and Biotechnology of the University of Ferrara, the Graduate School of Pharmacological Sciences of the University of Padova, and the Graduate School of Network Oncology and Precision Medicine of the University of Rome “La Sapienza”. Moreover, he is Associate Director of the European Academy for Tumor Immunology and Founding Member of the European Research Institute for Integrated Cellular Pathology.
Galluzzi is best known for major experimental and conceptual contributions to the fields of cell death, autophagy, tumor metabolism and tumor immunology. He has published over 450 articles in international peer-reviewed journals and is the Editor-in-Chief of four journals:
OncoImmunology (which he co-founded in 2011), International Review of Cell and Molecular Biology, Methods in Cell biology, and Molecular and Cellular Oncology (which he co-founded in 2013). Additionally, he serves as Founding Editor for Microbial Cell and Cell Stress, and Associate Editor for Cell Death and Disease, Pharmacological Research and iScience.
Affiliations and expertise
Assistant Professor of Cell Biology in Radiation Oncology, Department of Radiation Oncology, Weill Cornell Medical College, NY, USA
GK
Guido Kroemer
Guido Kroemer got his M.D. in 1985 from the University of Innsbruck, Austria, and his Ph.D. in molecular biology in 1992 from the Autonomous University of Madrid, Spain. He is currently Professor at the Faculty of Medicine of the University of Paris Descartes/Paris V, Director of the INSERM research team ‘Apoptosis, Cancer and Immunity’, Director of the Metabolomics and Cell Biology platforms of the Gustave Roussy Cancer Campus, and Practitioner at the Hôpital Européen George Pompidou (Paris, France). He is also the Director of the Paris Alliance of Cancer Research Institutes (PACRI) and the Labex 'Immuno-Oncology'. Dr. Kroemer is best known for the discoveries that mitochondrial membrane permeabilization constitutes a decisive step in regulated cell death; that autophagy is a cytoprotective mechanism with lifespan-extending effects; and that anticancer therapies are successful only if they stimulate tumour-targeting immune responses. He is currently the most-cited cell biologist in Europe (relative to the period 2007-2013), and he has received the Descartes Prize of the European Union, the Carus Medal of the Leopoldina, the Dautrebande Prize of the Belgian Royal Academy of Medicine, the Léopold Griffuel Prize of the French Association for Cancer Research, the Mitjavile prize of the French National Academy of Medicine and a European Research Council Advanced Investigator Award.
Affiliations and expertise
INSERM Cordeliers Research Cancer Paris; Hopital Europeen Georges Pompidou; Universite Paris Descartes, France
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