Cellular Signal Transduction in Toxicology and Pharmacology : Data Collection, Analysis, and Interpretation

Content: List of Contributors xvAbout the Editors xviiPreface xix1 Introduction to Cellular Signal Transduction: The Connection Between a Biological System and Its Surroundings 1Jonathan W. Boyd, Richard R. Neubig, Alice Han, and Maren Prediger1.1 Starting Big, but Ending Small 31.1.1 Key Features of Signal Transduction 31.2 Responding to Our Environment: Sensory Perception Begins and Ends with Signal Transduction 41.2.1 Taste (Gustation) 41.2.2 Smell (Olfaction) 51.2.3 Sight (Vision) 61.2.4 Sound (Audition) 61.2.5 Touch (Somatosensation) 81.3 Primary Transport Systems Involved in Signal Transduction 81.3.1 Ion Channels, Transporters, and Ion Pumps 91.3.2 Receptors 101.3.3 Endocytosis 101.3.4 Exosomes 111.4 Key Organelles Involved in Signal Transduction 121.4.1 Mitochondria 121.4.2 Endoplasmic Reticulum 141.4.3 Nucleus 15References 162 Mechanisms of Cellular Signal Transduction 21Richard R. Neubig, Jonathan W. Boyd, Julia A. Mouch, and Nicole Prince2.1 Posttranslational Modifications and Their Roles in Signal Transduction 222.1.1 Phosphorylation 222.1.2 Acylation 242.1.3 Alkylation 252.1.4 Glycosylation 262.1.5 Other PTMs 272.2 Receptors 272.3 Receptor Signaling Mechanisms 292.3.1 Basic Principles of Signal Transduction Mechanisms 292.3.1.1 Selectivity and Recognition 312.3.1.2 Flexible Modularity 312.3.1.3 Molecular Switches 342.3.1.4 GPCRs and Second Messengers 362.3.1.5 Amplification 392.3.1.6 Turn?Off Mechanisms 402.3.1.7 Localization 402.3.1.8 Biased Signaling/Functional Selectivity 412.4 Receptor Tyrosine Kinases 422.5 Steroid Receptors 432.6 Reactive Oxygen Species (ROS) 432.7 Summary 44References 443 From Cellular Mechanisms to Physiological Responses: Functional Signal Integration Across Multiple Biological Levels 49Robert H. Newman3.1 Introduction 493.2 Cellular Information Flow: Mechanisms of Cellular Signal Integration and Regulation 503.2.1 The InsR?aPKC?NF? B Signaling Axis 513.2.2 Modes of Regulation in InsR?PKC?NF? B Signaling Axis 543.2.3 Transcriptional Regulation 543.2.4 Regulating the Regulators: Phosphatase?Mediated Regulation of Signaling Molecules 593.3 Crosstalk and Functional Signal Integration in Response to Insulin in Hepatocytes 603.4 Systemic Signal Integration 653.4.1 Pancreatic  ?Cells 653.4.2 Skeletal Muscles 663.4.3 Adipose Tissue 673.5 Dysregulation of Insulin Signaling in the Etiology of Type 2Diabetes 67References 694 Signal Transduction in Disease: Relating Cell Signaling to Morbidity and Mortality 73Patricia E. Ganey and Sean A. Misek4.1 Introduction 734.2 Fibrosis as an Example of Complex Signaling 754.2.1 Development of Liver Fibrosis 754.2.2 Animal Models of Hepatic Fibrosis 764.2.3 Activation of Hepatic Stellate Cells 774.2.4 Epithelial?to?Mesenchymal Transition (EMT) 784.2.5 Other Cellular Interactions in Fibrosis 784.2.6 Intracellular Signaling Pathways Critical to Liver Fibrosis 804.2.6.1 TGF? 1 804.2.6.2 Kinase Pathways Involved in Fibrotic Responses 824.2.6.3 HIF?1  834.2.6.4 miRNA 844.2.6.5 Toll?Like Receptors (TLRs) 844.3 Cancer Drug Resistance: Complex Cellular and Population Changes 854.3.1 Genomic Resistance Mechanisms 854.3.2 Non?genomic Mechanisms 884.3.3 Non?cancer Drug Resistance Paradigms 884.3.4 Tumor Heterogeneity as a Driver of Drug Resistance 894.3.5 Mutational Drivers of Drug Resistance 904.3.6 Drug?Induced Rewiring of Signaling Networks as a Mechanism of Drug Resistance 914.3.7 Parallel Pathways and Combination Treatments 934.3.8 Epigenetic Mechanisms of Drug Resistance 954.3.9 Summary of Cancer Drug Resistance 974.4 Summary 98References 985 Experimental Design in Signal Transduction 113Weimin Gao, Meghan Cromie, Qian Wang, Zhongwei Liu, Song Tang, and Julie Vrana Miller5.1 Overview of Basic Experimental Design 1135.1.1 Independent Sample t Test 1145.1.2 Completely Randomized Analysis of Variance (ANOVA) 1145.1.3 t Test for Dependent Sample Design 1155.1.4 Randomized Block Design 1155.1.5 Completely Randomized Factorial Design 1165.1.6 Summary 1165.2 Aseptic Technique 1165.2.1 Sterile Work Environment and Laminar?Flow Hood 1175.2.2 Good Personal Hygiene Practices 1175.2.3 Sterile Reagents and Materials 1185.2.4 Sterile Handling 1185.3 Biological Sample Collection, Processing, and Pretreatment Technology 1195.3.1 Sample Collection 1195.3.1.1 Sample Collection In Vivo 1195.3.1.2 Cell Culture In Vitro 1205.3.2 Sample Processing 1215.3.2.1 DNA Isolation 1215.3.2.2 RNA Extraction 1215.3.2.3 Protein Extraction 1225.4 Sample Storage 1225.5 Common In Vitro Studies in Toxicology/Pharmacology 1235.5.1 Cytotoxicity Studies 1235.5.2 Viability Assays 1235.5.2.1 Trypan Blue 1235.5.2.2 Erythrosin 1245.5.2.3 Crystal Violet Staining 1245.5.2.4 Neutral Red Staining 1255.5.3 Survival Assays 1255.5.3.1 Clonogenic or Colony Formation Assay 1255.5.3.2 Cell Cycle Analysis: Flow Cytometry 1265.5.4 DNA Damage Assays 1265.5.4.1 Comet Assay 1275.5.4.2 Sister Chromatid Exchange Assay 1275.5.5 Southern Blot and DNA Sequencing 1275.5.5.1 Southern Blot 1275.5.5.2 DNA Sequencing 1285.5.5.3 Transfection and Gene Silencing 1285.5.6 RNA Quantification and Identification 1285.5.6.1 Northern Blot 1285.5.6.2 Promoter Deletion Analysis 1295.5.6.3 RNase Protection Assay 1295.5.7 Gene Expression 1295.5.7.1 Quantitative Real?Time Polymerase Chain Reaction (qRT?PCR) 1305.5.7.2 Microarray 1305.5.8 Protein?Related Assays 1315.5.8.1 Bradford Assay 1315.5.8.2 Enzyme-Linked Immunosorbent Assay (ELISA) 1315.5.8.3 Western Blot and 2D Gel Electrophoresis 1315.5.8.4 Immunolocalization 1325.5.8.5 Immunoprecipitation Assays 1325.5.8.6 Chromatin Immunoprecipitation (ChIP) 1325.5.9 Epigenetics 1335.5.9.1 Bisulfite Pyrosequencing 1335.5.9.2 ChIP?on?Chip 1335.5.9.3 Multiplex miRNA Profiling 1345.6 Common In Vivo Studies in Toxicology 1345.6.1 Toxicological Endpoints 1345.6.1.1 Maximum Tolerated Dose (MTD) 1345.6.1.2 Acute, Subchronic, and Chronic Toxicity 1355.6.1.3 Reproductive and Developmental Toxicity 1355.6.1.4 Genotoxicity and Carcinogenicity Studies 1365.6.2 Routes of Exposure 1365.6.2.1 Oral, Dermal, and Inhalation 1365.6.2.2 Exposure via Injection 1375.6.3 Animal Models 1375.6.3.1 Rodent Studies 1375.6.3.2 Other Studies 1385.7 Basic Advantages and Disadvantages Associated with Sample Types 1385.8 Human Epidemiology Studies 1385.8.1 Nonexperimental Studies 1395.8.2 Experimental Studies 1395.8.3 Molecular Epidemiology 1405.9 Examples of Tox? and Pharm?Based Experiments Relevant to Signal Transduction Endpoints 1405.9.1 Cytotoxicity 1415.9.1.1 Nicotine?Derived Nitrosamine Ketone (NNK) 1415.9.1.2 Doxorubicin (DOX) 1425.9.1.3 Curcumin 1425.9.1.4 Combination Effects of Cisplatin and/or Leptomycin B (LMB) 1435.9.2 DNA Damage 1435.9.3 Cell Cycle and Apoptosis 1455.9.4 ROS Induction in A549 Cells After LMB and Epigallocatechin Gallate (EGCG) Treatment 1465.9.5 Signaling Pathways 1465.9.5.1 Metabolizing Alterations After Chemical Exposure 1465.9.5.2 p53 Signaling Pathways 1485.9.6 Protein Kinase B (Akt/PKB)/Mechanistic Target of Rapamycin (mTOR) Pathway Analysis Using Multiblot 1505.9.7 Discovery of Unrecognized Pathways/Molecules Using Proteomics 1505.10 Coupling Experimental Results Within the Larger Literature Framework to Generate Information 1525.10.1 Cell Proliferation-EGFR Pathway 1525.10.2 Cell Cycle 1545.10.3 Signal?Mediated Cell Death 1565.10.4 Reactive Oxygen Species (ROS) 161References 1626 Techniques for Measuring Cellular Signal Transduction 171Julie Vrana Miller, Weimin Gao, Meghan Cromie, and Zhongwei Liu6.1 Introduction 1716.2 High?Throughput Versus High?Content Data 1726.2.1 Ergodic and Nonergodic Systems 1736.3 Methods to Measure Signal Transduction Data 1736.3.1 Microscopy 1736.3.1.1 Widefield Epifluorescence Microscopy 1736.3.1.2 Confocal Microscopy 1746.3.1.3 Immunohistochemistry 1756.3.1.4 FRET 1786.3.2 Enzyme?Linked Immunosorbent Assay (ELISA) 1796.3.2.1 Competitive ELISA 1796.3.2.2 Sandwich ELISA 1806.3.2.3 Direct Cellular ELISA 1806.3.2.4 Multiplex Suspension Array Assays 1816.3.2.5 Electrochemiluminescence (ECL) Array 1826.3.3 Gel Electrophoresis 1836.3.4 Western Blot 1836.3.5 Protein Nuclear Magnetic Resonance (NMR) 1866.4 Techniques to Generate Large Datasets for Signal Transduction Network Analysis 1876.4.1 '?omics Using Mass Spectrometry 1876.4.1.1 Separation Techniques 1886.4.1.2 Phosphoprotein Enrichment for Phosphoproteomics: IMAC, MOAC, and SMOAC 1896.4.1.3 Quantitation with Chemical Tags: iTRAQ and TMT 1906.4.2 RNA Sequencing (RNA?Seq) 1906.5 Bioenergetics 1916.5.1 Oxygen Consumption 1916.5.2 Reactive Oxygen Species (ROS) Fluorescent Probes 1926.5.3 ATP Assays 1936.5.4 Nicotinamide Adenine Dinucleotide (NADH) Assay 1936.5.5 Mitochondrial Membrane Potential 1946.6 Relating Signaling to Cellular Outcome Using Relevant Assays 1946.6.1 MTT/MTS/WST Assays 1946.6.2 LDH Assay 1956.6.3 Resazurin Assay (Alamar Blue) 1966.6.4 Cell Death: Plasma Membrane Degradation Assay 1966.7 Summary 196References 1977 Computational Methods for Signal Transduction: A Network Approach 201Giovanni Scardoni, Gabriele Tosadori, John Morris, Sakshi Pratap, Carlo Laudanna, and Alice Han7.1 Introduction 2017.2 Network Construction 2037.2.1 Introduction to Network Construction 2037.2.2 Network Construction from a Probe 2037.2.3 Mapping Methodology 2047.2.4 Small Networks 2087.2.5 Large Networks 2087.3 Facing the Network Analysis 2097.3.1 Centralities Definition and Description 2117.3.2 Global Parameters 2117.3.2.1 Diameter ( G) 2117.3.2.2 Average Distance 2127.3.3 Local Parameters 2137.3.3.1 Degree 2137.3.3.2 Eccentricity 2147.3.3.3 Closeness 2157.3.3.4 Radiality 2157.3.3.5 Centroid Value 2177.3.3.6 Stress 2197.3.3.7 S.?P. Betweenness 2197.3.3.8 Eigenvector 2207.3.3.9 Bridging Centrality 2217.3.3.10 Edge Betweenness 2217.3.3.11 Normalization and Relative Centralities 2227.3.4 Clusters 2227.4 Employing Centrality Analysis to Evaluate Stressed Biological Systems 2247.5 Interference Notion: How to Perform Virtual Knockout Experiments on Biological Networks 2267.5.1 Integrating Experimental Dataset into a Topological Analysis 2277.5.2 Integrating Expression or Activation Levels as Nodes Attributes 2287.5.3 Edge Attributes as Distance in a Computation 2287.6 Network Analysis Software 2297.6.1 Cytoscape and Its Apps 2297.6.1.1 structureViz/RINalyzer 2317.6.1.2 CentiScaPe 2317.6.1.3 PesCa 2317.6.1.4 Interference 2317.6.1.5 clusterMaker2 2327.6.1.6 chemViz 2337.6.2 Other Tools 2337.6.2.1 Gephi 2337.6.2.2 D3.js 2347.6.2.3 VisANT 2347.7 Conclusions 236References 2368 A Toxicological Application of Signal Transduction: Early Cellular Changes Can Be Indicative of Toxicity 239Julie Vrana Miller, Nicole Prince, Julia A. Mouch, and Jonathan W. Boyd8.1 Introduction 2398.2 Classification of Toxic Agent and Exposure Effects: A Toxicological Perspective 2408.2.1 Dose-Response for Chemical Exposure Toxicity Testing and Risk Assessment 2408.2.2 Chemical Mixtures 2418.2.3 Mode of Action Versus Mechanism of Action 2428.3 Early Cellular Changes Post?exposure 2448.3.1 Intracellular Signaling Perturbations Associated with Exposure 2458.3.2 Bioenergetic Changes Post?exposure 2488.3.3 Time Scale of Exposure Effects 2498.4 Experimentally Testing Early Cellular Changes that May Contribute to Exposure Sensing and Response 2508.4.1 Paradigm Shift Toward In Vitro Cell Culture 2508.4.2 Real?Time In Vitro Assays to Measure Early Cellular Changes 2518.4.2.1 Using NADH and Oxygen Consumption to Predict ATP Generation 2528.4.3 Prediction of Posttranslational Phosphorylation Response for Mixtures 2538.4.3.1 Using Bliss Independence (Response Addition) to Predict Relative Phosphorylation During Critical Signaling Events 253References 256Appendix A 2629 Future Research in Signaling 267Jonathan W. Boyd, Nicole Prince, and Marc Birringer9.1 Translational Research and a Spatiotemporal Understanding of Signal Transduction 2679.2 Integrating Second Messengers into Signal Transduction 2709.3 Understanding Crosstalk in Signal Transduction 2729.4 Posttranslational Modifications (PTMs) and Target Identification in Signal Transduction 2749.5 Epigenetic Endpoints in Signal Transduction 2769.6 The Integration of Nutrition and Signal Transduction 2789.6.1 Cellular AMPK Signaling 2819.6.2 Cellular TOR Signaling 2829.6.3 Gut Microbiota 2829.6.4 The Integration of Endocrine Gut Signaling 283References 284Index 291