HUMANA PRESS Methods in Molecular Biology TM Edited by David M. Terrian Cancer Cell Signaling HUMANA PRESS Methods in Molecular Biology TM VOLUME 218 Methods and Protocols Edited by David M. Terrian Cancer Cell Signaling Methods and Protocols Cancer Cell Signaling M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, S ERIES E DITOR 227. Membrane Transporters: Methods and Protocols, edited by Qing Yan, 2003 226. PCR Protocols, Second Edition, edited by John M. S. Bartlett and David Stirling, 2003 225. Inflammation Protocols, edited by Paul G. Winyard and Derek A. Willoughby, 2003 224. Functional Genomics: Methods and Protocols, edited by Michael J. Brownstein and Arkady Khodursky, 2003 223. Tumor Suppressor Genes: Volume 2: Regulation, Function, and Medicinal Applications, edited by Wafik S. El-Deiry, 2003 222. Tumor Suppressor Genes: Volume 1: Pathways and Isolation Strategies, edited by Wafik S. El-Deiry, 2003 221. Generation of cDNA Libraries: Methods and Protocols, ed- ited by Shao-Yao Ying, 2003 220. Cancer Cytogenetics: Methods and Protocols, edited by John Swansbury, 2003 219. Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications, edited by Joseph M. Metzger, 2003 218. Cancer Cell Signaling: Methods and Protocols, edited by David M. Terrian, 2003 217. Neurogenetics: Methods and Protocols, edited by Nicholas T. Potter, 2003 216. PCR Detection of Microbial Pathogens: Methods and Pro- tocols, edited by Konrad Sachse and Joachim Frey, 2003 215. Cytokines and Colony Stimulating Factors: Methods and Protocols, edited by Dieter Körholz and Wieland Kiess, 2003 214. Superantigen Protocols, edited by Teresa Krakauer, 2003 213. Capillary Electrophoresis of Carbohydrates, edited by Pierre Thibault and Susumu Honda, 2003 212. Single Nucleotide Polymorphisms: Methods and Protocols, edited by Pui-Yan Kwok, 2003 211. Protein Sequencing Protocols, 2nd ed., edited by Bryan John Smith, 2003 210. MHC Protocols, edited by Stephen H. Powis and Robert W. Vaughan, 2003 209. Transgenic Mouse Methods and Protocols, edited by Marten Hofker and Jan van Deursen, 2003 208. Peptide Nucleic Acids: Methods and Protocols, edited by Peter E. Nielsen, 2002 207. Recombinant Antibodies for Cancer Therapy: Methods and Protocols. edited by Martin Welschof and Jürgen Krauss, 2002 206. Endothelin Protocols, edited by Janet J. Maguire and Anthony P. Davenport, 2002 205. E. coli Gene Expression Protocols, edited by Peter E. Vaillancourt, 2002 204. Molecular Cytogenetics: Protocols and Applications, edited by Yao-Shan Fan, 2002 203. In Situ Detection of DNA Damage: Methods and Protocols, edited by Vladimir V. Didenko, 2002 202. Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201. Combinatorial Library Methods and Protocols, edited by Lisa B. English, 2002 200. DNA Methylation Protocols, edited by Ken I. Mills and Bernie H, Ramsahoye, 2002 199. Liposome Methods and Protocols, edited by Subhash C. Basu and Manju Basu, 2002 198. Neural Stem Cells: Methods and Protocols, edited by Tanja Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 197. Mitochondrial DNA: Methods and Protocols, edited by William C. Copeland, 2002 196. Oxidants and Antioxidants: Ultrastructure and Molecular Biology Protocols, edited by Donald Armstrong, 2002 195. Quantitative Trait Loci: Methods and Protocols, edited by Nicola J. Camp and Angela Cox, 2002 194. Posttranslational Modifications of Proteins: Tools for Functional Proteomics, edited by Christoph Kannicht, 2002 193. RT-PCR Protocols, edited by Joe O’Connell, 2002 192. PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen and Harry W. Janes, 2002 191. Telomeres and Telomerase: Methods and Protocols, edited by John A. Double and Michael J. Thompson, 2002 190. High Throughput Screening: Methods and Protocols, edited by William P. Janzen, 2002 189. GTPase Protocols: The RAS Superfamily, edited by Edward J. Manser and Thomas Leung, 2002 188. Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 187. PCR Mutation Detection Protocols, edited by Bimal D. M. Theophilus and Ralph Rapley, 2002 186. Oxidative Stress Biomarkers and Antioxidant Protocols, edited by Donald Armstrong, 2002 185. Embryonic Stem Cells: Methods and Protocols, edited by Kursad Turksen, 2002 184. Biostatistical Methods, edited by Stephen W. Looney, 2002 183. Green Fluorescent Protein: Applications and Protocols, edited by Barry W. Hicks, 2002 182. In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 2002 181. Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 2002 180. Transgenesis Techniques, 2nd ed.: Principles and Protocols, edited by Alan R. Clarke, 2002 179. Gene Probes: Principles and Protocols, edited by Marilena Aquino de Muro and Ralph Rapley, 2002 178. Antibody Phage Display: Methods and Protocols, edited by Philippa M. O’Brien and Robert Aitken, 2001 177. Two-Hybrid Systems: Methods and Protocols, edited by Paul N. MacDonald, 2001 176. Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A. Lieberman, 2001 175. Genomics Protocols, edited by Michael P. Starkey and Ramnath Elaswarapu, 2001 174. Epstein-Barr Virus Protocols, edited by Joanna B. Wilson and Gerhard H. W. May, 2001 173. Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J. Vogel, 2001 172. Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J. Vogel, 2001 171. Proteoglycan Protocols, edited by Renato V. Iozzo, 2001 170. DNA Arrays: Methods and Protocols, edited by Jang B. Rampal, 2001 Cancer Cell Signaling Methods and Protocols Edited by David M. Terrian Brody School of Medicine, East Carolina University, Greenville, NC M E T H O D S I N M O L E C U L A R B I O L O G Y™ Humana Press Totowa, New Jersey © 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology™ is a trademark of The Humana Press Inc. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. 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For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [1-58829- 075-1/03 $20.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging in Publication Data Cancer cell signaling : methods and protocols / edited by David M. Terrian. p. ; cm. (Methods in molecular biology ; 218) Includes bibliographical references and index. ISBN 1-58829-075-1 (alk. paper) 1-59259-356-9 (ebook) 1. Cancer cells Regulation Laboratory manuals. 2. Cellular signal transduction Laboratory manuals. 3. Cancer Genetic aspects Laboratory manuals. I. Terrian, David M. II. Methods in molecular biology (Clifton, N.J.) ; v. 218. [DNLM: 1. Neoplasms physiopathology Laboratory Manuals. 2. Neoplasms genetics Laboratory Manuals. 3. Signal Transduction physiology Laboratory Manuals. QZ 25 C215 2003] RC269.7 .C364 2003 616.99'407 dc21 2002027296 v Preface Cells respond to environmental cues through a complex and dynamic network of signaling pathways that normally maintain a critical balance between cellular proliferation, differentiation, senescence, and death. One current research challenge is to identify those aberrations in signal transduc- tion that directly contribute to a loss of this division-limited equilibrium and the progression to malignant transformation. The study of cell-signaling mol- ecules in this context is a central component of cancer research. From the knowledge of such targets, investigators have been able to productively advance many insightful hypotheses about how a particular cancer cell may misinterpret, or respond inappropriately to, growth regulatory cues in their environment. Despite these key insights, the rapidly evolving nature of cell signaling research in cancer has necessitated a continuous revision of these theoretical constructs and the updating of methods used in their study. One contemporary example of the evolution of this field is provided by an analysis of the Human Genome Project data, which reveal a previously unsuspected diversity in the multigene families encoding for most signaling pathway inter- mediates. In assessing the usefulness of a particular methodological approach, therefore, we will need to keep in mind that there is a premium on those pro- tocols that can be easily adapted for the analysis of multiple members within a gene family. Cancer Cell Signaling: Methods and Protocols brings together several such methods in cell signaling research that are scientifically grounded within the cancer biology field. The first part of this volume is generally concerned with methods and techniques for the investigation of apoptosis and cell death. The second part contains a complementary set of protocols for manipulating and/or monitoring oncogenic signals in cancer cells. In the third, methods for studying protein–protein interactions are covered. Finally, in part four, there is a detailed protocol for capturing pure samples of malignant cells from frozen tissue specimens and two alternative techniques for analyzing their genomic DNA. I thank the authors for providing such clear and detailed accounts of their experimental protocols and for the many useful hints they have gener- ously included in the notes to each chapter. David M. Terrian Contents Preface v Contributors xi PART I. MANIPULATION AND DETECTION OF SURVIVAL SIGNALS 1 Functional Analysis of the Antimitogenic Activity of Tumor Suppressors Erik S. Knudsen and Steven P. Angus 3 2 Rescue and Isolation of Rb -deficient Prostate Epithelium by Tissue Recombination Simon W. Hayward, Yuzhuo Wang, and Mark L. Day 17 3 Signal Transduction Study Using Gene-Targeted Embryonic Stem Cells Hideki Kawasome, Takashi Hamazaki, Tetsuo Minamino, and Naohiro Terada 35 4 The Use of the Yeast Two-Hybrid System to Measure Protein–Protein Interactions that Occur Following Oxidative Stress Richard A. Franklin 47 5 Differential Screening of cDNA Libraries for Analysis of Gene Expression During Tumor Progression Christopher W. Gregory 59 6 Mitogen-Activated Protein Kinase Signaling in Drug-Resistant Neuroblastoma Cells Raymond R. Mattingly 71 7 TUNEL and Immunofluorescence Double-Labeling Assay for Apoptotic Cells with Specific Antigen(s) Stephanie M. Oberhaus 85 PART II. MANIPULATION AND DETECTION OF ONCOGENIC SIGNALS 8 Kinetworks TM Protein Kinase Multiblot Analysis Steven Pelech, Catherine Sutter, and Hong Zhang 99 9 Protein Tyrosine Kinase and Phosphatase Expression Profiling in Human Cancers Wen-Chang Lin 113 vii viii Contents 10 Association of Nonreceptor Tyrosine Kinase c -Yes with Tight Junction Protein Occludin by Coimmunoprecipitation Assay Yan-Hua Chen and Qun Lu 127 11 Isolation of Novel Substrates Using a Tyrosine Kinase Overlay/ In Situ Assay Irwin H. Gelman 133 12 Manipulating Expression of Endogenous Oncogenic Proteins Using an Antisense Oligonucleotide Approach in Prostate Cancer Cells Daqing Wu, Ginger G. Wescott, and David M. Terrian 143 13 Measurements of Phospholipases A 2 , C, and D (PLA 2 , PLC, and PLD): In Vitro Microassays, Analysis of Enzyme Isoforms, and Intact-Cell Assays Julian Gomez-Cambronero, Joel Horwitz, and Ramadan I. Sha’afi 155 14 Detection of the Content and Activity of the Transcription Factor AP-1 in a Multistage Skin Carcinogenesis Model Yunfeng Zhao and Daret K. St. Clair 177 15 Fibroblastic, Hematopoietic, and Hormone Responsive Epithelial Cell Lines and Culture Conditions for Elucidation of Signal Transduction and Drug Resistance Pathways by Gene Transfer Linda S. Steelman, William L. Blalock, Xiao-Yang Wang, Phillip W. Moye, John T. Lee, John G. Shelton, Patrick M. Navolanic, Julianne M. Davis, Steven L. Knapp, Richard A. Franklin, Martyn K. White, and James A. McCubrey 185 16 Elucidation of Signal Transduction Pathways by Transfection of Cells with Modified Oncogenes Linda S. Steelman, William L. Blalock, Xiao-Yang Wang, Phillip W. Moye, John T. Lee, John G. Shelton, Patrick M. Navolanic, Julianne M. Davis, Steven L. Knapp, Richard A. Franklin, Martyn K. White, and James A. McCubrey 203 17 Elucidation of Signal Transduction Pathways by Retroviral Infection of Cells with Modified Oncogenes Linda S. Steelman, William L. Blalock, Xiao-Yang Wang, Phillip W. Moye, John T. Lee, John G. Shelton, Patrick M. Navolanic, Julianne M. Davis, Steven L. Knapp, Richard A. Franklin, Martyn K. White, and James A. McCubrey 221 Contents ix PART III. PROTEIN INTERACTIONS 18 Methods for the Study of Protein–Protein Interactions in Cancer Cell Biology Daniel Price, Iha Park, and Hava Avraham 255 19 Production of Ligand-Specific Mutants Using a Yeast Two-Hybrid Mating Assay André Nantel 269 20 Coimmunoprecipitation Assay for the Detection of Kinase–Substrate Interactions Lucio Comai 277 PART IV. GENOMIC REARRANGEMENTS 21 Mutational Analysis of the Androgen Receptor Using Laser Capture Microdissection and Direct Sequencing Sheila Greene, Patricia Stockton, Olga G. Kozyreva, Kris E. Gaston, Gordon P. Flake, and James L. Mohler 287 22 Clonality Analysis by T-Cell Receptor γ PCR and High-Resolution Electrophoresis in the Diagnosis of Cutaneous T-Cell Lymphoma (CTCL) Ansgar Lukowsky 303 Index 321 [...]... stromal-epithelial interactions (1–3) and genetic alterations of the epithelium resulting in activation of oncogenes (4–7) and inactivation of tumor-suppressor From: Methods in Molecular Biology, vol 218: Cancer Cell Signaling: Methods and Protocols Edited by: D M Terrian © Humana Press Inc., Totowa, NJ 17 18 Hayward, Wang, and Day genes (8,9) The involvement of multiple oncogenes and tumor-suppressor genes in... 1672–1677 8 Hanahan, D and Weinberg, R A (2000) The hallmarks of cancer Cell 100, 57–70 9 Evan, G I and Vousden, K H (2001) Proliferation, cell cycle and apoptosis in cancer Nature 411, 342–348 10 Peltomaki, P (2001) Deficient DNA mismatch repair: a common etiologic factor for colon cancer Hum Mol Genet 10, 735–740 11 Kolodner, R D (1995) Mismatch repair: mechanisms and relationship to cancer susceptibility... 5 Using the GFP fluorescence and phase contrast, determine the percentage of GFPpositive cells by counting random fields of cells 6 Compare the relative transfection efficiencies between vector control and antimitogen/tumor suppressor 3.2 Inhibition of BrdU Incorporation in Transiently Transfected Cells 3.2.1 Cell Culture 1 Culture cells at 60% confluence (approx 1 × 105 cells/well) on coverslips in... (H2B-GFP), and red (Red-X) channels to obtain photomicrographs of the same field 3.3 Cell- Cycle Arrest in Transiently-Transfected Cells 3.3.1 Cell Culture 1 Culture cells in 60-mm dishes at 60% confluence 2 Include a dish that will not be transfected 3.3.2 Cell Transfection 1 Use 8 µg of CMV-vector or CMV-RB and 0.5 µg of CMV-H2B-GFP (see Note 5) 2 Use 0.25 mL CaCl2 and 0.25 mL 2X BBS 10 Knudsen and Angus... Fig 3 and Note 8) 3 Analyze PI staining in GFP-positive cells 4 Perform ModFit analysis on PI histograms (see Fig 3) 3.4 Flat -Cell Assay/Colony Inhibition in Stably Transfected Cells 3.4.1 Cell Culture 1 Culture 1 × 105 cells in 60-mm dishes 2 Include a control plate that will not be transfected 3.4.2 Cell Transfection 1 Use 8 µg of CMV-vector or CMV-RB and 0.5 µg of pBABE-puro 2 Use 0.25 mL CaCl2 and. .. revolutionized the molecular analysis of cancer (1–3) By definition, tumor suppressor genes are genetically linked to a cancer For example, the retinoblastoma (RB) tumor suppressor was first identified as a gene that was specifically lost in familial RB (4–6) The majority of tumor suppressors From: Methods in Molecular Biology, vol 218: Cancer Cell Signaling: Methods and Protocols Edited by: D M Terrian ©... Kinzler, K W and Vogelstein, B (1996) Lessons from hereditary colorectal cancer Cell 87, 159–170 13 Levine, A J (1997) p53, the cellular gatekeeper for growth and division Cell 88, 323–331 14 Wang, J Y., Knudsen, E S., and Welch, P J (1994) The retinoblastoma tumor suppressor protein Adv Cancer Res 64, 25–85 15 Arap, W., Knudsen, E., Sewell, D A., Sidransky, D., Wang, J Y., Huang, H J., and Cavenee,... solution Inverted microscope with camera 3 Methods 3.1 Cell Culture and Transfection of Antimitogen/Tumor Suppressor 3.1.1 Cell Culture 1 Seed approx 1 × 105 cells per well of a six-well plate or 3 × 105 cells per 60-mm dish in DMEM supplemented with 10% FBS and penicillin-streptomycin 2 SAOS-2 cells should attach to the tissue culture dish within 4–6 h 3.1.2 Cell Transfection 1 Prepare purified plasmid... above line, GFP-negative cells below) Middle row, GFP-negative cells were analyzed for DNA content (PI) and ModFit analysis was performed to quantitate cell cycle distribution (% phase) as indicated Bottom row, GFP-positive cells were analyzed for DNA content (PI) and ModFit analysis was performed to quantitate cell cycle distribution (% phase) as indicated 3.4.3 Puromycin Selection and Staining 1 24 h... Surgery, Department of Cancer Biology, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt Prostate Cancer Center, Vanderbilt University Medical Center, Nashville, TN JOEL HORWITZ • Department of Pharmacology and Physiology, MCP Hahnemann University, Philadelphia, PA HIDEKI KAWASOME • Otsuka Pharmaceutical Co Ltd, Tokushima, Japan STEVEN L KNAPP • Department of Microbiology and Immunology, Brody . Terrian Cancer Cell Signaling Methods and Protocols Cancer Cell Signaling M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, S ERIES E DITOR 227. Membrane Transporters: Methods and Protocols, . John Swansbury, 2003 219. Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications, edited by Joseph M. Metzger, 2003 218. Cancer Cell Signaling: Methods and Protocols, edited by David. Methylation Protocols, edited by Ken I. Mills and Bernie H, Ramsahoye, 2002 199. Liposome Methods and Protocols, edited by Subhash C. Basu and Manju Basu, 2002 198. Neural Stem Cells: Methods and Protocols,