Cell Cycle Control and Dysregulation Protocols: Cyclins, Cyclin-Dependent Kinases, and Other Factors

182 Pages·2004·1.756 MB·English

by  Lucio Miele (auth.), Antonio Giordano, Gaetano Romano (eds.)

Cell Cycle Control and Dysregulation Protocols
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™
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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™
Cell Cycle Control
and Dysregulation
Protocols
Cyclins, Cyclin-Dependent Kinases,
and Other Factors
Edited by
Antonio Giordano
Sbarro Institute for Cancer Research and Molecular Medicine,
Center for Biotechnology, College of Science and Technology,
Temple University, Philadelphia, PA
Gaetano Romano
Department of Neurosurgery, Thomas Jefferson University,
Philadelphia, PA
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E-ISBN 1-59259-822-6
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Library of Congress Cataloging-in-Publication Data
Cell cycle control and dysregulation protocols: cyclins, cyclin-dependent kinases, and other factors/edited
by Antonio Giordano and Gaetano Romano.
p. ; cm. -- (Methods in molecular biology, ISSN 1064-3745 ; 285)
Includes bibliographical references and index.
ISBN 0-89603-949-8 (alk. paper)
1. Cell cycle--Laboratory manuals. 2. Cyclin-dependent kinases--Laboratory manuals. 3. Cyclins--Labo-
ratory manuals.
[DNLM: 1. Cell Cycle Proteins--genetics. 2. Cyclin-Dependent Kinases. 3. DNA. 4. Fluorescent Antibody
Technique. 5. Gene Expression. QU 55 C39265 2004] I. Giordano, Antonio, MD. II. Romano, Gaetano,
1959- III. Methods in molecular biology (Clifton, N.J.) ; v. 285.
QH605.C4257 2004
571.8'4--dc22
2004006931
Preface
Cell Cycle Control and Dysregulation Protocols focuses on emerging
methodologies for studying the cell cycle, kinases, and kinase inhibitors. It
addresses the issue of gene expression in vivo and in vitro, the analysis of
cyclin-dependent kinase inhibitors, protein degradation mediated by the
proteosome, the analysis of the transformed cell phenotype, and innovative
techniques to detect apoptosis. Because there are already many manuals and
protocols available, along with commercial kits and reagents, a variety of the
more common techniques have not been included in our book.
The protocols described, based on rather sophisticated techniques for in vivo
and in vitro studies, consist of molecular biology, biochemistry, and various
types of immunoassays. Indeed, the authors have successfully
accomplished an arduous task by presenting several topics in the simplest
possible manner.
We are confident that Cell Cycle Control and Dysregulation Protocols will
facilitate and optimize the work of practical scientists involved in researching
the cell cycle. We greatly acknowledge the extraordinary contribution of the
authors in writing this book.
Antonio Giordano
Gaetano Romano
v
Contents
Preface .............................................................................................................. v
Contributors .....................................................................................................xi
PART I. ANALYSIS OF CYCLINS AND CYCLIN-DEPENDENT KINASES
1 The Biology of Cyclins and Cyclin-Dependent Protein Kinases:
An Introduction
Lucio Miele...........................................................................................3
2 In Situ Immunofluorescence Analysis:
Immunofluorescence Microscopy
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................23
3 In Situ Immunofluorescence Analysis: Analyzing RNA Synthesis
by 5-Bromouridine-5'-Triphosphate Labeling
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................29
4 Immunofluorescence Analysis Using Epitope-Tagged Proteins:
In Vitro System
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................33
5 Analysis of In Vivo Gene Expression Using Epitope-Tagged Proteins
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................37
vii
viii Contents
6 Chromatin Immunoprecipitation
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................41
7 Protein–Deoxyribonucleic Acid Interactions Linked to Gene
Expression: Electrophoretic Mobility Shift Assay
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................45
8 Protein–Deoxyribonucleic Acid Interactions Linked to Gene
Expression: DNase I Digestion
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................57
9 Protein–Deoxyribonucleic Acid Interactions Linked to Gene
Expression: Ligation-Mediated Polymerase Chain Reaction
Amjad Javed, Sayyed K. Zaidi, Soraya E. Gutierrez,
Christopher J. Lengner, Kimberly S. Harrington,
Hayk Hovhannisyan, Brian C. Cho, Jitesh Pratap,
Shirwin M. Pockwinse, Martin Montecino,
André J. van Wijnen, Jane B. Lian, Janet L. Stein,
and Gary S. Stein............................................................................63
10 Assays for Cyclin-Dependent Kinase Inhibitors
Adrian M. Senderowicz......................................................................69
11 Protein Degradation Via the Proteosome
Henry Hoff, Hong Zhang, and Christian Sell.....................................79
PART II. ANALYSIS OF THE FACTORS INVOLVED IN CELLCYCLE DEREGULATION
12 The Transformed Phenotype
Henry Hoff, Barbara Belletti, Hong Zhang,
and Christian Sell............................................................................95
Contents ix
13 A Morphologic Approach to Detect Apoptosis Based
on Electron Microscopy
Martyn K. White and Caterina Cinti.................................................105
14 Detection of Apoptotic Deoxyribonucleic Acid Break by In Situ
Nick Translation
Carmela Trimarchi, Dario La Sala, Alessandra Zamparelli,
and Caterina Cinti........................................................................113
PART III. CELLULAR RESPONSE TO DEOXYRIBONUCLEIC ACID DAMAGE
15 Induction of Deoxyribonucleic Acid Damage by Alkylating Agents
Salvatore Cortellino, David P. Turner, Domenico Albino,
and Alfonso Bellacosa..................................................................121
16 Induction of Deoxyribonucleic Acid Damage by (cid:97)Irradiation
Salvatore Cortellino, David P. Turner
and Alfonso Bellacosa.......................................................................127
17 Ultraviolet Irradiation of Cells
David P. Turner, Anthony T. Yeung, and Alfonso Bellacosa............133
PART IV. RETROVIRIDAE-BASED VECTORS: PROTOCOLS
FOR LENTIVIRAL- AND RETROVIRAL-MEDIATED GENE TRANSFER
TO ENGINEER CELL CULTURE SYSTEMS
18 Transient Production of Retroviral- and Lentiviral-Based Vectors
for the Transduction of Mammalian Cells
Tiziana Tonini, Pier Paolo Claudio, Antonio Giordano,
and Gaetano Romano...................................................................141
19 Determination of Functional Viral Titer by Drug-Resistance
Colony Assay, Expression of Green Fluorescent Protein,
and (cid:96)-Galactoside Staining
Tiziana Tonini, Pier Paolo Claudio, Antonio Giordano,
and Gaetano Romano...................................................................149
20 Retroviral and Lentiviral Vector Titration by the Analysis
of the Activity of Viral Reverse Transcriptase
Tiziana Tonini, Pier Paolo Claudio, Antonio Giordano,
and Gaetano Romano.......................................................................155
PART V. DETECTION OF GENE EXPRESSION IN SUBCELLULAR COMPARTMENTS
21 Single and Double Colloidal Gold Labeling in Postembedding
Immunoelectron Microscopy
Nicoletta Zini, Liliana Solimando, Caterina Cinti,
and Nadir Mario Maraldi..............................................................161
x Contents
22 Multifluorescence Labeling and Colocalization Analyses
Massimo Riccio, Maja Dembic, Caterina Cinti,
and Spartaco Santi........................................................................171
Index.............................................................................................................179