Colorectal Cancer: Methods and Protocols (Methods in Molecular Medicine)

309 Pages·2001·1.295 MB·English

by  Steven M. Powell (Editor)

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Colorectal Cancer
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
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M E T H O D S I N M O L E C U L A R M E D I C I N ETM
Colorectal Cancer
Methods and Protocols
Edited by
Steven M. Powell,
MD
University of Virginia Health System,
Charlottesville, VA
Humana Press Totowa, New Jersey
© 2001 Humana Press Inc.
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Main entry under title: Methods in molecular medicine™.
Colorectal cancer: methods and protocols/edited by Steven M. Powell.
p. ; cm. —(Methods in molecular medicine; 50)
Includes bibliographical references and index.
ISBN 0-89603-767-3 (alk. paper)
1. Colon (Anatomy)—Cancer—Diagnosis—Laboratory manuals. 2.
Rectum—Cancer—Diagnosis—Laboratory manuals. I. Powell, Steven M. II. Series.
[DNLM: 1. Colorectal Neoplasms—pathology. 2. Colorectal Neoplasms—diagnosis. WI
529 C7191 2000]
RC280.C6 C668 2000
616.99'4347—dc21
00-022073
Preface
The contents of Colorectal Cancer: Methods and Protocols aim to
instruct investigators in all the key genetic, cellular, and molecular biological
methods of analyzing colorectal tumors. The focused techniques and assays
are described in sufficient detail to allow researchers to start an experiment on
colon tumors and proceed from beginning to end as if the expert in the field
who has performed these studies were guiding them at the bench. Of note,
most of the chapters in this volume are written by those scientists who pio-
neered these methods and assays in their respective fields.
The chapters in Colorectal Cancer: Methods and Protocolsdescribe “state
of the art” methods to analyze colorectal tumors, ranging from gross micro-
dissection of specimens to specific molecular analyses. Included are coverages
of mutational assays, instability testing, immunohistochemical assays,chromo-
somal studies, and gene expression analyses. The goal of our volume is to
facilitate the performance of colorectal tumor biological experiments by
investigators at various levels of training—from graduate students and
postdoctoral fellows to principal investigators who desire to advance our
understanding of colon cancer development.
Recent advances in the fields of molecular genetics, signal transduction,
DNA repair, and genomic instability—especially as they relate to colorectal
tumorigenesis—make this comprehensive coverage of molecular assays of this
cancer particularly timely. The initial section of the volume describes gross
microdissection of colon tumors and the establishment of cell lines and
xenografted tumors. The next section describes chromosomal analyses,
including comparative genomic hybridization and FISH assays. Mutational
analyses of colon tumors and of blood samples to determine whether they
have inherited a significant predisposition for colorectal cancer development
follows. Microsatellite instability testing is also presented. Gene expression
analyses including immunohistochemical assays, Western blotting, and
microarray assays are in the final section to complete the volume.
Steven M. Powell, MD
v

Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
1 Microdissection of Histologic Sections:
Manual and Laser Capture Microdissection Techniques
Christopher A. Moskaluk......................................................................1
2 Isolation of a Purified Epithelial Cell Population
from Human Colon
James K. Roche...................................................................................15
3 Xenografting Human Colon Cancers
Jeffrey C. Harper, Reid B. Adams,
and Steven M. Powell......................................................................21
4 Comparative Genomic Hybridization Technique
Wa’el El-Rifai and Sakari Knuutila.....................................................25
5 FluorescenceIn Situ Hybridization:
Application in Cancer Research and Clinical Diagnostics
Svetlana D. Pack and Zhengping Zhuang........................................35
6 Microsatellite Analysis to Assess Chromosome 18q Status
in Colorectal Cancer
Jin Jen...................................................................................................51
7 The SURF Technique: Selective Genetic Analysis
of Microscopic Tissue Heterogeneity
Darryl Shibata.......................................................................................59
8 Microsatellite Instability Testing
Yann R. Parc and Kevin C. Halling....................................................69
9 Immunohistochemical Analysis for hMLH1
andhMSH2 Expression in Colorectal Cancer
Kevin C. Halling and Patrick C. Roche.............................................81
10 Mutation Detection in Colorectal Cancers:
Direct Sequencingof DNA Mismatch Repair Genes
Julie M. Cunningham, David J. Tester,
and Stephen N. Thibodeau.............................................................87
vii
viii Contents
11 Mutation Detection in the TGF-β Receptors
William M. Grady, Lois L. Myeroff, Hongmei He,
and Sanford Markowitz...................................................................99
12 Direct Analysis for Familial Adenomatous Polyposis Mutations
Steven M. Powell................................................................................113
13 I1307K Mutation Detection by Allele-Specific PCR
in Familial Colorectal Cancer
Perry S. Chan and Steven L. Gersen...............................................129
14 Microsatellite Analysis of the Insulin-Like Growth
Factor II Receptor in Colorectal Carcinomas
Rhonda F. Souza and Stephen J. Meltzer.......................................137
15 Molecular Detection of Smad2/Smad4 Alterations
in Colorectal Tumors
Sam Thiagalingam.............................................................................149
16 Direct Sequencing for Juvenile Polyposis Gene
SMAD4/DPC4 Mutations
Lauri A. Aaltonen and Stina Roth....................................................167
17 Direct Sequencing for Peutz-Jehgers Gene
LKB1 (STK11) Mutations
Lauri A. Aaltonen and Egle Avizienyte...........................................175
18 Direct Sequencing for Cowden Syndrome Gene
PTEN (MMAC1) Mutations
Lauri A. Aaltonen, Stina Roth, and Charis Eng.............................185
19 Genetic Analysis Using Microarrays
Wa’el El-Rifai and Sakari Knuutila...................................................195
20 Cyclooxygenase-2 (COX-2) Protein Expression
by Western Blotting
Hossam M. Kandil and Raymond N. DuBois..................................205
21 The Immunohistochemical Method
Lisa A. Cerilli and Henry F. Frierson, Jr.........................................213
22 Immunohistochemical Detection and Quantitation
of Cell Surface Receptors for Prostanoids
Rosana Cosme and James K. Roche..............................................231
23 Adhesion Signaling Through Integrins
Reid B. Adams and Joshua D. Rovin..............................................239
24 Electrophoretic Mobility Shift Assay (EMSA)
Michael F. Smith, Jr. and Sandrine Delbary-Gossart....................249
Contents ix
25 Transfection Assays for Transformation
of Model Colonic Cell Lines
Mark T. Worthington and Roger Qi Luo..........................................259
26 Assessment of Intestinal Stem Cell Survival Using
the Microcolony Formation Assay
Kimberly S. Tustison, Joanne Yu,
and Steven M. Cohn......................................................................267
27 Gene Transfer into the Colonic Mucosa
Baçsak Çoruh and Theresa T. Pizarro..............................................275
Index............................................................................................................285
ix