Colorectal Cancer: Methods and Protocols

TABLE OF CONTENTS
Preface
Contributors
1 Microdissection of Histologic Sections: Manual and Laser Capture
Microdissection Techniques 1
2 Isolation of a Purified Epithelial Cell Population from Human Colon 15
3 Xenografting Human Colon Cancers 21
4 Comparative Genomic Hybridization Technique 25
5 Fluorescence In Situ Hybridization: Application in Cancer Research and
Clinical Diagnostics 35
6 Microsatellite Analysis to Assess Chromosome 18q Status in Colorectal Cancer
51
7 The SURF Technique: Selective Genetic Analysis of Microscopic Tissue
Heterogeneity 59
8 Microsatellite Instability Testing 69
9 Immunohistochemical Analysis for hMLH1 and hMSH2 Expression in Colorectal
Cancer 81
10 Mutation Detection in Colorectal Cancers: Direct Sequencing of DNA Mismatch
Repair Genes 87
11 Mutation Defection in the TGF-[beta] Receptors 99
12 Direct Analysis for Familial Adenomatous Polyposis Mutations 113
13 11307K Mutation Detection by Allele-Specific PCR in Familial Colorectal
Cancer 129
14 Microsatellite Analysis of the Insulin-Like Growth Factor II Receptor in
Colorectal Carcinomas 137
15 Molecular Detection of Smad2/Smad4 Alterations in Colorectal Tumors 149
16 Direct Sequencing for Juvenile Polyposis Gene SMAD4/DPC4 Mutations 167
17 Direct Sequencing for Peutz-Jehgers Gene LKB1 (STK11) Mutations 175
18 Direct Sequencing for Cowden Syndrome Gene PTEN (MMAC1) Mutations 185
19 Genetic Analysis Using Microarrays 195
20 Cyclooxygenase-2 (COX-2) Protein Expression by Western Blotting 205
21 The Immunohistochemical Method 213
22 Immunohistochemical Detection and Quantitation of Cell Surface Receptors for
Prostanoids 231
23 Adhesion Signaling Through Integrins 239
24 Electrophoretic Mobility Shift Assay (EMSA) 249
25 Transfection Assays for Transformation of Model Colonic Cell Lines 259
26 Assessment of Intestinal Stem Cell Survival Using the Microcolony Formation
Assay 267
27 Gene Transfer into the Colonic Mucosa 275
Index 285