Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas

Molecular Genetics, Gastrointestinal Carcinoma, and Ovarian Carcinoma

M. A. Hayat

• Publisher: Academic Press
• Year: 2006
• Print ISBN: 9780123694027
• eBook ISBN: 9780080457871
• Topic: Biological Sciences
• Subtopic: Human Anatomy & Physiology
• Index: Biological Sciences

Classical histology has been augmented by immunohistochemistry (the use of specific antibodies to stain particular molecular species in situ). Immunohistochemistry has allowed the identification of many more cell types than could be visualized by classical histology, particularly in the immune system and among the scattered hormone-secreting cells of the endocrine system. This book discusses all aspects of immunohistochemistry and in situ hybridization technologies and the important role they play in reaching a cancer diagnosis. It provides step-by-step instructions on the methods of additional molecular technologies such as DNA microarrays, and microdissection, along with the benefits and limitations of each method.* The only book available that translates molecular genetics into cancer diagnosis * Methods were developed by internationally-recognized experts and presented in step-by-step manner * Results of each Immunohistochemical and in situ hybridization are presented in the form of color illustrations

21

Role of Immunohistochemical Expression of DNA Methyltransferase 1 Protein in Gastric Carcinoma

Yae Kanai, Eri Arai and Tsuyoshi Etoh

Introduction

Deoxyribonucleic acid (DNA) methylation plays an important role in transcriptional regulation and chromatin remodeling in mammalian cells. Both overall DNA hypomethylation and more regional DNA hypermethylation have been well documented in various cancers (Jones and Baylin, 2002). Aberrant DNA methylation may be involved in carcinogenesis as a result of 1) increased gene mutagenicity from deamination of 5-methylcytosine to thymine; 2) a possible association of aberrant DNA methylation with genetic instability; and 3) repression of gene transcription through methylation of CpG islands in regulatory regions of specific genes, including tumor-suppressor genes. Furthermore, accumulating evidence suggests that aberrant DNA methylation is involved even in the early and precancerous stages of human carcinogenesis (Eguchi et al., 1997; Kanai et al., 1996, 1997, 1998, 1999, 2000; Kondo et al., 2000).

To date, three enzymes, DNA methyltransferase 1 (DNMT1) (Bestor et al., 1988), DNMT3a, and DNMT3b (Okano et al., 1998), have been confirmed to possess DNMT activity. Of these, DNMT1 is the most major and best known. Increased messenger ribonucleic acid (mRNA) or protein expression of DNMT1 has been reported in various human precancerous conditions and cancers. For example, we observed that mRNA expression of DNMT1 is significantly increased in chronic hepatitis or liver cirrhosis, which are considered to be precancerous conditions for hepatocellular carcinomas (HCCs), compared with normal liver tissues, and is further increased in HCCs (Saito et al., 2001; Sun et al., 1997). Immunohistochemically detected protein overexpression of DNMT1 in HCCs was significantly correlated with poorer tumor differentiation and with portal-vein involvement. Moreover, the recurrence-free survival rate and overall survival rate of patients with HCCs with increased protein expression of DNMT1 were significantly lower than those of patients with HCCs without such increased expression (Saito et al., 2003). Increased expression of DNMT1 is a biologic predictor both of the recurrence of HCCs and of a poorer prognosis for patients with HCC.

With regard to urothelial carcinogenesis, even noncancerous urothelia showing no marked histologic changes obtained from patients with bladder cancers can be considered precancerous because they may have been exposed to carcinogens in the urine. Indeed, increased protein expression of DNMT1 preceding an increase in proliferating cell nuclear antigen (PCNA) labeling index was observed in such noncancerous urothelia from patients with bladder cancers. The incidence and intensity of nuclear immunoreactivity for DNMT1 further and progressively increased from dysplastic urothelia to transitional cell carcinomas (Nakagawa et al., 2003). The increased protein expression of DNMT1 was particularly associated with the development of widely spreading flat carcinomas in situ of the urinary bladder, which are considered to be precursors of nodular invasive carcinomas with aggressive clinical courses (Nakagawa et al., 2003). Protein expression levels of DNMT1 correlated significantly with accumulation of DNA hypermethylation on multiple CpG islands during multistage urothelial carcinogenesis (Nakagawa et al., 2005). These data suggest that increased expression of DNMT1 may participate in both the precancerous stage and the malignant progression of human cancers originating from various organs.

In gastric carcinomas, we have reported that mRNA expression levels of DNMT1 were significantly higher than in noncancerous gastric mucosae and that the increased mRNA expression of DNMT1 correlated significantly with the CpG island methylator phenotype (CIMP) (Kanai et al., 2001). CIMP is defined as frequent DNA hypermethylation of C-type CpG islands, which are methylated in a cancer-specific but not an age-dependent manner (Toyota et al., 1999). In this chapter, we describe the methodology of immunohistochemistry for DNMT1 and discuss the significance of protein overexpression of DNMT1 in gastric carcinomas.

Materials

1. Five-μm-thick sections of formalin-fixed and paraffin-embedded tissue specimens mounted on silane-coated glass slides.

2. Xylene.

3. 100% Ethanol.

4. 3% and 30% H₂O₂; store at 4°C.

5. 0.3% H₂O₂: immediately before use, add 1 ml of 30% H₂O₂ to 99 ml of 100% methanol.

6. Phosphate buffer saline (PBS): dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g of KH₂PO₄ in 800 ml of distilled water. Adjust the pH to 7.4 and add water to 1 L.

7. 10 mM citric acid: dilute the commercially available stock solution of citric acid with distilled water.

8. 2% normal swine serum (NSS): add 1 ml of NSS (Dako Glostrup, Denmark) to 49 ml of PBS.

9. Anti-human DNMT1 antibody (goat polyclonal antibody, sc-10219; Santa Cruz Biotechnology, Santa Cruz, CA): for staining 50 slides at a dilution of 1:1000, add 12 μl of the antibody to 12 ml of 2% NSS. It is recommended that the primary antibody is diluted immediately before use.

10. Biotinylated anti-goat immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA): for staining 50 slides at a dilution of 1:200, add 60 μl of the antibody to 12 ml of 2% NSS. It is recommended that the secondary antibody is diluted immediately before use.

11. Avidin-biotin peroxidase complex (ABC) solution: add 50 μl of Reagent A and 50 μl of Reagent B (both supplied in the Vectastain Elite ABC kit, Vector Laboratories) to 10 ml of 2% NSS. Allow the ABC solution to stand at room temperature for 30 min before use.

12. 3,3′-Diaminobenzidine tetrahydrochloride (DAB) solution: immediately before use, dissolve 3 DAB-Tris tablets (MUTO Pure Chemicals, Tokyo, Japan) to 150 ml of distilled water. Keep the solution on ice throughout the incubation.

13. Hematoxylin solution.

Methods

1. Deparaffinize the sections in xylene for 30 min, and clear them through 5 changes of xylene for 1 min each.

2. Rehydrate the sections through 5 changes of 100% ethanol for 1 min each.

3. To block endogenous peroxidase activity, incubate the sections in freshly prepared 0.3% H₂O₂ solution in methanol at room temperature for 30 min.

4. Rinse the sections in PBS 3× for 5 min each.

5. For antigen retrieval, heat the sections in 10 mM citric acid for 10 min at 120°C in an autoclave.

6. Cool the sections, and rinse them in PBS 2× for 5 min each.

7. To block nonspecific reactions, apply 200 μl of 2% NSS to each section and incubate the sections at 4°C for 30 min.

8. Drain the serum solution from the sections. Do not rinse the sections between serum blocking and primary antibody incubation. Apply 200 μl of the diluted anti-DNMT1 antibody to each section and incubate the sections at 4°C overnight.

9. Rinse the sections in PBS 4× for 5 min each.

10. Apply 200 μl of the diluted biotinylated secondary antibody to each section, and incubate the sections at room temperature for 30 min.

11. Rinse the sections in PBS 4× for 5 min each.

12. Apply 200 μl of the freshly prepared ABC solution to each section, and incubate at room temperature for 30 min.

13. ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Handbook of Immunohistochemistry and in situ Hybridization of Human Carcinomas
• Front Matter
• Copyright page
• Dedication
• Authors and Coauthors of Volume 4
• Foreword
• Preface
• Selected Definitions
• Classification Scheme of Human Cancers
• Identification of Tumor-Specific Genes
• The Post-Translational Phase of Gene Expression in Tumor Diagnosis
• Role of Tumor Suppressor BARD1 in Apoptosis and Cancer
• Angiogenesis, Metastasis, and Epigenetics in Cancer
• Can Effector Cells Really “Effect” an Anti-Tumor Response as Cancer Therapy?
• Circulating Cancer Cells
• Circulating Cancer Cells: Flow Cytometry, Video Microscopy, and Confocal Laser Scanning Microscopy
• Gastrointestinal Carcinoma: An Introduction
• Role of Immunohistochemical Expression of p53 in Gastric Carcinoma
• Role of Immunohistochemical Expression of p53 and Vascular Endothelial Growth Factor in Gastric Carcinoma
• Role of Immunohistochemical Expression of p150 in Gastric Carcinoma: The Association with p53, Apoptosis, and Cell Proliferation
• Role of Immunohistochemical Expression of Ki-67 in Adenocarcinoma of Large Intestine
• Role of Immunohistochemical Expression of KIT/CD117 in Gastrointestinal Stromal Tumors
• Role of Immunohistochemical Expression of BUB1 Protein in Gastric Cancer
• Role of Immunohistochemical Expression of Epidermal Growth Factor in Gastric Tumors
• Role of Immunohistochemical Expression of Beta-Catenin and Mucin in Stomach Cancer
• Loss of Cyclin D2 Expression in Gastric Cancer
• Role of Immunohistochemical Expression of E-Cadherin in Diffuse-Type Gastric Cancer
• Role of Immunohistochemical Expression of Adenomatous Polyposis Coli and E-Cadherin in Gastric Carcinoma
• Immunohistochemical Expression of Chromogranin A and Leu-7 in Gastrointestinal Carcinoids
• Role of Immunohistochemical Expression of MUC5B in Gastric Carcinoma
• Angiogenin in Gastric Cancer and Its Roles in Malignancy
• Role of Helicobacter pylori in Gastric Cancer
• Protein Alterations in Gastric Adenocarcinoma
• Vesicle Proteins in Neuroendocrine and Nonendocrine Tumors of the Gastrointestinal Tract
• Role of Immunohistochemical Expression of Caspase-3 in Gastric Carcinoma
• Role of Immunohistochemical Expression of PRL-3 Phosphatase in Gastric Carcinoma
• Role of Immunohistochemical Expression of DNA Methyltransferase 1 Protein in Gastric Carcinoma
• Role of Immunohistochemical Expression of Cytoplasmic Trefoil Factor Family-2 in Gastric Cancer
• Role of Immunohistochemical Expression of Cytokeratins in Small Intestinal Adenocarcinoma
• Quantitative Immunohistochemistry by Determining the Norm of the Image Data File
• Ovarian Carcinoma: An Introduction
• Methods for Detecting Genetic Abnormalities in Ovarian Carcinoma Using Fluorescence in situ Hybridization and Immunohistochemistry
• Role of Immunohistochemical Expression of HER2/neu in High-Grade Ovarian Serous Papillary Cancer
• Role of Immunohistochemical Expression of BRCA1 in Ovarian Carcinoma
• Role of BRCA1/BRCA2 in Ovarian, Fallopian Tube, and Peritoneal Papillary Serous Carcinoma
• K-Ras Mutations in Serous Borderline Tumors of the Ovary
• Role of Immunohistochemical Expression of Ki-67 in Ovarian Carcinoma
• Role of Expression of Estrogen Receptor β, Proliferating Cell Nuclear Antigen, and p53 in Ovarian Granulosa Cell Tumors
• Role of Immunohistochemical Expression of Cyclooxygenase-2 in Ovarian Serous Carcinoma
• Role of Immunohistochemical Expression of Cyclooxygenase and Peroxisome Proliferator–Activated Receptor γ in Epithelial Ovarian Tumors
• CDX2 Immunostaining in Primary and Secondary Ovarian Carcinomas
• Epithelial Ovarian Cancer and the E-Cadherin–Catenin Complex
• Role of Cytokeratin Immunohistochemistry in the Differential Diagnosis of Ovarian Tumors
• Role of Immunohistochemical Expression of Cytokeratins and Mucins in Ampullary Carcinomas
• Role of Integrins in Ovarian Cancer
• Role of Immunohistochemical Expression of Vascular Endothelial Growth Factor C and Vascular Endothelial Growth Factor Receptor 2 in Ovarian Cancer
• Use of Microarray in Immunohistochemical Localization of SMAD in Ovarian Carcinoma
• Role of Immunohistochemical Expression of Fas in Ovarian Carcinoma
• Role of Immunohistochemical Expression of Lewis Y Antigen in Ovarian Carcinoma
• Role of Immunohistochemical Expression of ETS-1 Factor in Ovarian Carcinoma
• Role of MCL-1 in Ovarian Carcinoma
• Role of Elf-1 in Epithelial Ovarian Carcinoma: Immunohistochemistry
• Role of Immunohistochemical Expression of OCT4 in Ovarian Dysgerminoma
• Immunohistochemical Validation of B7-H4 (DD-O110) as a Biomarker of Ovarian Cancer: Correlation with CA-125
• Role of Immunohistochemical Expression of Alpha Glutathione S-Transferase in Ovarian Carcinoma
• Role of Immunohistochemical Expression of Aminopeptidases in Ovarian Carcinoma
• Expression of Angiopoietin-1, Angiopoietin-2, and Tie2 in Normal Ovary with Corpus Luteum and in Ovarian Carcinoma
• The Role of Immunohistochemical Expression of 1,25-Dihydroxyvitamin-D3-Receptors in Ovarian Carcinoma
• Role of Immunohistochemical Expression of Antigens in Neuroendocrine Carcinoma of the Ovary and Its Differential Diagnostic Considerations
• Role of Immunohistochemistry in Elucidating Lung Cancer Metastatic to the Ovary from Primary Ovarian Carcinoma
• Index