Skip Navigation



Japanese Journal of Clinical Oncology Advance Access published online on March 19, 2008
Japanese Journal of Clinical Oncology, doi:10.1093/jjco/hyn021
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
38/4/317    most recent
hyn021v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Kageyama, S.
Right arrow Articles by Sugimura, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kageyama, S.
Right arrow Articles by Sugimura, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author (2008). Published by Oxford University Press. All rights reserved

Fluorescence-labeled Methylation-sensitive Amplified Fragment Length Polymorphism (FL-MS-AFLP) Analysis for Quantitative Determination of DNA Methylation and Demethylation Status

Shinji Kageyama1,2, Kazuya Shinmura1, Hiroko Yamamoto1,2, Masanori Goto1, Koichi Suzuki3, Fumihiko Tanioka1,4, Toshihiro Tsuneyoshi2 and Haruhiko Sugimura1,

1 1st Department of Pathology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka
2 Department of Materials and Life Science, Shizuoka Institute of Science and Technology, Fukuroi, Shizuoka
3 Department of Surgery, Omiya Medical Center, Jichi Medical School, Saitama
4 Department of Pathology, Iwata City Hospital, Iwata, Shizuoka, Japan

For reprints and all correspondence: Haruhiko Sugimura, 1st Department of Pathology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi Ward, Hamamatsu, Shizuoka 431-3192, Japan. E-mail: hsugimur{at}hama-med.ac.jp

Received November 27, 2007; accepted February 16, 2008


   Abstract
TOP
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

Key Words: DNA fingerprinting • FL-MS-AFLP • gastric cancer • hypermethylation • hypomethylation


   INTRODUCTION
TOP
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
Transcription silencing by CpG island promoter hypermethylation has been linked to tumor suppressor gene inactivation (1–3), and DNA hypomethylation has been shown to play a role in cancer gene expression (4,5). Thus, to better understand the abnormal gene expression in human cancer, it is important to develop an effective system for detection of hypermethylation and hypomethylation in DNA. Since a recently developed PCR-based DNA fingerprinting method called methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis enables simultaneous genome-wide detection of DNA hypermethylation and hypomethylation in tumor tissue in comparison with adjacent normal tissue, it is one of the most useful approaches to cancer research related to DNA hypermethylation and hypomethylation (6,7). However, since MS-AFLP analysis is a radioisotope-based method, determinations of hypermethylation and hypomethylation are made by visual inspection of X-ray film, meaning there is subjective aspect to the determinations. Moreover, only about 150 CpG loci in one sample can be analysed per run (6,7). We therefore improved the original method of MS-AFLP analysis in order to enable evaluation of larger numbers of CpG loci in a quantitative fashion.


   MATERIALS AND METHODS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
Materials
Lung cancer cell line A549 was purchased from the American Type Culture Collection (ATCC). Blood samples were obtained from patients who had undergone surgery for gastric cancer at Iwata City Hospital. Gastric cancer tissue and adjacent non-cancerous tissue were obtained from the surgical specimens of patients who had undergone surgery for gastric cancer at Hamamatsu University School of Medicine, University Hospital. The study was approved by the Institutional Review Board (IRB) of Iwata City Hospital and the IRB of Hamamatsu University School of Medicine.

5-Aza-2'-Deoxycytidine Treatment
A549 cells were treated for 48 h with freshly prepared 5-aza-2'-deoxycytidine (Aza-dC) at a final concentration of 2 µM, as described previously (8).

Microdissection
Frozen sections of the gastric cancers were stained with hematoxylin and eosin (HE). Cancerous cell clusters in stained sections were examined and microdissected under an inverted microscope (Olympus IX71; Olympus, Tokyo, Japan) equipped with a microdissection device (MicroDissector; Eppendorf AG, Hamburg, Germany). Areas of interest were microdissected with an ultrasonically oscillating needle, and the tissue particles obtained were aspirated into a pipette tip with a piezo-driven micropipette.

Fluorescence-Labeled MS-AFLP (FL-MS-AFLP)
Genomic DNA was extracted with a DNeasy Kit (QIAGEN, Valencia, CA, USA) (9). A 500 ng sample of genomic DNA was digested overnight at 37°C with 5 U of the methylation-sensitive restriction enzyme NotI (New England Biolabs, Beverly, MA, USA) and 2 U of the methylation-insensitive restriction enzyme MseI (New England Biolabs) (Fig. 1a). Two pairs of oligonucleotides were annealed overnight at 37°C to generate a NotI-specific adaptor (5'-GAC GAT GAG TCC TGA G-3' and 5'-TAC TCA GGA CTC AT-3') and an MseI-specific adaptor (5'-CTC GTA GAC TGC GTA GG-3' and 5'-GGC CCC TAC GCA GTC TAC-3'). The digested DNA was ligated overnight at 16°C to the NotI adaptor and the MseI adaptor with 1 U of T4 DNA ligase (Promega, Madison, WI, USA) (Fig. 1b). The adaptor-ligated template DNA was amplified by PCR with AmpliTaq DNA polymerase (Applied Biosystems, Tokyo, Japan) and a set of primers consisting of fluorescein isothiocyanate (FITC)-labeled NotI primer (5'-FITC-GAC TGC GTA GGG GCC GCG-3') and non-labeled MseI primer (5'-GAT GAG TCC TGA GTA AC-3') (Fig. 1c). The PCR was started at 72°C for 30 s, then 94°C for 30 s, and followed by 36 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 2 min. The final extension was performed for 10 min at 72°C. After heat denaturing, the fluorescent PCR products were separated and analysed on an automatic DNA sequencer (DSQ-2000, Shimadzu Co., Kyoto, Japan). FITC-conjugated 400 bp marker and 1 kb marker (Bio Ventures, Murfreesboro, TN, USA) were used as DNA size standards.


Figure 1
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Schema of the fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis. (a) DNA is digested with NotI and MseI. (b) All the MseI sites are cleaved, and the MseI ends are ligated by MseI adaptors. The unmethylated NotI sites are cleaved by NotI, and the NotI ends are ligated by NotI adaptors, whereas the methylated NotI sites are resistant to cleavage and no adaptors are ligated. (c) NotI–MseI DNA fragments are amplified by polymerase chain reaction (PCR) with fluorescein isothiocyanate (FITC)-labeled NotI primer and non-labeled MseI primer. An asterisk indicates the FITC. (d) FL-MS-AFLP fingerprint of A549 cells treated (+) and not treated (–) with 5-aza-2'-deoxycytidine (Aza-dC). The panels on the right are electropherograms of the regions corresponding to the boxes on the left. The blue line is derived from cells treated with Aza-dC, and the black line is derived from cells not treated with Aza-dC. The arrows point to signals in which there was more than a two-fold difference between the peak height of the blue line and the black line.

 

   RESULTS
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 Funding
 Acknowledgements
 References
 
We developed a method of FL-MS-AFLP analysis by applying an FITC-labeled NotI primer and fluorescence-detecting electrophoresis apparatus (DSQ-2000) to the existing method of MS-AFLP analysis (6,7). In the new method, the FITC-labeled NotI primer is used in the PCR amplification after restriction enzyme digestion and adaptor ligation instead of the non-labeled primer with 32P radioisotope (Fig. 1a–c), and the FITC contained in the PCR product is analysed on a DSQ-2000 automatic DNA sequencer. To assess the effectiveness of the FL-MS-AFLP analysis, we first used it to analyse the genomic DNA of A549 cells treated with Aza-dC. Since Aza-dC is a demethylating agent, an increase in unmethylated NotI sites, i.e. increase in FITC-labeled PCR products, was expected. As shown in Fig. 1d, multiple CpG loci amplified by PCR were detected by the electropherogram as well as the electrophoretic ladder (fingerprint), and we were able to use the electropherogram to quantitatively evaluate alterations in the signal intensity of each CpG loci. As expected, many more CpG loci of signal intensity increase, i.e. hypomethylation, were detected in the sample treated with Aza-dC in comparison with the sample not treated with Aza-dC. Moreover, we were able to evaluate a total of more than 350 CpG loci between 60 bp long and 1000 bp long, a larger number than can be evaluated by MS-AFLP analysis (6,7). All of the results were highly reproducible.

Next, we attempted to determine whether FL-MS-AFLP analysis can be used to evaluate differences between the methylation status of blood DNA from gastric cancer patients. When the methylation status of blood DNA from a gastric cancer patient was compared with that of blood DNA from all the other gastric cancer patients (Fig. 2), different bands of signal intensity increase (hypomethylation) and signal intensity decrease (hypermethylation) were detected, suggesting that FL-MS-AFLP analysis enables evaluation of differences in the methylation level of the blood DNA among gastric cancer patients.


Figure 2
View larger version (68K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. FL-MS-AFLP fingerprint of blood DNA from gastric cancer patients. Blood DNAs from eight gastric cancer patients (Nos 1–8) were analysed. A 400 bp marker was used as a size standard. The panels on the right are electropherograms of the regions corresponding to the boxes on the left. Arrows point to the signals in which there was a more than two-fold difference between the peak height of the blue line and the black line.

 
Next, we attempted to determine whether FL-MS-AFLP analysis can be used to evaluate hypermethylation and hypomethylation in the DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue. As shown in Fig. 3, various bands of cancer-specific hypomethylation and hypermethylation were detected, suggesting that FL-MS-AFLP analysis makes it possible to evaluate the alterations in the methylation level of DNA from gastric cancer in comparison with adjacent non-cancerous tissue.


Figure 3
View larger version (65K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. FL-MS-AFLP fingerprint of sporadic gastric cancer. (a) Microdissection of gastric cancer tissue in hematoxylin and eosin (HE)-stained sections. The histological appearance of gastric cancer from two patients (nos 1 and 2) is shown in the upper panels and lower panels, respectively. The middle panels are higher magnifications of the boxed area in the left panels. The right panels show section in sequence after the section of the middle panel and microdissection under an inverted microscope (Olympus IX71) equipped with a microdissection device (MicroDissector). Scale bar: left, 200 µm; middle and right, 100 µm. (b) FL-MS-AFLP fingerprint. Two sets (nos 1 and 2) of gastric cancer tissue (T) and adjacent non-cancerous tissue (N) are analysed in duplicate. Five parts of FL-MS-AFLP fingerprint showing alterations in band signal intensity were selected and are shown. The panels on the right are electropherograms of the regions corresponding to the boxes on the left. The black and the red lines are derived from (N) and the blue line and the green line are derived from (T). The brown arrowheads and the violet arrowheads point to the signals in which there was a more than two-fold difference between the peak height of the lines derived from (N) and (T). The brown arrowheads point to hypermethylation in the cancer tissue, and the violet arrowheads point to hypomethylation in cancer. Note that the cases analysed in Fig. 3 are different from those in Fig. 2.

 

   DISCUSSION
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
Funding
 Acknowledgements
 References
 
In this study, we succeeded in developing a method of FL-MS-AFLP analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing MS-AFLP analysis method. The results showed that it was possible to quantitatively evaluate more than 350 random CpG loci per run by FL-MS-AFLP analysis. They also showed that this new method makes it possible to evaluate differences in the methylation level of the blood DNA of gastric cancer patients and to evaluate hypermethylation and hypomethylation in the DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

More than 350 random CpG loci per lane per run were quantitatively evaluated by FL-MS-AFLP analysis, and thus, its capacity to detect PCR-amplified bands corresponding to CpG loci is more than two-fold that of MS-AFLP analysis (6,7). The increase in number is attributable to the difference in gel size and electrophoresis apparatus. The glass plate is longer (61 versus 37 cm), and a fluorescence-detecting automatic DNA sequencer is used for FL-MS-AFLP analysis, whereas, a conventional sequencing gel electrophoresis apparatus designed for sequence determination of up to 250–350 bases is used for MS-AFLP analysis. The result is an increase in resolution and range of bands discernible by FL-MS-AFLP analysis, and in actual practice, a fragment as long as 1000 bases can be analysed. In addition, FL-MS-AFLP analysis allows determination of the presence or absence of alterations in signal intensity by comparing signal heights on the electropherogram. This means that FL-MS-AFLP analysis is a more quantitative method than MS-AFLP analysis, which requires alterations in the band signal intensity to be evaluated by visual inspection of X-ray film. With regard to sensitivity, since signals more than 1.4-fold higher than noise can be detected as a band by FL-MS-AFLP analysis, whereas signals more than approximately two-fold higher than noise can be detected as a band by MS-AFLP analysis, FL-MS-AFLP analysis is slightly more sensitive than MS-AFLP analysis. However, since the signals of the majority (≥95%) of the bands detected by FL-MS-AFLP analysis were more than 10-fold higher than noise, the difference in sensitivity makes only a small contribution to the increase in the bands detected. There are two other advantages to the use of the fluorescent tag. One is that FL-MS-AFLP analysis is safer and can be performed in the facilities that do not have a radioisotope center. The other is that the fluorescence-labeled primers have a long life in comparison with the short half-life of 32P (14.3 days). Thus, experiments can be performed more economically by FL-MS-AFLP analysis instead of radioactive MS-AFLP analysis. All of the above indicate that FL-MS-AFLP analysis is superior to MS-AFLP analysis.

Although an experimental trial of the use of fluorescence for MS-AFLP analysis of one tumor was briefly noted by Yamamoto et al. (6), only about 60 peak signals could be detected, perhaps because of the difference between their experimental system and ours. The advantages of our study compared with theirs are (i) a marked increase (more than five times) in the number of CpG loci evaluated, (ii) demonstration of the use of three different kinds of materials, (iii) a detailed explanation of method and results of FL-MS-AFLP analysis and (iv) demonstration that FL-MS-AFLP analysis can be used to evaluate differences in the methylation level of DNA in blood.

Recent studies have shown that subsets of genes are hypermethylated or hypomethylated in peripheral blood DNA, and these alterations are speculated to be associated with carcinogenesis. However, the studies have been performed only on certain genes, such as p53, CDH1, p16, MGMT and DAPK (10–12). The present study (Fig. 2) showed that multiple CpG loci in blood DNA can be evaluated quantitatively and simultaneously by FL-MS-AFLP analysis. If FL-MS-AFLP analysis is used to evaluate blood DNA in a large-scale cancer case-control series, methylated or demethylated genes associated with cancer risk may be found. In the past several years, rapid progress has been made in research on cancer-tissue-specific DNA hypermethylation and hypomethylation (1–5,13), however, not all of the hypermethylated or hypomethylated genes in cancer have been found. FL-MS-AFLP analysis may lead to the discovering of novel cancer-specific hypermethylated or hypomethylated genes.


   Funding
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Funding
Acknowledgements
 References
 
This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare for the Comprehensive 10-Year Strategy for Cancer Control (19-19), by a Grant-in-Aid from Japan Society for the Promotion of Science for Scientific Research (19790286), by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan on Priority Area (18014009), by the 21st century COE program ‘Medical Photonics’, and by the Smoking Research Foundation.

Conflict of interest statement

None declared.


   Acknowledgements
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
References
 
We acknowledge Dr F. Yamamoto (the first author of ref. 6) for useful discussion.


   References
TOP
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 Funding
 Acknowledgements
 References
 
1 Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer (2005) 5:223–31.[CrossRef][Web of Science][Medline]

2 Miyamoto K, Ushijima T. Diagnostic and therapeutic applications of epigenetics. Jpn J Clin Oncol (2005) 35:293–301.[Abstract/Free Full Text]

3 Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet (2007) 8:286–98.[CrossRef][Web of Science][Medline]

4 Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer (2004) 4:143–53.[Web of Science][Medline]

5 Hoffmann MJ, Schulz WA. Causes and consequences of DNA hypomethylation in human cancer. Biochem Cell Biol (2005) 83:296–321.[CrossRef][Web of Science][Medline]

6 Yamamoto F, Yamamoto M, Soto JL, Kojima E, Wang EN, Perucho M, et al. NotI-MseI methylation-sensitive amplified fragment length polymorphism for DNA methylation analysis of human cancers. Electrophoresis (2001) 22:1946–56.[CrossRef][Web of Science][Medline]

7 Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, et al. Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell (2006) 9:199–207.[CrossRef][Web of Science][Medline]

8 Wang J, Kataoka H, Suzuki M, Sato N, Nakamura R, Tao H, et al. Downregulation of EphA7 by hypermethylation in colorectal cancer. Oncogene (2005) 24:5637–47.[CrossRef][Web of Science][Medline]

9 Shinmura K, Tao H, Goto M, Igarashi H, Taniguchi T, Maekawa M, et al. Inactivating mutations of the human base excision repair gene NEIL1 in gastric cancer. Carcinogenesis (2004) 25:2311–7.[Abstract/Free Full Text]

10 Woodson K, Mason J, Choi SW, Hartman T, Tangrea J, Virtamo J, et al. Hypomethylation of. Cancer Epidemiol Biomarkers Prev (2001) 10:69–74.[Abstract/Free Full Text]

11 Reddy AN, Jiang WW, Kim M, Benoit N, Taylor R, Clinger J, et al. Death-associated protein kinase promoter hypermethylation in normal human lymphocytes. Cancer Res (2003) 63:7694–8.[Abstract/Free Full Text]

12 Russo AL, Thiagalingam A, Pan H, Califano J, Cheng KH, Ponte JF, et al. Differential DNA hypermethylation of critical genes mediates the stage-specific tobacco smoke-induced neoplastic progression of lung cancer. Clin Cancer Res (2005) 11:2466–70.[Abstract/Free Full Text]

13 Nomoto K, Maekawa M, Sugano K, Ushiama M, Fukayama N, Fujita S, et al. Methylation status and expression of human telomerase reverse transcriptase mRNA in relation to hypermethylation of the p16 gene in colorectal cancers as analyzed by bisulfite PCR-SSCP. Jpn J Clin Oncol (2002) 32:3–8.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
38/4/317    most recent
hyn021v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Kageyama, S.
Right arrow Articles by Sugimura, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kageyama, S.
Right arrow Articles by Sugimura, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?