Microarray analysis of promoter methylation in lung cancers.
Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated+methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated+methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.
Fukasawa
Masayuki
M
Laboratory of Genome Science, Biosignal Genome Resource Center, Department of Molecular and Cellular Biology, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.|PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Japan.
Kimura
Mika
M
Laboratory of Genome Science, Biosignal Genome Resource Center, Department of Molecular and Cellular Biology, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.|PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Japan.
Morita
Sumiyo
S
Laboratory of Genome Science, Biosignal Genome Resource Center, Department of Molecular and Cellular Biology, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan.|PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Japan.
Matsubara
Kenichi
K
DNA Chip Research Inc, Yokohama, Japan.
Yamanaka
Sumitaka
S
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.|Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan.
Endo
Chiaki
C
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Sakurada
Akira
A
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Sato
Masami
M
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Kondo
Takashi
T
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Horii
Akira
A
Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan.
Sasaki
Hiroyuki
H
Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, and Department of Genetics, School of Life Science, Graduate University for Advanced Studies, Mishima, Japan.
Hatada
Izuho
I
Laboratory of Genome Science, Biosignal Genome Resource Center, Department of Molecular and Cellular Biology, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. ihatada@showa.gunma-u.ac.jp.|PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi, Japan. ihatada@showa.gunma-u.ac.jp.
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012985:Software Design
D017931:DNA Primers; D004273:DNA, Neoplasm; D004262:DNA Restriction Enzymes