DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes.
The objectives of this study were to examine DNA demethylase (dMTase) expression in ovarian cancers and evaluate methylation of CpG sites in the promoter of the c-erbB-2 gene and survivin gene exon 1. Forty-three epithelial ovarian cancers and 43 non-cancerous ovarian tissues were studied for dMTase expression by RT-PCR. Genomic DNA was extracted and digested with HindIII and then HpaII. CpG site-sensitive primers were constructed to amplify the promoter of the c-erbB-2 gene and survivin gene exon 1. Immunohistochemical evaluation of ErbB-2 protein and RT-PCR for survivin were also performed. dMTase was positive in 88.4% of ovarian cancers but only in 9.3% of non-cancerous ovaries (P<0.001, Fisher's exact test). The expression was similarly observed in both early stage (stage I+II: 17/19) and advanced stage (stage III+IV: 21/24) groups of ovarian malignancy. It was found that 78.9% of dMTase-positive cancers had both c-erbB-2 promoter and survivin gene exon 1 unmethylated, whereas 40% of dMTase-negative cancers had both sites methylated. In non-cancerous ovaries, these sites were mostly methylated (90.6%) and the difference from cancer cases was highly significant (P<0.001). Immunohistochemical evaluation of ErbB-2 showed significant correlation of unmethylated c-erbB-2 promoter and ErbB-2 expression. The RT-PCR for survivin expression showed that 86% of cancers were positive and six cases were negative. Exon 1 was methylated in 83% of the survivin-negative cases. This is the first report of dMTase expression in ovarian cancers. The correlation of dMTase expression with unmethylation of c-erbB-2 promoter and survivin gene exon 1 suggests that these sites may be targets for demethylation by the enzyme. The up-regulation of oncogenes may be the consequence of epigenetic control of gene expression by the dMTase.
Hattori
M
M
Department of Obstetrics and Gynecology, Nihon University School of Medicine, 30-1, Oyaguchi, Kamimachi, Itabashi, 173-0861, Tokyo, Japan.
Sakamoto
H
H
Satoh
K
K
Yamamoto
T
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D018899:CpG Islands; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D008875:Middle Aged; D009363:Neoplasm Proteins; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism*; D010053:Ovary / Q000473:pathology; D010090:Oxidoreductases, O-Demethylating / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D013696:Temperature
D004247:DNA; D018076:DNA, Complementary; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D010090:Oxidoreductases, O-Demethylating; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D000077022:Survivin; C107489:BIRC5 protein, human; C117595:methyl-CpG DNA demethylase