Poster presentation, P137

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

Analysis of global 5-hydroxymethylcytosine in malignant melanoma and acquired melanocytic nevi

BACKGROUND: Epigenetic regulation, e.g. cytosine methylation, histone code, miRNA, plays a significant role in oncogenesis. Cytosine methylation in promoter regions suppresses gene expression in general, and the expression of several tumor suppressor genes, e.g. p16, CDH1, were down-regulated by cytosine methyltion in malignant melanoma. In 2009, another modification of cytosine, 5-hydroxymethylcytosine (5-hmC) was discovered. Although the role of 5-hmC in gene expression remains to be elucidated to date, its different function from 5-methylcytosine (5-mC) was speculated. We developed a novel method to measure 5-hmC, NICEA for 5-hmC, based on the global methylation analysis method, non-isotopic cytosine extension assay, and analyzed the 5-hmC in malignant melanoma and acquired melanocytic nevi. METHODS: Genomic DNA was extracted from malignant melanoma (n=9) and acquired melanocytic nevi (n=10). The extracted DNA was treated with the combination of T4-beta-glucosyltransferase and restriction enzymes MspI or HpaII, resulted in differentially created overhang at the recognition site CCGG. Incorporating biotinylated cytosine at the overhang, the biotinylated DNA was visualized on the nylon membrane, and underwent densitometry. RESULTS: The relative amount of unmodified cytosine (5-C) was identical in melanoma and nevi. The amount of 5-mC was significantly smaller and that of 5-hmC was larger in melanoma (p=0.01). CONCLUSIONS: We developed the novel method, NICEA for 5-hmC, for analyzing global 5-hmC, and reported the difference of 5-hmC between melanoma and nevi. Although the function of 5-hmC is yet to be determined, our data suggest the importance of 5-hmC in developing malignant melanoma.

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