Abstract
DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. In order to compare the methylation status in melanoma tissues and melanocytes from the same individuals, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports. In many normal melanocytes, NPM2 showed distinct immunohistochemical staining, while its expression was lost in malignant melanoma cells. In particular, intraepithelial lesions of malignant melanoma, an important challenge in clinical practice, could be distinguished from benign nevi. The present findings indicate the importance of using fresh melanoma samples, not melanoma cell lines and melanocytes in epigenetic studies. In addition, NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease.
Highlights
Malignant melanoma is one of the most fatal skin cancers, and continues to increase in prevalence [1]
After sheets of propagated cells were digested into a mixed, singlecell suspension, melanocytes should be isolated by flow cytometry using the antibody against c-Kit, melanocyte cell surface marker [14] (Figure 1B)
To avoid artificial selection by c-kit methylation status, by isolating only c-kit positive melanocytes, we optimized an antibody-free sorting method in which a 642-nm semiconductor laser would excite some component of the melanocytes—likely melanin—that could be detected by a 670-nm detector (Figure 1C)
Summary
Malignant melanoma is one of the most fatal skin cancers, and continues to increase in prevalence [1]. We utillized an epidermal sheet culture technique, capable of propagating a sufficient number of melanocytes, even from less proliferative tissue from elderly patients, and isolating melanocytes using a laser sorting technique, which enables the preparation of melanocyte and melanoma sample sets derived from the same patients for differential analysis of gene expression and methylation. Our analyses revealed several genes showing differences in the methylation status between melanoma cell lines and fresh melanoma samples that emphasize the importance of assessing epigenetic changes in normal and malignant tissues derived from the same patient. Among these genes, we found that the loss of NPM2 could be a candidate immunohistochemical marker for differentiation of melanoma and melanocytes
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