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Summary Over-expression and abnormal intracellular location of the product of the oncogene c-myc in colonic dysplasia and neoplasia may be related to alterations in epigenetic mechanisms controlling the functioning of this gene. We Methylation of cytosine residues of DNA, especially in CpG dinucleotide sequences, is thought to be closely involved with gene expression (Razin & Riggs, 1980;Bird, 1986). It has been established for some time that both the extent and the specific pattern of DNA metlylation may be altered in human tumours (Feinberg & Vogelstein, 1983); treatment of cultured T-lymphoma cells with the hypomethylating agent 5-azacytidine causes them to become invasive and metastatic in vivo (Habets et al., 1990). Hypomethylation of the 5' end of a gene tends to be associated with its expression; altered methylation patterns of genes involved in regulation of the cell cycle and proliferation may thus be directly related to the mechanism of malignancy. Significantly, in vivo methylation of oncogenes after experimental transfection into cells reverses their tumorigenicity (Renzo et al., 1989), and many reports have demonstrated aberrant patterns of methylation of growth-related genes such as c-myc (Cheah et al., 1984;Ohtsuki et al.,1991), N-myc, K-ras, Ha-ras (Barbieri et al., 1989), c-abl (Weitzman et al., 1989), erb-Al (Lipsanen et al., 1988, and epidermal growth factor receptor (Kaneko et al., 1985) al., 1991), and the translocated c-myc gene in plasmacytoma (Dunnick et al., 1985). In contrast, the methylatable sites at the 5' end of the gene are usually unmethylated, even in copies of the gene which are transcriptionally silent (Mango et al., 1989).In the study presented in this paper we demonstrate that normal colonic epithelium maintains a low but detectable level of hypomethylation of the third-exon CCGG sequence; hyperplasia is associated with a moderate loss of methylation at this site, while much greater levels of hypomethylation occur in adenomas, carcinomas and metastases. Low or moderate hypomethylation of the 3' end of the c-myc gene may thus be associated with proliferating cells in normal and hyperplastic tissue; however, the quantitative difference in hypomethylation between hyperplastic and adenomatous (dysplastic) polyps suggests that high levels of demethylation of the third exon, perhaps involving both copies within each cell, may reflect or contribute to deregulation of proliferation at an early stage in tumorigenesis. Materials

We have investigated the expression of c-myc in 24 ocular melanomas by immunohistochemistry, using two monoclonal antibodies raised against a mid-sequence portion of the c-myc product (6E10) and against the C-terminus (9E10). The results were compared with other putative prognostic factors, including tumour size, cell type, proliferation index (determined by flow cytometry), and ploidy, as well as immunohistochemical staining for HMB-45 and S-100 antigens. Staining, often focal, for c-myc was found in both the nucleus and the cytoplasm of a proportion of the cells in most tumours studied. Total cell staining for myc protein correlated with proliferative index in diploid tumours; seven out of nine aneuploid and mixed aneuploid/diploid cells showed strong staining in at least one cellular compartment. A positive correlation with myc expression was also found for HMB-45 staining, but not for cell type or staining for S-100. The results support the hypothesis that myc protein is involved in cellular proliferation in uveal melanomas and indicate that immunohistochemistry for myc antigen may be a useful prognostic marker in these tumours.

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