HYPERMETHYLATION OF CALCITONIN-GENE REGULATORY SEQUENCES IN HUMAN BREAST-CANCER AS REVEALED BY GENOMIC SEQUENCING

DNA methylation has been studied intensively during the past years in order to elucidate its role in the regulation of gene expression, gene imprinting and cancer progression. Earlier studies have shown that a general genomic under-methylation is associated with chronic lymphocyt ic leukemia and metastatic prostate cancer. Site-specific methylation changes, as revealed by the use of methylation-sensitive restriction e nzymes, have been reported to occur in the promotor region of the calc itonin gene in chronic myeloid leukemia as it progresses from the chro nic phase to blast crisis, in non-Hodgkin's lymphoid neoplasms and in non-lymphocytic leukemia. We have now explored possible methylation ch anges associated with benign and malignant breast tumors. Two approach es were employed: (i) chemical determination of general genomic methyl ation status and (ii) base-specific analysis of the methylation change s in the promoter of the calcitonin gene with the aid of genomic seque ncing. The results did not reveal any changes of total DNA 5-methylcyt osine content in ductal carcinoma of breast in comparison with benign tumors. There was a small, yet significant, increase in 5-methylcytosi ne content in lobular carcinoma. Genomic sequencing of the promoter re gion of the calcitonin gene, however, revealed a striking hypermethyla tion at or around the transcription start site of the gene in ductal c arcinomas. In benign tumors and lobular carcinomas, this region was ei ther entirely unmethylated or only slightly methylated. The latter cha nges may reflect a regional hypermethylation of the short arm of chrom osome 11, which harbors, in addition to the calcitonin gene, a number of putative or established tumor-suppressor genes. Our results demonst rate that genomic sequencing in its present form can be used for a rel iable and precise DNA methylation analysis of primary human tumors. (C ) 1996 Wiley-liss, Inc.