The expression of
metallothionein-I (MT-I), a known
antioxidant, was suppressed in a transplanted rat
hepatoma because of promoter methylation and was induced by
heavy metals only after demethylation by
5-azacytidine (5-AzaC). Treatment of the
tumor-bearing rats with 5-AzaC resulted in significant regression of the
hepatoma. When the inhibitor-treated
tumor was allowed to grow in a new host, MT-I promoter was remethylated, which suggested de novo methylation. The activities of both de novo (3-fold) and maintenance
DNA methyltransferases (DNMT) (5-fold) were higher in the
hepatoma than in the host liver. The
mRNA levels of the de novo
methyltransferases DNMT3a and DNMT3b were 3- and 6-fold higher, respectively, in the
tumor implicating transcriptional up-regulation of these two genes in this tissue. Immunohistochemical analysis showed exclusive localization of DNMT3a in the nuclei of both the liver and
hepatoma, whereas DNMT3b was detected in the nuclei as well as the cytoplasm. Immunoblot assay showed that the levels of DNMT1, DNMT3a, and DNMT3b
proteins in the
hepatoma were 5-, 10-, and 4-fold higher, respectively, than in the liver. The
mRNA level of the major methyl CpG-
binding protein (MeCP2) was 8-fold higher in the
tumor compared with the liver. Immunohistochemical studies showed that MeCP2 is localized exclusively in the nuclei of both tissues. A
chromatin immunoprecipitation assay demonstrated that MeCP2 was associated with the MT-I promoter in the
hepatoma implicating its involvement in repressing the methylated promoter. Analysis of the
DNA isolated from the liver and
hepatoma by RLGS-M (restriction landmark genomic scanning with methylation-sensitive
enzyme) (NotI) showed that many genes in addition to MT-I were methylated in the
hepatoma. These data demonstrate suppression of the MT-I gene and probably other genes in a solid
tumor by promoter methylation and have provided potential molecular mechanisms for the altered methylation profile of the genes in this
tumor.