Metallothionein I can be induced in response to a variety of agents that include
heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived
cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat
tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the
tumor, which was consistent with the efficient induction of the gene in this tissue by
zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the
hepatoma. The absence of footprinting was not due to inactivation of the
metal regulatory
transcription factor MTF-1, because it was highly active in the
hepatoma. Treatment of the
hepatoma bearing rats with
5-azacytidine, a demethylating agent, induced basal as well as
heavy metal-activated MT-I gene expression in the
hepatoma, implying that methylation was indeed responsible for silencing the gene.
Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanning MT-I promoter in the
hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of
DNA methyl
transferase activity as well as enhanced expression of its gene in the
hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid
tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some
tumors.