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J Biol Chem, Vol. 275, Issue 1, 539-547, January 7, 2000

Suppression of Metallothionein Gene Expression in a Rat Hepatoma Because of Promoter-specific DNA Methylation^*

Kalpana Ghoshal, Sarmila Majumder, Zhiling Li, Xiaocheng Dong, and Samson T. Jacob^§

From the Department of Medical Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210

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.

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^* This work was supported in part by U. S. Public Health Service Grant CA 61321 from the National Cancer Institute (to S. T. J).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally.

^§ To whom correspondence should be addressed: Dept. of Medical Biochemistry, College of Medicine, Ohio State University, 333 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210. Tel.: 614-688-5494; Fax: 614-688-5600.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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