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PMID: 11844796 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S. Retracted Publication

Role of de novo DNA methyltransferases and methyl CpG-binding proteins in gene silencing in a rat hepatoma.

The Journal of biological chemistry ·Vol. 277 ·No. 18 ·2002-05-03 ·Pages 16048-58

Majumder S, Ghoshal K, Datta J, Bai S, Dong X, Quan N, Plass C, Jacob ST

Abstract

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.

MeSH Terms
Animals Azacitidine/pharmacology Base Sequence Carrier Proteins/metabolism Cell Nucleus/enzymology DNA (Cytosine-5-)-Methyltransferase 1 DNA (Cytosine-5-)-Methyltransferases/metabolism DNA-Binding Proteins/metabolism DNA-Cytosine Methylases/metabolism Dinucleoside Phosphates/metabolism Gene Expression Regulation, Neoplastic/drug effects,genetics Gene Silencing/physiology Liver/enzymology Liver Neoplasms, Experimental Metallothionein/genetics Molecular Sequence Data Promoter Regions, Genetic Rats Reverse Transcriptase Polymerase Chain Reaction Substrate Specificity
Chemicals
Carrier Proteins DNA-Binding Proteins Dinucleoside Phosphates cytidylyl-3'-5'-guanosine Metallothionein DNA-Cytosine Methylases DNA (Cytosine-5-)-Methyltransferase 1 DNA (Cytosine-5-)-Methyltransferases Dnmt1 protein, rat Azacitidine
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Majumder Sarmila
Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.
Ghoshal Kalpana
Datta Jharna
Bai Shoumei
Dong Xiaocheng
Quan Ning
Plass Christoph
Jacob Samson T
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M111662200
Published
2002-05-03
Epub
2002-00-13
Pages
16048-58
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2241740
Subset
IM
Grants
NCI NIH HHS · R01 CA081024-03 · United States
NIEHS NIH HHS · ES10874 · United States
NIEHS NIH HHS · R01 ES010874-02 · United States
NCI NIH HHS · R01 CA081024-01A2 · United States
NCI NIH HHS · CA81024 · United States
NIEHS NIH HHS · R01 ES010874-01 · United States
NIEHS NIH HHS · R01 ES010874 · United States
NIEHS NIH HHS · R01 ES010874-05 · United States
NCI NIH HHS · R01 CA081024 · United States
NIEHS NIH HHS · R01 ES010874-04 · United States
NCI NIH HHS · R01 CA081024-02 · United States
NCI NIH HHS · R01 CA081024-04 · United States
NIEHS NIH HHS · R01 ES010874-03 · United States
Corrections
RetractionIn
PMID: 30120152
External Links
PubMed source DOI: 10.1074/jbc.M111662200 PMC Full Text View journal details
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