This laboratory has demonstrated that rat fibroblast cells that overexpress the large subunit of the protein Ku (Ku-80) repress metallothionein (MT) induction by heavy toxic metals, whereas the parental cells (Rat 1) or cells that overexpress the small Ku subunit (Ku-70) of both subunits as a heterodimer (Ku-7080) conintue to induce MT. Further study revealed that this repression is caused by hypermethylation of MT promoter, and probably by the induction of a diffusible repressor (R) that can function independent of the chromatin. The present proposal is aimed at elucidation of the mechanisms responsible for hypermethylation of MT-1 promoter and the mechanisms by with the methyl C binding protein (MeCP) and R mediate the rpression of Mt-1 gene expression. Bisulfite genomic sequencing showed that unlike Rat-1 cells, all 21 CpG dinucleotides in MT-1 immediate promoter are methylated in Ku-80 cells. This work will identify the nature of the methyl C-binding protein (MeCP) (activated or induced) by UV crosslinking and Southwestern blotting analysis using methylated DNA as probe, and Western blot analysis. If the MeCP activated or induced in Ku-80 cells is distinct from the two characterized MeCPs, it will be purified and cloned, the repressor domain identified, the effect of the recombinant MeCP on transcription from methylated MT promoter determined, and the mechanism by which it inhibits transcription will be elucidated. Next, the investigators will explore the signal transduction mechanisms by which overexpression of Ku subunit enhances methylation of MT-1 promoter, particularly with regard to enhancement of DNA methyltransferase activity and the probably role of H ras in mediating the increase in this enzyme activity. R will be characterized in addition to MeCP, and attempts will be made to elucidate the mechanisms by which it represses MT-1 transcription. Furthermore, tumor cells that do not expresss MT and exhibit higher levels of P80 compared to p70 will be investigated. MT is known to protect cells from damage against toxic metals and agents that generate free oxygen radicals and cause oxidative stress. Since the basal level of MT is negliglible in most tissues, its dramatic induction (as much as 50-100 fold, depending upon the inducer and the cell type) appears to be crucial for the survival of cells against the harmful effects of these agents. It is hoped that the present study will help design ways to augment MT induction by the inactivation of the signal transduction reactions that lead to hypermethylation of the MT promoter. Because MT induction is repressed in many cancer cells due to hypermethylation, the re-expresssion of MT may be one effective means to arrest malignant growth of these cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA081024-02
Application #
6377093
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Project Start
2000-05-01
Project End
2004-04-30
Budget Start
2001-05-01
Budget End
2002-04-30
Support Year
2
Fiscal Year
2001
Total Cost
$297,675
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
Majumder, Sarmila; Kutay, Huban; Datta, Jharna et al. (2006) Epigenetic regulation of metallothionein-i gene expression: differential regulation of methylated and unmethylated promoters by DNA methyltransferases and methyl CpG binding proteins. J Cell Biochem 97:1300-16
Motiwala, Tasneem; Jacob, Samson T (2006) Role of protein tyrosine phosphatases in cancer. Prog Nucleic Acid Res Mol Biol 81:297-329
Ghoshal, Kalpana; Datta, Jharna; Majumder, Sarmila et al. (2005) 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25:4727-41
Datta, Jhrana; Majumder, Sarmila; Bai, Shoumei et al. (2005) Physical and functional interaction of DNA methyltransferase 3A with Mbd3 and Brg1 in mouse lymphosarcoma cells. Cancer Res 65:10891-900
Bai, Shoumei; Ghoshal, Kalpana; Datta, Jharna et al. (2005) DNA methyltransferase 3b regulates nerve growth factor-induced differentiation of PC12 cells by recruiting histone deacetylase 2. Mol Cell Biol 25:751-66
Jacob, Samson T; Motiwala, Tasneem (2005) Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy. Cancer Gene Ther 12:665-72
Motiwala, Tasneem; Kutay, Huban; Ghoshal, Kalpana et al. (2004) Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer. Proc Natl Acad Sci U S A 101:13844-9
Ghoshal, Kalpana; Majumder, Sarmila; Datta, Jharna et al. (2004) Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J Biol Chem 279:6783-93
Datta, Jharna; Ghoshal, Kalpana; Sharma, Sudarshana M et al. (2003) Biochemical fractionation reveals association of DNA methyltransferase (Dnmt) 3b with Dnmt1 and that of Dnmt 3a with a histone H3 methyltransferase and Hdac1. J Cell Biochem 88:855-64
Majumder, Sarmila; Ghoshal, Kalpana; Summers, Dennis et al. (2003) Chromium(VI) down-regulates heavy metal-induced metallothionein gene transcription by modifying transactivation potential of the key transcription factor, metal-responsive transcription factor 1. J Biol Chem 278:26216-26

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