DNA methylation in mammals plays a crucial role in development, X chromosome inactivation, epigenetic silencing, aging, carcinogenesis and certain diseases. Although methylation at CpG islands is known to occur in several genes that leads to suppression of their expression, the molecular mechanism of silencing the genes by this process and its potential role in neoplastic transformation have not been completely elucidated. We showed that (a) unlike the parental tissues (thymus and liver), mouse lymphosarcoma cells P1798 and rat hepatoma 3924A are incapable of inducing metallothionein-I and II (MT-I and II) by heavy metals as a result of hypermethylation of their promoters and (b) P1798 cells contain a unique mentyl C-binding protein, designated MeCP-L that is distince from the well characterized MeCP2 and the partially characterized MeCP1. The long term goal of this proposal is to elucidate the molecular mechanisms by which DNA methylation suppresses gene expression in the cancer cells, its functional and potential clinical implications. The present study will (a) purify the unique MeCP- L from the nuclear extracts of the tumor cells and characterize it biochemically, clone the cDNA for the factor, express it in suitable expression system, study the mechanism by which this MeCP-L inhibits the MT-I promoter activity by an in vitro transcription assay (b) identify the polypeptides that specifically associate with MeCP-L to form a functional complex and identify the repressor domain of this protein in vitro (c) explore the role of MeCP-L in silencing the methylated genes in vivo by expressing MeCP-L in drosophila SL2 cells (that contains insignificant amounts of MeCPs) and methylated MT-I promoter reporter construct, and also using the antisense MeCP-L for stable transfection of the P1798 cells, (d) elucidate the molecular mechanism by which DNA methylation of different regions of the MT-I gene represses its expression in vivo using transient transfection assay, (e) study the effect of MT overexpression in P1798 cells on the growth and transformation potential of these cells,(f) investigate whether silencing of MT genes is a unique characteristic of tumors of lymphoid and hepatic orgin, and test the possibility that glucocorticoid receptor (GR) promoter methylation is repsonsible for the resistance of some lymphoid cancer cells to glucocorticoid, a potent drug used in the treatment of the cancers of lymphatic orgin. It is hoped that this study could provide the impetus to explore ways to reactivate these repressed genes that may result in the arrest of specific neoplastic growth.
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