DNA METHYLATION AND GENE EXPRESSION IN CANCER CELLS

哺乳动物的DNA甲基化在发育、X染色体失活、表观遗传沉默、衰老、癌症发生和某些疾病中起着至关重要的作用。虽然已知CpG岛上的甲基化发生在几个基因中，导致其表达被抑制，但通过这一过程沉默基因的分子机制及其在肿瘤转化中的潜在作用尚未完全阐明。我们发现：(A)与亲本组织(胸腺和肝脏)不同，小鼠淋巴肉瘤细胞P1798和大鼠肝癌3924A由于其启动子的高甲基化而不能被重金属诱导金属硫蛋白-I和金属硫蛋白-II(MT-I和II)；(B)P1798细胞含有一个独特的甲硫基C结合蛋白，命名为MeCP-L，它不同于特征良好的MeCP2和部分特征的MeCP1。这项建议的长期目标是阐明DNA甲基化抑制癌细胞中基因表达的分子机制，及其功能和潜在的临床意义。本研究将(A)从肿瘤细胞的核提取液中提纯独特的MeCP-L，并对其进行生化鉴定，克隆该因子的c DNA，在合适的表达系统中进行表达，通过体外转录实验研究这种MeCP-L抑制MT-I启动子活性的机制(B)鉴定与MeCP-L特异结合的多肽，并在体外鉴定该蛋白的抑制域(C)通过在果蝇SL2细胞(含有少量MeCP)和甲基化MT-I启动子报告载体中表达MeCP-L，以及利用反义MeCP-L稳定转染P1798细胞，探讨MeCP-L在体内沉默甲基化基因的作用。(D)利用瞬时转染实验阐明MT-I基因不同区域的DNA甲基化在体内抑制其表达的分子机制；。(E)研究MT在P1798细胞中过表达对这些细胞的生长和转化潜能的影响；(F)研究MT基因沉默是否淋巴和肝脏肿瘤的独有特征，并测试糖皮质激素受体(GR)启动子甲基化是否与某些淋巴癌细胞对糖皮质激素的耐药性有关。希望这项研究能够提供动力，探索重新激活这些被抑制的基因的方法，从而可能导致特定的肿瘤生长受阻。

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.