MOLECULAR MECHANISMS OF DIET-INDUCED CARCINOGENESIS

流行病学和临床研究表明，叶酸缺乏可能导致人类易患某些类型的癌症。补充叶酸可逆转癌前发育不良。因此，阐明叶酸缺乏和癌症易感性的分子机制对于确定叶酸和其他饮食元素在癌症预防中的作用至关重要。研究低蛋氨酸、胆碱和叶酸饮食（脂质缺乏或LD饮食）在没有任何外源性外源药物的情况下诱导肝癌发生的作用，有一个很好的大鼠模型系统。众所周知，在低脂饮食诱导的肝癌中，一个关键的抑癌基因（p53）被甲基化。利用这个模型系统，我们将(a)通过亚硫酸氢盐基因组测序和Ms-SNuPe（甲基化敏感单核苷酸引物延伸）探索肝癌发生不同阶段p53启动子上CpG二核苷酸的甲基化状态；(b)通过瞬时转染试验研究特定CpG二核苷酸甲基化在p53启动子失活中的作用；(c)研究甲基化介导的染色质结构改变的机制，并确定参与其中的关键因素通过限制性内切酶接近性实验和甲基CpG结合蛋白（MeCPs）特异性抗体的染色质免疫沉淀（ChIP）研究p53启动子在肿瘤发生过程中的沉默(d)研究LD饮食诱导肝癌发生过程中不同DNA甲基转移酶同工酶（参与维持和新生甲基化）的表达和活性调控(e)鉴定与新生甲基化酶相互作用的蛋白质(f)克隆基因。特别是RLGS技术检测到的三个在肿瘤前肝脏中甲基化的基因，鉴定它们，研究它们在肿瘤发生不同阶段的表达水平，并研究这些基因沉默在肿瘤发生中的功能意义。希望这项研究能够提供叶酸/甲基缺乏与生长/肿瘤抑制基因或编码抑制肿瘤发生不同阶段生长调控基因的蛋白质的区域高甲基化的关系，以及导致肿瘤形成的这些基因的沉默的重要信息。

Epidemiological and clinical studies have demonstrated that folate deficiency in humans could lead to susceptibility to certain types of cancers. Premalignant dysplasia could be reversed by folate supplementation. Elucidation of the molecular mechanisms underlying folate deficiency and predisposition to cancer is, therefore, of critical importance in determining the role of folate and other dietary elements in cancer prevention. An excellent rat model system is available to study the role of diet low in methionine, choline and folate (lipotrope-deficient or LD diet) on the induction of hepatocarcinogenesis in the absence of any exogenous xenobiotic agents. It is known that a key tumor suppressor (p53) gene is methylated in the hepatoma induced by LD diet. Using this model system, we will (a) explore by Bisulfite genomic sequencing and Ms-SNuPe (Methylation- sensitive single nucleotide primer extension) the methylation status of CpG dinucleotides on p53 promoter at different stages of hepatocarcinogenesis (b) investigate the role of methylation of specific CpG dinucleotides in p53 promoter inactivation by transient transfection assay (c) investigate the mechanism of methylation-mediated alteration in chromatin structure and identify the key factors involved in consequent silencing of p53 promoter with progression of tumorigenesis, by restriction endonuclease accessibility assay and chromatin immunoprecipitation (ChIP) with antibodies specific to methyl CpG binding proteins (MeCPs) (d) study the regulation of expression and activity of different DNA methyltransferase isozymes (involved in maintenance and de novo methylation) during hepatocarcinogenesis induced by LD diet (e) identify the proteins that interact with de novo methylases (f) clone the genes, specifically the three genes that are methylated in preneoplastic liver as detected by RLGS technique, identify them, investigate their expression levels at different stages of tumorigenesis and study functional significance of silencing of these genes in tumorigenesis. It is hoped that this study will yield important information concerning the relationship of folate/methyl deficiency to regional hypermethylation of growth/tumor suppressor genes or genes encoding proteins that suppress the growth regulatory genes at different stages of tumorigenesis, and their silencing that lead to tumor formation.