MECHANISMS OF CHEMICALLY INDUCED DIFFERENTIATION

化学诱导分化的机制

• 批准号: 2095778
• 负责人: SHIRLEY M TAYLOR
• 金额: $ 9.36万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2095778
• 关键词: DNA methylation; SDS polyacrylamide gel electrophoresis; azacitidine; cell differentiation; cell line; chondrocytes; complementary DNA; disease /disorder model; drug carcinogenesis; drug metabolism; enzyme inhibitors; enzyme substrate; gene expression; laboratory rabbit; messenger RNA; methyltransferase; molecular cloning; neoplasm /cancer chemotherapy; nonhuman therapy evaluation; nucleic acid sequence; polymerase chain reaction; protein purification; protein sequence; transfection

Analogs of cytidine modified in the 5-position, such as 5-azacytidine were originally developed as anticancer agents, and have been of some utility in treatment of certain childhood leukemias. More significantly however, their effect on the differentiated state of cultured cells, and their ability to cause oncogenic transformation in these same cells, have allowed the development of powerful in vitro models for studying the processes of differentiation and transformation. The overall goal of this proposal is to define the mechanism underlying the inhibition of DNA methyltransferase by 5-azacytidine, to understand the early changes in DNA methylation and specific mRNA, transcription that occur during and immediately following treatment with 5-aza-2'-deoxycytidine (5-aza-CdR), and to relate these changes to the processes of differentiation and oncogenic transformation. Specifically, we propose to use define DNA substrates and affinity-purified DNA methyltransferase to study the interaction between 5-azacytosine and the enzyme. We will identify a chondrocyte-specific determination gene using substractive cDNA cloning techniques with mRNA from a stable chondrogenic cell line. mRNA species whose expression changes immediately following drug treatment will be isolated using differential cDNA cloning and polymerase chain reaction (PCR) amplification. The timing of replication of these genes and temporal order of their expression will be determined in order to identify the primary target gene(s) of 5-aza-CdR and whether the drug initiates a cascade of gene activation events, ultimately leading to the development of multiple cell lineages within treated cultures. The existence of this type of relationship will be verified by examining the effects of these genes on cellular differentiation after transfection into C3H1OT1/2 cells. The experiments described in this proposal will significantly enhance our understanding of the regulatory events that occur during normal development. Furthermore, since oncogenic transformation occurs in the same experimental system, it will be possible to elucidate the role of these events in the process of transformation. Finally, the limitations of differentiation therapy will be better understood with a more sophisticated knowledge of the processes of development and differentiation.

在5-位修饰的胞苷类似物，如5-氮杂胞苷， 最初被开发为抗癌剂，并且已经在 治疗某些儿童白血病。 然而，更重要的是， 它们对培养细胞的分化状态的影响，以及它们的 在这些细胞中引起致癌转化的能力， 开发强大的体外模型，用于研究 分化和转化。 本提案的总体目标是 确定抑制DNA甲基转移酶的机制 通过5-氮杂胞苷，了解DNA甲基化的早期变化， 特异性mRNA，在转录过程中和紧接在转录过程中 用5-氮杂-2 '-脱氧胞苷（5-aza-CdR）处理，并将这些 分化和致癌转化过程的变化。 具体而言，我们建议使用定义的DNA底物和亲和纯化的 DNA甲基转移酶，研究5-氮杂胞嘧啶与 酶 我们将确定一个软骨细胞特异性决定基因 使用消减cDNA克隆技术， 软骨细胞系 表达立即改变的mRNA种类 将使用差异cDNA克隆分离药物治疗后的 和聚合酶链反应（PCR）扩增。 的时机 这些基因的复制和它们表达的时间顺序将被 测定以鉴定5-aza-CdR的主要靶基因， 药物是否会引发一连串的基因激活事件， 导致在治疗的细胞内形成多种细胞谱系， cultures. 这种关系的存在将通过以下方式进行验证： 研究这些基因对细胞分化的影响， 转染入C3 H1 OT 1/2细胞。 本提案中描述的实验将大大提高我们的 了解正常情况下发生的监管事件 发展 此外，由于致癌转化发生在 同样的实验系统，将有可能阐明的作用， 这些事件在转变的过程中。 最后， 分化治疗将更好地理解与更复杂的 了解发展和分化的过程。