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.

5-位修饰的胞苷类似物，例如 5-氮杂胞苷 最初是作为抗癌剂开发的，并在以下方面具有一定的用途： 治疗某些儿童白血病。 然而更重要的是， 它们对培养细胞分化状态的影响及其 在这些相同的细胞中引起致癌转化的能力，使得 开发强大的体外模型来研究过程 差异化和转型。 该提案的总体目标是 确定 DNA 甲基转移酶抑制的机制 通过 5-氮杂胞苷，了解 DNA 甲基化的早期变化和 特定 mRNA，在期间和之后立即发生的转录 用 5-aza-2'-脱氧胞苷 (5-aza-CdR) 治疗，并将这些联系起来 分化和致癌转化过程的变化。 具体来说，我们建议使用定义的 DNA 底物和亲和纯化的 DNA 甲基转移酶用于研究 5-氮杂胞嘧啶和 酶。 我们将鉴定软骨细胞特异性决定基因 使用消减 cDNA 克隆技术，从稳定的 mRNA 中提取 软骨形成细胞系。 表达立即改变的 mRNA 种类 药物治疗后将使用差异 cDNA 克隆进行分离 和聚合酶链反应（PCR）扩增。 的时间安排 这些基因的复制及其表达的时间顺序将是 确定的目的是为了鉴定 5-aza-CdR 的主要靶基因，以及 最终药物是否会引发一系列基因激活事件 导致治疗后多种细胞谱系的发展 文化。 这种关系的存在将通过以下方式验证 检查这些基因对细胞分化的影响 转染至C3H1OT1/2细胞。 本提案中描述的实验将显着增强我们的能力 了解正常期间发生的监管事件 发展。 此外，由于致癌转化发生在 相同的实验系统，将有可能阐明 这些事件都处于转变的过程中。 最后，限制 通过更复杂的方法可以更好地理解分化治疗 发育和分化过程的知识。