MECHANISMS OF CHEMICALLY INDUCED DIFFERENTIATION

在5-位修饰的胞苷类似物，如5-氮胞苷 最初是作为抗癌剂开发的，在 某些儿童白血病的治疗。然而，更重要的是， 它们对培养细胞分化状态的影响，以及它们的 在这些细胞中引起致癌转化的能力，已经允许 功能强大的体外模型研究进展 分化转型。这项提案的总体目标是 确定抑制DNA甲基转移酶的机制 通过5-氮胞苷，了解DNA甲基化的早期变化和 发生在过程中和紧随其后的特定的mRNA、转录 5-氮杂-2‘-脱氧胞苷(5-aza-CDR)治疗及其相互关系 分化和致癌转化过程的变化。 具体地说，我们建议使用定义DNA底物和亲和纯化 DNA甲基转移酶研究5-氮胞嘧啶与DNA甲基转移酶的相互作用 这种酶。我们将鉴定一种软骨细胞特异性决定基因 利用消减克隆技术从马厩中克隆出mRNA 成软骨细胞系。表达立即变化的信使核糖核酸 在药物治疗后，将使用差异cDNA克隆进行分离 聚合酶链式反应(PCR)扩增。时间的选择 这些基因的复制及其表达的时间顺序将是 确定为鉴定5-氮杂-镉R的主要靶基因(S)和 药物最终是否会引发一连串的基因激活事件 导致治疗中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进行验证 检测这些基因对细胞分化的影响 转染C3H1OT1/2细胞。 这项提案中描述的实验将显著增强我们的 了解在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，就有可能阐明其作用 这些事件发生在转型的过程中。最后，它的局限性 辨证治疗将更好地理解更复杂的 了解发展和分化的过程。

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.