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.
