Activation and demethylation of the intracisternal A particle genes by 5-azacytidine.

Treatment of C3H 10T1/2 mouse embryo fibroblasts with the cytidine analogues 5-azacytidine and 5-aza-2-deoxycytidine causes altered gene expression and results in the manifestation of phenotypic changes and altered cell morphology. This includes the conversion of these cells to adipocytes, chondrocytes and myotubes. The effects of these analogues on intracisternal A particle (IAP) gene expression in mouse C3H 10T1/2 cells have been examined. Treatment with either 3 microM 5-azacytidine or 0.3 microM 5-aza-2-deoxycytidine for 24 h was associated with an immediate increase in IAP gene transcription, and with the subsequent appearance of IAPs in the cisternae of the cells 24 h after removal of the drug. Control cells contained no, or very few, IAPs and IAP mRNA. Analysis of the methylation status of the IAP genes, using the restriction endonucleases HpaII, MspI and HhaI, showed that these genes were already demethylated at the end of the 24-h treatment period. IAP gene transcripts were detectable even after a 16-h drug treatment period, at which stage the genes were not yet fully demethylated. After further growth in fresh medium for 90 h, the levels of IAP RNA started to decline, but the demethylated CpG sites were not yet remethylated. These results suggest the involvement of other factors, in addition to methylation, in the regulation of IAP gene expression. These drugs were found to have no stimulatory effect on several oncogenes examined in this study.