5-azacytidine reactivates the erythroid differentiation potential of the myeloid-restricted murine cell line 32D Ro

32D cells grown for 1 year in interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) generated the 32D Ro cell line which retained the parental mast cell phenotype but lost ability to generate erythroid cells in response to erythropoietin (EPO). In order to clarify the mechanisms underlying such restriction, we compared 32D and 32D Ro cells for their capacity to express erythroid-specific transcription factors (Gata1, Gata2, Scl, Nef2, Eklf, and Id) and the capacity of short exposure to 5-azacytidine (5-AzaC) to reactivate erythroid differentiation potential in 32D Ro cells. By Northern analysis, the two cell lines expressed similar levels of all these genes. However, after being treated with 5-AzaC, 32D Ro cells acquired the ability to generate EPO-dependent clones (1 clone/10(4) cells) which gave rise to EPO-dependent cell lines. All the 10 EPO-responsive cell lines independently isolated from 5-AzaC-treated 32D Ro cells had erythroid morphology and expressed high levels of alpha- and beta-globin. In contrast, none of the IL-3-dependent and granulocyte/macrophage colony-stimulating factor-dependent clones concurrently isolated, as a control, showed erythroid properties. Therefore, 5-AzaC treatment reactivates the potential of the myeloid-restricted 32D Ro cells to generate EPO-responsive erythroid clones suggesting that gene methylation played an important role in the G-CSF-mediated restriction/activation of the differentiation potential of these cells.