Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7

Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number on UT-7 cells (2400 ± 3 vs. 13,800 ± 300). This difference in the number of binding sites could be due to differences in cell surface UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of β- and γ-globin but not α-globin. In comparison, UT-7 Epo cells expressed a-globin and higher levels of γ-globin (5-fold) and β-globin (from barely to clearly detectable). Globin chains (α, β and γ) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed β- and γ-globin genes in the two cell populations was measured by immunofluorescence with β- and γ-specific antibodies. The number of γ-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing γ-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3). Growth factor starvation (24 hours) increased by several fold the expression of both GATA-1 and Epo-R, consistent with the coordinated expression of these two genes. Under these conditions, however, levels of expression of the globin genes were not affected. These results suggest that possession of Epo receptors per se (even in high numbers) is not sufficient to deliver a growth signal in just any cell; other elements of the erythroid differentiation program determine whether the Epo receptor can deliver an adequate growth signal.