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 eryth roid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulate d by the transcription factor GATA-1. In order to understand the relat ionship, if any, between expression of GATA-1, response to Epo and ery throid differentiation, we analyzed the expression of GATA-1, Epo-R an d globin genes in an Epo-dependent human cell Line, UT-7 Epo. The resu lts were compared to those obtained with the parental granulocyte-macr ophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, w hich has a predominantly megakaryoblastic phenotype and is unable to p roliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expr essed similar levels of GATA-1 mRNA and binding activity. The two line s also expressed comparable levels of Epo-R mRNA while the number of E po-binding sites on UT-7 Epo cells was one-sixth the number on UT-7 ce lls (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 Northem analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells e xpressed alpha-globin and higher levels of gamma-globin (5-fold) and b eta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expre ssed beta- and gamma- globin genes in the two cell popula tions was me asured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity w ere higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive ce lls and 23 to 40% clearly positive cells, respectively), indicating th at 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 cel ls containing gamma-globin. The levels of GATA-1, Epo-R and globin mRN A expressed were not affected by a 24-hour incubation of either cell l ine with Epo, GM-CSF or interleukin-3 (IL-3). Growth factor starvation (24 hours) increased by several fold the expression of both GATA-1 an d 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 diffe rentiation program determine whether the Epo receptor can deliver an a dequate growth signal.