Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

ts-Oncogene-Transformed Erythroleukemic Cells: A Novel Test System for Purifying and Characterizing Avian Erythroid Growth Factors

“…Three days later, a small aliquot of each clone was stained for hemoglobin with acid benzidine [9]. Clones exhibiting> 95% benzidine-positive cells were centrifuged onto slides, stained with neutral benzidine and histological dyes, and evaluated for the presence of terminally differentiated cells, as described earlier [1]. Finally, those clones that contained more than 95% erythrocytes plus late reticulocytes [1] were tested for their response in both EPO assays (see below) and for their in vitro lifespan by repeated passage (1 in 3) in CFU-E medium.…”

“…Clones exhibiting> 95% benzidine-positive cells were centrifuged onto slides, stained with neutral benzidine and histological dyes, and evaluated for the presence of terminally differentiated cells, as described earlier [1]. Finally, those clones that contained more than 95% erythrocytes plus late reticulocytes [1] were tested for their response in both EPO assays (see below) and for their in vitro lifespan by repeated passage (1 in 3) in CFU-E medium. One clone (clone 30), which showed a particularly prominent response to anemic serum in both assays and exhibited a lifespan of > 45 generations in vitro, was frozen in many aliquots in liquid nitrogen and used for the experiments described in this paper.…”

“…Pilot experiments designed to test the usefulness of these cells in possible assay systems for erythroid growth factors quickly demonstrated that ts-sea erythroblasts were much more suitable for such assays than ts-erbB cells (probably because of the presence of v-erbA in the latter) [13]. In addition, it soon became evident that suitable test-cell clones had to be selected, exhibiting the potential for complete in vitro differentiation as well as an extended in vitro lifespan [1] without anemic serum revealed that the cells continued to proliferate in the presence of anemic serum for at least 2 days, whereas they rapidly withdrew from the cell cycle in the absence of anemic serum, being essentially at rest after 3~8 h. Furthermore, in the presence of anemic serum, very few cells died during differentiation, whereas anemic serum-deprived cells mostly disintegrated within 24-48 h after shift, then being at the early-to-Iate reticulocyte stage (Fig. 1).…”

“…Upon a shift to the nonpermissive temperature, ts-AEV and ts-S13 erythroblasts are induced to differentiate synchronously into erythrocytes. At the same time, the cells regain their dependence on a factor(s) from anemic chicken serum [1][2][3] (H. Beug et al, unpublished work).…”

“…The transformed cells, however, retain the ability to undergo termi-nal differentiation at low frequency [3,7,12]. At the same time, they become independent of an activity present in anemic chicken serum that induces CFU-E-like colonies in chicken bone marrow and probably represents avian erythropoietin (EPO) [1,21]. Recently, our laboratory described the use of erythroblasts transformed with temperature-sensitive mutants of v-erbB (ts AEV) and v-sea (ts S13) containing retroviruses as novel systems for studying differentiation of normal and leukemic erythroid cells.…”