Erythropoietin binding and induced differentiation of Rauscher erythroleukemia cell line red 5-1.5.
We isolated from the Rauscher erythroleukemia cell line (Red 5), a subclone (Red 5-1.5), which contains erythropoietin (epo) binding sites and demonstrates an epo-dependent erythroid differentiation. One class of high affinity binding sites was detected with a Kd (+/- S.D.) of 0.43 +/- 0.09 nM and a mean density/cell of 1200 +/- 311. The cell-associated 125I-epo was displaced by nonlabeled epo but not by other hormones or factors. The 125I-epo binding to Red 5-1.5 cells was maximal within 3 h at 15 degrees C and 1 h at 37 degrees C and proportional to cell number. The addition of epo increased [3H] uridine incorporation into RNA by 6 h and [3H]thymidine incorporation into DNA by 60 h followed by 59Fe incorporation into protein, cell proliferation, and formation of hemoglobin-containing colonies. The incorporation of 59Fe into protein demonstrated a linear dose response (from 0.002 to 1.5 units of epo/ml) beginning 60 h after addition of the hormone to the cultures, and there was a dose-dependent increase (from 0.1 to 1.0 unit of epo/ml) in the formation of hemoglobin-containing colonies. We concluded that the binding of 125I-epo to Red 5-1.5 suggests the presence of specific epo receptors. The sequence of the epo-induced proliferation and differentiation events is similar to primary erythroid cultures but requires longer epo exposure. Receptor occupancy correlates with the induced biological response.
Weiss
T L
TL
Department of Dermatology, University of California, San Francisco 94121.
Barker
M E
ME
Selleck
S E
SE
Wintroub
B U
BU
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D008854:Microscopy, Electron; D012313:RNA / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D017467:Receptors, Erythropoietin; D013936:Thymidine / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D012313:RNA; D004247:DNA; D013936:Thymidine; D014529:Uridine