Hypoxia Induces Erythropoietin Receptor Expression on K562 Cell Line

Abstract

ABSTRACTThe erythropoietin receptor (EpoR) and erythropoietin (Epo) mediate erythropoietin-induced erythroblast proliferation, differentiation and survival. This study examined the effects of the expression of EpoR on K562 (erythroleukemia) cells upon normoxia and hypoxia. In addition, the impact of the combined effect of recombinant human Epo and hypoxia was investigated. K562 (erythroleukemia) cells and as control group HL60 (promyeloblast) cells were cultured. Hypoxic incubation was performed with 5% O2, 5% CO2 and balance Nitrogen for 24 hours. After 24 hours, K562 and HL60 cells were transferred to normoxic and 5% hypoxic conditions. Recombinant Erythropoietin-alpha was added (10 U/ml) to the cells at the beginning of the experiment. Cultured cells were subjected to viability analysis, total RNA isolation, and protein isolation at three timepoints: 3 h, 6 h, and 24 h. Viability was analysed with a trypan blue exclusion using the Vi-Cell automated cell viability system. RT-PCR and western blot results of normoxic and hypoxic K562 and HL60 cell lines with/without rhEPO were compared.We showed that hypoxia upregulates the expression of EpoR on K562 erythroleukemia cells, and longer exposition to hypoxia turns the upregulated EpoR expression to basal levels. Recombinant human Epo (rhEpo) did not produce further impact in hypoxia-induced upregulation of EpoR neither in normoxia nor in hypoxia. Although addition of the rhEpo caused change in expression of cell-cycle regulators, it seems not to effect cell proliferation or cell viability. In the HL60 cell line, however, we detected EpoR mRNA, but not Epo mRNA by RT-PCR. Hypoxia did not alter the level of EpoR mRNA expression.The results of this study suggest that the expression of EpoR might be regulated by hypoxia, but not by Epo, and the expression of cell-cycle regulators might be regulated by both hypoxia and Epo.