Involvement of H-rasin Erythroid Differentiation of TF1 and Human Umbilical Cord Blood CD34+++Cells

Abstract

ABSTRACT: To investigate the role of therasgene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-rasgene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-rasgene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-rascDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-rasin vitro. The mRNA levels of β- and γ-, but not α-, globin genes were significantly higher in H-rastransduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-rastransduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34+++cells which are highly enriched for hematopoietic stem / progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-rasin erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34+++cells transduced with the H-rascDNA, compared to that from mock transduced cells. These data suggest a possible role forrasin erythroid differentiation.