Molecular mechanisms underlying erythropoiesis: Cycling activity of adult BFU-e relates to their requirement for c-myb function and potential for HbF synthesis

Abstract

Highly purified erythroid burst-forming units (BFU-e) from human embryonic liver, adult marrow and blood were manipulated in vitro by cytokine addition in order to explore their requirements for c-myb function and potential for fetal hemoglobin (HbF) synthesis, particularly as related to their cycling activity. c-myb is expressed at a minimal level and functionally required to a limited extent in quiescent adult BFU-e. However, c-myb is actively transcribed and stringently required for differentiation of actively cycling progenitors (embryonic BFU-e, embryonic and adult erythroid colony-forming units). The cycling activity of highly purified adult BFU-e, gradually enhanced by interleukin 3 (IL-3) addition, is strictly and directly related to both their functional requirements for c-myb and the level of myb mRNA expression in the progenitor population. It may be concluded that the transcriptional activity and the functional role of c-myb in early erythropoiesis are dependent upon the cycling activity of the erythroid progenitors. The reactivation of HbF synthesis in normal adult bursts, observed in the standard fetal calf serum-rich (FCS+) clonogenic system, is suppressed in cultures with a drastically limited growth of accessory cells (i.e., in FCS- or FCS+ Mo- conditions). In these cultures, addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3 induces a dose-related rise of gamma-chain synthesis, at least in part via a direct action at the BFU-e level. Preliminary studies involving priming of adult BFU-e with IL-3 in liquid phase suggest that the HbF potential is relatively low in quiescent BFU-e, but distinctly higher in actively cycling ones. It is postulated that the in vivo reactivation of HbF synthesis in bone marrow regeneration may be mediated via increased IL-3 and GM-CSF activity, leading to enhanced cycling and differentiation of BFU-e.

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis

“…Cells were then plated in clonogenetic culture in triplicate dishes as described elsewhere (Labbaye et al, 1995;Valtieri et al, 1991;Care et al, 1994).…”

Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation.

“…The amplification procedure included denaturation at 95°C for 30 sec, annealing at 54°C for 30 sec, and extension at 72°C during 40 PCR cycles-i.e., within the range of linear amplification. Methodology for c-myb mRNA RT-PCR has been reported (31).…”

Transcriptional elongation of c-myb is regulated by NF-κB (p50/RelB)

“…High levels of c-myb expression are found in immature, proliferating cells of all hematopoietic lineages and c-myb expression sharply declines with di erentiation (Introna et al, 1994). Inhibition of c-myb expression by antisense oligodeoxynucleotides (oligodNTs) results in inhibition of hematopoietic precursor cell proliferation and c-myb (7/7) mice demonstrate an abrupt failure of fetal liver hematopoiesis, suggesting that c-myb is required for maintaining the proliferative state of hematopoietic progenitor cells (Caracciolo et al, 1999;Valtieri et al, 1991;Mucenski et al, 1991). Forced c-myb overexpression suppresses terminal di erentiation in a number of leukemic cell lines including erythropoietin-induced di erentiation of SKT6 erythroleukemia cells, indicating that down-regulation of c-myb is a prerequisite for terminal di erentiation (Introna et al, 1994;Danish et al, 1992;Todokoro et al, 1988;Clarke et al, 1988;McClinton et al, 1990).…”