Alterations of the phosphoinositide 3-kinase and mitogen-activated protein kinase signaling pathways in the erythropoietin-independent Spi-1/PU.1 transgenic proerythroblasts.

During the cell transformation processes leading to erythroleukemia, erythroid progenitors often become erythropoietin (Epo)-independent for their proliferation. The biochemical events that could lead an erythroleukemic cell to growth factor-independence were investigated using spi-1 transgenic poerythroblasts. Spi-1/PU.1 is a myeloid and B-cell transcription factor of the ETS family and is activated by insertional mutagenesis during Friend erythroleukemia. Its overexpression in proerythroblasts induces their differentiation arrest without altering their erythropoietin requirement for proliferation (HS1 cells). At a later step, genetic alterations most probably occur allowing spi-1 transgenic poerythroblasts to proliferate in the absence of erythropoietin (HS2 cells). The signaling transduction pathways in HS1 and HS2 proerythroblasts were analyzed. The authors have previously shown that the Jak/STAT pathway was not activated in Epo-independent cells, but remained sensitive to Epo stimulation. In the present study, it is shown that the Epo-independent proliferation of HS2 cells requires active phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. In these cells, PI3K was constitutively associated with the molecular adapters Grb2 and Gab1, and with the phosphatases SHP-2 and SHIP. Moreover, PI3K activity was correlated with the constitutive phosphorylation of serine-threonine protein kinase (AKT) in HS2 cells. Lastly, a constitutive activation of the MAPKs extracellular signal-regulated kinases (ERK1/2) in HS2 cells was observed that occurs in a PI3K-independent manner, but depends strictly on the activity of the protein kinase C (PKC). These results suggest that constitutive activations of PI3K/AKT and PKC/MAPK pathways can act in synergy to lead a proerythroblast to proliferate without Epo.