EPO receptor, Bax and Bcl-xL expressions in murine erythropoiesis after cyclophosphamide treatment

The effect of a single dose of cyclophosphamide (CY, 150 mg/kg i.p.) on the erythropoiesis using an 'in vivo' murine model in a time course protocol (0-10 days) was studied through several experimental approaches. Total and differential bone marrow cellularities, apoptosis (TUNEL assays), bone marrow hematopoietic architecture (scanning electronic microscopy), proliferation (DNA assay), BM erythroid progenitors growth (semisolid clonogenic assays) and protein expressions for erythroid commitment and survival: erythropoietin receptor (EPO-R), Bcl-xL, Bax (immunoblottings) were performed on the scheduled days. Most of the experiences were conducted comparing spontaneous with human recombinant (hr EPO) 'ex vivo' stimulated bone marrow (BM) cells. Erythropoiesis was extremely affected by CY. Maximum apoptosis, minimal cellularities and severe disturbances of BM niche were noticed on the second day. During spontaneous recovery post-CY; EPO-R was expressed between 4 and 5 days. Following BM cells 'ex vivo' hr EPO stimulation (2 U/ml) EPO-R was expressed throughout the study except the period between the first and fourth day. Bax was noticeable all along the experience with and without hr EPO stimulation. Bcl-xL was barely detectable without hr EPO, but its expression showed a gradual enhancement from the fifth day onwards in hr EPO stimulated cells. This fact might be related to the end of the erythroid inhibitory stage and to the recovery of BM EPO-dependent proliferation between the fourth and fifth day, and the further recuperation of BFU-E and CFU-E colonies on days 6 and 7 post-CY, respectively. These findings suggest that the proliferation and differentiation of erythroid progenitor cells after the acute early injury inflicted by CY, is associated with changes in EPO-R expression during spontaneous recovery in this particular experimental system