Induction of Gamma Globin and Erythrocyte Anion Exchane Protein in BFU-E Cultured in the Presence of Hydroxyurea.

Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell hemoglobinopathy and beta-thalassemias. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined.

Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we have examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors.

To investigate HU induced protein synthesis as a function of HU concentration, erythroid progenitors were cultured in semisolid media containing different concentrations of HU [0–40 micromolar] then assayed for AE1(band 3) and gamma globin. BFU-E were scored and harvested after 15 days in culture, then assayed. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry, these cells were then assessed by laser scanning confocal microscopy. Results show that the frequency of cells positive for band 3 protein was greater in colonies grown in hydroxyurea as compared to controls. The band-3 upmodulation appears to plateau at 12.5 micromolar HU. These cells were assessed for dual and single stains by laser scanning confocal microscopy. Both band-3 protein and spectrin were detected. Laser scanning confocal microscopy revealed spectrin in the majority of cells; both band 3 and spectrin were detected in forty percent of the cells cultured in 12.5 micromolar HU. Band 3 protein detected by Western blots was increased [1–1.5 fold] over untreated control BFU-E harvested during the same time period.

Secondly, the presence of band 3 protein and gamma globin in BFU-E was detected using two-color flow cytometry. BFU-E were cultured in increasing concentrations of HU. Colonies were permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were subsequently labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. Results show 2–3 fold increase in the % band 3 plus gamma globin positive cells over untreated cells. Collectively, these results suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.