Hydroxyurea Induced Expression of gamma Globin and Erythrocyte Membrane Proteins in Cultured Erythroid Progenitor Cells.

BACKGROUND: Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell anemia and beta-thalassemia. 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 low dose HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. The frequency of cells expressing greater levels of (AE1) per cell was increased in each serial blood sample. 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 examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors. METHODS: HU induced protein synthesis was investigated as a function of HU concentration. Erythroid progenitors were cultured in serum free semisolid media containing different concentrations of HU [0–40 micromolar]. BFU-E were scored and harvested at day15 in culture, then assayed. BFU-E derived cells were first labeled with monoclonal anti-band 3 antibody. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry. In separate studies BFU-E derived cells were fixed, permeabilized, labeled with anti-spectrin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. The labeled cells were assayed by confocal microscopy. For the next set of experiments, BFU-E cultured in increasing concentrations of HU were assayed for the presence of band 3 protein and gamma globin. BFU-E cells were fixed, permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody; then assayed by flow cytometry. RESULTS: Results show that the frequency of cells positive for band 3 protein [AE1] was increased at increasing concentrations of HU as compared to controls. The band-3 upmodulation appears to start to plateau at 12.5 micromolar HU. Band 3 protein derived from BFUE was confirmed by Western blot analysis. The frequency of cells positive for both band 3 and spectrin was similarly increased in cells cultured in the presence of increasing concentrations of HU. In separate studies, cells were assayed for the expression of both gamma globin and AE1. Results show a 2–3-fold increase in the % band 3 [AE1] plus gamma globin positive cells in HU [5–40 micromolar] treated cells as compared to untreated controls. CONCLUSIONS: These results demonstrate that HU induces the expression of band 3 and gamma globin in cultured erythroid progenitors. These findings 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.