Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (

Mol Ther. 11:627–637. 2005
). Suppression of cell growth and erythroid colony formation correlated with the suppression level of RPS 19, indicating that these cell lines are useful to determine the mechanisms of RPS19 deficient DBA. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed cell cycle of Dox induced RPS19 deficient TF-1 cells. RPS19 deficient TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumlation of p21 and p27, and apoptotic cells detected by Annexin-V analysis also increased compared to control Dox induced TF-1 cells (13% vs 3.1%, p<0.05). Increase of apoptotic cells in RPS19 deficient cells was confirmed by TUNEL assay. Western blot analysis of apoptotic related protein showed that the level of bcl-2 and Bad was decreased in RPS19 deficient TF1 cells compared to control cells. This down-regulation of apoptotic related protein was improved by transduction with lentiviral vector expressing modified RPS19, which is not affected by siRNA but produce normal functional RPS19 protein and rescues the DBA phenotype. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generate a high number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). These findings indicate that erythroid progenitor cells are more sensitive to apoptosis than other hematopoietic progenitors and that RPS19 deficiency causes apotosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.

Topics:
apoptosis, bcl2 gene, diamond-blackfan anemia, erythroid progenitor cells, ribosomal proteins, rna, small interfering, annexin a5, cd34 antigens, doxycycline, in situ nick-end labeling

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2005, The American Society of Hematology
2005
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