| Budget Amount *help |
¥3,750,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
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 previously established in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19. 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 Here we show that while RPS19 silencing reduces erythropoietin (EPO) induced development of erythroid progenitors expressing Glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. This finding suggests that a deficiency of RPS19 may negatively affect development of cells corresponding to proerythroblasts. To further elu
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cidate molecular mechanisms in RPS19 deficient DBA, we analyzed the effects of RPS19 deficiency on EPO induced signal transduction, cell cycle and apoptosis in TF-1 cells. We did not find any abnormality in ERO induced signal transduction. However, RPS19 deficient TF-1 cells showed G0/G1 arrest (82% vs. 58%, p<0.05) together with accumulation of p21 and p27., The fraction of apoptotic cells detected by Annexin-V analysis also increased compared to control cells (13% vs. 3.1%, p<0.05). The 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 bc1-2 and Bad was decreased in RPS19 deficient TF1 cells compared to control cells. 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 apopto.sis and accelerated loss of erythroid progenitors in RPS19 deficient DBA. Less
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