RPS19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia

¹ Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden; Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan
² Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden
³ Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden; Clinic for Internal Medicine C, Department of Hematology and Oncology, Ernst-Moritz-Arndt University of Greifswald, Greifswald, Germany
⁴ Department of Safety Research on Blood and Biologics, National Institute of Infectious Diseases, Tokyo, Japan

^* To whom correspondence should be addressed. E-mail: stefan.karlsson{at}med.lu.se.

	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 erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed the effects of RPS19 deficiency on EPO induced signal transduction, cell cycle, and apoptosis in RPS19-deficient TF-1 cells. We did not find any abnormality in EPO 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). Western blot analysis of apoptotic related proteins showed that the level of bcl-2 and Bad was decreased and Bax was increased in RPS19-deficient TF1 cells. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generated a higher number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). Finally, we show that while RPS19 silencing reduces EPO induced development of erythroid progenitors expressing Glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.

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