Apoptotic Change Is a Major Reason for Defect in Early Erythroid Development in Cell Line Models for Ribosomal Protein (RP) S19 Deficient Diamond-Blackfan Anemia.
Authors: Miyake Koichi , Utsugisawa Taiju , Flygare Johan , Kiefer Thomas , Richter Johan , Wiznerowicz Maciej , Trono Didier , Karlsson Stefan 
Published: April 2020

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. The majority of cases appear to result from an intrinsic disorder of the erythroid progenitor that involves its inability to respond normally to inducers of erythroid proliferation and differentiation. Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. However, so far, no receptor-ligand defects have yet been identified. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Blood102:504a, 2003). To analyze the effect of suppression of RPS19 for erythroid differentiation, we used cytokine dependent TF-1 cell lines (CD34+, CD71+, GFAlow) together with new established cytokine independent K562 cell lines (CD34−, CD71+, GFAhigh). We established two types of cell lines (TF-1A, K562A and TF1B, K562B) using different siRNA against RPS19. Five days after Dox induction, RPS19 protein level was suppressed to 45–55% in TF-1A and K562A, 65–75% in TF1B and K562B compared to control scramble transduced cell lines (TF1S, K562S) by western blot analysis. Suppression of cell growth and colony formation correlated with the suppression level of RPS 19 in TF1A, B and K562A, B cells. In contrast, after stimulation with Epo, glycophorin A expression, measured by flow cytometry, and hemoglobin content (DAF staining) showed suppression of erythroid differentiation only in TF1A and TF1B but not in K562A and K562B. Since Epo induces the stimulation of Jak2 tyrosine kinase which leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated such as Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. Therefore, we analyzed Epo stimulated signal transduction in these cell lines. However, no abnormal signal transduction could be detected in any of the cell lines. Cell cycle analysis showed that the percentage of cells in the G0/G1 phase increased and apoptotic cells detected by Annexin-V analysis also increased in TF1 (A&lt;B) and K562 (A&lt;B) cells. Western blot analyzed p21 showed that the level of p21 was increased in TF1 (A&lt;B) and K562 (A&lt;B) cells. These results indicate that Epo triggered onset of terminal maturation is intact in RPS19 deficient DBA cell line models. We speculate that apoptotic change by suppressed RPS19 may be one of the major reason for the accelerated loss of erythroid progenitor clonogenicity in RPS19 deficient DBA. These cell lines are useful to determine the mechanisms of RPS19 deficient DBA.