Distinct Differences in in Vitro Erythroid Proliferation and Differentiation, p53 and Apoptosis Pathways in Diamond-Blackfan Anemia in Conjunction with Depletion of RPS19, RPL5 and RPL11 mRNA.

Abstract 176

Identification of mutations in a number of ribosomal genes including RPS19, RPL5 and RPL11 in Diamond-Blackfan anenia has established it as a disease of aberrant ribosome biogenesis. To date, it has not been possible to determine if a common cellular mechanism accounts for the erythroid defect in DBA in conjunction with various ribosomal gene mutations identified. To address this issue, we infected normal CD34⁺ cells from cord blood with specific short hairpin (sh) RNA against RPS19 (shRPS19C), RPL5 (shRPL5A, B, C) and RPL11 (shRPL11A, B, C) and determined their ability to undergo erythroid differentiation in a liquid culture system. Efficiency of each shRNA was verified at the RNA level in UT7 cell lines and CD34⁺. They all decreased dramatically mRNA expression of RPS19, RPL5 and RPL11 from 90% to 50% depending on the shRNA and the cells, except shRPL11B and C, which reduced RPL11 mRNA expression from 40% to 20%. Erythroid precursors after sh RNA infection were harvested at D7, D9, D11, D14 and D16 and analyzed for cell clonogenicity, erythroid cell differentiation (May Grünwald Giemsa or flow cytometry), apoptosis (Tunel assay and flow cytometry), and mRNA and protein expression. During in vitro erythroid differentiation, we noted a decrease in the extent of cell amplification, from D0 (CD34⁺ cells) to D7 (CFU-e/proerythroblasts), to D9 (basophilic erythroblasts), to D11, D14, D16 (orthophilic erythroblasts) for all the shRPS19C, shRPL5 (A, B, C) and shRPL11A infected cells compared to uninfected or scramble infected cells. The decrease in erythroid proliferation was less important (50%) in shRPS19 infected cells compared to shRPL5A,B,C and shRPL11A infected CD34⁺ cells (90–95%). In association with documented decrease in expression of RPS19 mRNA after shRPS19 CD34⁺ infection, we did not find either an alteration in erythroid differentiation or increased apoptosis at any stage of cell differentiation. In marked contrast, CD34⁺ cells infected with either shRPL5A,B,C or shRPL11A exhibited importantly a delayed erythroid differentiation with a marked increase in apoptosis. These findings have enabled us to document that while decreased cell proliferation is a feature of all CD34⁺ cells with decreased expression of different ribosomal proteins, defective differentiation and increased apoptosis of erythroid cells is a distinguishing feature of only CD34⁺ cells with decreased expression of RPL5 and RPL11.

To obtain mechanistic insights into the observed differences in apoptotic phenotype, we analyzed p53 and apoptosis pathways of CD34⁺ following infection with shRPS19, shRPL5A-B-C, and shRPL11A. All the proteins tested: p53, p21, caspase 3, Bax, Noxa exhibited a decreased expression levels following infection with shRPL5A,B,C and shRPL11A, while exhibiting an increased expression at the mRNA level, suggesting a general inhibition of protein synthesis following RPL5 and RPL11 mRNA depletion. Strikingly, RPS19 depletion resulted increased levels of p53 and p21 expression at both mRNA and protein levels, while Bax and Noxa (proapoptotic) mRNA were as the same level compared to the controls. These different patterns of alteration in p53 and apoptotic protein levels can account for different apoptotic phenotype observed following depletion of RPS19 and of RPL5 and RPL11.

In summary, we identified for the first time distinct erythroid proliferation and differentiation defects in conjunction with different ribosomal protein defects. These findings may have implications for future genotype-phenotype relationships in DBA patients.