The SBDS Gene Regulates Viability of Myeloid Precursor Cells and Is Associated with High ROS Production and Telomere Shortening in the SBDS Downregulated Cells.

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11 and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene resulting in a truncated and inactive protein. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues and its function is still unknown. In order to investigate the function of the protein, we have established two stably infected 32Dcl3 cell lines, expressing two different shRNAi against SBDS. The downregulation of SBDS was confirmed by Western blotting with anti human SBDS antibody. The growth of 32Dcl3 cells was significantly decreased in cells with down regulation of SBDS cells when compared to that of cells stable transfected with a control shRNAi cells. The maximum cell density of mIL-3 dependent cell growth was about 50% in SBDS downregulated cells (2.9x10⁵ cells/ml vs 1.3x10⁵ cells/ml, respectively). The SBDS downregulated cells were sensitive to serum starvation. The Annexin V binding was same as control in the presence of 10% FCS. However, after exposure to 1 % FCS, apoptotic cells were much increased in SBDS downregulated cells(16.45±4.2% in control cells vs 25.19±0.2% in SBDS downregulated cells, p=0.036). Fas and Fas ligand was not detected in SBDS downregulated cells, and both Bcl2 and BclxL expression was same between the cells. To further characterize the potential mechanism for cell kill as a consequence of SBDS knockdown, we examined the telomere length by Flow-FISH. The telomere length of SBDS downregulated cells was significantly shorter than control cells after 2 weeks infection (13.3±2.85 kb in SBDS knockdown cells vs 15.6±3.19 kb in control cells, p=0.013. Cells were passaged 6 times after infection). In addition, reactive oxygen species (ROS) production of SBDS downregulated cells was much higher than control infected cells. This high ROS production correlated with the rapid telomere shortening, and predisposition of the cells to apoptosis. We also characterized the consequence of SBDS protein knockdown on neutrophil differentiation. Knockdown cells showed normal differentiation as defined by morphology and the neutrophil maturation markers, Mac1, CD11b but proliferation was decreased by 90%. In summary, we have an established SDS model cell line and have demonstrated that SBDS is not required for neutrophil maturation. After loss of SBDS, ROS production was increased, and high concentration of ROS resulted in increased sensitivity to apoptosis stage and abnormal telomere shortening. These data give insights into the pathways affected by loss of SBDS function, together with mechanistic hypotheses that can be tested in primary samples form patients with SDS.