Depletion of the Shwachman-Diamond Syndrome Protein in Hematopoietic Progenitor Cells Impairs Growth, Colony Formation, and Ribosome Function.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, skeletal abnormalities, and bone marrow failure, which can evolve to leukemia. Mutations in SBDS have been shown to cause SDS. We and other investigators have suggested that SBDS orthologs in yeast play a role in biogenesis and function of the 60S ribosomal subunit. To clarify the unknown function of SBDS in hematopoiesis, human erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing the green fluorescent protein (GFP), neomycin phosphotransferase, and small interfering RNA (siRNA) against SBDS. After transduction, cells were selected for neomycin resistance and then sorted by flow cytometry. To probe for SBDS, an antibody against the carboxyl-terminus of human SBDS was generated, and individual TF-1 cell clones expressing different siRNAs were confirmed to knock down SBDS expression by Western blot analysis. Our experiments were aimed at analyzing the cellular effects of SBDS knockdown. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with siRNA against a scrambled SBDS sequence. Using propidium iodide staining and flow cytometric analysis, we found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether TF-1 cells expressing siRNA against SBDS have a selective deficiency of 60S ribosomal subunits, cell extracts were prepared and polysome profiles examined after sucrose gradient centrifugation. In preliminary experiments, TF-1 cells expressing siRNA against SBDS appeared to show a reduction in free 60S subunits and 80S subunits with a shift toward smaller polysomes, compared to cells expressing the scrambled sequence siRNA. We conclude that depletion of SBDS results in a significant growth and clonogenic defect in TF-1 hematopoietic cells. Our preliminary results also suggest defects in ribosome function and cell cycle transit, which may provide an integrated molecular explanation for the hematopoietic defect in SDS since nucleolar stress has been linked to cell cycle arrest and p53 stabilization.