Characterization of siRNA-Mediated SBDS-Knockdown Cells: Specific Hypersensitivity to Fas Stimulation.
Authors: Watanabe Ken-ichiro , Dror Yigal 
Published: October 2019

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and a marked propensity for myelodysplastic syndrome and leukemia. Approximately 90% of the patients have mutations in the SBDS gene. Recent studies suggested a role of the yeast SBDS homologue, YLR022C, in RNA processing and ribosomal biogenesis. However, the function of the human SBDS has not been clarified yet. We previously showed that marrow cells from SDS patients are characterized by accelerated apoptosis, overexpression of Fas and hypersensitivity to Fas stimulation. To study the function of SBDS and determine whether the above abnormalities are caused by deficiency of SBDS, we established stably transfected Hela cell lines expressing two different siRNAs against SBDS and lines expressing scrambled siRNA control. SBDS-knockdown was confirmed by Western blotting using polyclonal chicken anti-human SBDS antibody. The SBDS expression in the scrambled siRNA control cells was comparable to that of wild-type Hela cells. DNA content analysis by propidium iodide staining showed a prominent increase in sub-G1 population in asynchronous, non-treated SBDS-knockdown cells, suggesting that these cells are prone to cell death, however, no cell cycle arrest was noted. To further characterize the SBDS-knockdown cells, we examined their sensitivity to four groups of cell death inducers: DNA damaging agents (etoposide, cisplatin, and doxorubicin), transcriptional inhibitors (actinomycin D and α-amanitin), translation blocker (cycloheximide), and Fas pathway activator (agonistic anti-Fas antibody CH-11). Dose-response curves were obtained by MTT assay performed 48 hrs after treatment of the cells with the reagents. Interestingly, SBDS-deleted cells showed marked hypersensitivity to CH-11; while 3 μ g/ml of CH-11 reduced the survival fraction to 50% in wild-type and control cells, a similar effect was obtained at 0.02 μ g/ml in the SBDS-deleted cells. The hypersensitivity to Fas stimulation was also demonstrated by DNA content analysis. Based on the possible role of the yeast SBDS orthologue in RNA metabolism, we anticipated that the SBDS-deficient cells would be hypersensitive to the transcription inhibitors. However, even at concentrations which completely abolished RNA polymerase I or RNA polymerase II activity as determined by BrUTP labeling, the sensitivity of the SBDS-knockdown cells to the transciptional inhibitors was not remarkably different from that of the control or wild type cells. Similarly, the sensitivity to the genotoxic agents and protein synthesis blocker was not obviously different between the SBDS-deficient and proficient cells. To study the mechanism for Fas hypersensitivity, we analyzed Fas expression by flow cytometry using Cy5-conjugated anti-CD95 antibody and found overexpression of Fas in the SBDS-deleted cells in comparison with the Fas expression in the wild-type and control cells. Although further investigation is needed, these results suggest that the SBDS protein might be involved in cell death pathway, especially in the regulation of Fas-mediated apoptosis. The siRNA-mediated SBDS knock-down Hela cells duplicate important features of SDS cells, and may serve as a useful model to investigate the function of the human SBDS protein.