SBDS-deficiency results in deregulation of reactive oxygen species leading to increased cell death and decreased cell growth.
BACKGROUND
Shwachman-Diamond syndrome (SDS) is characterized by reduced hematopoietic and exocrine pancreatic cell numbers and a marked propensity for leukemia. Most patients have mutations in the SBDS gene. We previously reported that SBDS-deficient cells overexpress Fas, undergo accelerated spontaneous and Fas-mediated apoptosis and grow slowly. However the mechanism of how SBDS regulates apoptosis remains unknown. Several studies have shown that reactive oxygen species (ROS) regulate cell growth and spontaneous and Fas-mediated cell death. Therefore, we hypothesized that SBDS-deficiency disrupts ROS regulation and subsequently increases sensitivity to Fas stimulation and reduced cell growth.


METHODS
SBDS was knocked down in HeLa cervical cancer cells and TF-1 myeloid cells using short hairpin RNA. ROS levels were evaluated by oxidation of 2',7'-dichlorodihydrofluorescein diacetate. Apoptosis and cell growth were evaluated with and without antioxidants by annexin V/propidium iodide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, respectively.


RESULTS
We found that shRNA mediated SBDS-knockdown resulted in a significant increase in ROS levels compared to control cells. Fas stimulation further increased ROS levels in the SBDS-knockdown HeLa cells more than in the controls. Importantly, balancing ROS levels by antioxidants rescued SBDS-deficient cells from spontaneous and Fas-mediated apoptosis and reduced cell growth.


CONCLUSIONS
ROS levels are increased in SBDS-deficient cells, which leads to increased apoptosis and decreased cell growth. Increased baseline and Fas-mediated ROS levels in SBDS-deficient cells can enhance the sensitivity to Fas stimulation. By balancing ROS levels, antioxidants can improve cell growth and survival in SBDS-deficient cells.
Cell Biology Program, Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.
Chhaya
C
Ambekar
Bikul
B
Das
Herman
H
Yeger
Yigal
Y
Dror
D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D022423:Myeloid Cells / Q000378:metabolism / Q000473:pathology; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism
C110400:2',7'-dichlorodihydrofluorescein diacetate; C491901:FAS protein, human; D005452:Fluoresceins; D011506:Proteins; D017382:Reactive Oxygen Species; C470521:SBDS protein, human; D019014:fas Receptor