Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome
Karyn M. Austin, … , David Pellman, Akiko Shimamura
Karyn M. Austin, … , David Pellman, Akiko Shimamura
Published March 6, 2008
Citation Information: J Clin Invest. 2008;118(4):1511-1518. https://doi.org/10.1172/JCI33764.
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Research Article Hematology

Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome

Abstract

Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.

Authors

Karyn M. Austin, Mohan L. Gupta Jr., Scott A. Coats, Asmin Tulpule, Gustavo Mostoslavsky, Alejandro B. Balazs, Richard C. Mulligan, George Daley, David Pellman, Akiko Shimamura

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Figure 2

SBDS localizes to a region corresponding to that of the mitotic spindle.

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SBDS localizes to a region corresponding to that of the mitotic spindle....
(A) SBDS colocalizes with the spindle by immunofluorescence. Primary BMSCs in metaphase (upper 2 panels) or in interphase (lower 2 panels) from normal control or SBDS–/– patients were fixed and stained with antibodies against SBDS (green), α-tubulin (red), and DAPI (blue). SBDS spindle staining was not detectable in any of the 6 SDS patients exhibiting low SBDS protein expression as analyzed by western blot. (B) The spindle-staining pattern is SBDS dependent. DF259 primary BMSCs were infected with a retroviral vector carrying SBDS cDNA or an empty vector control, as indicated. Cells were lysed or fixed for immunofluorescence studies 48–72 h after infection. Western blot analysis for SBDS expression is shown on the right. Tubulin was stained to control for equal sample loading. Fixed cells were stained for SBDS (green), α-tubulin (red), or DAPI (blue) and visualized by fluorescence microscopy. (C) Loss of SBDS abrogates the spindle-staining pattern. GM00038 skin fibroblasts were infected with lentiviral siRNA vectors against either SBDS or a LUC control and analyzed by immunofluorescence for SBDS (green).