Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome
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
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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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 1

SBDS loss promotes mitotic abnormalities.

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SBDS loss promotes mitotic abnormalities. (A) SBDS–/– cells exhibit mito...
(A) SBDS–/– cells exhibit mitotic abnormalities. Primary BMSCs from at least 3 different SDS patients were fixed and stained with antibodies against pericentrin (green) and α-tubulin (red) and with DAPI (blue). The top row illustrates normal control metaphase staining, while the middle and bottom rows illustrate the aberrant mitotic figures observed in SDS patient cells. Note multiple centrosomes, multipolar spindles, and broad DNA distribution. The percentage of abnormal mitotic cells in controls versus SBDS–/– cells is noted on the right (P < 0.01). A minimum of 200 cells were counted per sample in a blinded fashion in 3 independent experiments. Original magnification, ×60. (B) Targeted SBDS loss results in aberrant mitosis. GM00038 or GM00637 skin fibroblast cell lines immortalized with SV40 T antigen were infected with dual expression cassette lentiviral constructs encoding both GFP and siRNA sequences, the latter targeted against either SBDS or a LUC control. GFP-positive cells were sorted and analyzed for SBDS expression by western blot. SBDS protein expression was markedly reduced by 3 days following infection (upper panel). On day 5 and day 21 following infection, the cells were fixed and stained with antibodies against pericentrin and tubulin. At least 150 cells per sample were scored for abnormal mitoses (centrosomal amplification and multipolar spindles as illustrated in A) in a blinded fashion in at least 3 independent experiments, and the percentage of abnormal mitoses were tabulated in the histogram. *P = 0.01, LUC siRNA compared with SBDS siRNA on day 21. (C) SBDS loss results in aneuploidy. GM00038 cells from B were infected with lentivirus vectors as described in B. These immortalized GM00038 cells failed to exhibit p53-dependent p21 upregulation following exposure to ionizing radiation. GFP-positive cells were gated and analyzed for DNA content by flow cytometry on the indicated days following infection. DAPI staining shows enlargement of nuclei for cells lacking SBDS. Cells were visualized under ×40 magnification, and a scale bar (arbitrary units) is shown in C for comparison of the top and bottom panels.