Our study demonstrates that SBDS promotes spindle stability and chromosome segregation. The spindle stability defect in SBDS cells may explain the high frequency of chromosomal abnormalities, commonly monosomy 7, observed in the bone marrows of SDS patients. Another human disease characterized by a mitotic defect and cancer susceptibility is the mosaic variegated aneuploidy syndrome (13
) resulting from BUB1B mutations that are associated with premature chromatid separation and disruption of the spindle checkpoint. To our knowledge, SBDS provides the first reported example of a defect in a microtubule binding protein associated with cancer predisposition and marrow failure.
It is important to point out that SBDS deficiency does not result in immediate global mitotic disruption, but rather spindle abnormalities whose consequences appear to be cumulative over time. Our observations that multipolar spindles and centrosomal amplification were not observed immediately after SBDS knockdown but grew increasingly prominent over time raise the likely possibility that these abnormalities result from additional secondary events following spindle destabilization by SBDS loss. Previous studies have shown that cells with spindle abnormalities can escape from mitotic arrest to re-enter the G1
phase of the cell cycle without completing cytokinesis, through a process called “mitotic slippage” (reviewed in refs. 14
). Such cells would harbor twice the normal number of chromosomes and centrosomes, thus promoting aberrations of subsequent mitoses such as those observed here. Although such polyploid cells may be eliminated through mechanisms such as apoptosis, such cells have been shown to propagate under certain conditions such as the loss of p53, which would impair both checkpoint activation and apoptosis (16
). Interestingly, tetraploidy is also associated with an increased incidence of chromosomal breaks and translocations through mechanisms that are currently unclear (15
). Elucidation of the subsequent events contributing to the development of multipolar spindles and centrosomal amplification following SBDS loss will be an important direction for future studies. Because patients with SDS do not exhibit constitutional aneuploidy, the relatively high incidence of mitotic abnormalities observed in vitro may reflect culture conditions that might allow the amplification of mitotically abnormal cells that would have been largely eliminated in vivo.
The mitotic defect of SBDS cells might lead to several downstream effects. In cells containing functional apoptotic mechanisms, we find that loss of SBDS results in apoptosis. Indeed, the bone marrow of SDS patients exhibit increased apoptosis (17
) and elevated p53 expression (18
). Thus, the spindle defect of SBDS cells may explain, at least in part, the bone marrow failure in SDS patients. Alternatively, in cells with compromised apoptotic or checkpoint pathways, loss of SBDS might enable more extensive genetic alterations that would promote tumorigenesis. Consistent with this idea, we find that SBDS depletion in cells lacking functional p53 results in polyploidy and aneuploidy. Similar divergent effects dependent upon the cellular or genetic context have been proposed for telomerase defects, which may either limit cell proliferation or result in genomic instability (19
). Chromosome segregation errors can lead to cytokinesis failure and tetraploidy that in turn can facilitate tumorigenesis and additional chromosomal aberrations (16
). Prior passage through an unstable tetraploid intermediate could also explain the extra centrosomes that we observed in SBDS–/–
cells, although at this point, a role for SBDS in centrosome duplication cannot be entirely excluded. Such events may contribute to the complex chromosomal aberrations typically seen in leukemia cells from SDS patients, and studies of SBDS function in hematopoietic systems to pursue this hypothesis are ongoing.
Although SDS may affect many organ systems, the reason why SBDS mutations particularly affect the bone marrow and the pancreas remains unclear. Indeed, the tissue-specific clinical phenotypes and tissue-specific malignant manifestations exhibited following disruption of fundamental cellular processes pose a puzzling question for many of the inherited marrow failure syndromes. It is possible that some tissues are particularly dependent on the SBDS pathway to maintain spindle integrity, perhaps due to the selective deficiency of additional spindle-stabilizing factors. Another possibility is that perhaps levels of functional SBDS protein are limiting in specific tissues during mitosis. It is also possible that certain tissues are particularly susceptible to endogenous or exogenous mitotic stressors. Additional functions of the SBDS protein might also contribute to the clinical phenotype.
Current evidence indicates that SBDS is likely a multifunctional protein. Based on inferences from orthologs, SBDS has previously been hypothesized to function in rRNA processing. (1
) Recent data link SBDS and 60S ribosome maturation (26
). In interphase cells, SBDS shuttles in and out of the nucleolus, the major cellular site of ribosome assembly (6
). It is possible that differential cellular localization might reflect different SBDS functions. Additional proteins, such as nucleophosmin (28
) and Rrp14 (29
), have also previously been shown to play roles in both ribosome biogenesis and mitosis. Such multiplicity of a given protein’s cellular function may additively contribute to disease phenotype. Dyskeratosis congenita (30
), another bone marrow failure syndrome with leukemia predisposition, provides an illustrative example of a disease in which phenotype may be affected by the multifunctional role of a single protein, dyskerin (32
). Dyskerin plays a role both in rRNA pseudouridylation (33
) and ribosomal function (35
) as well as in telomerase function (34
). Patients with dyskerin mutations typically exhibit a more severe phenotype than patients with mutations in TERC
, which affect only telomerase.
In summary, we have demonstrated that the SBDS protein has a critical role in stabilizing the mitotic spindle. Further study of the potential role of microtubule stabilizing agents in the treatment of SDS patients with bone marrow failure is warranted.