Unscheduled Sister-Chromatid-Separation in the Presence of Spindle Disruption in Acute Myelogenous Leukemia

Abstract

Mitosis is known to be one of the most critical events in the cell cycle. The spindle assembly checkpoint (SAC) is required for proper chromosome segregation during mitosis. The SAC serves as a mitotic surveillance mechanism responsible for detection of misassembly of chromosomes to the mitotic spindle. Lack of chromosome attachment and spindle tension generate a specific „wait-anaphase-signal“. This particular signal interferes with proteolysis, depending on the ubiquitin-ligase APCCdc20, thereby inhibiting mitotic progression through stabilization of mitotic regulators. We found several AML cell lines to be incapable of properly accumulating in mitosis upon nocodazole-induced spindle disruption when compared to a set of Burkitt's lymphoma cell lines. This result was further supported by the degradation of the mitotic regulators Cyclin B and Securin in synchronized Kasumi-1 cells in the presence of nocodazole shortly after entering mitosis. Interestingly, the SAC proteins BubR1 and Bub1 were found at low expression levels in AML cell lines in comparison to Burkitt's lymphoma cell lines. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found in AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Our findings support the view that BubR1 repression alone is sufficient to confer SAC deficiency. To determine the frequency of BubR1 repression in patient-derived primary cells, AML blasts were cytokine-stimulated to enter the cell cycle. Flow cytometry-based G2/M-specific expression analysis of BubR1 in primary AML blasts revealed lower expression in most analyzed cell populations. To further test the hypothesis that AML cells override the metaphase-to-anaphase-transition despite spindle damage, we performed Giemsa staining of cells that were incubated in nocodazole containing growth medium. In AML cell lines, unlike the analyzed Burkitt's lymphoma cell lines, a significant number of metaphase-like cells contained single chromatids, suggesting premature sister chromatid separation in the presence of spindle damage. Premature sister-chromatid-separation in the presence of chromosomal misalignment would lead to aneuploidy and favor the onset of genomic instability. Our recent efforts focus on high-throughput automated live cell scanning, promising a better understanding of cell division and chromosome separation in the context of different challenges, such as spindle damage. This powerful tool allows a more precise characterization of our knockdown phenotypes in the double-knockdown system, which is a prerequisite for comparison of our model system with AML cell lines. Finally, this new technique might also prove useful to extend our analyses to patient derived AML blasts. As we observed deregulation of SAC protein levels in AML cell lines and primary AML blasts, our findings of premature degradation of cell cycle regulators and unscheduled sister-chromatid-separation suggest an important role for SAC malfunction in the development of AML with karyotypic abnormalities. Mitotic kinases, such as Plk1 and Aurora, are already promising targets for modern antineoplastic therapies. A deeper understanding of mitotic control in AML might contribute to even more sophisticated targeted therapeutic approaches.