It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations.

No abstract

ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n 2 0 1 3 blasts investigated. By performing functional studies both in non-responsive myeloblastic and responsive lymphoblastic cells, we investigated how reconstitution of the SAC and interference with SAC activity translate into response to spindle poison. Using live-cell imaging, retrovirus-delivered inducible knockdown and overexpression, we demonstrate that re-expression of BubR1 in myeloblastic cells confers an improved response to spindle poison. Methods Cell culturesCell lines were cultured as described in the DSMZ (Human and Animal Cell Lines Database, Braunschweig, Germany) datasheets. Synchronization procedures, interference with microtubule kinetics, proteasome inhibition and antibiotic selection were performed as outlined in the Online Supplementary Appendix.Primary blasts from AML patients were isolated and cultured as described in the Online Supplementary Appendix. The analyses were carried out after approval by the local Ethics Committee according to the guidelines of the Declaration of Helsinki and good clinical practice. Informed consent was obtained from the patients. Live-cell imaging kineticsCells were seeded in eight-well microscope chambers (Ibidi) that were coated with fibrinogen or collagen IV, at a concentration of 30,000 and 60,000 cells per well and imaged as previously described. 13 Retroviral transductionRetroviral particles were produced using Phoenix alpha packaging cells and inducible cell lines were established according to the manufacturer's instructions. Detailed information concerning oligonucleotide sequences and the backbones used is provided in the Online Supplementary Appendix. In vitro ubiquitinationThe APC/C was immunopurified from DG-75 and Kasumi-1 cells using an anti-Cdc27 antibody (Sigma-Aldrich) and Protein Gagarose. Ubiquitination reactions using precipitated APC/C were performed in the presence of in vitro transcribed/translated 35S-marked cyclin B as described in detail in the Online Supplementary Appendix. Western blot analysisWestern blotting was performed as described previously 13 using the antibodies specified in the Online Supplementary Appendix. Flow cytometryCell cycle distribution and BubR1 expression in single cells were determined using a FACSCalibur (Becton Dickinson). Microarray data and data processingMicroarray datasets were obtained from the open-access NIH Gene-Expression Omnibus (GEO) database. Datasets used in our analysis were GSE30029 18 and GSE13204.19 Data analysis and statistical analysisTIFF image stacks were analyzed using LSM Image Browser (v.2.80.1123) (Carl Zeiss). Calculations and statistics were done in Microsoft Excel 2002 and/or GraphPad Prism V5.03 (GraphPad Software Inc.). P values <0.05 were considered statistically significant. The statistical test used was an unpaired t-test (two-tailed) with a confidence interval of 95%. Results The mitotic checkpoint protein BubR1 is repressed in acute myeloid leukemiaTo address the expression levels of the regulatory proteins BubR1, ...

The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase controlling mitosis and G 1 phase of the cell cycle. The APC/C is activated by two regulatory subunits Cdc20 (APC/C Cdc20 ) and Cdh1 (APC/C Cdh1 ) to target securin, mitotic cyclins and other cell cycle regulatory proteins. Cdc20 is essential for sister chromatid separation at the meta-to anaphase transition in yeast, drosophila and perhaps mouse embryos. However, whether Cdc20 is essential for mitotic control of human somatic cells is uncertain. Therefore, we used a lentiviral vectormediated inducible RNA interference (RNAi) system to generate strong downregulation of Cdc20 expression in clonal cells to further elucidate the role of human Cdc20. Here we show, that even an almost complete knockdown of Cdc20 below the detection limit in western blots does neither cause a mitotic block nor significant stabilization of the APC/C Cdc20 substrates cyclin B and securin. Thus, there may be redundant mechanisms of mitotic control in the human somatic cell cycle.

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.