BubR1 is frequently repressed in acute myeloid leukemia and its re-expression sensitizes cells to antimitotic therapy

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 3' Untranslated Region of the Cyclin B mRNA Is Not Sufficient to Enhance the Synthesis of Cyclin B during a Mitotic Block in Human Cells

“…Mitotic exit can be more efficiently blocked by direct inhibition of APC/C-dependent proteolysis, while mitotic slippage may occur after SAC activation following spindle poison treatment [24-26]. Furthermore, an increase in expression levels of the APC/C substrate protein cyclin B can boost a mitotic block in spindle poison treated cells by attenuating mitotic slippage [10,18,19]. Since synthesis of cyclin B takes place in mitosis in murine and human cells [14,15] we assumed that conserved networks control the strength of a mitotic block.…”

“…Cytoplasmic polyadenylation leads to poly(A) tail elongation which promotes translation and requires both a hexanucleotide sequence (AATAAA) and a cytoplasmic polyadenylation element (CPE; T 4-5 A 1-2 T) in the 3'UTR of the mRNA [17]. We and others could show that increased cyclin B levels contribute to a mitotic block [18,19]. These findings suggest that the strength of a mitotic block is governed by the cyclin B expression level, which represents an equilibrium between the synthesis and slow degradation of cyclin B [7,18].…”

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo

“…Mcl-1-inducible Kasumi-1 cells were grown in the above-mentioned complete medium in the presence of 2 μg/ml puromycin and 2400 μg/ml geneticin (as described elsewhere [26]). Kasumi-1 cells expressing H2B mCherry were grown in the presence of 5 μg/ml blasticidin.…”

BUBR1 Insufficiency in Mice Increases Their Sensitivity to Oxidative Stress

“…Dominik et al identified that a low expression of BUBR1 affected the sensitivity to antimitotic therapy of many acute myeloid leukemia cell lines (23). However, the relationship between the expression level of BUBR1 and the occurrence of leukemia remains unclear.…”