A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy by Anita Boyapati, Ming Yan, Luke F. Peterson, Joseph R. Biggs, Michelle M. Le Beau, and Dong-Er Zhang Blood Volume 109(9):3963-3971 May 1, 2007 ©2007 by American Society of Hematology

AEtr cells have reduced mitotic index in nocodazole. AEtr cells have reduced mitotic index in nocodazole. (A) K562 cells stably expressing AEtr or vector control cells were treated with nocodazole for 18 hours. Propidium iodide (PI)–stained cells were detected by flow cytometry. Representative PI histogram of vector control or AEtr cells following nocodazole treatment for 18 hours. (B) Graph indicates the percentages of vector control and AEtr cells that contained either 2N or 8N DNA after nocodazole treatment as determined by PI staining. The averages for 3 to 5 independent experiments with independent pools are indicated. The standard deviation is indicated above each bar. (C) Mitotic index of control or AEtr cells following nocodazole addition. Phospho–histone H3 staining was performed 0, 4, 8, 14, or 18 hours after nocodazole addition. The error bars in the 18-hour time point indicate the average of 3 independent experiments. (D) The graph indicates the percentage of cells in G0/G1 phase of the cell cycle determined by ModFiT analysis of histograms of PI-stained cells following nocodazole treatment for 0, 8, or 12 hours. (E) K562 vector or AEtr-expressing cells were treated with nocodazole for 18 hours followed by addition of 10 μM BrdU for 24 hours. Cells were fixed and stained with anti–BrdU-FITC antibody and analyzed by flow cytometry to determine the percentage of S phase cells. The average BrdU incorporation percentage is shown for 3 independent K562 vector control or AEtr-expressing pools. Error bars show standard deviation (SD) of 2 different AEtr pools. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

AEtr reduces mitotic arrest in several spindle poisons and in primary cells. AEtr reduces mitotic arrest in several spindle poisons and in primary cells. (A) K562 cells stably expressing AEtr or vector control cells were treated with 0.1 μg/mL colchicine for 18 hours. PI-stained cells were detected by flow cytometry, and the percentage of mitotic cells is indicated in the bar graph for 2 independent experiments. (B) K562 cells stably expressing AEtr or vector control were treated with 100 nM paclitaxel for 24 and 40 hours. PI-stained cells were detected by flow cytometry, and the percentage of mitotic cells is indicated in the bar graph for 2 independent experiments. (C) Primary bone marrow cells transduced with control EGFP virus or virus encoding AEtr and EGFP were treated with (+) and without (−) 0.1 μg/mL nocodazole for 20 hours. Mitotic index was measured after Hoechst 33342 staining of cells to gate the cells in G2/M phase of the cell cycle. The bar graph shows the flow cytometry data averaged for 3 independent experiments. The asterisk indicates a significant difference between AEtr and control cells after nocodazole treatment. Error bars show SD of the mean from 2 or 3 independent experiments as indicated in each panel. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

Abnormal morphology of interphase and mitotic AEtr cells. Abnormal morphology of interphase and mitotic AEtr cells. (A-C) Giemsa staining of interphase vector control nuclei. (D) Chromosome spread of a prometaphase vector control cell. (E) Giemsa staining of interphase AEtr nuclei. Arrows indicate chromosome bridges. (F) Polyploid AEtr interphase cells (indicated by arrows). (G) Lagging chromosomes (indicated by arrow) during anaphase of an AEtr cell. (H) Premature sister chromatid separation in an AEtr prometaphase cell. Images were captured with a Leica DMLB microscope with a 100×/_ oil objective and a Spot 2 digital camera (Diagnostic Instruments, Sterling Heights, MI) using Spot v.4.64 software. Images were processed with Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA). Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

AEtr cells are aneuploid. AEtr cells are aneuploid. Mitotic chromosome counts from vector control or AEtr cells. Cells from vector control cells or 2 AEtr lines (AEtr#1, AEtr#2) were used to generate metaphase chromosome spreads. Chromosomes from 54 metaphase cells per line were counted. The histogram indicates the number of vector control or 2 different AEtr pools containing various ranges of chromosomes per cell. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

Primary AEtr leukemia cells are aneuploid. Primary AEtr leukemia cells are aneuploid. (A) Gains and losses of chromosomes determined by SKY. A total of 88 metaphase cells from 8 independent leukemias was analyzed. The number of independent clones that displayed a particular loss or gain of each chromosome is indicated in the bar graph. (B) The number of clones displaying different modal numbers is indicated. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

Spindle checkpoint proteins are reduced in AEtr cells. Spindle checkpoint proteins are reduced in AEtr cells. (A) Western blot analysis of BubR1 and securin in K562 vector or AEtr-expressing cells treated with 0.025% DMSO (−) or 1 μg/mL nocodazole (+) for 18 hours. Immunoblot for tubulin indicates relative protein loading. Anti-HA Western shows AEtr expression. The same lysates were also analyzed for cohesin subunit Rad21 and tubulin to show relative protein loading. (B) Western blot analysis of separase full length and the autocleaved form in control or 2 different AEtr pools treated with nocodazole. The same membrane was probed for cohesin, which indicates the cleaved cohesin form is detectable only after a long exposure compared with cohesin in panel A. (C) K562 vector or AEtr-expressing cells were transfected with plasmids encoding EGFP or securin-EGFP. At 48 hours after transfection, cells were treated with 1 μg/mL nocodazole for 18 hours. Cells were monitored for endogenous securin and transfected securin-EGFP expression by immunoblotting, which detected expression of full-length securin-EGFP and C-terminal degraded forms (indicated by asterisks) as well as endogenous securin. (D) Cells were stained with propidium iodide to measure DNA content by flow cytometry. The percentage of cells in mitosis following transfection of EGFP or securin-EGFP for 3 independent experiments is shown. Error bars show SD of the average from 3 independent experiments. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology

t(8;21) cell lines have disrupted spindle checkpoint. t(8;21) cell lines have disrupted spindle checkpoint. (A) Immunoblot of lysates from K562, Kasumi-1, and SKNO-1 cells treated without (−) or with (+) 1 μg/mL nocodazole for 18 hours. Membranes were probed with antibodies for BubR1, Bub3, separase, cohesin subunit Rad21/scc1, and securin. Tubulin immunoblot shows relative protein loading. (B) Kasumi-1, SKNO-1, and K562 cells were treated with nocodazole for 18 hours and labeled with 10 μM BrdU for 24 hours. Cells were fixed and stained with anti–BrdU-FITC and analyzed by flow cytometry. The percentage of cells stained positive for BrdU is shown. Anita Boyapati et al. Blood 2007;109:3963-3971 ©2007 by American Society of Hematology