Aurora B kinase phosphorylates and instigatesdegradation of p53Chris P. Gullya,b,c, Guermarie Velazquez-Torresa,c,d,1, Ji-Hyun Shina,c,d,1, Enrique Fuentes-Matteic,1, Edward Wanga,c,d,Colin Carlocka,b,c, Jian Chenc, Daniel Rothenbergc, Henry P. Adamse, Hyun Ho Choia,c,d, Sergei Gumaa,c,d, Liem Phana,c,Ping-Chieh Choua,c,d, Chun-Hui Sua,b,c, Fanmao Zhanga,c, Jiun-Sheng Chenc, Tsung-Ying Yangc, Sai-Ching J. Yeunga,f,g,2,and Mong-Hong Leea,b,c,d,2

aGraduate School of Biomedical Sciences, University of Texas, Houston, TX 77030; bThe M. D. Anderson Cancer Center, Program in Genes and Development,University of Texas, Houston, TX 77330; cDepartment of Molecular and Cellular Oncology, The M. D. Anderson Cancer Center, University of Texas, Houston, TX77030; dThe M. D. Anderson Cancer Center, Program in Cancer Biology, University of Texas, Houston, TX 77330; eDepartment of Genetics, The M. D. AndersonCancer Center, University of Texas, Houston, TX 77030; fDepartment of Emergency Medicine, The M. D. Anderson Cancer Center, University of Texas, Houston,TX 77030; and gDepartment of Endocrine Neoplasia and Hormonal Disorders, The M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030

Edited by Moshe Oren, Weizmann Institute of Science, Rehovot, Israel, and accepted by the Editorial Board April 23, 2012 (received for review June 24, 2011)

Aurora B is a mitotic checkpoint kinase that plays a pivotal role inthe cell cycle, ensuring correct chromosome segregation andnormal progression through mitosis. Aurora B is overexpressedin many types of human cancers, which has made it an attractivetarget for cancer therapies. Tumor suppressor p53 is a genomeguardian and important negative regulator of the cell cycle.Whether Aurora B and p53 are coordinately regulated during thecell cycle is not known. We report that Aurora B directly interactswith p53 at different subcellular localizations and during differentphases of the cell cycle (for instance, at the nucleus in interphaseand the centromeres in prometaphase of mitosis). We show thatAurora B phosphorylates p53 at S183, T211, and S215 to acceleratethe degradation of p53 through the polyubiquitination–protea-some pathway, thus functionally suppressing the expression ofp53 target genes involved in cell cycle inhibition and apoptosis(e.g., p21 and PUMA). Pharmacologic inhibition of Aurora B incancer cells with WT p53 increased p53 protein level and expres-sion of p53 target genes to inhibit tumor growth. Together, theseresults define a mechanism of p53 inactivation during the cell cycleand imply that oncogenic hyperactivation or overexpression ofAurora B may compromise the tumor suppressor function of p53.We have elucidated the antineoplastic mechanism for Aurora Bkinase inhibitors in cancer cells with WT p53.

DNA damage | chromosome passenger complex | centromere protein A |AZD1152

Aurora kinases are serine/threonine kinases essential for cellcycle control and mitosis (1–7). Mammals have three Au-

rora kinase family members (A, B and C), and these kinases areexpressed at maximum levels during mitosis. Although all threekinases regulate mitosis, Aurora A and Aurora B differ in sub-cellular localization, and each kinase performs a distinct task(1). Aurora A is located at the centrosomes at prophase, and asmitosis progresses, it is located at the spindle poles duringprometaphase and metaphase. In contrast, Aurora B, part of thechromosome passenger complex (CPC), is located on the chro-mosome arms during prophase and at the centromeres duringprometaphase and metaphase (1). Aurora B subsequently local-izes to the midbody during cytokinesis. A recent report showedthat Aurora C behaves like Aurora B and is also a chromosomepassenger protein (8). These different subcellular localizationssuggest that Aurora kinases may recruit different substrates toregulate different processes during mitosis. Whereas many Au-rora A substrates have been characterized (2), few substrates forAurora B have been identified.Tumor suppressor gene p53 is a guardian of the genome and

an important negative regulator of the cell cycle; p53 delays orarrests cell cycling at DNA damage checkpoints precedingDNA replication (the G1/S checkpoint) as well as inhibits

damaged cells from entering mitosis (the G2/M checkpoint) (9).However, it is not clear whether and when p53 has functionduring mitosis. A recent report shows that cells lacking p53function lose spindle assembly checkpoint control (10), whichsuggests a role for p53 in mitosis. How the information on thestatus of the spindle assembly is relayed to p53 is, however, notknown. Importantly, activity of tumor suppressor p53 is lost in50% of human cancers by mutation, deletion of the p53 gene, orloss of cell signaling upstream or downstream of p53 (11).Aurora B has been shown to be overexpressed in many types

of cancers, including multiple myeloma, colorectal, prostate, andpancreatic cancers (12). This overexpression has made Aurora Ban attractive target for therapeutic cancer drugs (13). Althoughhigh levels of Aurora B are associated with advanced clinicalstage and poor prognosis in several cancers, the functional sig-nificance of aberrant overexpression of Aurora B remains un-clear. Many small-molecule inhibitors of Aurora kinases arebeing investigated as potential drugs for cancer therapy (13) intranslational studies and early-phase clinical trials (12). Thesesmall molecules can inhibit autophosphorylation of Aurora Band phosphorylation of histone H3 on Ser10 (7), but it is notclear how these small molecules can affect the uncharacterizedfunctions of Aurora B. It is also not known whether Aurora Band p53 are coordinately regulated during the cell cycle. Eluci-dating the mechanisms that coordinate Aurora B and p53 wouldhelp us to better understand the regulation of p53 during mitosisand may lead to the development of more effective or moreindividualized cancer therapies targeting Aurora B. Thus, wesought to investigate these mechanisms by investigating the in-teraction and any mutual regulation of Aurora B and p53 usingvarious cell cycle synchronizations, biochemical interactiontechniques, and animal models. Our findings show that p53 isa substrate of Aurora B and that Aurora B directly interacts withp53 at the CPC during mitosis. We also indicate that Aurora Bnegatively regulates p53 and that a specific Aurora B inhibitor

Author contributions: C.P.G., S.-C.J.Y., and M.-H.L. designed research; C.P.G., G.V.-T.,J.-H.S., E.F.-M., E.W., C.C., J.C., D.R., H.P.A., H.H.C., S.G., L.P., P.-C.C., C.-H.S., F.Z., J.-S.C.,and T.-Y.Y. performed research; C.P.G. and M.-H.L. analyzed data; and C.P.G., S.-C.J.Y.,and M.-H.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.O. is a guest editor invited by the EditorialBoard.

Freely available online through the PNAS open access option.1G.V.-T., J.-H.S., and E.F.-M. contributed equally to this work.2To whom correspondence may be addressed. E-mail: syeung@mdanderson.org ormhlee@mdanderson.org.

See Author Summary on page 9232 (volume 109, number 24).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1110287109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1110287109 PNAS | Published online May 18, 2012 | E1513–E1522

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can antagonize this impact. Our studies provide a mechanism-based application for effective cancer therapy when using AuroraB kinase inhibitors in cancers with WT p53 status.

ResultsAurora B Associates with p53. Previously, we discovered that thespecific Aurora B kinase inhibitor AZD1152 (13–15) reduced thesteady-state protein level of Aurora B (16). We also reportedthat AZD1152 reduced the protein level of Aurora B and con-currently elevated the protein level of p53 in cancer cells. Fromthis finding, we hypothesized that Aurora B and p53 have aninteractive and/or regulatory relationship. Indeed, coimmuno-precipitation (co-IP) experiments showed their association inintact cells and in vitro (Fig. 1 A and B). This association wasshown to be independent of the presence of Novel inhibitor ofhistone acetyltransferase (INHAT) Repressor (NIR), which waspreviously shown to associate with both Aurora B and p53 (Fig.S1A) (17). Next, we mapped the structural regions of p53 re-quired for the association with Aurora B. The results showedthat Aurora B was bound to the C terminus of p53 (amino acids160–393) but not the N terminus (amino acids 1–160) (Fig. 1Cand Fig. S1 B and C). To examine whether the interaction wascell cycle regulated, we collected cell lysates from synchronizedHCT-116 cells at various time points after release from doublethymidine block (Fig. 1D). Aurora B protein was detected inevery cell cycle phase, but it was reduced after mitosis (Fig. 1E).

Co-IP of Aurora B and p53 showed that they interacted duringmost of the cell cycle but not in the late M phase when Aurora Bis degraded (Fig. 1E). These results suggest that Aurora B mayhave an uncharacterized function in interphase that involvesinteraction with p53. Phosphorylation of H3 (HH3), a substrateof Aurora B (18, 19), was elevated during mitosis and then di-minished after the down-regulation of Aurora B after mitosis.The interaction pattern between p53 and Aurora B did notfollow the temporal pattern of HH3 phosphorylation, whichsuggests that p53 was not involved in the regulation of phos-phorylation of HH3 by Aurora B (Fig. 1E). Noticeably, the ex-pression levels of p53 and Aurora B seemed inversely correlatedduring the cell cycle (Fig. 1E).

Aurora B Interacts with p53 During both Interphase and Mitosis. Toinvestigate whether the Aurora B–p53 interaction could occursubcellularly during mitosis, we used immunofluorescence mi-croscopy to visualize colocalization of endogenous p53 and Au-rora B. The images indicated that these two proteins colocalizedat the midzone in anaphase and telophase (Fig. 2A). To provedirect molecular interaction between Aurora B and p53 in wholecells, we used the method of bimolecular fluorescent comple-mentation (BiFC) (Fig. 2B). Cells were cotransfected with aplasmid expressing the chimera of Aurora B and the C-terminalportion of Venus (an enhanced YFP) and a plasmid expressingthe chimera of p53 and the N-terminal portion of Venus (20)

+ - - - - - - + + + + + - + - - - - + - + - - - - - - + - - - - - - + - - - - - - +

- + + - + -+ - +

A

In vitro translated AurBGSTGST-p53TT

PD: GST IB: AurB

IB: AurB

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Vector VV Flag-AurB

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GST-p53 (1-160) TTGST-p53 (160-393) TTGST-p53 (320-393) TT

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Fig. 1. Aurora B associates with p53 at various phases of the cell cycle. (A) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53antibodies (AB-2) or preimmune IgG (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. (B) GSTpull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53was detected by IB. Coomassie staining of GST andGST-p53 inputs are in C. In vitro translated Aurora Bwas detected by immunoblot (Lower). (C) As presented inB, results of in vitro GST pull-down assay of immunopurified Flag-Aurora Bwith GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate thestained bands of GST, GST-p53, and GST-p53 deletion mutants. (D) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeledtime points after release of thymidine blockwere stainedwith PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time pointsas labeled. (E) Lysates of synchronized Hct116 cells from Dwere analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phasesof the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).

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(Fig. 2B). Consistent with the co-IP results from synchronizedcells (Fig. 1E), fluorescent microscopy of MCF7 cells transfectedwith such plasmids showed direct intermolecular interaction be-tween Aurora B and p53 (pseudocolored green), whereas thecontrol plasmids (Venus-N-Term and Venus-C-Term) did not

produce fluorescence (Fig. 2C). The fluorescence can be observedin prometaphase and interphase (Fig. 2C). Synchronization studyrevealed that p53 and Aurora B colocalized during differentphases of mitosis (prometaphase, metaphase, anaphase, andtelophase) (Fig. S1D). Close examination of mitotic cells revealed

B

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Fig. 2. Aurora B colocalizes and directly interacts with p53 in interphase and mitosis. (A) MCF7-Her18 cells were stained with DAPI, mouse anti-p53, andrabbit anti-Aurora B antibodies. Confocal immunofluorescence images of cells in anaphase and telophase are shown. (B) Schematic diagram to explain the useof Venus fusion proteins for BiFC. (C) Plasmids containing the N terminus of Venus fluorescent protein fused to p53 (Venus N-term-p53) and the C terminus ofVenus fused to Aurora B (Venus C-term-AurB) were cotransfected in MCF7. Cells were synchronized to prophase by thymidine–nocodazole block. Venusfluorescence is pseudocolored green. (D) Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus from C is shown at a high magnification.Arrows indicate p53 and Aurora B direct intermolecular interaction (BiFC) at the centromeres. (E) 293T cells transfected with Venus C-term-AurB and VenusN-Term-p53 and immunostained for the CPC member Survivin (red). The merged image shows that BiFC (pseudocolored green) colocalizes with at least someof the Survivin immunofluorescence (red). (F) MCF7 cells were transfected with Venus C-term-AurB and Venus N-term-p53 and then treated with thymidine–nocodazole for synchronization. Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus showing colocalization of Venus (Aurora B–p53 in-teraction) and the centromere marker CENP-A (red).

Gully et al. PNAS | Published online May 18, 2012 | E1515

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that the Aurora B–p53 interaction (BiFC signal) was located inthe middle part of each chromosome at prometaphase (Fig.2D, arrows). Given that Aurora B is located at the centromeres(21–23), it is likely at this location that the p53–Aurora B in-teraction occurs. To verify this location, we cotransfected cellswith Venus C-term-AurB and Venus N-Term-p53 and thenimmunostained for the CPC member Survivin (24), which isdynamically localized at the centromeres to activate Aurora B(22, 25). We observed that Aurora B, p53, and Survivin allcolocalized to the DNA of prometaphase cells, suggesting thatAurora B and p53 interact at the centromeres (Fig. 2E). Fur-thermore, we cotransfected Venus plasmids (as previously de-scribed) into synchronized cells and immunostained for thecentromere marker CENP-A. We observed that the Venus in-teraction of Aurora B and p53 occurred at distinct points thatalso stained clearly for CENP-A (Fig. 2F). Similar results wereobtained with other cell lines: BiFC was observed in interphaseU2OS cells (Fig. S1E) and also, at a distinct foci on eachchromosome in prometaphase 293T cells (Fig. S2A). These dataindicate that the interaction between Aurora B and p53 occursat different subcellular locations (e.g., interphase nuclei and thecentromeres) and during different phases of the cell cycle.

Aurora B Phosphorylates p53.After establishing that Aurora B andp53 interact directly, we investigated whether p53 is a kinasesubstrate for Aurora B. Using an in vitro kinase assay (Fig. 3A),we found that purified recombinant GST-p53 was phosphory-lated by Aurora B. As negative controls, Flag-tagged Aurora BK106R (kinase dead) or Aurora B (WT) were immunopurifiedfrom transfected cell lysates and assayed for their ability tophosphorylate recombinant GST-p53. Aurora B WT, but notAurora B K106R, efficiently phosphorylated p53 (Fig. 3B),confirming that phosphorylation of p53 in these reactions re-quired the presence of the functioning kinase domain of AuroraB. The specific Aurora B kinase inhibitor AZD1152-hydrox-yquinazoline pyrazole anilide (HQPA) (active form) blockedAurora B-mediated p53 phosphorylation in a dose-dependentmanner in vitro (Fig. 3C). Taken together, these results suggestthat Aurora B has kinase activity to p53.To further investigate Aurora B-mediated phosphorylation of

p53, we used different p53 fragments in another series of in vitrokinase assays. We found that Aurora B specifically phosphory-lated the fragment of p53 containing the DNA binding domain(amino acids 160–393) but not the fragment containing thetransactivating domain (amino acids 1–160) or the C-terminalfragment (amino acids 320–393) (Fig. 3D). To determine the p53phosphorylation sites, we scanned the p53 sequence using theNetPhos algorithm (http://www.cbs.dtu.dk/services/NetPhos/)and identified five potential Aurora B phosphorylation sites(S183, T211, S215, S269, and T284) that were conserved acrossdifferent species and shared the Aurora B phosphorylation siteconsensus motif (Fig. 3E and Fig. S2B). In vitro kinase assaysusing alanine mutants of potential phosphorylation sites in-dicated that several sites (S183, T211, and S215) were phos-phorylated by Aurora B (Fig. 3F).

Aurora B Enhances p53 Ubiquitination. Because the stability of p53 isregulated by its phosphorylation (11), we hypothesized that p53phosphorylation by Aurora B accelerated p53 protein turnover bypolyubiquitination and subsequent proteasomal degradation. Wefound that expression of Aurora B reduced the steady-state pro-tein level of p53 in a dose-dependent manner (Fig. 4A). Fur-thermore, in the presence of the de novo protein synthesisinhibitor cycloheximide, Aurora B overexpression accelerated theturnover rate of p53 (Fig. 4B). Consistently, Aurora B knockdownusing siRNA reduced the turnover rate of p53 (Fig. 4C and Fig.S2C). Real-time quantitative PCR (qPCR) analysis showed thatp53 mRNA levels were not affected by Aurora B overexpression

(Fig. S3B), suggesting that Aurora B down-regulated p53 at theposttranscriptional level. We also found that Aurora B-mediatedp53 down-regulation was antagonized by the proteasome inhibitorMG341 (bortezomib), indicating the involvement of ubiquitin–proteasome degradation in this process (Fig. 4D). The murinedouble minute 2 (MDM2) is the E3 ubiquitin ligase for p53, andwe found that Aurora B kinase potentiated MDM2-mediated p53ubiquitination (Fig. 4E). Importantly, AZD1152-HQPA, whichspecifically inhibits the kinase activity of Aurora B, was able to

A + - + - - + - + + + - - - - + +

Vector

Flag-AurB

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Coomassie

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32 P GST-p53

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+ + + + + + + + - 0.3 10 50

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+ - + + + + + - - - - - - + + - - - - - - + - - - - - - + - - - - - - +

Rec. AurB-GST GST

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GST-p53 (160-393) GST-p53 (320-393)

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32 P GST-p53 32 P GST-AurB

F

ESite

S183T211S215S269T284

Score

0.993 0.951 0.933 0.957 0.866

Sequence

HERCS DDRNT TFRHS LGRNS RDRRT

Cons.Seq.(R/K)1-3X(S/T)

1.0 0.16 0.22 0.001 0.51 1.2 Ratio

32 P Aurora B

Fig. 3. Aurora B phosphorylates p53 at multiple serine/threonine residuesin the DNA binding domain. (A) Flag-Aurora B was immunoprecipitatedwith anti-Flag antibody from lysates of 293T cells transfected with Flag-Aurora B or vector (negative control). The anti-Aurora B immunoblot isshown in A Middle. Coomassie blue-stained SDS/PAGE gel of GST and GST-p53 is shown (A Bottom). Phosphorylation resulting from Flag-Aurora Bcatalyzed in vitro kinase reactions using GST or GST-tagged p53 as thesubstrate was detected by autoradiography (A Top). (B) Flag-Aurora B orFlag-Aurora B K106R was expressed in 293T cells and immunoprecipitatedto catalyze in vitro kinase reactions as in A. Phosphorylation results areshown in a similar manner to A. (C) Recombinant GST-tagged Aurora B wasused in an in vitro kinase assay with GST or GST-p53 as the substrate in thepresence of an increasing dose of the specific Aurora B inhibitor AZD1152-HQPA. Phosphorylation results are shown in a similar manner to A. (D)Recombinant GST-Aurora B was used in an in vitro kinase assay as beforewith GST-tagged deletion mutants of p53. Phosphorylation results areshown in a similar manner to A. *Phosphorylated GST-p53 (amino acids160–393) fragment. (E) Consensus phosphorylation sequence and Netphos2.0 scores for potential Aurora B phosphorylation sites in human p53. (F) Invitro kinase assay with recombinant GST-Aurora B and various GST-p53 DNAbinding domain mutant substrates. Ratio of phosphorylation relative tocontrol is indicated above each lane.

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block p53 ubiquitination in a dose-dependent manner (Fig. 4F).To investigate the role of Aurora B-mediated p53 phosphoryla-tion in regulating p53 stability, we constructed a p53 triplephosphorylation site mutant, S183A/T211A/S215A → (AAA),and we found that, compared with the turnover rate of WT p53,

the turnover rate of this p53 AAA mutant was not influenced byAurora B (Fig. 4G). Together, these results indicate that Aurora Bkinase plays a role in enhancing the degradation of p53 throughthe ubiquitination–proteasome pathway, therefore reducing p53steady state in the cell.

- + + + +

- - - - + + + + 0 0.5 1 2 0 0.5 1 2

+ + + + - + - + - - + +

- - - - + + + + - - - + + + + + - - + + + + + + - + + + + + + +

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+ + + - - - - - - + + + 0 1 2 0 1 2

+ + + + - - - - + + + + - - - - + + + + - - - - + + + + + + + + - - - - - - - - - - - - + + + + 0 0.5 1 2 0 0.5 1 2 0 0.5 1 2

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Fig. 4. Aurora B phosphorylates p53 at multiple sitesresulting in polyubiquitination and degradation by theproteasome. (A) H1299 cells were transfected witha fixed amount of plasmid expressing GFP-p53 and anincreasing amount of plasmid expressing Flag-Aurora B.Immunoblots for GFP-p53, Flag-Aurora B, and Actin areshown. (B) U2OS cells were transfected with Flag-AuroraB or control vector and then exposed to cycloheximidefollowed by immunoblot for p53 turnover. (C) 293T cellswere infected with lentivirus expressing shRNA 468AurB or control lentivirus (shRNA luc). Infected cellswere exposed to cycloheximide and then analyzed byimmunoblot for p53 turnover. The asterisk (*) representsnonspecific band. (D) H1299 cells were cotransfectedwith GFP-p53 and Flag-Aurora B in the presence andabsence of the proteasome inhibitor MG341. (E) 293Tcells were transfected as indicated and immunoprecipi-tated with anti-HA antibody followed by immunoblotwith anti-GFP antibody to detect ubiquitinated p53. (F)293T cells were transfected as indicated, treated for 24 hbefore harvest with AZD1152-HQPA, and immunopreci-pitated with anti-HA antibody followed by immunoblotwith anti-p53 antibody to detect polyubiquitinated p53.(G) H1299 cells were transfected with plasmids as in-dicated and treated with cycloheximide to evaluate theturnover of GFP-tagged p53 or GFP-p53 AAA. Relativeremaining GFP-p53 expression value is indicated in aline graph.

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Aurora B Affects p53 Transcriptional Activity.Because Aurora B wasshown to down-regulate p53 protein level, we examined theimpact of Aurora B on the transcriptional activity of p53 usinga p53-responsive luciferase reporter gene assay. We observedthat Aurora B expression impaired p53 transcriptional activity(Fig. 5A), whereas Aurora B knockdown enhanced p53 tran-scriptional activity (Fig. 5B). A control experiment using a p53-independent promoter is shown in Fig. S3A. Next, we examinedthe expression of p53 target genes in Aurora B-overexpressingcells. Our results showed that expressions of p53 transcriptionaltargets MDM2 and p21 were lower in Aurora B-overexpressingcells than control cells (Fig. 5C and Fig. S3B). Because p53 isimportant in inducing apoptosis genes to regulate cell deathunder genotoxic stress, we used detection of sub-G1 DNA con-

tent in flow cytometry to analyze the impact of Aurora B ongenotoxic stress-induced apoptosis. Compared with findings incontrol cells that did not overexpress Aurora B, Aurora Boverexpression decreased apoptosis caused by the DNA-dam-aging agent cisplatin (CDDP) (Fig. 5D and Figs. S3C and S4A).Additional analysis of the functional effect of Aurora B-medi-ated p53 phosphorylation on transcriptional activity was carriedout using p53 phosphorylation mutants S183A, T211A, S215A,S183A/T211A, or S183A/T211A/S215A (the AAA mutant) ina p53-responsive luciferase reporter gene assay. T211A andAAA mutants were resistant to Aurora B-mediated repression ofp53 transcription, whereas S183A and S215A as well as S183A/S215A mutants remained sensitive to the presence of Aurora B

- -

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CDDP(20µM)

- + - + - - + +

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P=0.27

U2OS P=0.65

Vector

Flag-Aurora B

*: p < 0.05

* *

*

*

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Fig. 5. Phosphorylation of p53 by Aurora B inhibits p53 transcriptional activity. (A) 293T cells were transfected with fixed amounts of plasmids expressingGFP-p53, p53-Luciferase reporter plasmid (BDS2-3x-luc containing a p53-responsive element), and increasing doses of plasmid expressing GFP-Aurora B. p53transcriptional activity was measured by luciferase activity. All of the error bars in the bar charts of this figure represent 95% confidence intervals. (B) p53-Luciferase reporter assay (BDS2-3x-luc) in 293T cells transfected with increasing dose of shRNA 468-AurB plasmid. (C) qRT-PCR analysis was performed tomeasure p53 and p53 target gene mRNAs in U2OS transfected with vector or Flag-Aurora B. (D) Hct116 cells were transfected with Flag-Aurora B, treated withcisplatin (CDDP), and analyzed by FACS after PI staining. Percentage of apoptotic cells (sub-G1 fraction) was plotted. *P < 0.05 compared with CDDP alone byone-way ANOVA posthoc intergroup comparison by Tukey test. Analysis of cleaved PARP and Caspase 3 are shown in Fig. S3C. (E) H1299 cells were transfectedwith indicated GFP-p53 phosphorylation site mutants and p53-luciferase reporter plasmid (MDM2-luc containing a p53-responsive element) in the absence(vector) or presence of Flag-Aurora B. Relative luciferase activity of each p53 construct in the presence or absence of Aurora B was measured. Error barsrepresent 95% confidence intervals. (F) qRT-PCR analysis was used to measure p21 mRNA in Hct116 p53−/− cells transfected with Aurora B and either GFP-p53or GFP-p53 AAA mutant. *P < 0.05 by one-way ANOVA posthoc intergroup comparison by Tukey test.

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(Fig. 5E). A Western blot of GFP-p53 construct expression levelsis shown in Fig. S4B. In agreement, we found that, comparedwith WT p53, the p53 AAA mutant was resistant to AuroraB-mediated repression of p21, a p53 target gene (Fig. 5F).Therefore, T211 seems to be the most influential site phos-phorylated by Aurora B in terms of p53 transcriptional activity.Because the p53 AAA mutant was resistant to the Aurora B-mediated increase of turnover (Fig. 4G), this mutant was, thus,resistant to Aurora B-mediated repression of its transcriptionalactivity. Taken together, these results suggest that overexpressionof Aurora B led to phosphorylation of p53 at multiple sites, whichincreased p53 turnover, resulting in the repression of p53-medi-ated transcriptional activity.

Aurora B Kinase Inhibitor Potentiates p53 Stabilization and p53Transcriptional Activity in Vivo. To evaluate the in vivo relation-ship between Aurora B and p53, we used Aurora B-specific in-hibitor AZD1152-HQPA against aggressive human breast cancerMCF7-Her18 cells (WT p53). Our results showed that, afterAZD1152-HQPA exposure, p53 levels in these cells increased ina dose-responsive fashion concurrent with the increase of p53target genes (MDM2 and p21) (Fig. S4C), whereas phosphory-lation of H3 (substrate of Aurora B) showed a decrease (Fig. 6A).In agreement with these findings, immunoblots for p53 in tumorsamples from xenografted MCF7-Her18 cells (16) showed that,after a dose of AZD1152, p53 levels increased compared withlevels in vehicle-treated mice (Fig. 6B). Accordingly, MDM2,Bax, and p53 up-regulated modulator of apoptosis (PUMA)levels in these tumor samples increased compared with levels incontrols. Immunohistochemical staining for p53 in these tumorsamples indicated that both low- and high-dose AZD1152 treat-ments increased the number of p53-positive cells and increasedp53 staining intensity compared with the respective values incontrols (Fig. 6C). The p53 expression level determined on thebasis of staining intensity was quantitated by ACIS III and imageanalysis software (Dako) and presented as a bar graph (Fig. 6D).In addition, qRT-PCR indicated that AZD1152-treated tumorshad higher mRNA levels of p53 target genes, including PUMAand p21, than control tumors (Fig. 6E). These data suggest thatinhibition of Aurora B in vivo increased p53 protein level, whichin turn, increased target gene expression to inhibit tumor growth(∼50% reduction in tumor weight) (16).

DiscussionAs an important tumor suppressor, p53 needs to keep manyoncogenic signals or gene mutation events at bay. For example,DNA damage will activate ataxia telangiectasia-mutated (26, 27)or Chk (28) kinase to modify and/or strengthen the stability andactivity of p53 for initiating DNA repair or causing apoptosis.However, during tumorigenesis, p53 is also a target of manyoncogenic signals, such as Akt (29, 30), MDM2 (31, 32), COP1(33–35), or CSN6 (36), and these oncogenic signals will decreasethe protein level of p53. Aurora B is a protein overexpressed inmany types of cancer. Here, we report a critical role for AuroraB in controlling p53 homeostasis by regulating its phosphoryla-tion and subsequent ubiquitin–proteasome degradation. Ourresults provide a mechanism to explain the role of Aurora Bexpression in carcinogenesis and cancer progression. This layerof p53 posttranslational regulation also provides an example ofan interphase function for mitotic kinase Aurora B. Our dataindicate that p53 and Aurora B directly interact during in-terphase (Fig. 1E). This finding suggests a functional role forAurora B outside mitosis. It is important to point out that Au-rora A, another mitotic kinase, was shown by other investigatorsto phosphorylate p53 at Ser315 (37), which is not a consensus sitefor Aurora B and can also cause p53 destabilization (37). How-ever, the phase of the cell cycle on which the regulation of p53 byAurora A occurs remains to be determined. Our data show that

Aurora B was able to regulate p53 stability during interphase,thereby decreasing p53 transcriptional activity, facilitating cellcycle progression, and antagonizing apoptosis.The significance of Aurora B-regulated p53 destabilization

during interphase could serve to drive the cell cycle through thefollowing mechanisms by (i) blocking p53-mediated expressionof p21 (38, 39), an important cyclin-dependent kinase inhibitorthat blocks G1/S phase progression, and (ii) alleviating p53-mediated suppression of Cyclin B/Cdc2 and Survivin (40–44).These effects will aid cell cycle progression through interphase toenter mitosis.As for the role of Aurora B–p53 interaction during mitosis, it

has been shown that general suppression of transcription is ob-served during mitosis (45, 46); therefore, Aurora B-mediated p53transcriptional suppression will not play a role during mitosis.The work by Cross et al. (10) showed that p53 is involved infacilitating chromosome segregation to ensure the maintenanceof diploid cells, because p53 deficiency leads to tetraploidy invivo. It is possible that Aurora B coordinates with p53 to mediatethe spindle checkpoint (10) and aid progression through mitosis.Given that Aurora B deregulation also results in polyploidy, theinterplay between p53 and Aurora B could conceivably be im-portant for spindle checkpoint; however, this issue remains to beinvestigated. Nevertheless, our data fill an important gap in theknowledge regarding the localization of p53 during differentstages of mitosis—p53 associates with the CPC (Fig. 2E) and islocated at the centromeres (Fig. 2F) at prometaphase (Fig. 2D)or the mid-zone/cleavage furrow at anaphase/telophase (Fig.2A). The functional significance of Aurora B–p53 interactionduring different stages of mitosis also remains to be investigated,but p53 is definitely involved in the spindle checkpoint (10), andour data serve to confirm the presence of this tumor suppressorin the spindle checkpoint machinery. It is important to point outthat the binding between Aurora B and p53 decreases after theend of mitosis because of the down-regulation of Aurora B byanaphase promoting complex (47). After mitosis, Aurora B isrecovered from degradation, and binds again to p53 (Fig. 1E)and thus, potentially preventing p53 from arresting the cell cycleat G1 (48) or causing cell death by maintaining negative phos-phoregulatory control of p53. Future experiments will focus ondissecting the role of p53 within the CPC complex and the role ofAurora B-mediated p53 phosphorylation in this context. Giventhat overexpression or depletion of p53 will disturb the cell cycle,investigating this regulation during the mitosis window has majortechnical challenges. Nevertheless, our observations open a re-search avenue for additional studies in this area.On the basis of our biochemical studies, we propose the model

that Aurora B phosphorylates p53, leading to p53 ubiquitination/degradation and loss of p53 transcriptional activity as well asp53-mediated cell cycle suppression and apoptosis (Fig. 6F).Conversely, inhibition of Aurora B can reverse this effect and issufficient to inhibit tumor growth. These findings indicate thatAurora B negatively regulates p53 and that Aurora B inhibitorsmay have value as effective therapeutic agents against cancerswith WT p53 status.

Materials and MethodsTissue Culture. MCF-Her18 cells, a stably transfected subline of MCF7 thatoverexpresses Her2, have been described previously (49). These cells werecultured in DME/F12 (Sigma) supplemented with either 5% or 10% (vol/vol)FBS (Gemini). U2OS, 293T, H1299, MCF7, and HeLa cells were obtained fromthe ATCC and maintained in DME/F12 media (in-house supplier) supple-mented with 5% or 10% (vol/vol) FBS. Hct116 p53+/+ and Hct116 p53−/− (giftsfrom Bert Vogelstein, The Johns Hopkins University, Baltimore, MD) (39)were cultured in McCoy’s 5A media (HyClone) supplemented with 5% or10% (vol/vol) FBS, 2 mM L-glutamine (Cellgro), and 1% antibiotic–anti-mycotic solution (Invitrogen). All cells were incubated in a humidified in-cubator at 37 °C with 5% (vol/vol) CO2.

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Fig. 6. Inhibition of Aurora B induces p53 in a breast cancer xenograft model. (A) MCF7-Her18 cells were treated with increasing concentrations of AZD1152-HQPA as labeled for 48 h. Antigens for the immunoblots shown are labeled on the left. Mdm2 is indicated as full length (90 kDa) and cleavage product (60kDa). (B) MCF7-Her18 xenografts from nude mice (indicated by mouse number) treated with AZD1152 were immunoblotted for p53 and p53 target genes.The protein expression of Bax, Puma, and Actin was quantified from Western blot films using Image J program. Bax/Actin and Puma/Actin ratios werecalculated. Bar graphs and one-way ANOVA statistic analyses with Turkey test were done with GraphPad Prism 5.0c. (C) Representative photomicrographs areshown for immunohistochemical staining of p53 in MCF7-Her18 xenograft tumors treated with AZD1152. (D) Percentage p53-positive (Upper) and averagep53 immunostaining intensity (Lower) from automated quantitative image analysis of immunohistochemical staining of MCF7-Her18 nude mouse xenograftstreated with AZD1152. *P < 0.05 compared with vehicle control by one-way ANOVA posthoc comparison by Tukey test. Error bars represent 95% confidenceintervals. (E) qRT-PCR analysis was used to measure p53 and p53 target gene mRNA in MCF7-Her18 cells treated with 20 nM AZD1152-HQPA for 48 h. Errorbars represent 95% confidence intervals. (F) Model of p53 regulation by Aurora B.

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Drugs and Reagents. AZD1152 (orally bioavailable prodrug) and AZD1152-HQPA (active specific inhibitor of Aurora B kinase) were provided by KirstenMundt (Astra Zeneca, Cheshire, United Kingdom) (50). AZD1152-HQPA wasdissolved in 100% DMSO at a 10 mM concentration as a stock solution be-fore further dilution in appropriate aqueous solutions/media at final con-centrations as indicated. De novo protein synthesis inhibitor cycloheximideand proteasome inhibitor MG132 were obtained from Sigma and used at100 and 5 μg/mL, respectively. MG341 (bortezomib) was purchased fromMillenium Laboratories and used at 70 ng/mL overnight (8–12 h). Cisplatinwas purchased from Bedford and used as indicated.

Immunofluorescence. Indicated cell lines were grown on chamber slides, tissueculture dishes, or cover glasses to 50–75% confluence. Cells were treatedwith vehicle (20 nM AZD1152-HQPA), treated with indicated transfection, ornot treated for 48 h. Cells were rinsed two times with cold PBS and thenfixed with 3% (vol/vol) paraformaldehyde (Electron Microscopy Sciences)solution for 15 min at room temperature. Cells were then rinsed three timeswith cold PBS for 10 min, and each rinse was followed by permeabilizationwith 0.2% Triton X-100 (Sigma). Plates were blocked in either 5% BSA(Sigma) or 5% (vol/vol) normal goat serum (a gift from Elsa Flores, TheUniversity of Texas M.D. Anderson Cancer Center, Houston, TX) diluted inPBS for 1 h. Fixed and blocked cells were stained with antibodies dilutedappropriately in PBS and applied for a period of 1 h to overnight. Cells werestained with DAPI and mounted on microscope slides using Fluoromount G(Southern Biotech) or Prolong Gold (Invitrogen) that contained DAPI.Immunostained cells were visualized with an Olympus IX81 confocal micro-scope, an Olympus IX70 fluorescent microscope, a PerkinElmer Ultraview ERSspinning disk confocal microscope, or a Nikon Ti with a Photometric Cool-Snap HQ2 camera driven by Nikon Elements software. Deconvolved imageswere processed using AutoQuant ×2 software (Media Cybernetics).

Immunoblotting and Immunoprecipitation. Cells for Western blot or immu-noprecipitationwere collected from tissue culture dishes after two rinses withcold PBS. Cells were centrifuged at low speed for 10 min, and supernatantswere discarded. Pellets were then either frozen at −80 °C for additionalprocessing later or lysed with 100–300 μL 1× lysis buffer [0.5-L batch: 7.5 g1 M Tris (Fisher), 15 mL 5 M NaCl (Fisher), 0.5 mL Nonidet P-40 (USB Corp.),0.5 mL Triton X-100 (Sigma), 1 mL 0.5 M EDTA (Fisher)] for 20 min at 4 °C.Lysis buffer also contained a mixture of protease/phosphatase inhibitors:5 mM NaV, 1 mM NaF, 1 μM DTT, 0.1 mg/mL Pepstatin A, 1 mM PMSF, and1,000× Complete Mixture Protease Inhibitor (Roche). Lysates were centri-fuged at high speed for 10 min, and cell debris was discarded. Proteinconcentration was measured using the Bradford method with protein assayreagent (Biorad) and read on a Powerwave XS (Biotek) spectrophotometerat 595 nm. Protein samples were standardized and mixed with 5× loadingdye [100-mL batch: 3.78 g Tris base, 5 g SDS, 25 g sucrose (Sigma), 0.04 gbromophenol blue (Sigma), pH adjusted to 6.8], and they were boiled 5 minbefore SDS gel analysis. SDS/PAGE was performed according to standardprocedures. All gels were 10% (vol/vol) polyacrilamide except MDM2 (8%),p-HH3 (15%), Bax (15%), oligomerization of p53 (6%), p53 ubiquitination(6%), and p21 (15%). Transfer of proteins was performed using 1× transferbuffer [1-L batch: 30.3 g Tris base, 144 g glycine (Fisher), 10 g SDS, pH ad-justed to 8.3] to PVDF membrane (Millipore). For immunoprecipitation, celllysates were prepared and standardized as before, and 1 mg protein wasimmunoprecipitated with appropriately diluted antibody in lysis bufferovernight. Antibody was pulled down with 50 μL either Protein A or G beads(Santa Cruz Biotechnology) for 1 h. Beads were centrifuged at low speed for10 min, and the supernatant was discarded. Dried beads were mixed with 2×loading dye and boiled for 5 min. Lysate samples were loaded onto gels, andSDS/PAGE was performed as before.

Fluorescence Cell Sorting. Cells to be analyzed were plated in six-well tissueculture dishes and grown to log phase. Appropriate treatments by eithertransfection of plasmids or treatment with cisplatin (indicated dose) wereperformed for 24–48 h. Monolayers were rinsed two times with PBS, and thecells were scraped into microcentrifuge tubes. Cells were centrifuged at lowspeed and rinsed one time with PBS. Pellets were resuspended in 0.5 mLhypotonic propidium iodide (PI) solution [0.85 mg/mL sodium citrate(Sigma), 0.1 mg/mL RNase A (Qiagen), 0.1% Triton X-100, 20 mg/mL PI(Roche)] and incubated in the dark for 30 min. Cell cycle/PI analysis wasperformed using a FACScalibur flow cytometer (Becton Dickinson).

Cell Cycle Synchronization. Hct116, MCF7, U2OS, HeLa, or 293T cells wereplated in appropriate complete media and grown to 25–30% confluence; 2mM thymidine (Sigma) was added to the media for 18 h followed by block

release by washing two times with warm PBS and refeeding with freshcomplete media. Cells were allowed to grow for 9 h and then retreated with2 mM thymidine for 17 h (second block). After the second block, cells werereleased to cycle as before.Thymidine–nocodazole block. Hct116, MCF7, U2OS, HeLa, or 293T cells weresynchronized by plating to 40% confluence in normal complete media; 2 mMthymidine was added for 24 h followed by cell release by washing two timeswith warm PBS and refeeding with fresh complete media. Cells were releasedfor 3 h, and then, 100 ng/mL nocodazole (Sigma) was added to the media for12 h. Cells were then released again by washing two times with PBS andchanging to fresh complete media.

Dual Luciferase Reporter Assays. Analysis of p53 transcriptional activity wasperformed by transfecting log-phase cells cultured in 12-well dishes witheither a p53 luciferase reporter plasmid containing the three copies of thep53 binding sites from the 14-3-3σ or MDM2 promoters. Cells were alsocotransfected with the Renilla luciferase reporter plasmid. After trans-fection, cells were treated with either AZD1152-HQPA or vehicle and in-cubated for 24 h. At harvest, cells were collected using passive lysis buffer(Promega) and analyzed according to the manufacturer’s protocol for theDual Luciferase Reporter Assay kit (Promega).

Bimolecular Fluorescence Complementation Assays. C- and N-terminal Venusplasmids were provided by Gordon Mills (51) and modified to contain AuroraB or p53 by subcloning. Primers for Aurora B and p53 were designed tocontain AscI and EcoRV restriction sites (Table S1). Cells were transfected bythe liposome method with Venus plasmids containing Aurora B, p53, orempty Venus plasmids as appropriate controls. After 24 h, cells were imagedby fluorescent microscopy as described above.

Construction of Mutants. Aurora B and p53 mutants were constructed usinga site-directed mutagenesis technique. Forward and reverse primers (com-plementary) that were ∼30 bases long were used in a PCR with PFU Turbopolymerase (Stratagene) to amplify plasmids in their entirety. The primerswere designed to change one or two bases to effect the change in the aminoacid sequence. Table S1 shows the sequences of the primers used for thispurpose. After amplification, plasmids were treated with restriction enzymeDpnI (New England Biolabs) to digest any remaining template. Plasmidswere then transformed into DH5α Escherichia coli competent cells and se-lected with appropriate antibiotics.

In Vitro Kinase/Binding Assays. Immunopurified Aurora B (IP as describedpreviously) or recombinant Aurora B (Cell Signaling) was incubated in 1×kinase buffer [80 mM Mops (Sigma), 7.5 mM MgCl2 (Fisher), pH 7.0] withGST-purified p53 substrates, cold ATP, and γ32 ATP (Perkin-Elmer) at 30 °C for15 min. Kinase reactions were mixed with loading dye and analyzed by SDS/PAGE as described before. SDS/PAGE gels were dried and imaged usinga phosphoimager cassette (Molecular Dynamics) and a Typhoon Trio variablemode imager. Images were processed using Image Quant 5.1 software.Recombinant p53 substrates were produced by growing BL-21 E. coli bac-teria transformed with the GST-p53 plasmid of interest in 250 mL LB for1 h followed by induction of expression with 1 mM isopropyl β-D-1 thio-galactopyranoside (IPTG) (Fisher). Cells were grown for 4 h and harvested bycentrifugation. Cells were lysed with NaCl, EDTA, Tris, NP40 buffer (NETN)buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% (vol/vol)Nonidet P-40) plus protease–phosphatase inhibitor mixture and sonicatedfor 5 min. Cell debris was removed by centrifugation (10,000 × g), and then,200 μL GST beads (GE Healthcare) were added. Lysates were incubated withbeads overnight at 4 °C. The next day, the beads were washed three times inNETN plus inhibitors followed by one wash in kinase buffer. Recombinantp53 substrates for in vitro binding assays were prepared as for kinase assays.Substrates were incubated with IP-purified Flag-Aurora B overnight in 1 mLlysis buffer. GST-tagged substrates were pulled down using GST beads fol-lowed by SDS/PAGE and Western blotting with anti-Flag antibody.

Real-Time qPCR. Total RNA was extracted from 293T, MCF7-Her18, or Hct116p53−/− cells with TRIzol (Invitrogen) according to the manufacturer’s instruc-tions. RT was performed according to the manufacturer’s instructions usingthe iScript cDNA synthesis kit (BioRad); 1 µL per reaction of cDNA product wasused in real-time qPCR according to the manufacturer’s instructions with theiQ SYBR Green Supermix (BioRad) and iCycler (BioRad) thermocycler. The fol-lowing cycle was used: 95 °C for 10 min (1 cycle), 95 °C for 15 s, 60 °C for 1 min,95 °C for 15 s for 40 cycles and then, 95 °C for 15 s and 60 °C for 1 min. Nu-cleotide sequences of forward and reverse primers are listed in Table S2.

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Antibodies. The following antibodies were used in this study: 14-3-3σ(1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Bio-sciences), Aurora B (Ab2254; Abcam), Bad (B36420; BD Biosciences), Bax(B73520; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245;Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz),Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His(SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; SantaCruz), Mouse IgG (488; Alexa; A11029; Molecular Probes), Mouse IgG (568;Alexa; A11031; Molecular Probes), p53 for IP (AB-1, PAB1801; OncogeneScience), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53(p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA(SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes),rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; CellSignaling).

ACKNOWLEDGMENTS. We thank Shirl Ware-Gully and Diane Hackett forediting and Dr. R. Legerski, M. Cho, Dr. M. Blonska, and Dr. H.-K. Lin for materialsupport. We also thank Drs. K. Mundt and E. Anderson of Astra Zeneca forsupplying AZD1152, Dr. Subrata Sen for supplying Aurora B plasmids, andDr. Gordon Mills for supplying Venus plasmids. G.V.-T. is supported by a cancerprevention fellowship from National Cancer Institute Grant R25T CA57730(principle investigator Shine Chang), and E.F.-M. is supported by The M. D.Anderson Cancer Center Training Grant Program in Molecular GeneticsT32CA009299. This research was supported in part by the National Institutes ofHealth through The M. D. Anderson Cancer Center, University of Texas SupportGrant CA016672, National Institutes of Health Grant 5P30CA016672-29, DirectedMedical Research Programs Department of Defense Synergistic Idea Develop-ment Award BC062166 (to S.-C.J.Y. and M.-H.L.), the Susan G. Komen BreastCancer Research Foundation Promise Grant (to S.-C.J.Y. and M.-H.L.), NationalCancer Institute Grant R01CA 089266 (to M.-H.L.), and Department of GeneticsMicroscopy Core at The University of Texas M. D. Anderson Cancer Center.

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