Tumor and Stem Cell Biology Cancer Research Low ......Materials and Methods Plasmids pTRE-tight and pcDNA3.1-EL and pcDNA3.1-LMW-E were each digested with Nhe1 and EcoRV. The insert

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Published OnlineFirst June 8, 2010; DOI: 10.1158/0008-5472.CAN-09-4094

Tumor and Stem Cell Biology
Cancer
Research

Low Molecular Weight Cyclin E Overexpression ShortensMitosis, Leading to Chromosome Missegregation andCentrosome Amplification

Rozita Bagheri-Yarmand1, Anna Biernacka1, Kelly K. Hunt2, and Khandan Keyomarsi1

Abstract

Authors' Aand 2SurgCenter, Ho

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Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability;however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL andLMW-E overexpression impairs the G2-M transition differently and leads to different degrees of chromosomeinstability in a breast cancer model system. First, the most significant difference is that EL overexpressionprolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosisand accelerates mitotic exit. Second, LMW-E–overexpressing cells are binucleated or multinucleated withamplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third,LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphasebridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitoticdefects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E–overexpressing cells failto arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led tofailed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomalinstability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues cor-relates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E over-expression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomicinstability. Cancer Res; 70(12); 5074–84. ©2010 AACR.

Introduction

Themajority of solid tumors display abnormal chromosomalsegregation at cell division (1, 2). These abnormalities play arole in tumorigenesis and possibly metastasis by increasingthe rate of chromosome mutations, including deletion andamplification of genes involved in cellular proliferation and/or survival (1). Chromosome aberrations can be caused byeither the loss of chromosomes at metaphase/anaphase ormultipolar divisions associated with an abnormal number orstructure of centrosomes (2). During the progression towardhigher stages of malignancy, chromosome aberrations be-come more frequent. This may be explained by a process ini-tiated by telomeric dysfunction and anaphase bridging, whichmay give rise to an increased frequency of multinucleated cells

ffiliations: Departments of 1Experimental Radiation Oncologyical Oncology, University of Texas M.D. Anderson Canceruston, Texas

lementary data for this article are available at Cancer Research://cancerres.aacrjournals.org/).

ding Authors: Rozita Bagheri-Yarmand orKhandan Keyomarsi,f Texas M.D. Anderson Cancer Center, 1515 Holcombe Boule-on, TX77030.Phone: 713-792-4845; Fax: 713-794-5369; E-mail:mdanderson.org or [email protected].

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because of cytokinesis failure (3). These multinucleated cellscontain an abnormal number of centrosomes, leading to mul-tipolar mitotic figures at the next cell division. A key questionin understanding genomic instability is to delineate the eventscausing the initial abnormalities in the centrosome number.Cyclin E, a regulatory subunit of cyclin-dependent kinase 2

(CDK2), is an important regulator of entry into the S phase inmammalian cells (4–6). Using cell-free and Xenopus modelsystems, several independent groups have provided evidenceshowing that in normal proliferating cells cyclin E/CDK2 isan important regulator of the initiation of both centrosomeduplication and DNA replication during the G1 phase (7–9).Constitutive overexpression of cyclin E does not significantlyincrease the frequency of gene amplification but does in-crease the frequency of polyploid cells (10). Inhibition ofcyclin E degradation in cell culture models has also been as-sociated with the formation of micronuclei, which are sug-gestive of the generation of aneuploid cells (11). Thesestudies suggest that maintaining normal levels of wild-typecyclin E is critical for preserving an unperturbed genome.In many aggressive and highly metastatic cancers, full-

length cyclin E is proteolytically cleaved to its low molecularweight isoforms (LMW-E; ref. 12). The tumor-specific over-expression of LMW-Es was originally discovered in breast can-cer cell lines; however, these isoforms are also overexpressedin some ovarian cancers (13, 14), melanomas (15), colorectal

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LMW-E Overexpression Shortens Mitosis


cancers (16), and renal cell carcinomas (17). These LMW-Eisoforms are generated from the NH2-terminal elastase cleav-age of the 50-kDa, full-length cyclin E1 protein (termed EL;ref. 18). LMW-Es are tumor specific, are predominantly cyto-plasmic (19), and have enhanced biochemical and biologicalproperties that differ from EL. LMW-Es are also resistantto inhibition by the CDK inhibitors p21 and p27 and thegrowth-inhibitory effects of antiestrogens (20, 21).Whereas the action of EL on the transitions between dif-

ferent phases of the cell cycle has been established, the roleof the tumor-specific LMW-Es in maintaining a genomic bal-ance and how they may differ from that of EL has not yetbeen investigated. In addition, no model systems have beendeveloped to delineate how EL and LMW-E overexpressiondifferentially alter cell cycle progression. In this report, weshow that induction of LMW-E in breast cancer is muchmore potent than EL at inducing supernumerary centro-somes, abnormal mitotic spindles, chromosome segregationproblems, and multinucleated cells. LMW-E primes the cellsto accrue genomic instability by shortening the length of mi-tosis, whereas EL-overexpressing cells have a lengthened Mphase. Human breast cancers that overexpress LMW-E har-bor supernumerary centrosomes, in contrast to tumors thatonly express EL. These results collectively suggest that over-expression of LMW-E (but not EL) can induce chromosomalinstability and supernumerary centrosomes by shorteningthe timing of M-phase progression.

Materials and Methods

PlasmidspTRE-tight and pcDNA3.1-EL and pcDNA3.1-LMW-E were

each digested with Nhe1 and EcoRV. The insert encoding ELor LMW-E was ligated into the Nhe1 and EcoRV site of pTRE-tight to generate pTRE-tight EL and pTRE-tight LMW-E.

Cell cultures and transfectionsTetracycline-inducible cell lines expressing Flag-tagged EL

and LMW-E were generated by transfection of MCF-7 cells.Plasmid transfections were performed using FuGENE(Roche) according to the manufacturer's protocols. MCF-7cells were transfected with the doxycycline-inducible vectorpTRE-tight (Clontech) harboring constructs expressing Flag-tagged EL or the EL-5/6 LMW-E isoforms. For all experi-ments herein, we used a construct representing the smallestLMW-E isoform, T2, as it does not contain additional elas-tase cleavage sites. For simplicity, we have referred to thisform as LMW-E throughout the manuscript. Stable transfor-mants were established by selection for 2 weeks with 1 μg/mLG418 (Invitrogen) and 100 μg/mL hygromycin (Merck). Thesecells were isolated, propagated, and analyzed by Westernblotting for cyclin E expression in the presence or absenceof doxycycline. Representative clones for EL and LMW-Ewere then selected and used for analysis.The immortalized human mammary epithelial cell lines

76NE6 and 76NF2V were a generous gift from Dr. Vimla Band(University of NebraskaMedical Center, Omaha, NE); these cells

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were maintained in DCFI-1 media (D1-media; ref. 22). MCF-7cells stably expressing a small interfering RNA vector targetingp53 were a generous gift from Dr. Xinbin Chen (University ofCalifornia at Davis, Davis, CA); these cells were maintained inα-MEM supplemented with 0.5 μg/mL puromycin (Sigma).

Immunofluorescence stainingMCF-7 (induced or uninduced) or HeLa, 76NE6, 76NF2V,

and MCF-7 short hairpin RNA (shRNA) p53 (infected withEL or LMW-E adenovirus) cells were grown in six-well plateswith cover slides and fixed in cold 4% neutral paraformalde-hyde in PBS for 20 minutes at room temperature. The cellswere then washed in PBS, permeabilized in cold methanol for5 minutes, and blocked in 5% bovine serum albumin in PBS.Incubation with a primary antibody was carried out for 1 hourat 37°C. Incubation with a secondary antibody was carried outfor 1 hour at room temperature, followed by staining of DNAwith 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes.Slides were mounted with Vectashield antifade medium (Vec-tor Laboratories) after three washes with PBS and examinedusing a Leica DM4000B microscope equipped with ×63 Plan-Apochromat oil immersion objective.

Generation of recombinant adenovirusesCyclin E adenoviruses were constructed according to the

manufacturer's protocols using the AdEasy XL adenoviralvector system (Stratagene). Tumor cells were infected at amultiplicity of infection selected to ensure >70% transduc-tion efficiency.

SDS-PAGE electrophoresis, Western blotting,immunoprecipitation, and kinase assaySDS-PAGE electrophoresis, Western blotting, immunopre-

cipitation, and kinase assays were performed as previouslypublished (19).

Live cell microscopyWe used H2B-GFP HeLa cells, which allow high-resolution

imaging of both mitotic chromosomes and interphase chro-matin in live cells as described (23). Briefly, H2B-GFP HeLacells were treated with 2 mmol/L thymidine for 18 hours at37°C, washed, released for 8 hours, and treated again with2 mmol/L thymidine for an additional 18 hours before re-lease to a thymidine-free medium. Cells were then trans-duced with 1,000 virus particles (EL or LMW-E) per cell incomplete medium for 24 hours during the second thymidinetreatment. Cells were followed for 24 hours, starting 4 hoursafter release from the block, and photographed once every5 minutes. We set the 0-minute time point at nuclear enve-lope breakdown (NEB; ref. 24) and the end point at the com-pletion of anaphase. For these experiments, three differentfields per condition were photographed, and a total of20 cells were followed in three independent experiments.The apparatus for time-lapse experiments consisted of anOlympus IX81 spinning disc confocal microscope and aCO2-equilibrated chamber heated to 37°C and equippedwith ×40 and ×60 objectives. Movies were generated fromthe acquired images.

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Tissue samples and immunoperoxidase stainingTumor samples were obtained from patients who under-

went surgery at The University of Texas M.D. AndersonCancer Center. Sequential sections from formalin-fixed,paraffin-embedded tissue blocks were cut 4-μm thick witha microtome, fixed to charged slides, and immunostainedwith a centrosome-specific antibody to pericentrin (Abcam)or cyclin E antibody (Santa Cruz Biotechnology). To mea-sure centrosomal aberrations, at least 10 different high-powered fields per case were examined by light microscopy(Leica), photographed with a SPOT digital camera, and an-alyzed with ImagePro image analysis software. Centrosomeswere considered abnormal if they had a diameter at leasttwice that of centrosomes in adjacent nonneoplastic mam-mary gland tissue, if there were more than two per cell, orif the ratio of the greatest to the smallest diameter ex-ceeded 2. Mean percentages of centrosomal abnormalitieswere calculated by including all cells harboring centrosomesper sample. Cyclin E staining intensity and percent positiv-ity were evaluated both in the nucleus and in the cytoplasmin invasive carcinoma and in nonneoplastic breast epithelialcells as previously described (25).

Patient characteristicsPatient characteristics are described in the legends of

Supplementary Table S1.

Statistical analysisStatistical analyses were performed using GraphPad Prism

software with two-way ANOVA or StatsDirect software. Allstatistical tests were two-sided and were considered to bestatistically significant at P < 0.05.

Results

Induction of LMW-E expression causescentrosome amplificationWe initially set out to address whether EL and LMW-E have

different effects on the induction of chromosome instability bymeasuring the number of centrosomes in cells upon EL orLMW-E induction. For these analyses, we generated MCF-7cells that can inducibly express Flag-tagged EL (Fig. 1A, left)or LMW-E (Fig. 1A, right) upon treatment with doxycycline.In induced cells, the CDK2 kinase activity associated withFlag-LMW-E was 1.5-fold higher than that of Flag-EL despitesimilar levels of EL and LMW-E (Fig. 1B). We used this induc-ible system to explore whether induction of EL and LMW-Edifferentially affects centrosome numbers. Centrosomes werestained with anti-γ-tubulin antibody. Induction of EL did notresult in a significant increase in the number of cells withmorethan two centrosomes (Fig. 1C and D). In contrast, upon in-duction of LMW-E, there was a 2.5-fold increase in the numberof cells with more than two centrosomes (Fig. 1C and D).

Spindle defects and chromosome missegregation incyclin E–overexpressing cellsWe next set out to examine whether there are mitotic

defects associated with centrosome amplification in EL-

Cancer Res; 70(12) June 15, 2010


overexpressing or LMW-E–overexpressing cells using anti-bodies to β-tubulin (green) to stain microtubules andγ-tubulin (red) to stain centrosomes (Fig. 2). Among the un-induced EL and LMW-E cells, 90% to 95% of the cells in mi-tosis showed normal chromosome condensation andcongression on a bipolar spindle (Fig. 2A, −Dox). After induc-tion of EL, only 20% of the mitotic cells had defects, whereasafter induction of LMW-E, 56% of the mitotic cells had de-fects associated with abnormal spindles, including branchedand splayed spindles (71%), chromosome alignment defects(9%), and abnormal centrosome numbers (19%; Fig. 2A andB). Furthermore, cells overexpressing LMW-E had 3-foldmore mitotic defects than EL-overexpressing cells (Fig. 2B).We also found highly aberrant structures, including chromo-some missegregation (57%), anaphase bridges (75%), andfailed cytokinesis (12%) in LMW-E–expressing cells com-pared with only chromosome missegregation in 16% andanaphase bridges in 7% of EL-expressing cells (Fig. 2B andD). One of the most significant mitotic defects in LMW-Ecells were abnormal spindles with defects in chromosomealignment (i.e., chromosome missegregation), suggesting thatthere were defects in attachment of the chromosomes to thespindle microtubules. These results suggest that LMW-E ismore likely than EL to result in mitotic defects that could leadto genomic instability.

Cyclin E expression cooperates with p53 loss in causingmitotic defects and chromosome missegregationThe presence of the tumor suppressor p53 is a crucial com-

ponent of a checkpoint that limits the accumulation of cellswith supernumerary centrosomes (24). To examine whetherp53 loss cooperates with cyclin E overexpression (EL orLMW-E) to induce mitotic defects, we introduced EL andLMW-E by adenoviral infection into humanmammary epithe-lial 76NF2V and 76NE6 cells (Fig. 3A). The 76NE6 cell line wastransfected with the E6 gene of human papillomavirus; thisimmortal phenotype lacks p53 due to E6-directed proteaso-mal degradation (26). The 76NF2V cell line was transfectedwith a mutant E6 gene (F2V) incapable of degrading p53but still able to immortalize cells (27). Mitotic defects wererecorded by staining the cells with antibodies to β-tubulinand γ-tubulin (Fig. 3C). Although, in response to LMW-E,both 76NE6 and 76NF2V cells underwent mitotic defects,there was a significantly higher number of mitotic defectsin 76NE6 cells overexpressing LMW-E compared with76NF2V cells (Fig. 3C and D, left). Specifically, 48% of 76NE6cells overexpressing LMW-E contained mitotic defects com-pared with 34% of 76NF2V cells overexpressing LMW-E (P <0.01). The mitotic defects included monopolar, tripolar, andmultipolar cells as well as those with centrosomes clustering,lagging chromosomes, and spindle polarity defects (Fig. 3C).Similarly, EL-overexpressing 76NE6 cells incurred more mito-tic defects (28%) compared with EL-overexpressing 76NF2Vcells (17%; P < 0.05; Fig. 3C and D, left). The same experimentwas repeated in another model system in which p53 was sta-bly downregulated with p53 shRNA in MCF-7 cells (Fig. 3Band C) We found that both EL and LMW-E cooperate withthe loss of p53 to induce mitotic defects, but the percentage of

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mitotic defects was higher in the LMW-E–overexpressingcells (66%; P < 0.001; Fig. 3D, right) than the EL-overexpressingcells (40%; P < 0.01, Fig. 3D, right). These results show that theloss of p53 cooperates with LMW-E and, to a lesser extent,with EL to induce mitotic defects in both immortalized andbreast cancer cell lines.

LMW-E overexpression causes multinucleation,polyploidy, and compromised spindlecheckpoint activationWe next examined the effect of EL and LMW-E on genomic

instability by measuring the number of multinucleated andpolyploid cells on induction of either EL or LMW-E in twodifferent systems (Fig. 4, right). Using the MCF-7–induciblesystem, we found a significant difference in the number ofmultinucleated cells between the EL-induced and LMW-E–induced cells. Specifically, 12% of all LMW-E–overexpressingcells and 95% of those with multiple centrosomes were binu-cleated ormultinucleated versus only 4% of EL-overexpressingcells (Fig. 4A). These results were reproduced in HeLa cellsinfected with adenoviral EL or LMW-E (Fig. 4B), suggestingthat expression of LMW-E produces aneuploid progeny morefrequently than expression of EL, owing to high rates of chro-mosomemissegregation. Next, we asked whether the accumu-lation of multinucleated cells would lead to live polyploid cellsover time. To this end, we cultured EL-inducible and LMW-



E–inducible MCF-7 cells in the presence or absence of doxycy-cline for 14 days and subjected these cells to metaphasespread, which we then enumerated to measure the numberof diploid cells (Fig. 4C). These analyses revealed that therewas no difference in cell number between the induced and un-induced EL and LMW-E cells at the end of 14 days (data notshown). However, LMW-E–overexpressing cells had 20% fewerdiploid cells, whereas the percentage of diploid cells wasunchanged in the EL-overexpressing cells (Fig. 4C). We nextassessed the integrity of the mitotic spindle checkpoint inEL-induced and LMW-E–induced MCF-7 cells by measuringthemitotic index in cells treated with nocodazole for 16 hours.The control cells showed a robust mitotic arrest, with 28% ofthe cells arrested by 16 hours following nocodazole treatment(Fig. 4D). However, the induction of EL versus LMW-E resultedin different mitotic indices. For example, whereas the induc-tion of EL slightly increased themitotic index, the induction ofLMW-E resulted in a 60% drop in the mitotic index (to 8%)compared with that of control or EL cells (Fig. 4D). Thus, in-duction of LMW-E compromised the spindle checkpoint ac-tivation, as shown by a strongly reduced mitotic indexfollowing 16 hours of nocodazole treatment. These resultscollectively show that genomic instability, as measured bythe extent of multinucleated and polyploid cells and compro-mised mitotic index, was significantly altered on induction ofLMW-E but not EL.

Figure 1. LMW-E overexpression causes centrosome amplification. A, pTRE-EL–transfected or pTRE-LMW-E–transfected MCF-7 cells were incubated withor without doxycycline (Dox) at the concentrations indicated for 24 h and subjected to Western blot analysis with cyclin E and vinculin. B, cell lysateswere immunoprecipitated with either a Flag or CDK2 antibody and kinase activity was measured using histone H1 as a substrate. Bands corresponding toH1 phosphorylation were quantified by densitometry, and the ratio of the activity in the uninduced versus induced cells was plotted (right). Columns,mean (n = 3 independent experiments); bars, SD. C, MCF-7 cells were treated with or without doxycycline (1 μg/mL) for 24 h and stained for γ-tubulin (green)or DNA (blue) and analyzed by immunofluorescence microscopy. D, cumulative data were obtained from three independent experiments, with at least200 cells counted per experiment. Data are presented as the percentage of cells with more than two centrosomes. Columns, mean of three independentexperiments; bars, SD. *, P < 0.01.

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LMW-E overexpression causes cells to enter andexit mitosis prematurelyThe studies on spindle checkpoint activation showed that

induction of LMW-E induces more mitotic defects than EL.We next asked if the mitotic defects observed were due to thealteration of the transition of the LMW-E–induced cellsthrough mitosis using real-time microscopy. HeLa cells ex-pressing green fluorescent protein (GFP)–tagged histoneH2B were transduced with EL, LMW-E, or control adenovirusand imaged as they progressed through mitosis. We detectedEL and LMW-E expression within 24 hours of adenoviral in-fection by Western blot analysis (Fig. 5A). After synchroniza-



tion in G1-S, the cells expressing EL and LMW-E enteredmitosis earlier than control cells, with mitotic entry starting4 to 16 hours (EL), 4 to 12 hours (LMW-E), and 10 to 16 hours(control) after release from G1-S arrest, respectively (Fig. 5B).In addition, the LMW-E–expressing cells exited mitosis4 hours earlier than control cells (Fig. 5B), whereas the EL-expressing cells exited mitosis 2 hours later than control cells(Fig. 5B). To dissect the timing of mitotic exit in EL-expressing and LMW-E–expressing cells, we measured thetime from NEB until anaphase onset in these cells. We foundthat in EL-expressing cells the time from NEB to the ana-phase transition ranged from 305 to 645 minutes, with a

Figure 2. LMW-E overexpression leads to mitotic defects. A, cells were treated with doxycycline (1 μg/mL) for 24 h; fixed; stained for β-tubulin (green),γ-tubulin (red), or DNA (blue); and analyzed by immunofluorescence microscopy. B, quantification of spindle defects in 200 mitotic cells per experiment from10 independent experiments. Columns, percentage of cells with mitotic defects; bars, SD. *, P < 0.001. C, pictorial presentation of various mitoticerrors (micronuclei, chromosomes missegregation, anaphase bridges, and cytokinesis failure) in LMW-E–overexpressing cells. D, quantification of mitoticerrors in control, EL-expressing, and LMW-E–expressing cells. Graphs indicate the percentage of cells with micronuclei, metaphase chromosomemissegregation, anaphase bridges, and cytokinesis failure. Columns, mean (n = 3); bars, SD. *, P < 0.01; **, P < 0.001.

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median of 415 minutes (n = 20) compared with a median of130 minutes in control cells (Fig. 5C and D). In contrast, inLMW-E–expressing cells, the time from NEB until anaphaseonset ranged from 60 to 135 minutes, with a median of80 minutes (Fig. 5C and D). The differential mitotic exit timesmediated by LMW-E versus EL are also depicted in severalmovies taken from normal cells (Movie 1), EL-overexpressingcells showing premature mitotic entry and prolonged mitoticexit (Movie 2), LMW-E–overexpressing cells showing a fastermitotic transition (Movie 3) and mitotic defects (Movie 5),



and binucleated cells (Movie 6). Because EL is not modifiedby proteolytic cleavage in HeLa cells, we asked whether ex-pression of LMW-E could rescue the mitotic arrest inducedby EL. To this end, we overexpressed both EL and LMW-Ein HeLa H2B-GFP cells (Fig. 5A, lane 4). The median time fromNEB to anaphase was ∼120 minutes (Fig. 5C and D; Movie 4),suggesting that the presence of LMW-E overrides the mitoticblock induced by EL. Lastly, we observed that some mitoticcells expressing LMW-E (7%, n = 40) had multipolar spin-dles, resulting in a longer time from NEB to anaphase onset

Figure 3. Cyclin E overexpression cooperates with p53 loss to induce mitotic defects. A and B, 76NF2V (p53 positive) and 76NE6 (p53 deficient), as wellas MCF-7 breast cancer cells, parental and stably transfected with either control or p53 shRNA, were infected with the adenoviruses indicated. p53,cyclin E, and vinculin expression were detected by Western blotting. C, cells treated as in A and B were fixed and then stained for γ-tubulin (red) or β-tubulin(green), counterstained with DAPI (blue), and analyzed by immunofluorescence microscopy. Representative images of mitotic abnormalities observedin 76NE6 and MCF-7 p53 shRNA cells infected with LMW-E–expressing adenovirus are shown. D, quantification of mitotic defects in LacZ control,EL-expressing, and LMW-E–expressing 76NF2V/76NE6 cells (right) and control shRNA/p53 shRNA MCF-7 cells (left). Two hundred and fifty mitotic figureswere counted for each condition in two independent experiments. *, P < 0.01; **, P < 0.001.

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(195 min), but were able to still divide (Fig. 5D). Theseresults show that although overexpression of both EL andLMW-E results in faster mitotic entry, LMW-E–expressingbut not EL-expressing cells exit mitosis much faster thancontrol cells, suggesting that LMW-E expression altersmitotic exit differently than does EL expression.

LMW-E expression is positively correlated withcentrosome amplification and nuclear gradein breast cancer specimensBoth chromosomal instability and centrosome defects are

common features of epithelial cancers (28–30). BecauseLMW-E overexpression causes centrosome amplification incell lines, we examined whether a correlation exists betweencentrosome abnormalities in human breast tumors and ex-pression of LMW-E isoforms. To this end, we used Westernblotting to analyze cyclin E expression in 30 primary invasivebreast carcinomas and adjacent nonneoplastic breast tissuesamples as well as in normal breast tissues from reductionmammoplasty specimens. The tissue samples were also ana-



lyzed by immunohistochemistry for pericentrin, whichdetects centrosome abnormalities (Fig. 6A and B; Supple-mentary Fig. S2). The clinicopathologic characteristics of allpatients are summarized in Supplementary Table S1. Wequantitated EL and LMW-E levels by densitometry, normal-ized those levels to the EL level in adjacent normal breasttissue, and analyzed the correlation between EL and LMW-E levels and the degree of centrosome abnormalities. Theseanalyses revealed that the invasive tumor tissues had 20%more centrosome abnormalities, including supernumerarycentrosomes, abnormally shaped centrosomes, and centro-somes of larger diameter, than tissue from normal adjacentepithelium (Fig. 6A). Multiple linear regression analysis re-vealed that LMW-E expression, but not EL expression, is anindependent predictor of centrosome alteration (P > 0.0005;Fig. 6C). In addition, centrosome abnormalities (r2 = 0.137779,P = 0.0434) and LMW-E expression (r2 = 0.18627, P = 0.0172)were positively correlated with a higher modified Black's nu-clear grade (Supplementary Fig. S1B and C). The data fromthe breast cancer patients suggest that overexpression of

Figure 4. LMW-E overexpression leads to polyploidy. A, pTRE-LMW-E–expressing MCF-7 cells were treated with or without doxycycline (1 μg/mL) for 24 h;stained with β-tubulin (red), phalloidin (green), or DAPI (blue); and analyzed by immunofluorescence microscopy. Right, the percentages of binucleated andmultinucleated cells are plotted. Columns, mean from three independent experiments; bars, SD. B, HeLa cells infected with EL or LMW-E adenovirusfor 24 h and stained with β-tubulin (red), phalloidin (green), or DAPI (blue). Right, the percentages of binucleated and multinucleated cells are plotted.One thousand cells were analyzed for each group for binucleated or multinucleated cells. Columns, mean from three independent experiments; bars, SD.C, pTRE-EL and pTRE-LMW-E cells were treated with doxycycline (1 μg/mL) for 14 d and subjected to metaphase spread. The percentage of diploid cellswas counted. Columns, mean from three independent experiments; bars, SD. D, MCF-7 EL and MCF-7 LMW-E cells were treated with or withoutdoxycycline for 24 h and nocodazole for 16 h, and the number of mitotic cells was counted microscopically. *, P < 0.01.

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LMW-E, but not EL, is associated with centrosome abnor-malities and higher grade of tumors.

Cyclin E localizes to the cytoplasm in cells withcentrosome amplificationWe recently tested the hypothesis that LMW-E isoforms

have an altered subcellular localization because they lack aportion of the EL NH2 terminus containing a nuclear locali-zation sequence (19). Here, we asked whether patient tumorsamples overexpressing LMW-E have cytoplasmic expressionof LMW-E and if the cytoplasmic staining of cyclin E is pre-



dominant in patients with high pericentrin levels. Immu-nostaining of tumor tissues showed high cytoplasmiclocalization of cyclin E in samples with high degree of cen-trosome abnormalities detected by immunohistochemistryfor pericentrin (Supplementary Fig. S1A). We also analyzed10 reduction mammoplasty samples from nontumor-bearingpatients by immunohistochemistry for pericentrin and byWestern blot and immunohistochemistry for cyclin E (Sup-plementary Fig. S2). We found an absence of LMW-E onWestern blot of normal breast tissues, nuclear localizationof cyclin E immunostaining, and pericentrin in centrosomes

Figure 5. Expression of EL and LMW-E induces premature mitotic entry and differential mitotic exit times. A, Western blot analysis of HeLa cells infectedwith adenovirus expressing LacZ control, EL, LMW-E, or a combination of EL and LMW-E using a cyclin E antibody. B, HeLa H2B-GFP cells weresynchronized by double thymidine block; infected with adenovirus expressing LacZ, EL, or LMW-E during the second thymidine block; and analyzed bytime-lapse microscopy 4 h after release for 24 h. Cells were photographed every 5 min from three different fields. Mitotic cells were counted and plotted ascumulative numbers. Data are percentage of mitotic cells (mitotic index) at different time points after thymidine release. C, the time from NEB toanaphase onset was measured by time-lapse microscopy in control cells and in cells infected with EL, LMW-E, or both EL and LMW-E adenoviruses. Eachsymbol in the scatter plot represents a single cell. D, representative fluorescence videomicroscopy series from cells described in C; numbers denotedare times (min).

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normal in number and structure. These data suggest that cy-toplasmic cyclin E is tumor specific and linked to aberrantcentrosomes.

Discussion

In this report, we have shown that expression of LMW-Einduces genomic instability by causing cells to progress fas-ter through mitosis without proper chromosome segregation



and with failure of cytokinesis and the generation of multi-nucleated cells with supernumerary centrosomes. As previ-ously suggested, failure to execute a crucial step in the celldivision process, such as late anaphase and cytokinesis, leadsto the formation of cells with two nuclei; these 4N cells canthen undergo either bipolar or multipolar mitosis with am-plified centrosomes (31). We observed that most cells withmore than two centrosomes were binucleated or multi-nucleated, and DNA content analysis showed polyploid cells,

Figure 6. Centrosome abnormalities correlate with LMW-E expression. A, human breast cancers and adjacent nonneoplastic breast tissues wereimmunostained with a pericentrin antibody to visualize centrosomes. Nuclei were stained light blue with hematoxylin. Numbers above the panels denotedifferent surgical cases. Magnification, ×1,000. Insets are digitally magnified views of centrosomes. In normal epithelia, centrosomes are uniform and small,whereas in tumors they are larger, multiple, or structurally abnormal. Top row images, normal epithelium (left) and tumors with low LMW-E expression;bottom row images, normal epithelium (left) and tumors with high expression of LMW-E. B, Western blot analysis of EL and LMW-E expression in humanbreast cancer cell lines, human breast cancer tissue samples, and adjacent normal breast tissue samples. Vinculin was used as loading control. C, positivecorrelation between LMW-E expression and centrosome abnormalities. To examine centrosomes, at least 10 different high-power fields per case wereanalyzed. Centrosomes were considered abnormal if they had a diameter at least twice that of centrosomes in adjacent nonneoplastic mammary glandtissue, if they were present in numbers >2, or if the ratio of their greatest and smallest diameters exceeded 2. Mean percentages of centrosomalabnormalities were calculated by including all cells harboring centrosomes per sample. EL and LMW-E protein levels were measured by densitometry ofbands on Western blot. Quantification of centrosome abnormalities was correlated with ratio of tumor EL to normal EL or tumor LMW-E to normal EL.Multiple linear regression analysis revealed that LMW-E expression, but not EL expression, is an independent predictor of centrosome alteration.

Cancer Research





suggesting that LMW-E triggers accumulation of cells withcentrosome amplification by abortive mitosis rather thanby affecting centrosome fragmentation or duplication.Time-lapse microscopy data suggest that overexpression ofLMW-E forces cells to escape mitotic arrest, reenter G1 witha 4N DNA content, survive, and increase the population ofpolyploid cells. However, overexpression of EL results in amuch longer/delayed mitosis, suggesting that these cellsdo not have a growth advantage. The results we present withEL agree with previous studies showing that overexpressionof EL alone does not cause centrosome amplification andleads to prolonged mitotic arrest due to inhibition of theanaphase-promoting complex (APCcdh1) and accumulationof cyclin B1 and securin (32). Prolonged mitosis in EL-overexpressing cells leads to complete alignment of chromo-somes at the metaphase plate. Only cells that fail to dividetheir chromosomes will become polyploid, an event that israre in EL-overexpressing cells but one that is significantlyenhanced in LMW-E–overexpressing cells. What is noveland clinically relevant in the present study is that, in contrastto EL, LMW-E–overexpressing cells exit mitosis faster thanthe EL-overexpressing cells, leading to a failure of cytokinesisand higher levels of polyploidy. Thus, we propose that thegeneration of LMW-E isoforms drives chromosomal insta-bility by perturbing the G2-M transition and the G1-Scheckpoint.LMW-E–overexpressing cells that prematurely exit mitosis

must bypass the subsequent G1-S checkpoint before the nextDNA replication cycle to become polyploid (33, 34). It has beensuggested that this G1-S checkpoint is dependent on p53 func-tion (33) and that loss of p53 function induces phenotypessimilar to those observed with LMW-E overexpression: centro-some amplification and aneuploidy (35, 36). We have shownthat cyclin E overexpression cooperates with loss of p53 inboth an immortalized and a breast cancer cell line to increasemitotic defects and chromosome missegregation. These re-sults suggest that the LMW-E–induced mitotic defects con-comitant with downregulation of p53 allow cells to bypassthe G1-S checkpoint and enter the next round of DNA replica-tion, thus becoming more likely to generate polyploid cells.



We also translated our cell line observation to the clinic.Using breast cancer specimens, we showed a positive corre-lation between the level of centrosome amplification, thepresence of LMW-E, and the nuclear grade in human breastcancer. Centrosome defects are present in low-grade tumorsbut increase in more aggressive carcinomas (37). In addition,LMW-E was absent in normal adjacent tissues of breast can-cer patients or normal tissues obtained from reduction mam-moplasty samples and was not a function of the levels of EL.LMW-E is preferentially accumulated in the cytoplasm intumors with centrosome amplification. This is consistentwith our recent finding that the subcellular localization ofLMW-E is altered in cell lines in vitro (19). This tumor-specific cytoplasmic LMW-E expression suggests that cyclinE may be deregulated during tumorigenesis through alteredcellular localization and suggests that centrosome abnormal-ities associated with LMW-E expression can predict thedevelopment of high-grade cancer and may provide amuch-needed surrogate marker for aggressive disease.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Angela Nanos-Webb for generating the EL-inducible and LMW-E–inducible MCF-7 cell lines, Said Akli and Yan Liu for generation of the cyclinE–expressing adenovirus, Tuyen Bui for Western blot analysis of tumor tissues,Jared K. Burks for help with time-lapse microscopy, Marcin Dobaczewski forhelp with the statistical analysis, Geoffrey M. Wahl for providing the HeLaH2B-GFP, Dr. Vimla Band for 76NE6 and 76NF2V, and Dr. Xinbin Chen forthe MCF-7 p53 shRNA cell lines.

Grant Support

NIHgrants CA87458 andP50CA116199 andClaytonFoundation (K. Keyomarsi)and Susan G. Komen grant PDF0707621 (A. Biernacka and K. Keyomarsi).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/06/2009; revised 03/29/2010; accepted 04/21/2010; publishedOnlineFirst 06/08/2010.

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2010;70:5074-5084. Published OnlineFirst June 8, 2010.Cancer Res Rozita Bagheri-Yarmand, Anna Biernacka, Kelly K. Hunt, et al. Centrosome AmplificationMitosis, Leading to Chromosome Missegregation and Low Molecular Weight Cyclin E Overexpression Shortens

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