Seminars in Cancer Biology 15 (2005) 25–32

Review

Centrosomal amplification and spindle multipolarity in cancer cells

William Saunders∗

Department of Biological Sciences, 258 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, USA

Abstract

Recent developments have highlighted the important role centrosomal defects play in the cellular changes associated with tumorigenesis.This article reviews recent developments addressing the impact of numerical centrosomal amplification on chromosomal segregational defectsin the cancer cell. Probably, the most significant is the change to the structure of the spindle that leads to increased numbers of spindle polesand abnormal partitioning of the chromosomes in mitosis. I address how centrosomal changes are initiated and how they may lead to spindlemultipolarity.© 2004 Elsevier Ltd. All rights reserved.

Keywords:Centrosome; Spindle; Cytokinesis

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1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2. Runaway centrosomal duplication and tumor suppressors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3. Centrosome amplification and DNA damage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4. Centrosome replication defects versus failure of cytokinesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

5. Centrosomal amplification and spindle multipolarity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

6. Virally induced centrosomal amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

7. Future directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

. Introduction

Chromosomal instability, defined as a continuous change

receiving growing appreciation as central to our understing of the cellular mechanisms behind tumorigenesis.impact of chromosomal instability is a varied and ongo

n the transcriptional capacity of the cell caused by al-erations in the structure or number of chromosomes, is

∗ Tel.: +1 412 624 4320; fax: +1 412 624 4759.E-mail address:wsaund@pitt.edu.

RL: http://www.pitt.edu/∼biohome/Dept/Frame/Faculty/Saunders.htm.

change in the expression of genes controlling cell division,mobility, survival, and the many other cellular and tissuealterations needed for tumor formation. One of the majorcause of chromosomal instability in cancer cells appears tobe segregational errors during the mitotic division of thecell.

044-579X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.oi:10.1016/j.semcancer.2004.09.003

26 W. Saunders / Seminars in Cancer Biology 15 (2005) 25–32

Chromosomes are segregated by attachment to the highlyorganized microtubule spindle, which forms uniquely duringdivision, replacing the long monoastral array of the interphasecell. The spindle in a normal cell has mirror symmetry withthe now duplicated microtubule asters connected by cross-linked polar microtubules from each spindle pole. To thishighly symmetrical structure, the duplicated chromatids at-tach and are diametrically separated in anaphase to give equalproportions to each daughter cell – or at least that is how itis supposed to work. In cancer cells, spindles can often havemore than two poles causing the chromosomes to segregateasymmetrically, and often chaotically, leaving the daughtercells with abnormal numbers of chromosomes.

The microtubule organizing center in mammalian cells istypically associated with the centrosome. The centrosomecontains a highly organized centriolar core composed of apair of barrel-shaped orthogonally arranged cylinders of mi-crotubules. The surrounding amorphous pericentriolar regionis where microtubules are nucleated and organized into theastral array critical for directional movement in the cell. Cen-trosomes are not essential for spindle formation[1,2] but areimportant for normal chromosome segregation. Centrosomesalso play other key roles controlling cell division includinginitiation of cytokinesis and the entry into S-phase of thecell cycle [1,3,4]. Centrosomes also influence cell motility[ ort iv-a rolei ngt of thecd weenc les inc

eachd cen-t ruc-t repli-c andi p-a atesb Du-p l1,PC tro-s witht s-sr pa-rDc lin-d egge c-tD du-

plication[29]. Some these kinases may be hyperactive in tu-mor tissue as centrosomes are hyperphosphorylated in breasttumor compared to normal breast tissue[30].

During G2, a variety of additional proteins are recruitedto centrosomes and presumably play roles in maturation, in-cluding members of the Polo-like, Aurora family, and Cdk1kinases[31–33]. In mitosis, nucleophosmin is again observedat the spindle poles and associates with the centrosomal or-ganizing protein NuMA[34]. Also during mitosis, the tumorsuppressor proteins p53, TACC, and BRCA1 are found atthe centrosomes[35,36]. Centrosomes are segregated duringcell division by positioning the cell cleavage furrow near theequator of the spindle, allowing the contractile ring to sepa-rate them to the daughter cells.

Centrosomal defects were originally proposed to lead toaneuploidy and cancer in 1914 by Boveri[37,38]. He saw thatcancer cells commonly have centrosomal defects includingincreased centrosome number and postulated that changes incentrosome functionality may be key to cancer formation.Centrosome defects are indeed seen in many types of carci-nomas including breast, gall bladder, lung, bone, pancreas,colorectal, prostate, head, and neck cancers[10,30,39–45].For example, nearly 80% of invasive breast tumor cells haveamplified centrosome numbers[46]. Centrosomal changesare strongly linked to aneuploidy and chromosomal instabil-i

ells?O s ac-c stingt pro-g mald stageo riort la ans-g ) E6o ci-no tiona rly int rapido

pro-g n-t sedfi 0%i forc somald tabil-i otis morshg ors,a

5], polarity, and shape[6], which may also be important fumorigenesis[7]. In yeast, the centrosomal functional equlent, which is called the spindle pole body, plays a key

nitiating exit from mitosis[8]. Thus, scientists are beginnio appreciate the centrosome as a central componentell regulatory machinery[9–13]. This review will primarilyiscuss recent literature dissecting the relationship bethanges in centrosomes number and multipolar spindancer cells.

For chromosome segregation to proceed accurately,aughter must receive only a single centrosome, and this

rosome must only replicate once per cell cycle. Ultrastural studies have shown that centriole and centrosomalation begins near the G1/S boundary of the cell cycles completed by G2[14–16]. Replication begins by the seration of the centriolar pair. Each centriole now duplicy growth of a procentriole at a right angle to the parent.lication is controlled by members of the Cdk, Aurora/Ipolo-like, and NIMA families of cell cycle kinases[17–22].dk2 bound to cyclins A or E acts to stabilize the cenomal kinase hMps1 allowing its continued associationhe centrosome in mice[23] while phosphorylating and diociating nucleophosmin/B23[24]. Knockdown of Aurora-Aeduces�-tubulin accumulation and prevents centriolar seation during replication inCaenorhabditis elegans[25] androsophila melanogaster[26]. Work inXenopushas impli-ated a role for calcium, calmodulin and the calmoduependent protein kinase II in centrosomal replication inxtracts[27]. In budding yeast, mitotic cyclins also funion to inhibit re-replication of the spindle pole body[28]. Inrosophila, the Zyg-1 kinase is required for centrosomal

ty in a variety of studies[30,41,43,47].When do centrosomal changes first occur in tumor c

ne study showed that centrosomal defects were alwayompanied by complex chromosomal changes suggehat centrosomal changes occur relatively late in tumorression[48]. Other studies have indicated that centrosoefects were found to occur at a very early premalignantf tumor formation in a mouse mammary tumor model, p

o the appearance of detectable lesions[49]. Centrosomamplification was also seen in premalignant lesions in trenic mice expressing the human papilloma virus (HPVr E7 oncoprotein[50] and in 30–72% of preinvasive caroma in situ of cervix, breast, and prostate[46,47]and rarelybserved in normal tissue. Thus, centrosomal amplificand other centrosomal changes appear to occur very ea

umorigenesis, but are apparently associated with thenset of complex chromosomal changes.

Centrosomal defects increase in severity during tumorression[10,41,47,48,51]. For example, the frequency of ce

rosomal amplification in cervical carcinoma cells increarom nearly zero in normal epithelium, to∼20% of the cellsn grade 1 tumors,∼50% in grade 2 tumors, and nearly 7n grade 3 tumors[47]. Similar results were observedhanges in the shape and size of centrosomes. Centroefects are also correlated with increased genetic ins

ty and aneuploidy[41,43,48,52], although apparently nn HPV virally induced centrosomal amplification[53]. In atudy of breast tumors, genetically unstable aneuploid tuad∼6.8 centrosomes per cell, compared to∼3.6 per cell inenetically stable aneuploid tumors, 2.8 in diploid tumnd 1.5 in normal tissues[46].

W. Saunders / Seminars in Cancer Biology 15 (2005) 25–32 27

2. Runaway centrosomal duplication and tumorsuppressors

Under some conditions, centrosome replication canbecome uncoupled from the cell cycle allowing multiplecentrosomes to form in a single cell. In tumor cell lines,over-replication is correlated with reduced activity of thep53 tumor suppressor pathway, commonly by mutationalinactivation of theTP53gene[42,54–56]. For example, p53mutant mice had a marked increase in sensitivity to papillomaformation following exposure to chemical carcinogens, with75% of the tumor cells containing three or more centrosomes[56]. A reduction in p53 activity in tumors can also beachieved by overexpression of the p53 inactivating proteinMdm2 [42] or reduced activity of the downstream targetsof p53, p21Cip1/waf1 [55,57] and Gadd45[58,59] and all ofthese are correlated with centrosomal amplification.

Why is loss of p53 important? As I discuss further below,centrosomal amplification is often correlated with an increasein ploidy from cytokinesis defects. It has been suggested thatloss of p53 activity allows cells with polyploidy to proliferateinstead of apoptose[60–63]. Treatment of cells with actininhibitors like cytochalasin induces polyploidy by blockingcytokinesis[64]. When primary rat embryo fibroblasts cellswere treated with cytochalasin, they arrested indefinitely with4 thes sint NArH avear enceo s toc ulatec crip-t mesi smi ectso

fica-t ationa withp

ors lossoo toc no cen-t theu bi s inH orE y actt ssiono rip-

tion regulators[68]. Taken together, these results suggestthat loss of Rb alone is insufficient to induce centrosomalamplification, but that loss of Rb may play an important rolein allowing centrosomal amplification by other mechanisms.Thus, it is likely loss of Rb and/or p53 may promote centro-somal amplification by setting up a permissive condition forcells with amplified centrosomes to proliferate.

3. Centrosome amplification and DNA damage

There are many observations linking DNA damage tochanges in centrosome number. For example, incompletelyreplicated or damaged DNA leads to centrosomal splitting inDrosophilaand mammalian cells[69] and�-irradiation canlead to centrosome amplification in tumor cells[70]. Also,overexpression of the DNA damage sensors ATM and ATRcan lead to centrosomal amplification[71], as can mutationsin BRCA1 or BRCA2[72,73], Xrcc2 and Xrcc3[72], andin the presence of a dominant negative mutant of the Rad51DNA repair protein[74]. However, some of these proteinshave pleiotropic effects on the cell cycle, apoptosis, chro-matin and other cellular processes that may indirectly influ-ence centrosome number.

A particularly well-described example of centrosome de-fE n ofD g-i mi-tr ncet ucest rasei tro-s ore,i -i losso n ina witha cen-t rticalm ucleif Ad nasea losso tos Chk1o spin-d agentc cen-t andm ytoki-n olars , butp nd int

n DNA. When the same cells were transformed withimian virus-40 large T antigen to inhibit p53, cytochalareatment did not arrest the cells but allowed further Deplication to 8n cells, followed by severe aneuploidy[60].uman colorectal cancer cell lines with p53 mutations hn increased tendency towards polyploidy[65], which mayesult from increased survival of these cells in the absf p53-mediated genomic surveillance. p53 localizeentrosomes before and after duplication and can regentrosome replication independent of its role as transion regulator[66]. p53 disassociates from the centroson cells with mitotic defects and is stabilized in the cytoplanducing G1 arrest in the next cell cycle. When mitotic defccurred in cells without p53, no arrest occurred[35,36].

Is loss of p53 an essential part of centrosomal ampliion? The answer appears to be no. Centrosomal amplificnd other centrosomal changes can be seen in tumorsersistent p53 activity[46,47,51].

There is still uncertainty about the role of the Rb tumuppressor protein in centrosomal amplification. Thef Rb itself, or reduced expression of p16ink4a or inductionf cdk4, both of which block Rb function, did not leadentrosomal alterations[42,54]. However, while expressiof a mutant HPV E7, unable to bind Rb, decreased

rosomal amplification by E7 by three-fold, similar tontransfected control[67], inhibitory phosphorylation of R

s required to allow over-replication of the centrosomeU treated cells[68]. Overexpression of either cyclin A2F1 can compensate for loss of Rb, suggesting Rb ma

o regulate centrosome number by transcriptional repref the cyclin A gene, a target of the E2F family of transc

ects and DNA damage has been demonstrated inDrosophila.mbryos induced to undergo mitosis prior to completioNA replication, or following treatment with DNA dama

ng X-rays and UV light show centrosomal defects inosis, including loss of the microtubule nucleating�-tubulining complex[75,76]. Takaeda et al. have gathered evidehat DNA double strand breaks are the signal that indhe centrosomal changes. DNA break-forming topoisomenhibitors or the drug bleomycin produced similar cenome changes in 100% of injected embryos. Furthermnjection of restriction-digested�-X174 DNA induced simlar changes as DNA damaging agents, including thef �-tubulin staining from the centrosomes, a reductiostral microtubules and disorganization of the spindle,ccompanying chromosomal segregational defects. The

rosomal changes led to loss of the nucleus from the coonolayer of the embryo and prevented the damaged n

rom being included in the developing embryo. Like DNamaging agents, mutational inactivation of the Chk1 kilso led to spontaneous centrosomal inactivation withf �-tubulin. The Chk2 DNA damage kinase is requiredignal the centrosomal changes associated with loss ofr DNA damage. Chk2 localizes to the centrosome andle, a response that is enhanced by the DNA damagingamptothecin or loss of Chk1. In the absence of Chk2rosomal inactivation, the damaged cells were not lostitosis proceeded, but chromosome segregation and cesis failed, leading to multiple centrosomes and multippindles in the next mitosis. Chk2 acts upstream of p5353 was not required for the centrosomal changes fou

he Chk2 mutant.

28 W. Saunders / Seminars in Cancer Biology 15 (2005) 25–32

Fig. 1. A pathway for multipolarity is shown. A cell with normal centrosome numbers can amplify the centrosomes by over-replication or failure of cytokinesis.Alternatively, centrosomes may fragment into multiple microtubule organizing centers. The multiple centrosomes may initially be clustered in a bipolar spindleand require further steps to overcome this clustering mechanism. Alternatively, the amplified centrosomes may form multipolar spindles without further changeand cluster only in unusual circumstances (not shown).

Spindle multipolarity is strongly correlated with anaphasebridges, which frequently result in DNA breaks, in tumorsof the head and neck (p< 0.05) and bone and soft tissue(p< 0.0006)[48,77]. The authors suggest this linkage couldbe due to bridges blocking cytokinesis and leading to cen-trosome amplification. The previously cited work from fliessuggest that multipolarity may also be triggered in some casesby DNA damage in cells with anaphase bridges via a Chk2-related pathway.

Cells from BRCA1 or BRCA2 deficient mice have cen-trosomal amplification[72,73]and BRCA1 binds to centro-somes in a�-tubulin-dependent manner. Expression of theBRCA1 �-tubulin binding domain alone reduces BRCA1 atthe centrosome and results in multipolar spindles. BRCA1 isphosphorylated in S-phase and in cells with DNA damage,and a reduction in BRCA1 phosphorylation results in reducedbinding of BRCA1 to�-tubulin and centrosomes and an in-duction of multipolar spindles.

4. Centrosome replication defects versus failure ofcytokinesis

Centrosome amplification could proceed through eitherover-replication of the centrosomes or by a cell division orcytokinesis defect leading to amplified centrosome numbers( o beda de-f ationi e ofc -A isfia am-p NAr isa ctingt of

the cells with centrosomal amplification were multinucleatedand 20% were delayed in late mitosis or cytokinesis consis-tent with a cytokinesis defect. Surprisingly, centrosomal am-plification was still observed in mutants with sharply reducedor absent kinase activity. Similar observations were madewith overexpression of Plk1 or Aurora-B kinases. p53−/−mutants showed a 66% correlation between cells with multi-polar spindles and multinucleation. These observations sug-gest that multipolarity in these mutants is a result of a failureof cytokinesis rather than a centrosomal replication defect.Further study is needed to define fully the relative role of thesetwo mechanisms in centrosomal amplification in tumor cells.

5. Centrosomal amplification and spindlemultipolarity

The main impact of centrosome amplification on cancercells is most likely the formation of multipolar spindles. Howtightly linked are centrosomal amplification and spindlemultipolarity? Both the defects are generally observedtogether in tumor tissue[47,48] however, the frequencyof mitotic cells in tissue is typically quite low, and strongcorrelations are difficult to make. We know that spindlemultipolarity can be induced in the absence of centrosomalamplification. For example, overexpression of the centroso-m andm theto mesa (N.Q ) anda mals

mala hea ncesa TheN dis-

Fig. 1). Centrosomal amplification is often suggested tue to centrosomal replication defects[42,54,57], yet ex-mples of over-replication in the absence of cytokinesis

ects are rare, therefore at least some of the amplificnterpreted as replication defects may be due to failurytokinesis. The centrosomal-associated kinase Aurorarequently amplified in tumors and cancer cell lines[78] ands associated with centrosomal amplification[79]. Meraldi etl. recently showed that Aurora-A induced centrosomallification is blocked in cells arrested in S-phase by the Deplication inhibitor hydroxyurea, suggesting Aurora-Acting at another point of the cell cycle and may not be a

o over-replicate centrosomes[80]. Seventy-five percent

al Nek2 kinase elicits a splitting of the centrosomesultipolarity [21] as does treatment of neutrophils with

umor promoter 12-0-tetradecanoylphorbol-13-acetate[81]r DNA damaging conditions. However, most centrosoppear to be unfragmented in cancer cells in cultureuintyne and W. Saunders, unpublished observationsfurther consideration of the relevance of centroso

plitting in tumor cells is needed.Are additional changes in the cell other than centroso

mplification required to induce spindle multipolarity? Tnswer remains unclear. It is known that in some instamplified centrosomes fail to form multipolar spindles.E-115 neuroblastoma cell line has large numbers of

W. Saunders / Seminars in Cancer Biology 15 (2005) 25–32 29

persed centrosomes in interphase, but at mitosis the cen-trosomes coalesce allowing a bipolar spindle to form[15].Induction of extra centrosomes with the actin inhibitor cy-tochalasin B in BSC-1 cells still lead to bipolar division in30–50% of the cells, showing that some mechanism acts toprevent full centrosome separation following amplification[83]. Bill Brinkley has recently proposed that this may repre-sent a centrosomal coalescence mechanism that may preventmultipolarity in some cells[82]. However, the nature of thisputative coalescence activity remains unknown. Specifically,it is unclear if the mechanism of clustering of complete cen-trosomes such as observed in the NE-115 cells is related tothe cohesion of the two centrioles found in the normal cen-triolar pair. Another possibility is that clustering may merelyrepresent the proximity of replicated centrosomes[83].

Ch-TOG belongs to the XMAP215 family of microtubulebinding proteins known to play a key role in microtubule sta-bility in Xenopusegg extracts[84]. Binding of ch-TOG to thecentrosome is mediated by a set of three TACC proteins[85].Changes in both TACC and ch-TOG human homologs havebeen implicated in cancer[36]. siRNA knockdown of humanTOG caused showed little or no change in microtubule stabil-ity but a dramatic rise in multipolar spindles to approximately40% of the metaphase population. Thus, in human cells thispathway may play an important role in multipolarity in tumorc

6

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fica-t tin-d andt mi ba auses b.H stilli thef notrT mpli-fi Rb,b lved[

mec t theym st ap-

pearance of structurally immature centriolar cores[67]. E6,on the other hand, takes weeks to amplify the centrosomesand typically results in multinucleated cells. These results canbe interpreted to indicate that E7 acts primarily to interferewith centrosomal duplication while E6 may interfere with celldivision[92]. However, E7 expression also increases the fre-quency of multinucleated cells, and cell cycle arrest preventsthe centrosomal amplification phenotype from cells express-ing E7 [67]. Thus, E7 apparently also acts at least in partthrough misregulation of cell division.

Recently, centrosomal amplification and spindle multipo-larity was also observed in cells expressing the Hepatitis Bvirus X (HBx) oncoprotein[93,94]. Forgues et al. found thatHBx bound to and sequestered the nuclear transport proteinCrm1 in the cytoplasm and that HBx mutants lacking nuclearlocalization failed to induce multipolarity. Furthermore, in-hibition of nuclear transport by leptomycin B also inducedspindle multipolarity while overexpression of Crm1 blockedHBx induction of multipolarity. Thus, HBx may act to blockCrm1 nuclear export function. HBx inactivates p53 and tran-scription of p21[95], and some Crm1 could be seen at thecentrosome and was frequently lost in cells treated with lep-tomycin B. Yun et al. suggest that cytoplasmic sequestrationof p53 may be the mechanism by which HBx induces cen-trosomal amplification[93]. Alternatively, centrioles in lep-t atedw ug-g mesi gt torsn g tom

7

cen-t lastf . Asw im-p fullyd s in-c somalf e ofc vesti-g ies ofs lls.O ulti-p fixedc encem like-l asa tipo-l ser-v ancyi les.

ells.

. Virally induced centrosomal amplification

It has been appreciated for many years that expref certain viral protein can induce mitotic errors includolyploidy [86]. Expression of variants of either HPV E67 in transgenic mice cooperatively lead to centrosomalification and spindle multipolarity in normal keratinocy

53,67,87]. Adenovirus E1A-expressing cells also show crosomal amplification and multipolar spindles[88].

What is the mechanism behind centrosomal ampliion from viral proteins? The E6 protein activates ubiquiependent degradation of p53 while the E7 protein binds

argets Rb for destruction[89]. E7 also prevents p21 fronhibiting Cdk2/cyclin E, thus interfering with both the Rnd p53 pathways. Thus, these viral oncoproteins may cpindle multipolarity by inhibiting the activity of p53 and Rowever, E7 variants that lack the pRb binding domain

nduce centrosomal amplification, albeit at only one thirdrequency[67]. Also, overexpression of Cdk4 alone caneplicate the centrosomal defects of E7 expressing cells[90].hese data suggests that E7 induction of centrosomal acation does not proceed simply by inactivating p53 andut that other unknown partners of E6/E7 are also invo88].

While both E7 and E6 can induce multipolarity, the tiourse of induction and the exact phenotypes suggesay act through different pathways[91]. E7 expression lead

o centrosome amplification in less than 48 h, with the

omycin B-treated cells were irregular, abnormally elongith extra “minicentrioles” separated from their parents, sesting a defect in centriolar replication. Some centroso

n HBx-expressing cells lacked�-tubulin staining suggestinhat HBx may interfere with Crm1-mediated export of facecessary to maintain centrosomal integrity thus leadinultipolarity [94].

. Future directions

Though Boveri suggested almost 100 years ago thatrosomal changes play a key role in cancer, only in theew years have we come to realize how right he wase continue to investigate this further, I suggest twoortant research issues to keep in mind. One is to careistinguish between centrosomal amplification, defined areased numbers of normal centrosomes, and centroragmentation or splitting. Furthermore, the significancentrosomal fragmentation in cancer cells needs to be inated. The second is to at least consider actual frequencpindle multipolarity and other mitotic defects in live ceur observations indicate that the real frequency of molarity may be substantially less than that observed inells, at least in some cancer cell lines. Real-time fluorescicroscopy of histone-GFP labeled cells showed that the

ihood that a cell will divide with a multipolar spindle wbout one half the frequency of metaphase cells with mul

ar spindles in fixed samples (Luo et al., unpublished obations). Preliminary analysis suggests that the discreps due to a delay in mitosis of cells with multipolar spind

30 W. Saunders / Seminars in Cancer Biology 15 (2005) 25–32

While the equipment for live cell microscopy may not be al-ways available, investigators need to keep in mind that the ac-tual frequency of mitotic defects like multipolar spindles maybe substantially different than that observed in fixed cells.

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