Aneuploidy: a matter of bad connections

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Vol.15 No.8 August 2005

Chromosome Segregation and Aneuploidy series

Aneuploidy: a matter of bad connections Daniela Cimini1 and Francesca Degrassi2 1

Department of Biology, University of North Carolina at Chapel Hill, 607 Fordham Hall, Chapel Hill, NC 27599, USA CNR Institute of Molecular Biology and Pathology, Department of Genetics and Molecular Biology, La Sapienza University, Via degli Apuli 4, 00185 Rome, Italy 2

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to the next and to prevent aneuploidy, the condition in which a cell has gained or lost one or several chromosomes during cell division. Aneuploidy is a hallmark associated with birth defects and cancer, and is observed at relatively high frequencies in human somatic cells. Recent studies in mammalian tissue culture cells suggest that the persistence of kinetochore–microtubule misattachments through mitosis is a major cause of chromosome mis-segregation and aneuploidy. Furthermore, studies in mice and humans suggest that small changes in the expression, rather than complete inactivation, of genes encoding specific proteins might be associated with aneuploidy in living organisms. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we survey the outcome of these studies, focusing on the importance of kinetochore misattachments in producing aneuploid cells.

Introduction Equal distribution of genetic material to the two daughter cells is the ultimate goal of mitotic cell division. Chromosome segregation errors during mitosis result in the production of aneuploid cells [i.e. cells in which the chromosome number is not a multiple of the haploid number of the species (the presence of one or more extra sets of chromosomes is known as polyploidy)]. Aneuploidy that originates in germ cells is the major cause of miscarriage and still birth in humans and is responsible for severe genetic diseases such as Down syndrome. In addition, abnormal numbers of chromosomes were recognized O100 years ago as a ubiquitous feature of human tumor cells, providing the basis for Boveri’s hypothesis that aneuploidy is a cause of cancer [1]. Although the relative contribution of gene mutations and abnormal numbers of chromosomes in human cancers is still a matter of debate [2], a crucial role of aneuploidy in tumorigenesis is now widely accepted [3–5]. Finally, recent work suggests an association between aneuploidy and other acquired diseases such as biliary cirrhosis [6] Corresponding author: Degrassi, F. ([email protected]). Available online 14 July 2005

and other autoimmune diseases [6,7] (Box 1). Despite the role that aneuploidy might have in cancer and other diseases, the occurrence and mechanisms of mitotic chromosome mis-segregation in human populations are still relatively unexplored issues. The rate of spontaneous aneuploidy in human somatic cells is largely unknown because cells and tissues from healthy individuals are rarely available for large-scale screens, and most tissues and/or cell types can be obtained only through extremely invasive methods. Because peripheral blood does not present such limitations, many studies investigating aneuploidy in humans have been performed in peripheral blood lymphocytes. Centromeric DNA probes have been used to determine aneuploidy frequencies in lymphocytes from healthy individuals in multicolor fluorescence in situ hybridization experiments. A normal diploid cell exhibits two fluorescent dots in its nucleus or two centromerically labeled homologous chromosomes in a metaphase spread for each probe used. Such an approach has provided estimates of between 0.1% and 0.8% of nuclei exhibiting trisomy (one specific chromosome is present in three copies rather than in two) for a specific chromosome in interphase cells [8], or even higher values for metaphase cells [9,10]. Furthermore, an age-dependent increase in aneuploidy of sex chromosomes has been found in both male and female donors [11]. Box 1. X-chromosome aneuploidy and autoimmunity Sex differences in autoimmune diseases have been known for O100 years, with women affected more often than men by most autoimmune diseases [75]. Such discrepancy has been explained by differences in hormone production, and this idea was supported by the observation that autoimmunity fluctuates during and after pregnancy [75]. However, a recent study suggests an alternative explanation. As for many other autoimmune diseases, there is a 9:1 female predominance of primary biliary cirrhosis (PBC) [6]. Invernizzi et al. assessed the rate of X-chromosome monosomy (having only one copy of the X chromosome) in 100 women with PBC and found it to be significantly higher than in controls [6]. The authors suggested that haploinsufficiency for specific X-linked genes might lead to female susceptibility to PBC. This study, however, does not explain why women affected by Turner’s syndrome do not commonly develop PBC, despite being frequently affected by other autoimmune diseases [6,7,76]. An alternative explanation is that X-chromosome monosomy could make women more likely to develop autoimmune diseases but that mutations in other specific genes determine what type of autoimmunity would be developed [7].

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If aneuploidy frequencies in other human tissues were comparable to those observed in peripheral blood lymphocytes, hundreds of thousands of aneuploid cells would be produced every day, considering that w2.5!108 cells are dividing in the human body at a given time [12]. One can speculate that high aneuploidy frequencies might be tolerated in terminally differentiated cells (such as lymphocytes) that will not go through further divisions, whereas mitosis of undifferentiated or stem cells should produce few aneuploid daughter cells. However, the actual frequencies of aneuploidy in different human tissues and cell types remain to be determined. Understanding the cellular mechanisms involved in the origin of aneuploid cells is one of the main goals of cell biology research, as shown by the growing number of studies investigating sister chromatid segregation in mitosis. Because of the limitations outlined for obtaining human cells or tissues, model systems such as mammalian

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tissue culture cells or knockout mice have been used frequently. Using such models, a wealth of new information has been collected during recent years. In this article, we review these discoveries and discuss how aneuploidy might originate in human tissues, focusing particularly on microtubule–kinetochore misattachments as a source of chromosome mis-segregation and aneuploidy.

Cell division and errors: deviant orientations For proper chromosome segregation to occur, multiple cellular structures must cooperate during mitosis to accomplish a series of highly coordinated events such as chromosome condensation, bipolar spindle formation, chromosome movements and cytokinesis (Box 2). The accuracy of chromosome segregation is also ensured by the existence of a control signal-transduction pathway – the mitotic spindle checkpoint – that prevents anaphase onset

Box 2. Chromosome segregation during mitosis During mitosis in vertebrates, the sister kinetochores – multiprotein complexes that assemble on each chromatid of a replicated chromosome – must interact with microtubules from opposite spindle poles so that the sister chromatids can be pulled to opposite directions after anaphase onset. After nuclear envelope breakdown, kinetochores interact with microtubules emanating from the two spindle poles. When the first sister kinetochore on a chromosome attaches to microtubules, the chromosome becomes mono-oriented and moves towards the pole to which it is attached (Figure Ia). When the unattached sister kinetochore interacts with microtubules from the opposite pole, the chromosome becomes bioriented and moves to the spindle equator (Figure Ia, top blue chromosome). Eventually, all of the chromosomes align at the spindle equator to form a metaphase plate (Figure Ib) until chromatid disjunction at the onset of anaphase.

(a) Prometaphase (checkpoint on)

The mitotic spindle checkpoint (Box 3) remains active as long as unattached kinetochores are present (Figure Ia). After all of the kinetochores become fully attached to microtubules and after chromosomes become aligned at the metaphase plate, the mitotic checkpoint is inactivated (Figure Ib) and the cell enters anaphase. During anaphase (Figure Ic), each chromatid (now a chromosome) migrates to its facing pole in a process known as anaphase A. In addition, the spindle poles move apart in a process – anaphase B – that increases the distance between the separating chromosome sets. When anaphase motion is nearly complete, the chromosomes progressively decondense and a new nuclear envelope forms around each chromatin mass. During this final stage of mitosis, called telophase, the cell also undergoes cytokinesis, the process that physically separates the two daughter cells by dividing the cytoplasm into two roughly equal parts (Figure Id).

(b) Metaphase (checkpoint off) Kinetochore fiber Spindle pole Astral microtubule

Non-kinetochore microtubule

(c) Anaphase

Kinetochore

Sister chromatids (d) Telophase–cytokinesis

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Figure I. Stages of mitosis. (a–d) If mitotic chromosome segregation occurs properly, the two daughter cells produced at the end of mitosis possess the same number and type of chromosomes as the mother cell. www.sciencedirect.com

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Box 3. The mitotic spindle checkpoint Considering the catastrophic consequences that might arise by precocious anaphase onset, eukaryotic cells have evolved a surveillance mechanism – the mitotic spindle checkpoint – that regulates the metaphase-to-anaphase transition. The main components of the mitotic checkpoint were identified through genetic screens in yeast. These initial screens identified six genes: MAD1–3, BUB1, BUB3 and MPS1. Highly conserved homologs of all of these genes have been identified in higher eukaryotes (in which MAD3 is called BUBR1). As described in Box 2, anaphase does not initiate when unattached kinetochores are present. All of the checkpoint proteins localize at unattached kinetochores in vertebrate cells and are thought to generate the so-called ‘wait anaphase signal’ (for review, see Refs [14,16]). Additionally, the mitotic spindle checkpoint is thought to sense the interkinetochore tension (for review, see Ref. [19]) generated by bipolar attachment (amphitelic orientation, see Figure 1 in main text). Checkpoint proteins prevent anaphase onset when the wait anaphase signal is on by sequestering Cdc20 (Figure Ia), an activator

of the anaphase-promoting complex, also known as the cyclosome, (APC/C). Unattached kinetochores are thought to function as activation platforms for binding between checkpoint components and Cdc20. As kinetochores bind to microtubules and as attachment sites become filled, checkpoint proteins leave the kinetochores and no longer bind to Cdc20, which becomes free to bind to and activate the APC/C (Figure Ib). Before anaphase onset, sister chromatids are held together by the ‘molecular glue’ cohesin. When all chromosomes have successfully attached to a full complement of microtubules and become bioriented, the APC/C is activated and its E3 ubiquitin-ligase activity targets securin and cyclin B for destruction by the proteasome. The release of the endopeptidase separase from the inhibitory interaction with securin enables the proteolytic cleavage of cohesin and separation of sister chromatids (Figure Ic). This determines anaphase onset and the movement of separated sisters to opposite poles. In addition, cyclin B destruction triggers mitotic exit.

(a) Prometaphase ‘Wait anaphase signal’

Checkpoint protein complex

Separase Securin

Cdc20

Cohesin

Inactive APC/C

Microtubules

(b) Metaphase

Checkpoint protein complex

Ub Separase Ub Cdc20 Securin

Ub

Active APC/C

(c) Anaphase

Ub Ub Ub

Separase

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Figure I. The mitotic spindle checkpoint.

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until all sister kinetochores interact properly with the spindle (Box 3). Vertebrate cell kinetochores have multiple microtubule-attachment sites, and the mitotic checkpoint is turned off when all kinetochores are attached to spindle microtubules, have achieved full occupancy and are under tension (for review, see Ref. [13]). For proper chromosome segregation to occur, sister kinetochores must biorient and attach to microtubules emanating from opposite poles. This amphitelic orientation (Figure 1) produces tension between sister kinetochores and stabilizes microtubule attachment. Tension is thought to have a role in the mitotic spindle checkpoint pathway, either directly or indirectly through its ability to promote stable microtubule attachments, and lead to acquisition of a full complement of microtubules by the kinetochore (up to O20 kinetochore–microtubules) (for review, see Ref. [13]). Thus, amphitelic orientation satisfies the mitotic checkpoint requirements and enables segregation of the two sisters to opposite poles (Figure 2a). Because of the stochastic nature of kinetochore– microtubule encounters during the early stages of mitosis, erroneous kinetochore attachments such as monotelic, syntelic and merotelic kinetochore orientation (Figure 1) can occur frequently. Monotelic orientation, the attachment of only one sister kinetochore to the spindle microtubules, is a frequent event in the early stages of chromosome orientation (Box 2). Unoccupied microtubule-attachment sites on unattached kinetochores accumulate mitotic checkpoint proteins and generate a signal that keeps the mitotic

(a)

Amphitelic kinetochore orientation

(b)

Monotelic kinetochore orientation

(c)

Syntelic kinetochore orientation

(d)

Merotelic kinetochore orientation

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Figure 1. Kinetochore–microtubule attachments. (a) In amphitelic kinetochore orientation, the two sister kinetochores are attached to microtubules from opposite poles; the chromosome is bioriented. (b) In monotelic kinetochore orientation, one sister kinetochore is attached to one pole, whereas the other kinetochore is unattached; the chromosome is mono-oriented. (c) In syntelic kinetochore orientation, both sister kinetochores are attached to microtubules from the same spindle pole; the chromosome is mono-oriented. (d) In merotelic kinetochore orientation, one kinetochore is attached to microtubules from both poles, instead of just one; the chromosome is bioriented. www.sciencedirect.com

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checkpoint active so that anaphase onset is delayed, even if only one unattached kinetochore is present (for review, see Refs [14–16]) (Boxes 2,3). Syntelic orientation is achieved when both sister kinetochores attach to microtubules from the same spindle pole (for review, see Ref. [17]). It is still a matter of debate whether syntelic kinetochore orientation is detected by the mitotic spindle checkpoint [13,15]. However, when syntelic kinetochore orientation is induced by chemical inhibition of spindle bipolarization, the mitotic checkpoint protein Mad2 accumulates on the kinetochores of syntelically oriented chromosomes [18], suggesting that the checkpoint remains active in the presence of syntelic orientations. What remains unclear is whether the lower tension experienced by syntelic kinetochores is detected directly by the mitotic spindle checkpoint or whether the reduced tension prevents sister kinetochores from maintaining stable microtubule attachment, thus reducing the level of kinetochore occupancy by microtubules and keeping the mitotic checkpoint active [13,19]. Cells that enter anaphase with monotelically or syntelically oriented chromosomes produce aneuploid daughter cells. Both monotelic and syntelic chromosomes are positioned close to the pole to which they are attached (Figure 2b,c, metaphase), whereas bioriented chromosomes align at the spindle equator (Figure 2a, metaphase). At the onset of anaphase, sister chromatids separate and syntelically oriented sister kinetochores are actively pulled towards the same pole (Figure 2b, anaphase). The unattached sister of a monotelic chromosome does not move poleward but persists in a position that is close enough to the pole to be included in the daughter nucleus, together with its sister, at the end of mitosis [20,21] (Figure 2c, anaphase). Alternatively, after sister chromatid separation, the unattached sister of a monotelic chromosome can establish attachment to microtubules and move poleward with its sister, as occurs with syntelic chromosomes [20,21]. Persistence of syntelic and monotelic orientation in anaphase produces what is generally known as a non-disjunctional event, resulting in a trisomic daughter cell and a monosomic (only one copy of a specific chromosome) daughter cell (Figure 2b,c, daughter cells). In the presence of a functional mitotic checkpoint and an efficient correction mechanism, monotelic and syntelic orientations do not persist until anaphase but, instead, are corrected and converted into bipolar attachments. An active correction process for syntelic orientation, involving Aurora B kinase, has been shown in both yeast [22] and mammalian cells [23]. Merotelic kinetochore orientation occurs when a single kinetochore becomes attached to microtubules from both spindle poles (Figure 2d, metaphase), rather than just one [21,24]. This orientation occurs frequently in early mitosis and does not activate the mitotic checkpoint; therefore, cells can initiate anaphase without complete correction [21,24–26]. Although most merotelically oriented chromosomes segregate properly during anaphase [26], a fraction of them induces anaphase lagging-chromosomes [21,24,26]: chromosomes that remain at the spindle equator as all of the other chromosomes move to the poles (Figure 2d, anaphase). Lagging chromosomes are found in w1% of

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(a) Amphitelic

+

(b) Syntelic

+

(c) Monotelic

+

(d) Merotelic +

+

Metaphase

Anaphase

Daughter cells TRENDS in Cell Biology

Figure 2. Chromosome segregation produced by different kinetochore–microtubule attachments. (a) Proper chromosome segregation occurs as a result of amphitelic orientation. (b–d) Chromosome mis-segregation and production of aneuploid daughter cells occur as a consequence of persistent kinetochore misorientation in anaphase. Chromosome orientation at metaphase is shown in the left column, anaphase chromosome segregation is shown in the middle column and the resulting daughter nuclei are shown in the right column. Each chromosome is shown in a different color and the two sister chromatids of one chromosome are shown in different shades of blue.

mammalian tissue culture cells undergoing anaphase [24]. When cell cleavage occurs, the lagging chromosome is included in one of the two daughter cells, depending on where the cleavage furrow ingresses, thus producing aneuploid daughter cells in w50% of cases (Figure 2d, daughter cells). Most lagging chromosomes become micronuclei (chromatin bodies separated from the main nucleus) when chromosomes decondense at the end of mitosis [25] (Figure 2d, daughter cells). Because merotelic kinetochore orientation is not detected by the spindle checkpoint, it is the biggest threat to the maintenance of an appropriate chromosome number in cells with normal spindle-checkpoint function. Inducing aneuploidy through persistence of kinetochore misattachments at anaphase If a disastrous result such as chromosome mis-segregation is produced by kinetochore misattachment, what induces cells to enter anaphase with misoriented chromosomes? Changes in the function of several classes of protein that localize mostly at kinetochores have been shown to induce errors in chromosome orientation (for review, see Ref. [27]). www.sciencedirect.com

Depletion of the outer-kinetochore Ndc80 protein complex, which is important for kinetochore–microtubule attachment, results in an irreversible mitotic block and cell death [13]. However, depletion of most mitotic proteins, although frequently inducing prolonged prometaphase delays, does not induce a permanent mitotic arrest, and cells exit mitosis exhibiting various types of segregation abnormality such as anaphase lagging-chromosomes, micronuclei and abnormally shaped nuclei (Table 1). Proteins whose inactivation promotes aneuploidy can be grouped into different classes: core kinetochore proteins, proteins involved in kinetochore–microtubule interaction, checkpoint proteins, proteins responsible for correction of misattachments and proteins involved in maintaining the general chromosome architecture. Disruption of core kinetochore proteins Depletion of either the core kinetochore protein CENP-A or the kinetochore protein hMis12 induces an increase in the number of unaligned chromosomes [28]. Surprisingly, cells possessing unaligned chromosomes do not undergo a mitotic delay – although the mitotic checkpoint protein

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Table 1. Chromosome segregation defects and kinetochore misattachments after mitotic protein depletion or inactivationa Protein class

Proven or putative

Mitotic defect when

KT–MT attachments at

Possible

function in mitosis

depleted

anaphase onset

mechanism

Kinetochore assembly

Chromosome mis-

Mono-orientation

and size determination

segregation

Model-system–method

Refs

HeLa–RNAi

[28]

HeLa–Ab-inj

[29]

CINC cells, 293–MinCE,

[30,32–34]

Core kinetochore proteins CENP-A, hMis12 CENP-I

Chromosome mis-

No sustained checkpoint

Mono-orientation

segregation

Defective accumulation of checkpoint proteins at KT, no sustained checkpoint

KT–MT interaction APC

Unstable attachments

Unknown

Microtubule plus-end

Chromosome mis-

dynamics

segregation,

ES–MinC/C,

aneuploidy, spindle

HCT116–MinC/C

defects CENP-E

Kinesin-like plus-end-

Chromosome mis-

directed motor

segregation

Few or no attached MTs

No sustained

ES–CD

[35,36]

checkpoint

Checkpoint proteins Mad2, BubR1

Bub3

Chromosome mis-

Monotelic, syntelic,

Premature anaphase

PtK1–Ab-inj

[37,38]

segregation,

merotelic

onset

PtK1–Prot-inj

[20,21]

aneuploidy

HeLa–RNAi

[39]

Binds to BubR1

Lagging chromosomes

ES–TD

[56,61–63]

Defective correction,

XTC–Ab-inj, HeLa–CI,

[41–43]

checkpoint override,

NRK–DN

Sequester Cdc20

Misorientation correction Aurora B

Syntelic

Multifunctional

Chromosome

passenger protein

mis-segregation,

Plus-end-destabilizing

Lagging

Merotelic, syntelic,

Defective correction of

kinesin

chromosomes,

merotelic–syntelic

improper attachments

Improper interactions,

Abnormal centromere

merotelic?

structure

cytokinesis failure

polyploidy MCAK

PtK2–Ab-inj

[49]

MRC5–CI, HeLa–CI

[52–55]

mis-segregation Centromere architecture Topoisomerase II,

Scaffold protein,

histone deacetylases

chromatin remodeling

Lagging chromosomes

a

Abbreviations: Ab inj, inactivating-antibody injection; CD, conditional disruption; CE, conditional expression; CI, chemical inhibition; CINC cells, chromosome instability colorectal tumor cells; DN, dominant–negative; ES, mouse embryonic stem cells; HCT116 and 293, tumor cells containing wild-type APC; KT, kinetochore; Min, C-terminal truncated APC; MT, microtubule; NRK, normal rat kidney; Prot inj, protein injection; PtK, potoroo kidney epithelial cell; TD, targeted gene disruption; XTC, Xenopus tissue culture cells.

Mad2 localizes at unattached kinetochores in a timely fashion – but progress through mitosis and exhibit anaphase lagging-chromosomes and telophase micronuclei [28]. Likewise, depletion of the kinetochore protein CENP-I induces an increase in the number of unaligned chromosomes [29]. Unlike CENP-A-depleted and hMis12depleted cells, CENP-I-depleted cells undergo a transient mitotic delay that is characterized by proper accumulation on unaligned kinetochores of the checkpoint proteins hBub1, hBubR1, hZw10 and hRod, but not Mad2 [29]. Such cells eventually exit mitosis and show defects in anaphase chromosome segregation [29]. As suggested by the prolonged persistence of unaligned chromosomes, cells deficient in CENP-A, hMis12 or CENP-I enter anaphase in the presence of monotelic and/or syntelic chromosomes. Disruption of kinetochore–microtubule interactions The adenomatous polyposis coli (APC) gene is mutated in several colorectal tumor cells that are characterized by numerical chromosome instability (CIN) [30]. Although the role of APC mutations in inducing CIN is controversial [31], C-terminal APC truncations in APC-mutated cell lines are associated with defects in attachments between microtubule plus-ends and kinetochores [30]: a phenotype that is recapitulated by conditional expression of the truncated gene in tumor cells containing wild-type APC [30]. Interestingly, a truncated form of the protein induces aneuploidy and defects in mitotic spindle organization or function when expressed in mouse embryonic stem cells [32,33] or non-CIN colon carcinoma cells [34]. A role in www.sciencedirect.com

establishing and/or maintaining microtubule attachment has also been proposed for CENP-E because its selective gene inactivation in mouse primary cells results in persistence of unaligned chromosomes with few or no attached microtubules [35]. Cells depleted of CENP-E and possessing unaligned chromosomes are delayed in mitosis but they eventually enter anaphase and exhibit chromosome segregation defects [36]. Interestingly, unaligned chromosomes observed in the absence of CENP-E recruit reduced amounts of mitotic checkpoint proteins, suggesting an indirect role of CENP-E in the mitotic checkpoint pathway [36]. Inactivation of checkpoint proteins Microinjection experiments in cultured mammalian cells using antibodies against checkpoint proteins [37,38] or dominant–negative mutant checkpoint proteins [20,21] have shown that microinjected cells can enter anaphase with unaligned chromosomes (monotelic and/or syntelic), resulting in the segregation of two sister chromatids to the same pole. In addition, a recent study using live-cell imaging and RNA interference (RNAi) for checkpoint proteins in HeLa cells showed that cells enter anaphase with unaligned chromosomes when checkpoint proteins are depleted and that mitosis is shortened when Mad2 or BubR1, but not other Mad or Bub proteins, are depleted [39]. In all of these studies, chromosomes failed to congress to the metaphase plate before anaphase onset, either because mitosis was no longer delayed in response to mono-oriented chromosomes or because of

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‘lack of time’ (as in the case of Mad2 or BubR1 inactivation). Moreover, this lack of time affects the ability of the cell to achieve correction of merotelic kinetochore orientation, resulting in higher frequencies of anaphase lagging-chromosomes [21,39].

Defects in misattachment correction The specific role of Aurora B in correcting syntelic chromosome orientation [23] and its possible role in correcting merotelic orientation [40] suggest that inactivation of this kinase might result in anaphase onset in the presence of misoriented chromosomes and, hence, chromosome missegregation. Indeed, Aurora B inactivation by different means produces multiple chromosome-segregation defects in mammalian cells [40–44], including non-disjunction [43] and lagging chromosomes [42]. Moreover, Aurora B seems to have other important regulatory roles in misattachment correction. Different groups have found independently that the microtubule-destabilizing mitotic centromere-associated kinesin (MCAK) is phosphorylated by Aurora B [45–48]. Inhibition of MCAK in cultured mammalian cells increases the frequency of both merotelic kinetochore orientation [49] and anaphase laggingchromosomes [49,50]. Interestingly, Kline-Smith et al. showed that specific inhibition of the centromeric function of MCAK produces severe chromosome segregation defects [49]. In the same study, electron microscopy analysis showed that merotelic and syntelic kinetochore orientations coexist in this situation [49], indicating that MCAK has a crucial role in correcting kinetochore misattachments. Finally, a recently identified protein, the inner centromere KinI stimulator (ICIS), has been shown to interact with Aurora B and inner centromere protein (INCENP), and stimulate the microtubuledepolymerizing activity of MCAK [51]. The authors proposed that ICIS and MCAK function coordinately to remove lateral kinetochore–microtubule attachments and to correct merotelic kinetochore orientations [51]. Exogenous factors such as microtubule drugs might also interfere with the process of misattachment correction. Microtubule-destabilizing drugs such as nocodazole, colchicine and vinca alkaloids are effective anticancer tools but they are also known to induce aneuploidy, although a clear understanding of the cellular mechanisms involved is lacking. In the presence of high doses of these drugs – and, hence, complete microtubule disassembly – aneuploidy could arise in cells that have lost the ability to remain arrested in mitosis in response to an activated mitotic checkpoint [15]. By contrast, the presence of low doses of these drugs or a decrease in their concentrations over time could cause cells to accumulate a large number of kinetochore misattachments that might lead to the cell ‘correction apparatus’ becoming saturated and failing to reach an appropriate level of correction before anaphase onset. In support of this idea, high levels of prometaphase merotelic orientations and, hence, anaphase lagging-chromosomes have been observed in cultured mammalian cells recovering from a transient nocodazole-induced mitotic arrest [21,24]. www.sciencedirect.com

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Regulation of chromosomal architecture Proteins that regulate chromosome structure, such as topoisomerase II [52,53] and histone deacetylase [54,55], have also been implicated in aneuploidy. Inhibition of these enzymes in mammalian cells induces defects in chromosome segregation, including lagging chromosomes [52,54,55]. It has been hypothesized that, under these conditions, a deformed architecture might result in curved or bent centromeres. Different regions of these deformed centromeres might face different spindle poles and, thus, easily become merotelically oriented [17,53]. How does aneuploidy originate in humans? As described, mitotic checkpoint inactivation or misfunction in experimental systems leads to persistence of syntelic and monotelic orientations and increased merotelic orientations in anaphase. Therefore, one might expect that altered expression of mitotic checkpoint genes is also responsible for the persistence of these misattachments in human tissues. However, complete loss of function of mitotic checkpoint genes seems unlikely to be the main mechanism of aneuploidy induction in cells and tissues of higher organisms because inactivation of mitotic checkpoint proteins in mice results in embryonic death after the blastocyst stage [35,56] and researchers have found only a small fraction of chromosomally unstable human tumor cells that harbor mutations in mitotic checkpoint genes [57,58]. Recently, a large PCR screen for mutations in 100 human homologs of yeast and Drosophila ‘chromosome instability’ genes (genes in which mutations induce variation in chromosome number) identified only 19 mutations distributed among three classes of gene in 24 human colorectal cancers [59]. Mutated genes included MRE11 (a gene known to be involved in DNA double-strand break repair [60]), hZW10, hZWILC, hROD (components of the tension-responsive branch of the mitotic checkpoint [19]) and DING (a previously uncharacterized gene selected on the basis of its sequence similarity with the yeast securin protein Pds1 [59]). This suggests that mutations in specific protein subsets might be compatible with cell survival, whereas complete loss of mitotic checkpoint function by inactivation of the genes encoding Mad or Bub proteins might lead to cell death. It seems, therefore, more likely that small changes in expression, rather than complete inactivation, of genes encoding mitotic checkpoint proteins might be responsible for persistence of monotelic, syntelic and merotelic attachments at anaphase in cells and tissues of higher organisms (Table 2). Indeed, haploinsufficiency for Mad2, BubR1, Bub3 or Rae1 (a protein whose function overlaps with Bub3 function) induces mitotic checkpoint defects and chromosome mis-segregation [56,61,62], and promotes the formation of lung or intestinal tumors in mice [63]. In addition, both mouse embryonic fibroblasts (MEFs) and mice harboring different combinations of hypomorphic and null BUBR1 alleles show increased aneuploidy and senescence, with the severity of the phenotypes relating to the protein level [63]. Epigenetic silencing (a change in gene expression that does not entail a change in DNA sequence) might be an additional

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Table 2. Genetic and epigenetic variations associated with mitotic chromosome mis-segregation and/or aneuploidy Gene MAD2, BUBR1, BUB3, RAE1 BUBR1 BUB1, BUBR1 CHFR hMAD1

Gene status Haploinsufficiency

Model system Mouse

Hypomorphic mutation Promoter hypermethylation Promoter hypermethylation Single nucleotide polymorphism

Mouse, MEFs Colon carcinoma Human cancers Breast cancer

BUBR1

Truncating, missense mutations

MVA patients

mechanism that induces subtle reductions in checkpoint protein levels, which might lead to chromosome missegregation. For instance, promoter hypermethylation of the genes encoding Bub1 and BubR1 has been found in aneuploid colon carcinoma [64], and CpG methylationdependent silencing of the gene checkpoint with forkheadassociated and RING finger (CHFR) has been found in w40% of analyzed human cancers [65]. The CHFR protein is responsible for delaying chromosome condensation and mitotic progression in response to mitotic stress induced by microtubule drugs [66] and, interestingly, its epigenetic inactivation results in defective response to spindle poisons [65]. Finally, a single nucleotide polymorphism in hsMAD1, the gene encoding the protein responsible for recruiting Mad2 to kinetochores, has recently been found in a human breast cancer [67]. This polymorphism was in the region of Mad2 binding and was shown to attenuate Mad2–Mad1 interaction and reduce the response to spindle poisons. These data suggest that, in addition to reduced gene expression due to hypomorphic mutations or epigenetic silencing, polymorphisms of allelic variants that slightly change the functional characteristics of proteins involved in mitosis might be responsible for the production of aneuploid cells. However, the presence and importance of such polymorphisms in healthy human populations are still unexplored issues. Two examples of human genetic syndromes that confer increased spontaneous aneuploidy are Roberts syndrome [68,69] and mosaic variegated aneuploidy (MVA) [70]. Roberts syndrome individuals are characterized by symmetrical limb reduction (tetraphocomelia), and both syndromes are associated with severe prenatal and postnatal growth retardation and craniofacial abnormalities [69,70]. MVA patients are also at increased risk of cancer, including Wilms tumor and rhabdomyosarcoma [70]. In Roberts syndrome, patients’ lymphocytes show various defects in anaphase chromosome segregation, including lagging chromosomes [68,69]; cells from MVA patients are predominantly trisomic and monosomic [70,71], indicating that they might enter anaphase in the presence of misoriented chromosomes. Interestingly, hereditary mutations in the BUBR1 gene were recently identified in five individuals with MVA [71]. As described earlier, BubR1 depletion leads to abnormal mitotic progression, anaphase onset in the presence of misoriented chromosomes and aneuploidy [39]. However, none of the MVA individuals studied was homozygous for the truncating mutation identified, indicating that complete loss of the protein might be too severe to be observed in living organisms [71]. Finally, no BUBR1 mutations were identified in other families analyzed, www.sciencedirect.com

Phenotype Mitotic checkpoint defects, chromosome mis-segregation, lung tumors, intestinal tumors Aneuploidy, senescence Aneuploidy Defective response to MT drugs Attenuated Mad1–Mad2 interaction, reduced response to spindle drugs Trisomy, monosomy

Refs [56,61] [63] [64] [65] [67] [71]

indicating that MVA is a heterogeneous condition and that mutations in other genes that have a role in mitosis might cause this disorder [71]. Finally, individual susceptibility to DNA-damaging agents that induce structural chromosome aberrations has been linked to polymorphic variants of DNA-repair genes or genes involved in metabolism of xenobiotics, resulting in increased metabolic activation or decreased deactivation of genotoxic agents [72]. Similar susceptibility variants have not yet been reported for environmental agents that induce aneuploidy, such as spindle poisons. However, a recent study investigated the presence of aneuploidy of chromosomes 8 and 21 in workers exposed to the leukemogen and aneuploidyinducing agent benzene, which requires metabolic activation to be transformed into reactive metabolites [73]. This study showed that specific genotypes of genes encoding metabolic enzymes such as cytochrome p450 and glutathione S-transferases were associated with increased frequencies of aneuploidy among benzeneexposed individuals, suggesting that specific combinations of polymorphic genes might confer a predisposition to aneuploidy [73]. Concluding remarks In this article, we have discussed how persistent kinetochore misattachment through mitosis can lead to aneuploidy. Kinetochore misorientations occur at each mitotic division. Consequently, even if some of these errors occur only rarely [42] or are efficiently corrected [21,26], they can still represent a major source of aneuploidy, considering the large number of mitotic divisions that occur in living organisms. Although the understanding of chromosome segregation and its errors has taken a big step forward in recent years, further studies are needed to identify the mechanisms of chromosome mis-segregation in human tissues. This is particularly true for stem cells, in which chromosome segregation is expected to be monitored thoroughly because aneuploidy in such progenitor cells would have catastrophic consequences for tissue development and organ preservation in living organisms. Cultured human embryonic stem cells have been found to be largely aneuploid [74] but it is possible that culture conditions affect fidelity of chromosome segregation. To address this question, it will be interesting to assess aneuploidy levels in uncultured embryonic and adult human stem cells. Genetic polymorphisms, or altered expression, of metabolic enzymes, structural proteins, mitotic checkpoint proteins and proteins correcting kinetochore

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misattachment might be involved in individual susceptibility to chromosome mis-segregation in vivo. Although some of this information is available for model organisms and tumor cells, one can only speculate that similar mechanisms are responsible for aneuploidy induction in human somatic cells. Additional information about the role of persistent kinetochore misattachment in the genesis of human aneuploidy will be obtained by analyzing chromosome segregation in cells from patients affected by aneuploidy-predisposition syndromes such as Roberts syndrome and MVA, and identifying other mutated genes that are associated with these diseases. Furthermore, future studies should aim to identify polymorphic variants and possible predisposition genes or genotypes that might be involved in aneuploidy induction. This will be the starting point for the potential development of diagnosis, prevention and intervention protocols. Acknowledgements We thank Ted Salmon, Maurizio Gatti and Andrea Musacchio for critically reading the manuscript and for helpful discussions and comments.

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