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Leukemia (2002) 16, 767–775
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REVIEW
Centrosome replication, genomic instability and cancer
A Krämer, K Neben and AD Ho
Medizinische Klinik und Poliklinik V, Ruprecht-Karls-Universität, Heidelberg, Germany
Karyotypic alterations, including whole chromosome loss or
gain, ploidy changes, and a variety of chromosome aberrations
are common in cancer cells. If proliferating cells fail to coordinate centrosome duplication with DNA replication, this will
inevitably lead to a change in ploidy, and the formation of monopolar or multipolar spindles will generally provoke abnormal
segregation of chromosomes. Indeed, it has long been recognized that errors in the centrosome duplication cycle may be
an important cause of aneuploidy and thus contribute to cancer
formation. This view has recently received fresh impetus with
the description of supernumerary centrosomes in almost all
solid human tumors. As the primary microtubule organizing
center of most eukaryotic cells, the centrosome assures symmetry and bipolarity of the cell division process, a function that
is essential for accurate chromosome segregation. In addition,
a growing body of evidence indicates that centrosomes might
be imortant for initiating S phase and completing cytokinesis.
Centrosomes undergo duplication precisely once before cell
division. Recent reports have revealed that this process is
linked to the cell division cycle via cyclin-dependent kinase
(cdk) 2 activity that couples centriole duplication to the onset
of DNA replication at the G1/S phase transition. Alterations in
G1/S phase regulating proteins like the retinoblastoma protein,
cyclins D and E, cdk4 and 6, cdk inhibitors p16INK4A and
p15INK4B, and p53 are among the most frequent aberrations
observed in human malignancies. These alterations might not
only lead to unrestrained proliferation, but also cause karyotypic instability by uncontrolled centrosome replication. Since
several excellent reports on cell cycle regulation and cancer
have been published, this review will focus on the role of centrosomes in cell cycle progression, as well as causes and
consequences of aberrant centrosome replication in human
neoplasias.
Leukemia (2002) 16, 767–775. DOI: 10.1038/sj/leu/2402454
Keywords: centrosomes; cell cycle; cyclin-dependent kinases;
cyclins; aneuploidy; genomic instability
Genomic instability
A relationship between genomic damage and cancer development has been suggested since the beginning of the 20th century.1 The genetic alterations occurring in human tumors can
be divided into four major categories: (1) subtle sequence
changes; (2) gene amplifications or deletions; (3) chromosome
translocations; and (4) alterations in chromosome number.2
Whereas subtle sequence instabilities represented by nucleotide-excision repair-associated instability (NIN) and microsatellite instability (MIN) are rare, chromosomal instability (CIN),
involving gains and losses of whole chromosomes, is likely to
occur in most human malignancies.2,3 It is estimated that
numeric chromosomal imbalances, referred to as aneuploidy,
are the most prevalent genetic changes recorded among over
Correspondence: A Krämer, Medizinische Klinik und Poliklinik V,
Ruprecht-Karls-Universität Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany; Fax: 49 6221 565721
Received 19 June 2001; accepted 7 January 2002
20 000 solid tumors analyzed thus far.3,4 Whether aneuploidy
is a cause or a consequence of cancer has long been debated.5
Several lines of evidence now argue in favor of aneuploidy
as a discrete chromosome mutation event that contributes to
malignant transformation and tumor progression. Most interestingly, in colorectal and endometrial cancers, there is an
inverse relationship between MIN and CIN.6 Tumors showing
microsatellite instability are diploid, whereas microsatellite
stable malignancies are usually aneuploid and exhibit
increased rates of chromosomal changes, suggesting that
MIN and CIN are alternative mechanisms of malignant
transformation.
Changes in DNA content and chromosome number have
been shown to occur already in preneoplastic lesions like oral
leukoplakia,7 early cervical neoplasias,8 and small benign
tumors of the colon,9,10 or breast11 and the deviations from a
normal karyotype increase as the tumors enlarge and eventually become malignant. Furthermore, it has recently been
demonstrated that the ploidy status in cells of oral leukoplakia
is a reliable predictor of progression into oral squamous cell
carcinoma.7 It has also been reported that an aneuploid DNA
content indicates a high risk of malignant transformation in
patients with ulcerative colitis12 and Barrett’s esophagus.13
These clinical observations are supported by in vitro studies in
which numeric chromosome aberrations are induced at early
stages of transformation.14,15 In a transgenic mouse model,
mice with a 1 Mb duplication of a portion of chromosome
11 synthenic with human chromosome 17 developed corneal
hyperplasia and thymic tumors.15 With regard to tumor progression and prognosis aneuploidy has been shown to be an
indicator of metastasis in patients with gastric carcinoma,16 as
well as papillary thyroid carcinoma17 and associated with a
poor outcome as compared to diploid neoplasms in a wide
variety of malignancies.16–19
Recent studies have revealed that specific nonrandom
numerical chromosome aberrations correlate with distinct
tumor phenotypes. Tumors from patients with hereditary papillary renal carcinomas commonly show a trisomy of chromosome 7, resulting in nonrandom duplication of a mutant MET
allele, thereby implicating this trisomy in tumorigenesis.20,21
In addition to trisomy 7, trisomy 17 in papillary tumors and
trisomy 8 in mesoblastic renal cell cancers are commonly
seen. In situ hybridization analysis of cervical intraepithelial
neoplasias has provided evidence that chromosome 1, 7, and
X aneusomies are associated with progression towards cervical carcinoma.8 Monosomy 5 or 7 seems to define a distinct
subgroup of acute myelogenous leukemia, commonly evolving from therapy-related myelodysplastic syndromes and
reflecting a poor prognosis.22 These findings support a role of
specific numeric chromosome aberrations in malignant
transformation.
Another argument in favor of a role of chromosomal instability in the process of malignant transformation is that aneuploidy seems to be a dynamic chromosome mutation event
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associated with a subsequently even further elevated rate of
chromosome instability. Lengauer and coworkers6 have
shown that in colorectal tumors with chromosomal instability
gains or losses of chromosomes occurred in excess of 10−2
per chromosome per generation. It has been estimated that
tumors of the colon, breast, pancreas and prostate may lose
an average of 25% of their alleles. In another study it was
found that the rate of chromosomal gains or losses is proportional to the degree of the pre-existing aneuploidy in the
transformed cells, hence that aneuploidy by itself further
destabilizes the karyotype, thereby altering the cellular phenotype and leading to tumor progression.23
There are many potential mitotic targets, which could cause
unequal segregation of chromosomes, among those chromosomal, spindle microtubule and centrosomal defects.5 Correct
sister chromid separation during metaphase–anaphase transition depends on the proper and timely degradation of a
group of proteins called securins that inhibit sister chromatid
separation. A human securin analogue has been identified to
be identical with the pituitary tumor transforming gene, which
is overexpressed in certain malignancies and exhibits transforming activity in vitro.24 Survivin, an inhibitor of apoptosis
protein that is selectively expressed in G2/M phase of the cell
cycle, specifically associates with microtubules of the mitotic
spindle, thereby preventing apoptosis induction at the G2/M
spindle checkpoint. Overexpression of survivin in tumor cells
may overcome this apoptotic checkpoint and favor aberrant
progression of aneuploid cells through mitosis.25 Furthermore,
in some cancers displaying chromosomal instability, the loss
of mitotic spindle checkpoint function is associated with the
mutational inactivation of genes controlling proper kinetochore-microtubule attachment.26 Normally, stable, bipolar
attachment of the kinetochores to spindle microtubules leads
to dissociation of the kinetochore-binding proteins BUB and
MAD from the kinetochore. If the kinetochore is not connected to the spindle microtubules, the BUB and MAD kinases
remain attached and arrest the cell in metaphase by delaying
activation of the cyclin destruction machinery. Cahill and
coworkers27 have found that a small fraction of human colorectal cancers display mutations in BUB proteins and that
induced overexpression of mutant hBUB1 induces chromosomal instability. Interstingly, truncations of the adenomatous
polyposis coli (APC) gene, whose protein product accumulates at the kinetochore during mitosis, is a high-affinity substrate for BUB kinases in vitro and has been shown to be
mutated in familial colon cancer and most early stages of
sporadic colorectal tumors, also lead to extensive chromosome and spindle aberrations and might contribute to
chromosomal instability in colorectal cancer28,29 Furthermore,
hMAD1 seems to be inactivated by the Tax protein of the
human T cell leukemia virus 1, leading to multinucleation and
genomic instability as well.30
Among the targets implicated in genomic instability and
malignant transformation, it has long been recognized that
errors in centrosome replication may be an important cause
of aneuploidy and might thus contribute to cancer formation.
The remaining part of this review will focus on causes and
consequences of aberrant centrosome duplication in human
neoplasias.
Centrosome structure and function
The centrosome was among the first organelles described by
early cell biologists at the end of the 19th century and was
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viewed to be as important as the discovery of the nucleus.31
Centrosomes were named during this time by Theodor Boveri
on the basis of their central position in the cell. The importance of centrosomes for cell division was well recognized,
although microtubules had not yet been discovered and it was
not yet known that centrosomes are the main microtubule
organizing centers. The sea urchin egg was one of the first
systems studied by Theodor Boveri, who elaborated an outstanding wealth of information on centrosomes by using iron
hematoxylin as the primary staining technique.32 After Boveri’s remarkable discoveries, research on centrosomes stagnated and was not persued for many years. The focus changed
back again when immunofluorescence microscopy and the
availability of immunological probes opened a renaissance of
centrosome research which is now being extended to the
molecular level.
As the primary microtubule organizing center of most eukaryotic cells, the mammalian centrosome functions as the site
for the nucleation and organization of microtubules and thereby plays a fundamental role in organizing the interphase
cytoskeleton and the mitotic spindle.33,34
The centrosome consists of three major domains, the most
prominent being the centriolar domain and the pericentriolar
domain35,36 (Figure 1). The centriolar domain defines the
center of the centrosome, and dependent of the stage of the
cell cycle contains either a single centriole or a centriole pair.
The pericentriolar domain encircles the centriole as a cloud
of pericentriolar material. These domains together have an
average diameter of approximately 1 ␮m in most animal cells.
The centrosome does not end at the edge of the pericentriolar
domain. Rather, it extends out and integrates with the surrounding cytoplasm and organelles through microtubules and
microfilaments. These outer reaches of the centrosome are
called outer centrosomal domain.
Figure 1
Schematic diagram of a centriole pair. Centrioles are
arranged at right angles and connected at their proximal ends by
fibrous material. The mature mother centriole consists of nine triplet
microtubules and has appendages and satellites at its distal end,
whereas the immature daughter centriole does not. Adapted with permission from Ref. 127.
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The centriolar domain contains the centriole, a barrelshaped, cylindrical organelle that is found in most, but not all
animal cells. Centrioles are structurally similar to basal bodies,
which are found at the base of eukaryotic cilia and flagella.37,38 In some organisms, the same structure acts as basal
body in interphase and centriole in mitosis. During fertilization, basal bodies of sperm function as templates for the
organization of maternal material into the first centrosome of
the egg.
A mature centriole consists of nine triplet microtubules. The
outer surface of the centriole is associated with matrix material
and electron-dense satellite structures along its length, a series
of appendages at the distal end, and during replication, an
immature or daughter centriole at its proximal end.
The functions of the centrioles within the centrosome
remain unclear. Plant cells, yeast, fungi and oocytes in some
embryonic systems are known to have centrosomes without
centrioles, suggesting that at least in some cases the centrioles
are not essential for centrosome organization and function.39
However, recent results demonstrate that centrioles are
required for organizing the centrosomal components into a
single stable structure in vertebrate cells.40 Since the reproductive capacity of a centrosome depends on its centriole
content,41 and since centrosomes lacking centrioles do not
reproduce in animal cells,42 centrioles likely play an
important role in centrosome reproduction, which must be
tightly controlled to maintain genetic stability.
Surrounding the centriole, the pericentriolar material contains a large number of coiled-coil proteins and functions as
the site for microtubule polymerization.36–38 One well-characterized component of the pericentriolar material is the ␥-tubulin ring complex, a conserved, ring-shaped multiprotein complex of all microtubule organizing centers that seems to serve
as a template for microtubule polymerization from closely
related ␣- and ␤-tubulins.43 The ␥-tubulin ring complex consists of ␥-tubulin and at least five other proteins, two of those
being Spc97p and Spc98p.44 Little is known about how ␥tubulin ring complexes are recruited to, and anchored at centrosomes in mammalin cells. However, pericentrin, a large
coiled-coil protein that is known to form a dynamic reticular
lattice in the pericentriolar material, has been implicated in
the recruitment of ␥-tubulin complexes to centrosomes in vertebrate cells.45 In addition to the ␥-tubulin ring complex
around 100 proteins have been described to be contained
within the centrosome. However, it is useful to consider only
those proteins as true components of the centrosome whose
localization is microtubule- and cell cycle-independent, since
increasing evidence suggests that several proteins may use
association with the centrosome to ensure proper segregation
at mitosis. The pericentriolar material is preferentially associated with mature centrioles. The size of this domain is cell
cycle-dependent, reaching its largest dimensions at the metaphase–anaphase transition and a low point at telophase.37
The centrosome has three important functions: (1) it
nucleates the polymerization of tubulin subunits into long
microtubule polymers; (2) it organizes the nucleated microtubules into useful arrays; and (3) it duplicates onces every cell
cycle, thereby licensing cell cycle progression from G1 into S
phase, as well as exit from cytokinesis. According to a current
model, it seems likely that microtubule nucleation and anchoring are two separate activities, mediated by different classes
of molecules. After being nucleated in the pericentriolar
material by ␥-tubulin ring complexes, microtubules are
released from their nucleating sites and translocated to sites
of anchoring at the subdistal appendages of the maternal cen-
triole, which contain ninein and centriolin as anchor proteins,
but lack microtubule-nucleating proteins.38,46,47
It has long been assumed that centrosomes are necessary for
mitotic spindle assembly. However, higher plants and some
developmental systems lack classical centrosomes but still
organize normal spindles48,49 and spindle assembly by noncentrosomal pathways has recently been demonstrated even
for mammalian cells.50,51 Therefore, centrosome-mediated
spindle assembly might provide a redundant pathway to
ensure high fidelity of chromosome segregation or guarantee
equal distibution of centrosomes to daughter cells for completion of other essential cellular functions by these
organelles.
In addition to classical centrosomal functions like microtubule nucleation and spindle assembly, recent studies with vertebrate cells suggest that centrosomes are required for somatic
cell cycle progression from G1 into S phase, as well as for
completion of cytokinesis. Microsurgical or laser ablation of
centrosomes from interphase cells does not prevent mitotic
spindle assembly, but leads to either incomplete cytokinesis
and, regardless of whether or not cytokinesis was completed,
to an arrest in late G1 phase of the following cell cycle.50,51
Another study also observed that the exit from cytokinesis in
Drosophila cells is dependent on presence and post-anaphase
repositioning of centrosomes.52 These studies indicate that
centrosomes might be involved in checkpoints that monitor
completion of mitosis prior to cytokinesis and the presence of
aberrant centrosome numbers or excess DNA prior to
initiation of DNA replication.53,54 Alternatively, centrosomes
might be directly required to activate final stages of cytokinesis and DNA replication.
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Centrosome replication
In animal cells, the interphase centrosome reproduces or
duplicates only once per cell cycle, thereby ensuring a strictly
bipolar mitotic spindle axis.55 Duplication of the single centrosome is initiated at the G1/S transition and completed before
mitosis, where the duplicated centrosomes play a role in
organizing the poles of the mitotic spindle. The centrosomes
are segregated at mitosis such that each of the two cells
resulting from division receives only one. Duplication of the
centrosome is semiconservative: the paired centriole splits and
a new centriole forms in association with each, creating two
centrosomes56 (Figure 2). Centrioles replicate during S phase
concurrent with DNA replication. During this time a small
procentriole bud forms adjacent to the proximal wall of each
parenting centriole, which then gradually elongates.57 By G2,
when the cell possesses a 4N DNA content, it also possesses
four centrioles arranged into two pairs referred to as diplosomes. Within each diplosome the mother and daughter centrioles are orthogonally oriented so that a line through the long
axis of the daughter points to the wall of the mother. This
relationship is then maintained through the ensuing mitosis.
During the next cell cycle, a slow process of maturation of
the daughter centriole takes place which transforms it into a
fully differentiated centriole apparently similar in all respects
to the mother centriole. In parallel, the centrosome matrix
changes in size and structure, displaying a varying number of
satellite structures, the significance of which is not clear.58,59
Recent studies indicate that many regulatory pathways
might control centrosome duplication. The most likely candidates for initiating centrosome replication during G1 phase are
extracellular signals. It seems reasonable to assume that the
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Figure 2
Centrosome reproduction cycle. Centrioles are represented as barrels. Grey shading represents the mature mother centriole (M). The immature daughter centriole (D) is displayed in white.
Adapted with permission from Ref. 36.
environmental signals responsible for activating centrosome
doubling would vary from cell to cell depending upon their
phenotypes. However, to date, the only extracellular ligand
that has been shown to affect centrosome doubling is epidermal growth factor (EGF) which has the capacity to activate
the entire repertoire of events necessary for centrosome replication in many cell types.60 The cellular pathways that are
activated following receptor stimulation remain to be defined.
However, recent studies suggest that the ultimate intracellular
target are cyclin/cdk2 complexes.61–64 The major difference
between studies utilizing embryonic cells and somatic cells is
the demonstration that cyclin E is the cdk2 partner in embryonic cells,61,62 while cyclin A is the cdk2 activating subunit
in somatic cells.63,64 This may simply indicate differences
between cell cycle regulation in embryonic and somatic cells.
For example, in embryonic cells, cyclin E is responsible for
activating DNA synthesis, whereas cyclin A fulfills that role in
somatic cell types.65 In addition, in embryonic cells cyclin E
levels do not oscillate during the cell cycle,66 an observation
that fits nicely with the demonstration that rounds of centrosome replication will occur in the absence of protein synthesis
in embryonic cells.67 In addition to cdk2 activation, centrosome duplication in somatic cells seems to be strictly dependent on phosphorylation of pRb and subsequent transcriptional activity of E2F64 (Figure 3).
One of the centrosomal substrates of cdk2 activity seems to
be nucleophosmin, a protein that associates with the centrosome during mitosis and thereby prevents centriole splitting
and duplication until late G1 phase, when cdk2 activity rises
and leads to phosphorylation and subsequent dissociation or
degradation of nucleophosmin from the centrosome.68
Another downstream phosphorylation target of cdk2 is the
mouse orthologue of yeast Mps-1p, whose kinase activity is
required for centrosome duplication.69 Phosphorylation of
Mps-1p by cdk2 leads to stabilization of Mps-1p protein levels
at the G1/S boundary, ensuring once more that centrosome
duplication is coordinated with S phase entry.
Apart from G1/S phase regulatory molecules, additional proteins appear to be involved in the decision to replicate centrosomes by somatic cells. Prominent among those are
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Figure 3
Regulation of the G1/S phase transition with subsequent
DNA replication and centrosome duplication in somatic cells. Phosphorylation of the retinoblastoma protein (Rb) by cyclin D-cdk4/6 and
cyclin E-cdk2 leads to transcriptional activation of E2F followed by
transactivation of the cyclin A promoter and other E2F target genes.
The activity of cdks is negatively regulated by cdk inhibitors like
p16INK4A, p21CIP1 and p27KIP1. Both DNA replication and centrosome
duplication are regulated through the pRb pathway. E2F transcription
factors are required for both processes. In addition to E2F, cyclin A
may be the preferred partner of cdk2 for centrosome duplication,
whereas cyclin E may be primarily responsible for promoting the G1/S
transition and initiating DNA replication.
NEK2/cNAP1, p53/p21KIP1, ZYG-1, Aurora kinases, and the
ubiquitin-mediated proteolysis pathway.
NEK2 is a centrosomal protein kinase that phosphorylates
cNAP1, a part of the structure linking parental centrioles in a
cell cycle-regulated manner, thereby inducing the splitting of
centrioles as the initial event of centrosome duplication in late
G1 phase.70,71 Mutational inactivation of the p53/p21CIP1 pathway,72,73 or STK15/BTAK/Aurora 2,74,75 a member of the Aurora family of protein kinases has been shown to induce multiple rounds of centrosome replication within a single cell
cycle, suggesting a role for these molecules in the regulation
of centrosome duplication. In Caenorhabditis elegans, a novel
kinase called ZYG-1 has been demonstrated to be necessary
for centrosome duplication.76 ZYG-1 mutant embryos failed
to form a bipolar spindle, even though cell cycle progression
appeared normal, resulting in the assembly of a monopolar
spindle at mitosis. Furthermore, it has been observed that
ubiquitin-mediated proteolysis is required for centrosome
duplication. Inhibition of the proteasome in Xenopus extracts
inhibited centrosome replication and centriole splitting, arguing in favor of the hypothesis that proteolysis is involved in
the degradation of connections between centrioles early in the
centrosome duplication process.77 On the other hand, contrasting data in mice and Drosophila have shown that knockouts of SCF ubiquitin ligase components result in centrosome
overreplication, suggesting that proteolysis prevents uncontrolled centrosome duplication.78,79 Additional studies are
needed to clarify the precise role of the proteins in the centrosome replication process.
Centrosome aberrations in human malignancies
Centrosomal abnormalities have been detected in several disease processes including neurodegeneration,80,81 autoimmune
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disorders,82 and viral or bacterial infections.83–85 They may
also play a role in aging.86 Most frequently, however, centrosome abnormalities are observed in cancer cells.
Karyotypic alterations, including whole chromosome loss or
gain, ploidy changes, and a variety of chromosome aberrations are common in cancer cells.3,87 If proliferating cells
fail to coordinate centrosome duplication with DNA replication, this will inevitably lead to a change in ploidy, and the
formation of monopolar or multipolar spindles will generally
provoke abnormal segregation of chromosomes. Indeed, it has
long been recognized that errors in the centrosome duplication cycle may be an important cause of aneuploidy and
thus contribute to cancer formation.1 This view has recently
received fresh impetus with the description of supernumerary
centrosomes in many solid human tumors including brain,88,89
breast,89–92 lung,89 colon,89 prostate,89 pancreas,93 bile duct,94
and head and neck.95 Hematological malignancies like nonHodgkin’s lymphomas and acute leukemias also display centrosome aberrations at high frequencies96,97 (Figure 4). Most
centrosome defects fall into three groups: aberrant microtubule nucleation, abnormal centrosome duplication, and a failure to correctly separate during mitosis. Centrosomes of tumor
cells display diverse structural alterations, including (1) an
increase in centrosome number and volume; (2) accumulation
of excess pericentriolar material; (3) supernumerary centrioles;
and (4) inappropriate phosphorylation of centrosome proteins.89,90 In addition, tumor centrosomes show functional
abnormalities characterized by increased microtubule
nucleation activity.90,98 There are at least two functional
consequences of centrosome defects that may play important
roles during neoplastic transformation and tumor progression.
First, aberrant centrosome duplication may adversely affect
maintenance of cell polarity in interphase cells because cytoplasmic architecture and directional vesicular trafficking may
be disorganized in a cell with multiple centrosomes. Second,
centrosome defects may increase the incidence of multipolar
mitoses that lead to chromosomal segregation abnormalities
Figure 4
Indirect immunofluorescence staining of normal bone
marrow (a) and acute myelogenous leukemia (b), mantle cell lymphoma (c) and Hodgkin’s lymphoma (d). Cells were immunostained
with an antibody to pericentrin, followed by a FITC-conjugated secondary antibody. In part adapted from Refs 96 and 97.
and aneuploidy. While normal-appearing metaphase plates
with bipolar spindles are typical in malignant tumors, aberrant
mitotic figures are not infrequent. A careful serial section electron microscopic study in which individual spindle microtubules were traced to the poles of breast cancer cells with
multipolar spindles revealed that particular spindle poles may
possess one, two, supernumerary, or no centrioles at all.91 In
some cases, individual tumor bipolar spindles were observed
to have microtubule foci containing supernumerary centrioles.
Thus, multiple centrosomes containing supernumerary centrioles can either lead to multipolar mitosis or through coalescence, may result in bipolar spindles. When a bipolar spindle
is produced, equal partition of sister chromatids is the anticipated outcome. On the other hand, multipolar or monopolar
spindles will result in aneuploid or polyploid daughter cells
with cell death being the most common outcome for aneuploid daughter cells that lack essential chromosomes. However, aneuploid cells with an appropriate complement of
chromosomes or parts thereof do survive and, in rare cases,
may acquire comparative growth advantage.99 Survival and
perpetuation of the transformed clone, however, requires an
additional step – the resumption of mitotic stability through
the assembly of a bipolar, not multipolar, spindle. Current
data support coalescence of multiple centrosomes into two
spindle poles as a mechanism for regulating the number of
functional centrosomes in tumor cells.91,98,100
The mechanistic significance of centrosome aberrations for
the induction of genomic instability has been a matter of
intensive debate. On one hand, it has been proposed that
abnormal centrosome numbers may directly cause mitotic
defects that lead to genomic instability. According to this
model, centrosome abnormalities emerge early during neoplastic progression. Alternatively, centrosome abnormalities
may simply reflect genomic instability and accumulate in parallel with other cellular abnormalities. These two models have
different implications for the diagnostic and prognostic value
of centrosome abnormalities in human tumors. Importantly,
it has been demonstrated recently that expression of human
papilloma virus (HPV) 16 E7 oncoprotein, which binds to and
destabilizes the Rb tumor suppressor protein, is associated
with an abnormal synthesis of centrioles and induces abnormal centrosome numbers and genomic instability early during
neoplastic progression in primary human epithelial
cells.101,102 Consistent with these observations, preinvasive as
well as invasive HPV-associated genital lesions contain abnormal centrosome numbers that are associated with mitotic
abnormalities and numeric chromosomal alterations.101 Similarly, the development of pancreatic cancer in transgenic mice
expressing the simian virus 40 tumor antigen, which inactivates Rb and p53 proteins, is characterized by supernumerary
centrioles and the sequential appearance of tetraploid and
then multiple aneuploid cell populations.103
As a second line of evidence, centrosome defects have been
found in a fraction of preinvasive cancers of the prostate and
breast, as well as in some adenomas of the pancreas.93,104 In
an orthotopic implantation model of human pancreatic cancer
cells into pancreata of nude mice centrosome abnormalities
were detected in only a small fraction of parental cells in culture, whereas the frequency markedly increased in cells isolated from the pancreatic xenografts.105 Abnormal centrosome
numbers were found at even higher frequencies in metastatic
foci, as compared to the pancreatic xenografts. In addition,
centrosome abnormalities occur at a higher frequency in
advanced stages as compared to early stages of different
malignancies.94,95,104 In mantle cell lymphoma, the frequency
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772
of centrosome aberrations correlates with tetraploidization of
clinically aggressive blastoid variants.96 Furthermore, centrosome abnormalities were detectable at a significantly higher
frequency in aggressive diffuse large B cell lymphomas as
compared to indolent follicular lymphomas, suggesting that
abnormal centrosomes may contribute to the aquisition of an
increasing karyotypic instability typically seen in aggressive
lymphomas.
A comparison of mismatch repair deficient diploid vs mismatch repair proficient aneuploid colorectal carcinoma cell
lines revealed the exclusive occurrence of centrosome amplification and instability in all of the aneuploid tumor cell lines
analyzed.106 All diploid tumors contained centrosomes that
were functionally and structurally indistinguishable from those
in normal human fibroblasts.
Taken together, these observations support the notion that
the integrity of the centrosome plays a central role in the
development of genomic instability.
Molecular scenarios for the origin of centrosome
aberrations
The detailed mechanisms by which centrosome aberrations
develop in malignant tumors are largely unknown. Because
the centrosome is the microtubule organizing center of
interphase cells, and because this organelle must duplicate
and separate in G1 phase ultimately to become the poles of
the mitotic spindle, several lines of evidence point to a G1
phase cell cycle checkpoint that ensures proper centriole
duplication and separation.61–64,107 Accordingly, defects of
G1/S phase regulatory proteins should not only lead to unrestrained proliferation, but also cause karyotypic instability by
uncontrolled centrosome replication.
A physiological example of centrosome hyperreplication
supporting this view are endomitotic cell cycles of megakaryocytes. In order to become polyploid, these cells replicate
their DNA without karyokinesis and cytokinesis. Megakaryocytes undergo a normal cell cycle progression with G1, S and
G2 phases, nuclear membrane breakdown, sister chromatid
separation, and chromosome pole to pole movement.108
However, late mitosis stages like anaphase B, telophase and
cytokinesis are skipped, resulting in cells that contain a single
polylobulated typical nucleus with a 2xN ploidy. Microscopic
examination shows that a large number of centrosomes is
present in polyploid megakaryocytes. These findings have
been explained by the sustained expression of the G1 cyclins
D3, A and E, accompanied by pRb hyperphosphorylation at
least in megakaryocytic cell lines.109,110 Most interestingly,
careful analysis of multipolar endomitotic spindles revealed
an asymmetrical, aneuploid distribution of chromosomes
between the spindle poles and, subsequently, the different
lobes of interphase megakaryocyte nuclei despite an intact
metaphase/anaphase checkpoint.111 These findings suggest
that centrosome hyperreplication during G1/S phase by upregulated function of cdk2 indeed leads to aberrant chromosome distribution during mitosis.
Recently, the multiple-centrosome phenotype has been
recapitulated in budding yeast by disrupting the activity of
specific cdk complexes.112 In budding yeast, nine different
cyclins which associate with a single cdk, p34Cdc28, can be
divided into three groups – three G1 cyclins, two S phase Bcyclins, and four mitotic B-cyclins. Analogous to the findings
in eukaryotic cells, spindle pole body (SPB) duplication is
initiated by G1 cyclins. For SPB reduplication, however, S
Leukemia
phase B-cyclin activity seems to be necessary, whereas mitotic
B-cyclins inhibit SPB reduplication to ensure the dependence
of SPB reduplication on the completion of mitosis and subsequent down-regulation of mitotic cyclin/cdk activity. Similarly, in Xenopus, centrosomes were found to reduplicate in
the continuous presence of cyclin E/cdk2 activity when the
mitotic cyclin A/cdk1 and cyclin B/cdk1 activities were
absent.50 Xenopus extracts depleted for cyclin E/cdk2 could
duplicate, but not reduplicate centrosomes, indicating that an
activity analogous to yeast G1 cyclins – most likely cyclin
D/cdk4 – may be responsible for the initial duplication of the
centrosome in Xenopus.
In line with these observations, both inactivation of pRb and
overexpression of cyclin E have been shown to induce abnormal centrosome synthesis and genomic instability.101,113,114
Overexpression of cyclin A, E2F-2, and E2F-3 also leads to
multiple rounds of centrosome duplication.64 Also, overexpression of HPV-16 E6 and E7 oncoproteins, which inactivate p53 and pRb, has been shown to induce centrosome
aberrations with subsequent genomic instability.101,102,115 On
the other hand, overexpression of p16INK4A, p21CIP1 and
p27KIP1 is able to block centrosome duplication.63,64,116
Mutational inactivation of p53 has also been described to
initiate multiple rounds of centrosome replication within a single cell cycle.72,114,117,118 This effect seems to be mediated at
least in part through the p53/p21CIP1 axis, since induced
expression of the cdk2 inhibitor p21CIP1 in p53−/− cells partially restores centrosome duplication control, suggesting that
p21CIP1 is one of multiple effector pathways of the p53mediated regulation of the centrosome duplication cycle.73 In
line with these reports, reduced p21CIP1 expression as well as
overexpression of the p53 inhibitor mdm2 results in centrosome overduplication, gross nuclear abnormalities and polyploidy in human cells.106,117–119 In addition, centrosome aberrations and chromosome instability resulting from mutational
inactivation of the tumor suppressors BRCA1 and BRCA2
might also be mediated by interaction and inactivation of the
p53/p21CIP1 axis.120–124 Interestingly, in prolonged cell culture,
primary epithelial cells from p53-null mice display centrosome hyperamplification and numerical chromosome instability at high frequency in early to mid passage cells.118 Both,
heterogeneity in the number of chromosomes and centrosome
amplification diminish in late passage cells, giving rise to distinct subpopulations of cells which might have aquired chromosome compositions that promise an optimal growth in a
given environment.
Defects which lead to centrosome amplification, aneuploidy and transformation in vitro without aparent association to
G1/S phase regulatory events include overexpression of centrosome-associated kinases, such as breast tumor amplified
kinase (STK15/BTAK/Aurora 2).74 This protein is found to be
overexpressed in multiple human tumor types.125 Overexpression of the centrosomal protein pericentrin in prostate
cell lines has also been shown to lead to severe centrosome
and spindle defects, cellular disorganization, enhanced soft
agar growth and genomic instability.104 Highly significant
increases in chromosome missegregation associated with centrosome fragmentation have also been described in cell lines
lacking the mammalian DNA damage repair genes XRCC2
and XRCC3.126
In summary, centrosomes are structurally and numerically
abnormal in the vast majority of human malignancies. Observation of centrosome aberrations in preinvasive cancers, correlation of the extent of centrosome abnormalities with the
extent of chromosome instability, and centrosome aberrations
Centrosomes and cancer
A Krämer et al
preceding other manifestations of genomic instability in
experimental systems all argue in favor of a role of centrosome
abnormalities in the development of karyotype instability in
human cancers. Since centrosome replication has been found
to be ultimately controlled by pRb and cdk2 activity, the commonly observed abrogation of the pRb pathway in human
tumors will not only facilitate progression towards DNA replication, but may also deregulate the centrosome duplication
cycle and thereby lead to genomic instability. However, a
direct link between centrosome aberrations and cancer has
still to be established. A better understanding of how centrosomes contribute to cell cycle regulation and aneuploidy may
help to define novel therapeutic strategies to suppress genomic instability at early stages of malignant transformation.
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