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Leukemia (2002) 16, 767–775 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu 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 Centrosomes and cancer A Krämer et al 768 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 Leukemia 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. Centrosomes and cancer A Krämer et al 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. 769 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 Leukemia Centrosomes and cancer A Krämer et al 770 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 Leukemia 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 Centrosomes and cancer A Krämer et al 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 771 Leukemia Centrosomes and cancer A Krämer et al 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. 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