Mechanisms of chromosome instability in cancers

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1 Critical Reviews in Oncology/Hematology 59 (2006) 1 14 Mechanisms of chromosome instability in cancers Charles Edward Jefford a,b, Irmgard Irminger-Finger a,b, a Biology of Aging Laboratory, Department of Geriatrics, University Hospitals Geneva HUG, Switzerland b Department of Gynecology and Obstetrics, Maternité, University Hospitals Geneva HUG, Switzerland Accepted 22 February 2006 Contents 1. Introduction Cancer is a complex disease Why cancer occurs in humans Principles of cancer development Causes of tumour formation, mutation rate and clonal origin of tumours Genetic instability Genetic instability as the cause of tumorigenesis Types of genetic alterations in tumours Types of genetic instability Microsatellite instability Chromosome instability Mechanisms maintaining chromosome integrity Cellular mechanisms involved in maintaining genome stability DNA repair pathways involved in genome stability Telomere maintenance as a mechanism of chromosome stability Role of telomeres and telomerase in senescence The role of telomerase in immortalization Telomeres and telomerase in tumours Telomere instability Maintenance of chromosome integrity by mitotic checkpoints Defects of chromosome segregation Centrosome aberrations Conclusion Reviewers Acknowledgements References Biographies Abstract Most tumours arise through clonal selection and waves of expansion of a somatic cell that has acquired genetic alterations in essential genes either controlling cell death or cell proliferation. Furthermore, stability of the genome in cancer cells becomes precarious and compromised because several cancer-predisposing mutations affect genes that are responsible for maintaining the integrity and number of chromosomes during cell division. Consequently, the archetypical transformation in tumour cells results in aneuploidy. Indeed, almost all tumour cells display a host of karyotype alterations, showing translocations, gains or losses of entire or large parts of chromosomes. Cancers do not necessarily have a higher mutation rate than normal tissue at the nucleotide level, unless they have gained a mutator phenotype through exposure to Corresponding author at: Department of Gynecology and Obstetrics, Maternité, University Hospitals Geneva HUG, 30 Blvd de la Cluse, 1211 Geneva, Switzerland. addresses: Charles.E.Jefford@hcuge.ch (C.E. Jefford), Irmgard.irminger@hcuge.ch (I. Irminger-Finger) /$ see front matter 2006 Published by Elsevier Ireland Ltd. doi: /j.critrevonc

2 2 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) 1 14 environmental stress, but rather exhibit gross chromosomal changes. Therefore, it appears that the main mechanism of tumour progression stems from chromosome instability. Chromosomal instability prevailing in tumour cells arises through several different pathways and is probably controlled by hundreds of genes. Therefore, this review describes the main factors that control chromosome stability through telomere maintenance, mechanisms of cell division, and the mitotic checkpoints that govern centrosome duplication and correct chromosome segregation Published by Elsevier Ireland Ltd. Keywords: Cancer; Chromosome instability; Aneuploidy; Telomeres; Centrosomes; Cell-cycle checkpoints; DNA repair 1. Introduction 1.1. Cancer is a complex disease There are probably more than a 100 different types of cancer emerging in numerous ways, forming many distinct subtypes in each affected organ. The rate and scope of tumour progression may evolve according to the biological, immunological, environmental and genetic background of the person afflicted, thereby adding to the intricacy of the aetiology of the disease. Furthermore, the complexity of the disease increases with the continuous discovery of novel tumour suppressor genes, mutations, molecular pathways and tumour markers Why cancer occurs in humans Several hundred genes, representing 1% of the human genome, have been implicated in tumorigenesis and progression [1]. Yet, looking at the chaotic genome and the aberrant gene expression pattern of cancer cells, one can assume that tumour cells probably require only a small number of essential genes for cell proliferation. Lower multicellular organisms, such as Drosophila melanogaster and Caenorhabditis elegans, do not develop cancers, because cells rapidly acquire a post-mitotic phenotype. On the other hand, evolution has created higher complex organisms that are composed of both post-mitotic and mitotic somatic tissues. Mitotic tissues are renewable, permitting development of differentiated tissue, and the replacement of pathological and dying cells. Furthermore, embryogenesis, tissue development and cellular differentiation require precise expression of a complex array of genes, tightly controlling cellular proliferation, mitosis, chromosome segregation, cell survival mechanisms, cell-cycle arrest and programmed cell death. However, the high cellular proliferation rate in renewable tissues jeopardizes the integrity of the genome, because the probability of acquiring and propagating deleterious mutations contributing to malignant transformation increases over time. Therefore, complex organisms are prone to cancer. They will inexorably be afflicted with cancer in advanced age, since cancer incidence increases with age [2,3]. In order to curb uncontrolled continuous cellular proliferation and to preserve the genome from acquiring adverse mutations, complex organisms with renewable tissues requiring homeostasis through regulated cell death, have developed several tumour suppressor pathways. These tumour suppressor pathways are of two types, involving gatekeeper and caretaker mechanisms. Gatekeeper proteins prevent unwanted cellular growth by inducing the elimination of potential cancer cells, and caretaker (or stability) proteins protect the genome from accumulating oncogenic mutations [4]. Therefore, because human beings are composed of mitotic tissues and since mutations occur continuously, they are bound to develop at some point faulty gatekeeper or caretaker functions Principles of cancer development Despite the complexities inherent in tumour development, most cancers have several common traits and may be described in terms of a few simple underlying principles. Cancers are classically diagnosed from clinical symptoms, systemic factors, histological examination of tumour biopsies, cytogenetic analysis of metaphase chromosome spreads, cellular phenotypes, and molecular genetics. Indeed, human cancers may be studied and examined at various levels of investigation. At the clinical level, cancers are usually recognized by clinical symptoms, such as abnormal tissue function and size, and can be visualized by conventional radioscopic methods. At the tissue level, and after histological examination of sections from tumour biopsies, cancers are characterized by altered tissue morphology, structure and growth, metastasis, angiogenesis and lymphangiogenesis. At the systemic level, changes in leukocyte levels may be monitored, and circulating factors may be associated with tumours. At the cellular level, for instance, cancer cells can be distinguished by abnormal centrosome numbers and conspicuous morphological changes. Furthermore, many common traits are found also at the protein level, especially in pathways that regulate replicative lifespan of cells. Remarkably, most cancer cells have either telomerase activity (90%) [5,6], inactivated p53 and prb pathways (50%), or abrogation of the INK4a- ARF locus (30%) [7]. Most importantly, the final diagnosis for cancer lies at the nuclear level. Cytogenetic analysis of cultured tumour cells reveals that most solid tumours have aneuploid karyotypes, i.e. aberrant nuclei with altered chromosome numbers and structure. These karyotypic alterations can be of numerous types and have been well documented [8,9]. They will be discussed further below. All these characteristics are the hallmarks of neoplasia and are overt manifestations of cancer cell genotypes reflecting defects in cell physiology. These defects have been described elsewhere, and have been grouped in six main physiological changes which consist of novel capabilities acquired during tumour development: self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of programmed cell death or apoptosis, limitless replicative

3 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) potential, sustained angiogenesis, and lastly, tissue invasion by metastasis [10]. Moreover, tumour tissues are complex, since they are composed of other non-cancerous cells like fibroblasts, immune cells, and endothelial cells forming neovasculature, which are all subverted for neoplastic growth. Nevertheless, cancers are the result of uncontrolled cellular growth with catastrophic repercussions at the tissue level or at the systemic level with the development of metastasis. Although there are differences between malignant and benign cancers, all tumours have defects in cellular proliferation. Therefore, described simply, tumorigenesis at the cellular level can either occur through a loss of function of tumour suppressor proteins that trigger cell death (or apoptosis), or through a dominant gain-of-function of cell survival and/or cell proliferation signals, e.g. through overexpression of oncogenes [11] Causes of tumour formation, mutation rate and clonal origin of tumours In essence, the unifying trait of most cancer cells is their altered karyotype, but the unifying principle of tumour development is gene mutation. All phenotypic changes in cancer cells stem from aberrant gene expression. Neoplasia is undoubtedly a genetic disease arising either from inherited mutations or from de novo mutations in somatic tissue. It is commonly accepted that malignant transformation is a lengthy multi-step process and arises through an accumulation of mutations at various genetic loci. However, the causes for genetic change or for high mutation rates, accountable for increased tumour incidence, are of different types: environmental stress, cigarette smoke, irradiation, various genotoxic insults, such as chemical carcinogens, immunological deficiencies, hormones, growth factors, oncoviruses or bacteria. Some of these factors can considerably increase the incidence of tumours in a population, for instance lung cancer in smokers. Nevertheless, increased mutation rates at the nucleotide level or presence of mutator phenotypes are not a prerequisite for tumorigenesis, since they might be the same in tumours and normal tissue. It has been demonstrated that selection of pre-existing mutations, and not increased mutation rates, is sufficient to explain the evolution of tumours [12]. Even though DNA tends to maintain remarkable stability, the incessant process of evolution and natural selection would not be possible without a means of modifying the genome. The plasticity of the genome is a prerequisite for the evolution of species. A form of permissive mutability needs to occur at the nucleotide level and at the chromosome level in germ cells in order for evolution to proceed. This is processed by recombination events during meiosis. Therefore, speciation and species evolution could be a consequence of a mild form of genetic instability in germ cells. Nevertheless, survival of the fittest genes, and the fittest individual, or species, is the consequence of Darwinian natural selection of mutations that are carried down in germ cells. In the same way, natural selection might enrich advantageous somatic mutations into an organism. Although, cancer mutations usually occur in pair-wise fashion, cellular immortalization of cancer cells is a natural consequence of selecting viable mutations that favor cell proliferation. Hence, cancer may be regarded as an evolutionary consequence of genetic instability in somatic cells [13]. To summarize, tumour development might be a selective process for continued proliferation. Tumours are clonal because they may derive from a single progenitor cell [14]. 2. Genetic instability 2.1. Genetic instability as the cause of tumorigenesis One of the main causes of cancer, if not the common primordial cause, is high genetic change or genetic instability. But what is genetic instability? Genetic instability is a transient or a persistent state that causes a series of mutational events leading to gross genetic alterations. It is now clear that most cancers have altered genomes. Genetic instability has been found in many types of cancer. This instability can be observed by the heterogeneity in karyotypes within each tumour of the same type and in different parts of the same tumour. Other cancer cells may have a dynamic instability. They consist of cells with continuous transformation capabilities that may undergo clonal selection. Cancers may also be polyclonal [15]. The question whether genetic instability is a cause or a consequence of tumorigenesis has been debated for years. However, it now appears that genetic instability not only possibly drives tumorigenesis, but is at least a factor in tumour progression [16]. One could hypothesize that widespread genomic instability in a give tissue could rise to a clonal event of carcinogenesis (Fig. 1) Types of genetic alterations in tumours Most cancer cells harbour genetic alterations. These genetic alterations consist of abnormal chromosome numbers (aneuploidy, polyploidy) and structural changes. The structural changes may occur either at the chromosome level (translocations, gains or losses of large parts of chromosomes) or at the nucleotide level affecting gene structure or expression (mutations, deletions, gene amplifications, microsatellite amplification, gene silencing by epigenetic effects). The various types of genetic alterations responsible for tumour development may be divided into five groups: (a) At the molecular level, subtle sequence changes involve base substitution, deletions or insertions of nucleotides. For instance, 80% of pancreatic cancers have missense mutations in the K-ras gene. (b) At the genome level, faulty mismatch repair pathways (e.g. human hereditary colorectal cancers) generate microsatellite instability with the appearance of short DNA repeats that are scattered all over the human genome.

4 4 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) 1 14 Fig. 1. Diagram showing the pathways that link genotoxic stress and genomic instability to cancer. The accumulated mutations acquired over time due to increased genetic instability may be stored in either gametes or somatic cells. The somatic and inherited genetic alterations involved in genomic instability incur caretaker functions such as DNA repair and maintenance programs and cell-cycle and mitotic checkpoints which both preclude high telomerase activity but only somatic cells require gatekeeper dysfunctions for tumour propagation. The mutations allowing genomic instabilities to occur are selected in both gametes and somatic cells through Darwinian-like selection. (c) At the chromatin level, epigenetic changes may occur such as gene silencing by DNA methylation (e.g. inactivated BRCA1 in sporadic breast tumours by promoter methylation) or gene amplification (e.g. N-myc occurring in 30% of advanced neuroblastoma cells). (d) At the chromosome level, illegitimate mitotic inter- or intra-chromosome recombination may result in neomorphic (gain-of-function) or hypermorphic (overexpression) mutations that cause neoplasia. Most recombinations in cancers are translocations. In haematological malignancies, the phenotype at the molecular level stems either from the abnormal expression of fusion proteins or the overexpression of growth related factors when translocations relocate genes in the vicinity of highly expressing promoters. The best-known example is the Philadelphia chromosome in chronic myelogenous leukemia (CML), whereby a fraction of chromosome 9 is fused to chromosome 22, causing the aberrant expression of a neomorphic tyrosine kinase (BCR-ABL). (e) At the chromosome level, most tumours show structural and numerical chromosomal rearrangements resulting in gains or losses of large chromosomal parts or even whole chromosomes leading to aneuploid karyotypes. Some cancer cells may produce polyploid genomes Types of genetic instability The term genetic instability is generic, since it describes any genetic change over time, which encompasses nucleotide alterations, occurring at the gene level, and genomic instabilities, occurring at the chromosome level. Nucleotide alterations are mainly due to faulty or leaky DNA repair pathways such as nucleotide excision repair (NER) and base excision repair (BER), described as nucleotide instability (NIN). Genomic instability reflects the dynamic change at the genome level and refers mainly to two types of changes: (A) microsatellite instability (MIN) affecting chromatin structure and gene expression, and (B) chromosomal instabilities (CIN) that are responsible for aneuploidy and translocation events and account for most of the chromosomal defects in tumour cells [8]. Interestingly, genetic instability syndromes in humans are often related to progeria syndromes and predisposition to cancer [17,18] Microsatellite instability Microsatellite instability (MIN) is found in many tumour types, but has been studied mostly in familial colorectal cancers. It consists of an accumulation of a wide array of poly-a tracts and poly-ca repeats randomly inserted all over the genome disrupting gene sequences [19 21]. It was also discovered that most patients with hereditary non-polyposis colorectal cancer (HNPCC) acquire MIN. It was confirmed that HNPCC tumours have mutations in either hmsh2 on human chromosome 2 or in hmlh1 on chromosome 3 [22,23]. Several further studies have demonstrated that MIN has deficiencies in mismatch repair pathways [24,25]. Mutations due to faulty NER result in a nucleotide instability syndrome (NIN) as exemplified by patients with Xeroderma pigmentosum [18]. These patients have defects in DNA repair, are

5 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) sensitive to sun light and UV exposure, and are prone to skin cancers Chromosome instability Chromosome instability (CIN) is defined by continuous and conspicuous changes in chromosome structure and number. These mutations produce either gains or losses of whole or large portions of genomic material at an accelerated rate [26]. The genetic change leading to aneuploidy was termed chromosomal instability as an alternative and a specific form of genetic instability. This ongoing process of karyotypic change reflects high mutability of cancer cells sieved through the pressure of selective growth advantage. A single karyogram of a randomly selected human epithelial cancer cell, bearing an aberrant karyotype, may easily encompass the whole catalogue of structural and numerical chromosome anomalies encountered in human cancers [27]. These cancers can either show heterogeneous karyotypes within a single tumour sample or also include areas with homogeneous karyotypes, suggesting both a constant genetic change and a clonal evolution of cancers. The heterogeneity in the karyotypes of these cells would typically display a CIN phenotype. The mutation rate, which can be measured in a culture of tumour cell line evolving from a single clone, is accelerated in CIN. The accelerated rate of mutability of chromosome alterations, which defines CIN, alters dramatically the expression of thousands of genes and increases the probability of cellular dedifferentiation and malignancy. This fact could explain why CIN tumours have a poorer prognosis than MIN or NIN tumours. Strikingly, the most frequent type of chromosomal imbalances, due to CIN found in cancer genotypes, are translocations [1], evoking the propagation of breakagefusion-bridge cycles described by McClintock in It is noteworthy, that some karyotypic alterations initiated by CIN can remain stable, probably because they have acquired some form of balance between genomic stability and proliferation signals [28]. 3. Mechanisms maintaining chromosome integrity 3.1. Cellular mechanisms involved in maintaining genome stability Without making an exhaustive list of all the putative molecular protagonists responsible for genomic instability, we will review the main components and some of the pathways they involve. Many of the mechanisms underpinning chromosomal instability and aneuploid karyotypes in cancer cells still remain shrouded in mystery. However, a number of pathways implicated in cancer prevention through maintenance of chromosome stability, are well known and have been extensively described. These pathways concern basically two categories involving: (A) fidelity in DNA replication and (B) cell division pathways. Both categories may be subdivided in several distinct sub-groups: (A) DNA replication mechanisms that include: (a) DNA repair pathways responsible for keeping genes and genome intact; (b) chromatin epigenetic modification pathways; (B) maintenance of mitotic stability and chromosome integrity includes pathways implicated in: (a) telomere and centromere stability; (b) defects in cell cycle checkpoint proteins and kinases; (c) the regulation of those proteins by post-translational modification via ubiquitin pathways; (d) sister chromatid cohesion and chromosome segregation; (e) centrosome duplication. Furthermore, chromosome instability is also due to other various defective pathways implicated in chromosome segregation and mitotic checkpoint failures as well as telomere dysfunction, all of which will be discussed in detail below DNA repair pathways involved in genome stability Genome maintenance mechanisms prevent cancer through the well-characterized DNA repair pathways safeguarding the genome from deleterious mutations [29,30]. The main DNA repair mechanisms described are: (a) nucleotide excision repair (NER) after single strand breaks (SSB) and during transcription-coupled repair (TCR); (b) base excision repair (BER); (c) homologous recombination (HR); (d) non-homologous end joining (NHEJ); and mismatch repair (MMR). Most of these pathways are highly conserved through evolution, and across species, and must have arisen early in evolution. It should be noted that there is a considerable overlap of components in these aforementioned DNA repair pathways. For instance, NER and TCR share practically the same pathway. Furthermore, both HR and NHEJ pathways make use of the MRE11/RAD50/NBS1 complex, as well as the ATM and ATR, stress response kinases. Inherited defects in any of these pathways may severely predispose to genomic instability and to cancer predisposition, because they may impinge on several DNA repair mechanisms. Also, any mutation acquired in a somatic cell in one of the aforementioned pathway through favourable clonal selection could potentially increase mutation rate and produce a mutator phenotype. This would increase genomic instability and increase the probability of neoplastic transformation. However, the DNA repair pathways involving NER, BER, and MMR occur mostly in MIN and NIN, and probably do not account for CIN. It is likely that CIN accounts for almost all abnormal karyotypes found in tumour. Genomic instability can be produced through various defective pathways as indicated by the causal factors of several well-characterized genetic instability syndromes [29]. 4. Telomere maintenance as a mechanism of chromosome stability 4.1. Role of telomeres and telomerase in senescence Telomeres are complex ribonuclear protein structures, situated at the end of all functional eukaryotic chromosomes

6 6 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) 1 14 [31]. They were first described by Hermann J. Müller in 1938 and later by Barbara McClintock in 1941 as being essential structures for maintaining unity of chromosomes during segregation [32]. It was proposed that telomeres protect the ends of chromosomes from fusing, since dicentric chromosomes fused at their termini would break during segregation at various points. Fusion of non-protected DNA ends engages a breakage-fusion-bridge cycle either resulting in gene amplification or deletion. Therefore, telomeres are structures that may be distinguished from ordinary DNA breaks, and elude normal DNA repair pathways. Telomeres were first cloned and sequenced by Elizabeth Blackburn and colleagues and are composed of a high number of repeats of an hexanucleotide element, TTAGGG, stretching to about 4 15 kb in length in human cells and are capped with numerous associated proteins. The end-replication problem enunciated by Watson and Olovnikov in 1972 and 1973, reviewed recently [32], whereby a portion of telomeric DNA is lost after each round of cell division, is due to the inability of the DNA polymerase alpha to add nucleotides at the very ends of linear DNA molecules. The result is that telomeres lose about bp after each round of replication. It explains cellular replicative senescence as described by Hayflick in 1965 [33]. Subsequently it was shown that the onset of replicative senescence is imparted by persistent shortening of telomeres in the absence of functional telomerase after each round of cellular division [34]. Telomere erosion may also occur through other mechanisms, such as genotoxic insults, and oxidative stress [35 37]. Specifically, it is the G-rich (TTAGGG) 3 -single-strand overhang that is eroded through replicative senescence, rather than the overall shortening of the double-strand repeats of the telomere structure that signals cellular senescence [38]. Short telomeres are recognized by DNA damage checkpoints such as ATM/ATR kinases and trigger cell senescence pathways by eliciting p53 and p16 INK4a /prb activation and apoptosis [39,40]. The telomerase enzyme is composed of two main subunits necessary and sufficient for basic telomere elongation: the reverse transcriptase component (htert) and the ribonucleo-protein moiety (hterc or htr) containing an RNA strand, which partly serves as a template for the repeated addition of hexanucleotidic elements. The other proteins involved in the regulation of telomerase function (dyskerin, Est1 A/B) are not necessary for telomere elongation per se [41]. The RNA component (htr or TERC) of the complex is ubiquitously expressed in all human cells. On the other hand, whereas telomerase activity is a characteristic of germ cells, embryonic stem cells, some adult haematopoietic cells, and some human fibroblasts, it is repressed in normal somatic cells. Consequently, telomere length tends to decrease with age in somatic cells, but it may be maintained in human sperm [42]. However, telomerase activity may be found in normal haematopoietic and human fibroblasts demonstrating that telomerase may also be a central regulatory element in controlling the replicative lifespan of normal human cells [43,44]. Furthermore, in certain situations telomerase has no effect on telomere elongation. Indeed, in B and T lymphocytes, it was shown that telomere lengths continue to shorten even in the presence of active telomerase [45 47] The role of telomerase in immortalization It appears that telomerase activity is a necessity for the survival of the human species because it replenishes the chromosome ends in germ cells, stem cells and in certain highly proliferative tissues [48]. Telomerase activity specifically adds the G-rich single-strand overhangs to telomeres [38]. Most human tumour cells and immortal cell lines in vitro may proliferate indefinitely, i.e. beyond the natural Hayflick limit and beyond crisis by keeping their chromosome ends long enough for limitless rounds of replication. This is demonstrated by exogenous expression of htert, i.e. the catalytic subunit of the human telomerase holoenzyme, restores telomerase activity, induces telomere elongation, which extends replicative lifespan of normal human cells thus bypassing cell senescence and crisis pathways [49,50]. Moreover, the long term forced expression of telomerase is sufficient for immortalization, but does not induce a cancer phenotype such as karyotypic alterations or loss of contact inhibition [51]. In addition, it is possible to transform normal human fibroblasts into tumorigenic cells by transfecting them with htert, SV40 large-t antigen and Ras oncogene expression [52]. This experiment identifies htert as a purported component of an oncogenic transformation pathway. Especially since the transformed cells are thought to have functional p53, p16 INK4a, and prb pathways. Therefore, it is likely that htert expression contributes directly to oncogenesis by permitting pre-cancerous cells to proliferate beyond their natural Hayflick limit. It has also been shown that overexpression of htert in human cells with chromosomal instability or progeria syndromes, such as Roberts (RBS), Werner (WRN), Bloom (BLM), Xeroderma pigmentosum (XP), and Hutchinson-Guilford progeria syndrome (HGPS) leads to immortalization [53]. The establishment of these immortal cell lines was performed without any prior viral transformation. On the other hand, inhibition of telomerase expression through knockout of gene coding for the RNA component (mtr) of telomerase in mice is associated with severe telomere shortening and increased tumour formation [54]. Since mouse telomeres are long, several generations are required to induce cellular senescence and telomere instability syndromes. Similar results were obtained by knocking out the reverse transcriptase component (mtert ) in mice [55] Telomeres and telomerase in tumours Strikingly, it was found that about 85 90% of human cancers can maintain telomere length by abnormally reexpressing telomerase, regardless of the state of telomere

7 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) length [5,6,33,56]. Although most tumours possess telomerase activity, telomere length in tumour cells has been reported to be drastically shorter than that of surrounding cells from healthy tissue [57 59]. Telomere erosion normally leads to senescence and cell death, but in tumour cells telomeres become dysfunctional. Dysfunctional telomeres may recombine and fuse thereby initiating random chromosome breakage and formation of dicentric chromosomes, thus increasing chromosome instability and probability of oncogenesis [54,60]. The remaining cancers, which are negative for telomerase expression, also maintain telomere length, by using a different mechanism, appropriately named alternative lengthening of telomeres (ALT) [6,61 63]. The ALT mechanism probably takes advantage of DNA repair pathways and recombination events for maintenance of telomere length [64]. Telomere length is heterogeneous in ALT cell lines compared to telomerase positive cell lines [64]. The ALT mechanism has so far been identified in vitro but has been recently related to human tumours. It appears to be a dysregulated version of an aspect of normal mammalian telomere homeostasis which requires the MRE11/RAD50/NBS1 molecular components [65]. Nevertheless, it appears that maintenance of telomere length performed either by telomerase expression or by telomerase-independent mechanisms, is a prerequisite for tumour progression and cellular immortalization. Indeed, ALT fibroblasts immortalized with SV40 large T- antigen only require oncogenic Ras V12G to convert to a tumorigenic phenotype [66]. Thus, ALT cell lines do not need telomerase for malignant transformation. Although the ALT mechanism does not substitute for telomerase activity, it may contribute to chromosomal instability and tumour progression by a telomere length-independent mechanism [67,68]. The importance of telomerase activity in tumour cells raises hopes for novel cures, because telomerase inhibitors could be prospective drugs in cancer chemotherapy. The usefulness of telomerase inhibition has been demonstrated, because the abrogation of telomerase expression leads to telomere shortening, apoptosis and to inhibition of cancer growth [69 72]. However, one caveat is that abrogation of telomerase activity in mtr knockout cells are not resistant to oncogenic transformation by constitutive expression of Ras V12G [54], possibly limiting the effectiveness of these drugs. Moreover, telomerase is also a regulatory component of certain normal fibroblasts and other haematopoietic cells [44 46], which implies that telomerase inhibitors may have a detrimental effect on the lifespan of normal human cells. Nonetheless, a novel telomerase inhibitor, GRN163L, has shown to be a promising cancer therapy agent because it efficiently decreases telomere length and diminishes growth of breast cancer cells [73,74]. Other anti-telomerase drugs could target expression of negative regulators of htert transcription, such as MAD1, a repressor of c-myc, TGF-, which interacts with SIP1, and Menin, which binds directly to the htert promoter [75] Telomere instability Telomere maintenance is a vital mechanism in cellular immortalization and tumour progression, but is also required for chromosomal stability [76]. Since cancer cells are telomerase positive and develop inevitably into a genomic instability phenotype, one may ask whether telomerase ignition and maintenance is a consequence or a cause of genomic instability in tumour cells. It could be both. The pathways leading from telomerase re-expression in somatic tumour cells, to telomere dysfunction, to genomic instability and to tumorigenesis remain unclear and need to be explored further. Whereas telomerase expression prevents chromosome ends from shrinking after each successive round of replication, telomere structures on the other hand protect chromosomes from illegitimate recombination, breakage, degradation and fusion [41,77]. Therefore, telomere shortening and uncapping are crucial cellular events that may lead to loss of genome integrity and eventually to tumorigenesis [76]. Telomeres are protected from degradation and recombination by several elements. The telomere repeat binding factor, TRF2, is an essential component in telomere protection. It covers the double-strand DNA of the telomere repeats at all time during the cell cycle [78,79]. TRF2 also helps to maintain the formation of the t-loop structure. The t-loop is a specialized structure conferring maintenance of telomere stability and function in humans [80]. An estimated one hundred copies of TRF2 per telomere may be found. Inhibition of TRF2 exposes chromosome ends to the DNA damage response, because they may be perceived as DNA double stranded breaks, triggering apoptosis [81]. Cells mediate the apoptotic response through activation of ATM which normally senses double strand breaks (DSB), and leads to p53 activation and stabilization, via phosphorylation on serine 15 [81,82]. Apoptosis occurs also in telomerase-deficient mouse cells, once telomeres have attained a critically short length [83]. Moreover, cells expressing a mutant telomerase unable to bind to TRF2 also proceed to apoptosis. In general, cells lacking TRF2 undergo senescence and growth arrest followed by apoptosis [77]. Furthermore, when a conditional dominant negative mutant of TRF2 B M is transfected in vitro, the chromosomes fuse together at their ends in a string-like fashion [84]. The consequence of capping problems arising through TRF2 abrogation enables the TTAGGG repeat strand to ligate with another CCCTAA strand through the NHEJ pathway, thereby forming end-to-end chromosome fusion and visible anaphase bridges at mitosis [84]. In effect, the result in chromosome breaks may depend on whether the recombination events take place before or after the S phase. In fact, the many chromosomal aberrations found in cancer cells can be explained by end-to-end fusion and breakages that may be associated with dysfunctional telomeres [77]. Another element necessary in telomere protection is POT1, since it binds ssdna in the t-loop sub-structure which cooperates with TRF2 in the protection of telomere t-loop entity [85]. Loss of hpot1 promotes telomere instability because

8 8 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) 1 14 it stimulates the WRN and BLM RECQ helicases to unwind telomeric DNA structures [86,87]. Protection of telomeres requires additionally the three subunits of the DNA dependent kinase (DNA-PK), Ku70, Ku80 and the catalytic subunit (DNA-PKcs). It was shown that mouse embryo fibroblasts (MEFs) containing null mutations for either DNA-PKcs /, Ku70 / or Ku80 / show an increase in chromosome end fusions in metaphase spreads [88 91]. Since NHEJ pathways are severely impaired in these mice, ligation of unprotected telomeres must be performed through an alternative pathway. Furthermore, it was shown that DNA-PK binds physically with telomeres and its ancillary proteins [92]. Interestingly, several other proteins from DNA repair pathways such as MRE11/RAD50/NBS1, RECQ helicases, disrupted in WRN and BLM, are required for proper telomere t-loop structures and chromosome maintenance [93]. Dysfunctional MRE11/RAD50/NBS1 leads to chromosomal instability and tumour formation, probably through deregulation of telomere function and fusion of telomeres by faulty recombination pathways. Moreover, nucleotide excision-repair endonucleases ERCC1/XPF are also part of the telomere complex, the inactivation of which could lead to chromosomal and genetic aberrations. Chromosome instability may be mediated through dysfunctional telomere binding proteins, such as TRF1, RAP1, TIN2, TAZ1, or Tankyrase. These proteins are negative regulators of telomerase, and play a protective role on telomeres. The number of TRF1 proteins bound to the duplex telomeric TTAGGG sequence is proportional to the length of the telomere string. It was found that TRF1 overexpression decreased overall telomere length and that TRF1 levels do not affect telomere length in telomerase negative cell lines [94], implying a direct role on telomerase activity. It has been further shown that arrest of telomere elongation induced by TRF1 is a cis-acting mechanism, which occurs in individual telomeres [95]. It is not known whether TRF1 knockdown induces chromosome instability, but it could be assumed that inhibition of TRF1 may induce telomerase activation and possibly a lack of telomere protection hence inducing tumour formation. The binding partners of TRF1, such as Tankyrase 1 and 2, PINX1, and TIN2, that also affect telomere length, could also assume a role in chromosomal stability. Tankyrase inhibits TRF1 action by binding through its ankyrin domains. These proteins probably play numerous roles in other pathways. The TIN2 protein, which tethers TRF1 to the telomere complex, is a negative regulator of telomere length. A knockout of TIN2 in mice results in early embryonic lethality [96], but has not been evaluated for chromosomal alterations like end fusions and translocations. Other negative regulators of telomere length like RAP1, RIF1, and RIF2 are not known either for their role in chromosomal stability and will not be discussed further. However, it is noteworthy that TRF1 interacts with the spindle checkpoint protein MAD1 and NEK2 mitotic kinase [97], underpinning a link between mitotic checkpoints and telomere function. Since maintenance of telomere length by telomerase activity in somatic cells may induce cellular immortalization and tumour progression, it is reasonable to ask if telomerase expression per se is sufficient to initiate and drive genomic instability. It has been shown that primary cell lines transformed with the SV40 large T-antigen and Ras oncogene are sufficient for malignant transformation [52]. In addition, htert expression extends the lifespan of normal human cells without transforming them or affecting genome integrity [51]. Indeed, a study has shown that immortalization by htert transduction of human skin fibroblasts derived from patients afflicted with aging syndromes associated with chromosome instability, such as WRN, RBS, BLM, XP, and HGPS, are phenotypically and karyotypically stable [53]. Therefore, it appears that telomerase does not act as an oncogene that permits a hypermutability phenotype in order to drive chromosome instability and tumorigenesis [98]. Patients with Fanconi anaemia (FA) have short telomeres and active telomerase in blood lymphocytes that display show frequent dicentric and end-to-end fusion chromosomes [99 101]. Therefore, FA is a disease of chromosome instability that evolves progressively to bone marrow failure and acute leukaemia [102]. All proteins from the 11 complementation groups of FA are involved in the same DNA repair pathway with a compounded negative effect on telomere length [103,104]. Dyskeratosis congenita (DC) is a rare genetic disorder that affects the function of telomerase and the length of telomeres [105,106]. Patients with DC have either X-linked mutations in the dyskerin gene (DKC1), a protein implicated in both telomerase function and ribosomal RNA processing, or autosomal dominant mutations affecting the RNA component of telomerase (hterc) [107]. These patients have developmental problems, show signs of premature ageing, bone marrow failure and increased cancer incidence, probably because they are highly chromosomally instable due to dysfunctional telomerase and short telomeres [108]. DC and FA are examples of how problems in telomere metabolism can produce chromosome instability phenotypes and increased susceptibility to cancer. In addition, the function of telomeres is impaired in progeria syndromes, such as Ataxia telangiectasia, Nijmegen breakage syndrome, BLM and WRN, which also show telomere fusion and chromosome instability [99]. Cells lacking the tumour suppressors BARD1, BRCA1 or BRCA2 also show a CIN phenotype as well as malignant transformation. The BRCA1/BARD1 heterodimer is also involved in chromosome instability syndromes, because proteins interact in DNA repair pathways, FA proteins and telomere structures [109]. To conclude, telomere maintenance programs are important in tumorigenesis for two main reasons. Firstly, telomeres are responsible for maintaining integrity of chromosome ends during cell division and secondly because telomerase activity is reinstated in practically all human cancer cells. Telomere dysfunction occurs probably early in tumour development, whereas telomerase activation is a late event. Telomerase activation could be a prerequisite for tumour development

9 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) in the presence of short telomeres. In addition, short telomeres would normally signal cellular senescence and apoptosis through p53 and prb. However, telomerase is not considered as an oncogene, because reactivation alone is not sufficient to induce a malignant phenotype and genomic instability [98]. It appears that the combined telomere-associated functions could behave simultaneously as caretakers and gatekeepers of tumorigenesis. Therefore, dominant gain-of-function of telomerase-related activities in tumorigenesis act as the gatecrashers of the combined gatekeeper and caretaker parties, enabling the propagation of CIN events and oncogenesis. 5. Maintenance of chromosome integrity by mitotic checkpoints 5.1. Defects of chromosome segregation Cell division is the default mechanism for cell multiplication of all species. The only purpose of mitosis is the segregation of replicated DNA, primarily passing down an identical set of chromosomes to the next generation. However, mitosis is potentially a delicate process, since the chromosomes have to be perfectly duplicated and evenly segregated. Gross defects during mitosis, such as abnormal chromosome condensation, defective sister chromatid cohesion and segregation, impaired kinetochore assembly, and several spindle checkpoint deficiencies results in aberrant karyotypes and can lead to chromosomal instability (CIN) [110]. At metaphase, all sister-chromatids are maintained together through a multiprotein complex, cohesin, bridging the whole length of the chromosomes [111,112]. The anaphase-promoting complex or cyclosome (APC/C) is kept inactive through binding of checkpoint proteins MAD2, BUB3, and MAD3 (BUBR1), and because separase is held inactive by the inhibitor securin. At anaphase, securin is ubiquitinated by the APC/CDC20 complex which acts as an E3 ubiquitin ligase and securin is degraded through the ubiquitin-proteasome pathway [ ]. Active separase then cleaves the Scc1, a subunit of cohesin, permitting chromosome segregation. The main proteins of the spindle checkpoint response are MAD1, MAD2, MAD3, BUB1, BUB3, MPS1, and CDC20 [116,117]. These checkpoint elements are vital in cell division since abrogation of activity of any of these proteins leads to chromosome mis-segregation, with incapacity to arrest cells treated with microtubule stabilizing agents like paclitaxel. It has been shown that colorectal cancers with a massive CIN phenotype have inexistent cell-cycle arrest when treated with microtubule inhibitors, revealing deficient spindle checkpoint responses [118]. Some of theses tumours have indeed mutations in the human BUB1 gene [119]. Furthermore, other cancers with CIN may carry MAD mutations. For instance, MAD2 expression appears to be repressed in numerous solid tumours [120,121]. Interestingly, mice lacking the Brca2 tumour-suppressor gene have also frequent mutations in the Mad3L and Bub1 genes [122,123]. It is possible that Brca2 mutations in mice may foster formation of tumours through CIN, since knockout for this gene reveals problems at mitosis [124]. Chromosome segregation at mitosis and meiosis is enabled by retracting microtubules at sites of kinetochore attachment, which are positioned at a discrete location on each chromosome, named the centromere [117]. Loss of centromere integrity as well as improper kinetochore chromosome attachment may lead to loss of chromosomes during mitosis, producing aneuploid karyotypes and a chromosomal instability phenotype. However, the spindle checkpoint kinases are silenced through tension imposed by proper attachment of kinetochores to microtubules, CENP-E to microtubules releasing MAD2 from the APC/C, activating the ubiquitin ligase activity of the APC/CDC20 complex Centrosome aberrations Another characteristic of tumour cells is aberrant centrosome number. Most cancer cells have extra copies of centrosomes, resulting in the formation of multipolar spindles. Failure of centrosome separation results in monopolar asters. Evidently centrosome amplification can lead to genomic instability, since faulty centrosome metabolism can lead to chromosome mis-segregation. Defects in centrosome structure, function, duplication and regulation have shown chromosome abnormalities and genomic instability found in most tumours [125]. These defects have been observed in many different tumour types and first observed by Boveri in This has led to the hypothesis that centrosome dysfunction could be a leading cause of chromosomal instability [126]. Centrosomes are essential in chromosome segregation of animal cells. They were first described by Flemming in 1875 and named later by Boveri in The centrosome is also known as the Microtubule-Organizing Centre (MTOC). It directs microtubule assembly and coordinates all microtubule-associated functions. These functions include maintenance of cell architecture, cell motility, intracellular transport of organelles and cell division. They are formed from two orthogonal centrioles surrounded by a pericentriolar matrix (PCM) and contain -tubulin. They are duplicated prior to DNA replication, migrate on either side of the cell nucleus, form the spindle poles and nucleate the microtubule asters on which chromosomes anchor during mitosis. Centrosome function is therefore closely regulated by mitotic checkpoints. Cancer cells might arise when the centrosome cycle is uncoupled from the DNA replication cycle. The observation that most cancers host cells with centrosome aberrations, has led to the hypothesis that mutations in proteins involved in centrosome regulation hold the potential of a mutator phenotype and chromosomal instability. Actually, many pathways might regulate centrosome duplication [127]. Duplication of the single centrosome is

10 10 C.E. Jefford, I. Irminger-Finger / Critical Reviews in Oncology/Hematology 59 (2006) 1 14 semi-conservative and is initiated at the G1/S transition by CDK2/cyclinE and CDK2/cyclinD and completed before mitosis, where the duplicated centrosomes play a role in organizing the poles of the mitotic spindle. The cyclin dependent kinase 2, CDK2/CyclinE, is possibly the trigger in embryonic cells whereas CDK2/cyclinA could be the trigger in somatic cells [128]. However, phosphorylation of prb and subsequent transcriptional activity of E2F by prior CDK2 activation appears to be a prerequisite for centrosome duplication [129]. In mammals the centrosome duplication event requires two CDK2 substrates, NPM/B23 and the MPS1 protein kinase, which also plays a role in kinetochore function. MPS1 plays many roles since it is also a regulator of Aurora A [130]. Centriolar maturation is complete when it acquires markers such as ninein and Cenesin/Odf2 [131,132]. This process is distinct from centrosome maturation which happens when the PCM expands at G2-M to nucleate sufficient number of microtubules. Centrosome separation occurs normally at prophase. It is triggered by CDK1 phosphorylation and NEK2 kinase phosphorylation of C-NAP. Aurora kinases are also involved in centrosome duplication, since overexpression of Aurora A induces formation of multiple centrosomes and aberrant karyotypes. Aurora A, like other mitotic kinases including Polo, NIMA and CDK1 that are involved in the G2/M transition, locates to the MTOC [133]. Significantly, Aurora A also interacts in parallel with the APC/CDC20 complex involved in chromosome segregation. Aurora A has henceforth been classified as a bona fide oncogene and is believed to play a role in tumour initiation in vivo [134]. Compellingly, other tumour suppressors such as p53 and BRCA1 were involved in regulating the centrosome duplication cycle. The inactivation of p53 in knockout mouse embryo fibroblasts (MEFs) leads to repeated rounds of centrosome amplification [135]. The pathway to normal centrosome duplication may be restored by reintroducing the CDK2 inhibitor p21 waf/cip1. Overexpression of HDM2 or mdm2 may also result in the same p53 inactivation and centrosome amplification. It is worth noting that BRCA1 tumours, like p53 tumours, harbour chromosomal instabilities. Moreover, it has now clearly been shown that BRCA1 may bind to -tubulin during mitosis [136,137]. Deletion of Gadd45, like p21 abrogation, results also in defective centrosome duplication. Members of the transforming acidic coiled-coil (TACC) protein family have also been implicated in human cancers [138]. Overexpression of human papillomavirus (HPV) type 16 E6 and E7 oncoproteins by HPV infection induces abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability [139]. It is thought that HPV deregulates the cell cycle and centrosome duplication by inactivating p53 and prb pathways, via the Ran GTPase [140,141]. Moreover, ectopic overexpression of E2F-2 and E2F-3 also leads to multiple rounds of centrosome amplification with the identical end result [129]. Deregulated centrosome duplication, and production of aberrant centrosome numbers with formation of multipolar mitosis, is a consequence of tumour development. However, it is not known how centrosome malfunction contributes to the propagation of CIN events and tumour formation. Therefore, it is necessary to identify the specific proteins that regulate centrosome duplication in order to better understand development of human cancers and improve its treatment. 6. Conclusion Cancer cells evade normal growth controls either by ignoring death signals or by responding to survival and proliferative cues. The hallmark of cancer is defective tumour suppressor pathways either through p53 or prb inactivation, reactivation of telomerase, together with multipolar cell division and the formation of a wide array of abnormal karyotypes indicative of chromosomal instability. Pre-tumour lesions occur through several rounds of pair-wise mutations followed by waves of expansion after clonal selection of cells with definite growth advantages. The rate-limiting mutations may happen at a normal rate, although some mutations may impinge on DNA repair pathways directly, and therefore accelerate the rate of mutations so creating a mutator phenotype. Environmental stress factors also contribute to higher mutation rates, but nucleotide mutation is not the major factor of tumour progression. Indeed, CIN is the biggest contributing factor to genomic instability in tumours. Strikingly, it seems that the chief chromosomal instability events, such as translocations, deletions and chromosome losses or duplications could be explained by abnormal telomere metabolism. Impairment of telomerase function or telomere structure may contribute strongly to chromosomal instability. It appears that many mechanisms maintain chromosomal integrity through a number of different pathways. These pathways include DNA repair and telomere maintenance. These two processes converge because they use identical proteins. Telomeres and telomerase interact with tumour suppressor pathways (ATM, p53, and prb) and with DNA repair pathways (MRE11/RAD50/NBS1 complex). Telomeres function and cooperate with DNA replication mechanisms (Polymerase II), with recombination pathways such as the NHEJ ligation machinery (LigIV), and with various helicases (WRN, BLM). Telomeres utilize ubiquitination pathways (TRF1 ubiquitination and degradation). They rely on mitotic checkpoints necessary for progression through mitosis and sister chromatid segregation (with MAD1 and Tankyrase). Telomeres and microtubules may interact possibly through RIF1 which may also bind to telomeres [142]. Finally, telomeres also cooperate indirectly with centrosome duplication pathway via its association with BRCA1/BARD1 heterodimer. It could also play a role with the BRCA1- associated genome surveillance complex (BASC) through MRE11/RAD50/NBS1, ATM, replication factor C, BLM protein and hmsh2 and hmlh1 [30]. Therefore, telomere metabolic pathways are necessary for genome stability as a whole. Telomeres could provide the platform enabling the integration of all the aforementioned systems serving as