Biology of Telomeres

Telomeres are specific non-coding nucleotide sequences located at the ends of eukaryotic chromosomes. They are necessary for the complete replication and stability of the chromosome. The replication of the lagging strand of the double DNA helix requires the presence, at the replication fork, of a certain number of nucleotides in front of the sequence to be copied, which serve as a template for an RNA primer functional to the activity of the DNA polymerase. The nucleotide sequence which acts as a template for the primer is not replicated. Thereby, to duplicate at each cycle the entire sequence of the chromosomal DNA an additional non-coding sequence must be present at the end of each chromosome to allow replication of the last coding nucleotides. These additional sequences are telomeres, highly conserved in organisms as diverse as protozoa, fungi, plants and mammals; they consist of many tandem repeats of a short nucleotide sequence that contains a block of G nucleotides (Alberts B BD et al, 2009). In human cells telomeres consist of from hundreds to several thousands tandemly repeated hexameric sequences: TTAGGG on the main filament, CCCTTA on the lagging strand, for a total length of 5-12 kb (Figure I).

Cuneo_ROLE_OF_TELOMERES_ALTERATIONS_IN_CLL_FIGURE_1

Figure I: Structure of telomeres (see text for explanation).
Adapted from Calado RT and Young NS. Telomere diseases.

 

Due to the above-described mechanism telomeres are physiologically shortened of 50-100 bp at each cell division cycle; telomere length thus reflects the replicative history of a cell.
When telomeres are shortened below a threshold length, specific pathways, mediated by the tumor suppressor genes p53 and Rb, are activated and lead to the arrest of cell proliferation and to the so-called “cellular senescence” that precedes apoptosis (Calado RT and Young NS, 2009PubMed ; Young NS, 2010PubMed ).
In cells with high proliferative index telomeres are continuously elongated by an enzyme complex called telomerase. Telomerase activity is restricted to a few normal cytotypes in humans: germ cells, stem cells, activated lymphocytes and epithelial cells with high replicative activity. In addition, during germinal center formation in the lymph node, B cells restore a high level of telomerase activity that allows a significant lengthening of telomeres (Grabowski P et al, 2005)PubMed. The enzyme is also reactivated in over 80% of cancers, playing a key role in cell immortalization (Bechter OE et al, 1998)PubMed.
Telomerase is a particular reverse transcriptase that utilizes an RNA template to synthesize telomeric DNA. The telomerase complex is composed of a catalytic subunit with inverse transcriptase activitiy (telomerase reverse transcriptase: TERT) on a specific RNA template (telomerase RNA component: TERC or TR), a main protein component called dyskerin (encoded by the gene DKC-1) and proteins that stabilize the complex (Figure II).

 

Cuneo_ROLE_OF_TELOMERES_ALTERATIONS_IN_CLL_FIGURE_2

Figure II: Structure and elements of telomerasic complex (see text for explanation).
Adapted from Calado RT and Young NS. Telomere diseases.

 

The enzyme adds TTAGGG repeats to the 3′ end of the main filament; the complementary strand is then extended by the conventional replicative apparatus (Calado RT and Young NS, 2009PubMed ; Young NS, 2010PubMed). TERC gene expression is substantially similar in germ cells and somatic tissues while TERT expression is finely regulated, it is generally not measurable in most somatic cells and represents the limiting factor of telomerase activity (Gancarcikova M et al, 2010)PubMed .
An alternative mechanism of telomeres lengthening (ALT: alternative lengthening of telomeres), evident in tumor cells in which telomerase activity is not demonstrable, might entail homologous recombination events (telomeric DNA of homologous chromosomes is used as a template for the elongation) or intratelomeric duplication of DNA sequences, or other unknown mechanisms (Gancarcikova M et al, 2010)PubMed .

Another important function of telomeres is to confer genetic stability to the ends of chromosomes. The 3′ end of the telomeric sequence on the main filament ends with a single-stranded guanine-rich protrusion, which is folded into a loop that inserts itself in between the double helix of DNA, which in turn opens forming a triple helix structure called D-Loop (displacement loop). The greater ring generated by the folding of the telomeric end is called T-loop (telomere loop) (Figure I). This arrangement gives protection from exonucleases and from events of termino-terminal fusion, avoids the end of the chromosome being incorrectly recognized as breakage of the double-strand by DNA repair systems and is a limiting factor for the elongation of telomeres by telomerase (Calado RT and Young NS, 2009PubMed ; Young NS, 2010PubMed ; de Lange T, 2005PubMed ). In vertebrates, a complex of six proteins (TRF1, TRF2, TIN2, POT1, TPP1, RAP1), called “shelterin complex”, is specifically associated with telomeric TTAGGG repeats and promotes the formation and stability of the T-loop (de Lange T, 2005)PubMed (Figure I). Furthermore, the components of the shelterin complex recruit additional polypeptides to form a complex network that covers the chromosome ends. These polypeptides include several proteins involved in the response to DNA damage as the ATM kinase, the Ku70/80 heterodimer and the MRE11/RAD50/NBS1 (MRN) complex (de Lange T, 2005)PubMed. The telomeric sequences may further stabilize the ends of chromosomes generating unusual structures through interaction between four guanines and formation of a planar structure that stacks on top of other similar structures to obtain a stable filament defined G-quadruplex structure (Burge S et al, 2006)PubMed . These entities are stabilized by hydrogen bonds established between the tops of bases and by chelation with a metal ion, located at the center of each unit of four bases (Parkinson GN et al, 2002)PubMed.

 

Pathology of telomeres

 

Inherited altered expression of different components involved in normal telomere function is associated, to varying degrees, to diseases such as aplastic anemia, dyskeratosis congenita, idiopathic pulmonary fibrosis, cryptogenic cirrhosis of the liver and genetic susceptibility to the development of numerous types of neoplasia (Calado RT and Young NS, 2009PubMed ; Young NS, 2010PubMed).
The erosion of telomeres that occurs during each cell division, in the presence or absence of specific mutations of genes involved in telomere function, may contribute to chromosomal instability and neoplastic transformation, including both solid tumors and hematologic malignancies (Calado RT and Young NS, 2009PubMed ; Young NS, 2010PubMed).

The loss of telomeres’ protective functions, due to dysfunction of telomeric proteins or to critical shortening of telomeres, determines the exposure of chromosome ends, resulting in abnormal activation of DNA damage response machinery. The repair mechanisms may lead to the irreversible arrest of cell growth and proliferation, an event known as cell senescence, but may also induce genetic instability. The instability resulting from telomere dysfunction is associated with some typical chromosome aberrations: termino-terminal fusion, loss of telomeric signal on one or both chromatids of a chromosome, presence of extrachromosome telomere signals, telomeric duplication on one or both chromatids of a chromosome, fusion of sister chromatids (Brugat T et al, 2010bPubMed ; O’Sullivan RJ and Karlseder J, 2010PubMed ).
These events feature a phenomenon described in vitro as “telomere crisis,” observed after deletion of p53 and Rb specific pathways (a rather frequent event in cancer). The failure of these tumor suppressor pathways allows the cells to overcome the limiting step of cellular senescence and continue to divide despite the erosion of telomeres, until the so called “telomere crisis” occurs, in which a high genetic instability due to extremely short telomeres leads to cell death or neoplastic transformation (Baird DM, 2005)PubMed. In particular, a minimum threshold of telomere length has been identified, equal to approximately 13 TTAGGG repetitions, below which the telomere-telomere melting phenomena occur. These events were further characterized by long-range PCR (fusion PCR) and subsequent sequencing with demonstration that fusion is always associated (or preceded) by deletion of one or both telomeres on involved chromosomes and by micro-homology phenomena. These deletions may be a consequence of fusion mechanisms or may precede them, also in stochastic order, and subsequently favor them. The sporadic deletion of telomere sequences can then favor the chromosomal instability even in cells with normal or not excessively short telomeres (Capper R et al, 2007)PubMed .
Telomere-telomere fusion is considered an important mutational event inducing chromosomal rearrangements involved in neoplastic transformation. It generates dicentric chromosomes that undergo breakage during cell division with subsequent abnormal fusion of the stumps with other DNA sequences. The repetition of fusion/ break/ fusion cycles is associated with non-reciprocal translocations, gene amplification, and other chromosomal aberrations that overall characterize neoplastic transformation (Lin TT et al, 2010PubMed ; Murnane JP, 2010PubMed ; Artandi SE and DePinho RA, 2010PubMed ) (Figure III).

 

Cuneo_ROLE_OF_TELOMERES_ALTERATIONS_IN_CLL_FIGURE_3

Figure III: Mechanism of chromosomal instability due to telomeric loss and termino-terminal fusion (see text for explanation).
Adapted from Murnane JP. Telomere loss as a mechanism for chromosome instability in human cancer.

 

Methods of quantification of telomere length and telomerase activity

 

TRF (terminal restriction fragment) analysis (Southern blot)

The genomic DNA is digested with restriction enzymes that do not cut within the specific telomeric sequences, leaving intact the telomeres of each chromosome (terminal restriction fragment). The fragments are then separated by electrophoresis on agarose gel and TRF are identified by hybridization with specific probes in Southern blot analysis. This technique, which is the most widely used and is applicable to different tissues, both fresh and frozen, has however some disadvantages: 1) it requires amounts of DNA of the order of micrograms, then it takes at least 10^5 cells; 2) the TRF may also include, in addition to the purely telomere sequences, variable fragments of the pretelomeric region. This region is constituted by a 0-1 kb portion contiguous to the proximal end of the telomere containing repetitions of single nucleotide variants of telomeric sequences (TVR = telomere variant repeats) and by a proximal subtelomeric region that is one of the most dynamic and variable loci of the genome. The variable length of TRF may thus be significantly influenced by non-telomere components; 3) being the identification of telomere sequences based on a process of hybridization, the shorter the telomere the less effective the hybridization, up to a length threshold below which the telomere termination will not be detected. The method is thus not able to identify the shortest telomeres; 4) the results of the analysis are expressed as telomere peak length, or as an average of telomere lengths of different chromosomes belonging to all cell types present in the sample. At the same time, however, the method is able to describe the telomere length and its distribution in the whole genome (Baird DM, 2005)PubMed.

 

Q-PCR (quantitative-PCR)

This technique, referred to as Tel-PCR, amplifies all telomeric sequences of the studied cell population and expresses the total content of telomeric DNA in the sample as the ratio between the number of amplified copies of the telomeric repeats and the number of amplified copies of a control gene sequence, known to be present in the genome as a single copy. The result is expressed in terms of T/S value (= telomere / single copy gene ratio). This technique has some advantages: 1) speed and sensitivity (a DNA amount in the order of nanograms is sufficient); 2) effectiveness even in the presence of very short telomeres; 3) it can be performed on degraded or fixed material; 4) results are not distorted as subtelomeric regions are not amplified (Grabowski P et al, 2005)PubMed. Still, it does not provide any indication of telomere length distribution (Baird DM, 2005)PubMed.

 

Q-FISH (quantitative-FISH)

It allows a more specific analysis of telomere dynamics in a single cell or a small population. Metaphase chromosome preparations are hybridized with PNA (peptide nucleic acid) fluorescent labeled probes specific for telomeric sequences. The telomere length of each chromosome arm is expressed in TFU (telomere fluorescence units).
The main advantage consists in the direct visualization of telomeric sequences at the ends of each chromosome and therefore of the precise distribution of the different lengths.
Among the disadvantages: 1) the difficulty of translating the TFU in actual DNA length, and 2) the poor sensitivity for shorter telomeres, being the method based on hybridization. It may, however, quantify the SFE (signal free ends), ie the completely denuded chromosome ends or with telomeres below the threshold of detection; 3) it is applicable only to cells proliferating in vitro because it applies to metaphase cells (Baird DM, 2005)PubMed.

 

Flow-FISH

Adaptation of the Q-FISH in which a specific interphase telomere FISH is coupled to multiparameter flow cytometry, in order to show the telomere dynamics of specific hematopoietic subpopulations. It provides a quantitative assessment of the average telomere content of the single cell and of different cell populations as a whole. It is not widely used, also because it appears not to be reliable on stored samples (Grabowski P et al, 2005PubMed ; Baird DM, 2005PubMed ; Damle RN et al, 2004PubMed ).

 

STELA (single telomere length analysis)

It is a high resolution PCR for the amplification of telomeric sequences of a specific chromosome arm. It relies on the use of primers that bind to the 3′ single-stranded termination on the one hand, and to a specific sequence of the subtelomeric region of the chromosome of interest on the other. The amplified fragments then migrate on agarose gel electrophoresis and are identified by Southern blot analysis with telomere-specific probes. The method is applicable only if chromosome-specific primers are available and this is allowed by the high polymorphism of subtelomeric sequences (Baird DM, 2005)PubMed. Telomeres of the short arm of chromosomes X and Y are well suited for this purpose, in that they are preceded by a subtelomeric region, called pseudoautosomal region (PAR1), characterized by a high frequency of polymorphism as it is the obliged locus annealing of the two chromosomes during meiosis, and then of recombination events (Baird DM et al, 1995)PubMed .
The advantages of this investigation are evident: 1) the ability to accurately quantify the length of telomeres at a specific chromosomal end and to sequence the amplified fragments, permitting the definition of the exact length of telomeric sequences, of repeated telomeric variants and the subtelomeric region; 2) the ability to identify even very short telomeres. The disadvantages are represented by: 1) the limitation imposed by the fact that only some chromosomal terminations have unique subtelomeric sequences that enable the creation of specific primers; 2) the technical difficulties related to the highly sophisticated method; 3) the inapplicability to degraded or fixed material (Baird DM, 2005)PubMed.

 

Evaluation of telomerase activity

The better investigation to quantify telomerase activity is a functional assay of specific amplification of telomeric repetitions of a cell lysate (TRAP assay) and is expressed in TPG (total product generated) units, each TPG U corresponds to the number of specific primers elongated of at least 4 telomeric repeats in 30 minutes of incubation at 30 °C (Bechter OE et al, 1998PubMed ; Damle RN et al, 2004PubMed).
An indirect method of telomerase activity measuring has also proved reliable and potentially more feasible in the clinical practice. It consists of quantifying the h-TERT gene transcripts by real-time PCR. Studies comparing this method with the TRAP assay showed a direct correlation between h-TERT transcript levels and the telomerase activity measured by the functional assay (Terrin L et al, 2007)PubMed .

 

Telomeres and chronic lymphocytic leukemia

The relationship between telomere length, telomerase activity and cancer has become the subject of great interest and active scientific research over the past twenty years.

The results obtained to date globally agree on the presence in neoplastic cells of solid and hematological tumors of average shorter telomeres and increased telomerase activity, both features that correlate with advanced stage of disease and poor prognosis (Grabowski P et al, 2005PubMed ; Bechter OE et al, 1998PubMed ; Damle RN et al, 2004PubMed ; Terrin L et al, 2007PubMed Ohyashiki JH et al, 2002PubMed , Hackett JA and Greider CW, 2002PubMed ). Unlike what happens in normal cells, telomere length in tumor cells does not correlate with demographic parameters such as age and sex (Grabowski P et al, 2005PubMed ; Ricca I et al, 2007PubMed ).

The first work on the characteristics of telomeres in chronic lymphocytic leukemia (CLL) was conducted by Bechter and colleagues in 1998 (Bechter OE et al, 1998)PubMed and showed an inverse relationship between telomere length and telomerase activity and correlation of both parameters with Binet stages: stage A patients showed significantly longer telomeres and lower telomerase activity than patients in stage B/C. The short telomeres and high enzymatic activity were significantly associated with reduced overall survival (Bechter OE et al, 1998)PubMed.

 

a) Telomeres and IGVH mutation status

 

Subsequent studies have confirmed this data and observed a correlation between telomere dynamics and other recognized prognostic factors for CLL: CD38, ZAP-70, IGVH genes mutation status (variable region of the immunoglobulin heavy chain) and cytogenetic abnormalities (Terrin L et al, 2007PubMed ; Roos G et al, 2008PubMed ).

Of particular interest is the correlation between telomere length and IGVH mutation status: in all studies, the subset of CLL cases with unmutated IGVH has been shown to have significantly shorter telomeres compared to cases with mutated IGVH (Grabowski P et al, 2005PubMed ; Damle RN et al, 2004PubMed ; Ricca I et al, 2007PubMed ; Roos G et al, 2008PubMed ; Walsh SH et al, 2007PubMed ).

The rate of telomeres shortening in the two subgroups was, however, comparable and constant over time (Damle RN et al, 2004PubMed); moreover most circulating lymphocytes in CLL are in a quiescent replicative phase (G0). Therefore it is likely that the difference in telomere length between the two subgroups reflects the different replicative history of the cell before the leukemic transformation. This in turn is determined by the characteristics of antigenic stimulation of cells that then gave rise to clones of mutated or unmutated CLL (Grabowski P et al, 2005PubMed ; Damle RN et al, 2004PubMed ; Ricca I et al, 2007PubMed). The mutated CLL cases probably originate from B lymphocytes that have taken part in the events of the germinal center after T-dependent antigen stimulation. This justifies the somatic hypermutation of the IGVH genes, the brevity of telomeres compared to naive B cells and at the same time the greater length compared to cases with unmutated IGVH, since the germinal center reaction involves physiologically important telomerase activation. On the basis of telomere shortening, even cases with unmutated IGVH seem to derive from B cells that have encountered the antigen, possibly outside the germinal center, thereby without somatic hypermutation and telomerase activation. Functional assessment of enzyme activity has, however, demonstrated increased telomerase activation in the unmutated IGVH subgroup, presumably subsequent to the leukemic transformation. Telomere activation may reflect a compensatory mechanism of the excessive telomere shortening in this subgroup as only clones developing this compensatory activity may be able to survive and proliferate (Damle RN et al, 2004PubMed).
In other mature B lymphoproliferative disorders telomere length has been shown to correlate with the origin of the transformed cell from the germinal center (GC): the disorders arising from GC-experienced B cells have longer telomeres (follicular lymphoma, Burkitt lymphoma, marginal cells lymphoma, GC-like diffuse large B-cell lymphoma, multiple myeloma) compared to those originating from B lymphocytes that have not had relations with the germinal center (non GC-like diffuse large B-cell lymphoma, mantle cell lymphoma) (Grabowski P et al, 2005PubMed ; Ricca I et al, 2007PubMed, Walsh SH et al, 2007PubMed).

Initially, therefore, the independent prognostic significance of telomere length has been limited because it was considered a parameter directly related to the mutation status, as an equivalent marker of germinal center derivation and IGVH hypermutation (Grabowski P et al, 2005PubMed ; Ricca I et al, 2007PubMed).

However, some observations have challenged this paradigm: 1) mutation status proved to be a significant prognostic factor for patients with Binet stage A, but not for those with stage B/C, unlike telomere length which is predictive of better or worse overall survival both for stage A and B/C (Grabowski P et al, 2005)PubMed; 2) in the context of mutated and unmutated CLL some so-called “discordant” cases have been shown, harboring short telomeres while having mutated IGVH or longer telomeres with unmutated IGHV (Grabowski P et al, 2005PubMed ; Ricca I et al, 2007PubMed): the prognosis of these cases agreed with the telomere length rather than with mutation status, then the subgroup with mutated IGHV and short telomeres had unfavorable outcome, similar to that of patients with unmutated IGHV (Ricca I et al, 2007PubMed); 3) “discordant” cases have also been observed with regard to the mutation status and telomerase activity: the subgroup with unmutated IGHV but reduced telomerase activity showed better prognosis, while those with mutated IGHV and high telomerase activity showed a significantly worse outcome  (Terrin L et al, 2007)PubMed; 4) in-depth study of telomeres dynamics in mature B lymphoproliferative disorders showed cases of discrepancy between telomere length and germinal center origin of the transformed cell (Walsh SH et al, 2007)PubMed.

 

 

b) Prognostic significance of telomere aberrations

 

It was therefore evident that telomere length does not simply correlate with the replicative history of the cell before the transformation but rather it is a more dynamic factor, also influenced by the proliferative activity of the transformed cell, able to act as an independent prognostic factor (Terrin L et al, 2007PubMed ; Walsh SH et al, 2007PubMed).
In several studies multivariate analysis has been conducted that took into account other recognized CLL prognostic factors (mutational status, CD38, ZAP70, clinical stage, disease activity): telomere length and telomerase activity have proven independent prognostic factors in terms of overall survival (OS), treatment-free survival (TFS), progression-free survival (PFS) and risk of progression to a more aggressive histological form that is Richter’s syndrome (Grabowski P et al, 2005PubMed ; Bechter OE et al, 1998PubMed ; Terrin L et al, 2007PubMed ; Ricca I et al, 2007PubMed ; Roos G et al, 2008PubMed ; Rossi D et al, 2009PubMed ).
Analysis of cases of discrepancy between telomere length and other recognized prognostic factors have shown that the presence of telomeres shorter than a specific threshold (5,000 bp) identified a CLL group with reduced TFS and OS even if characterized by favorable prognostic factors (age <65, stage A, mutated IGVH, cytogenetics, negative CD38 and ZAP70); similarly, the presence of telomeres longer than the threshold identified a group with better prognosis even in the presence of an unfavorable risk profile (Rossi D et al, 2009)PubMed .
This prognostic data showed a significant accordance in the performed studies, although carried out with different methods of telomere length and telomerase activity evaluation, which strengthens the predictive significance of this parameter and its clinical applicability (Ricca I et al, 2007PubMed ; Rossi D et al, 2009PubMed).

 

c) Telomeres and molecular cytogenetic lesions

 

Several studies tried to address the question of how the shortness of telomeres could actively worsen the prognosis. An early observation was that a significant difference in terms of telomere length was present in CLL with normal karyotype or a single chromosomal aberration and CLL with two or more cytogenetic abnormalities (Roos G et al, 2008)PubMed. After exclusion of cases with isolated 13q deletion, every additional aberration increased the likelihood of association with short telomeres and short telomeres correlated more closely with prognostically unfavorable cytogenetic abnormalities (17p-, 11q-) (Roos G et al, 2008)PubMed. The explanation for these observations should probably take into account two major events: 1) genetic aberrations, especially of TP53 or RB, allow cellular senescence to be overcome and prolong survival and proliferative activity of a cell beyond the limits normally imposed by telomere shortening, thus causing a further extreme consumption of telomeric sequences; 2) cancer cells are able to resist death induced by critically short telomeres by up-regulation of telomerase activity, often induced by the same cytogenetic abnormalities. This is however insufficient to compensate for the genetic instability due to the critical shortness of telomeric sequences (Roos G et al, 2008)PubMed.

The ability of critically short telomeres to induce genetic instability and the appearance of chromosomal aberrations has been demonstrated and characterized in vitro by inducing the so-called “telomere crisis”. This has been obtained through suppression of p53 and Rb pathways mediated by forced expression of the HPV16 E6 and E7 oncoproteins (Capper R et al, 2007)PubMed . Lin and colleagues, using advanced high-resolution STELA and the extension of its applications (TVR-PCR, fusion-PCR), have recently shown that phenomena similar to those observed during the in vitro telomere crisis may characterize CLL progression in vivo (Lin TT et al, 2010PubMed). This work first gave direct evidence that the critical shortening of telomeres, their dysfunction and fusion phenomena contribute to disease progression. The sequencing analysis of telomere-telomere fusion processes in CLL showed features already described in the fusion processes occurring between short telomeres during the in vitro telomeric crisis, that are represented by the presence of a limited number of telomeric repeats at melting points, by the deletion of the telomeric and subtelomeric regions of at least one of the two involved chromosomes and by microhomology phenomena at melting points. Analysis of CGH-array (comparative genome hybridization) of cases with prominent telomere dysfunction showed large-scale genomic rearrangements concentrated in telomeric regions. This is not found in cases with longer telomeres. It is noteworthy that the frequency of telomere erosion, dysfunction and melting increased in advanced CLL stages. However, some cases were also found in early stages with poor prognosis, suggesting that these changes may actually precede and favor the progression of the disease (Lin TT et al, 2010PubMed).

Leukemic cells undergoing the telomeric crisis have lost earlier mechanisms regulating the cellular senescence checkpoint, such as p53 and ATM pathways. Cytogenetic abnormalities causing the loss of these pathways, respectively 17p- and 11q- are actually associated with very poor prognosis. Nevertheless, they may not be the only ones allowing a cell to overcome senescence, partly because they are rarely found in the early stages of CLL, before progression actually happens (Lin TT et al, 2010PubMed). It is therefore reasonable to assume that the unfavorable cytogenetic abnormalities are responsible for telomere shortening as they are associated with higher proliferative activity overcoming the cellular senescence, or that pathologically short telomeres due to other causes lead to genetic instability responsible for the accumulation of more cytogenetic abnormalities (Ladetto M, 2010PubMed ; Jahrsdorfer B and Weiner GJ, 2008PubMed ). The two hypotheses are probably not mutually exclusive according to a speculative model of CLL progression involving the loss due to different mechanisms of cellular senescence checkpoint, progression towards the telomeric crisis, subsequent genetic instability with merging of dysfunctional telomeres with telomeric sequences or other double-stranded DNA break points, cycles of anaphase bridges, breaks and new fusions. This results in more chromosomal rearrangements (non-reciprocal translocations, amplifications, etc.), frequently responsible themselves for the silencing of tumor suppressor genes such as TP53 and ATM, with further promotion of telomere dysfunction in a vicious circle that leads to disease progression (Lin TT et al, 2010PubMed ; Ladetto M, 2010PubMed) (Figure IV).

 

Cuneo_ROLE_OF_TELOMERES_ALTERATIONS_IN_CLL_FIGURE_4

Figure IV: Schematic depiction of the role played by progressive shortening of telomeres in CLL progression. Repeated cell division cycles, with other potential concurrent factors, induce progressive telomeres shortening. Inactivation of cell senescence checkpoint allows cells to reach a telomeric shortening such as to determine a condition of “telomeric crisis”, associated with genetic instability, possible clonal evolution and disease progression.
Adapted from Ladetto M. Telomere disrupts, CLL progresses.

 

Numerous studies have demonstrated an altered global expression of genes involved in normal telomere function in CLL cells, i.e. genes encoding the different subunits of the telomerase complex or constituents of the shelterin complex or other regulatory proteins. This results in a marked telomere dysfunction and a telomerase activation that can respectively contribute to the critical shortening of telomeres and to cell survival even in the presence of short telomeres, thus exposing the cell to the risks of genetic instability due to the telomeric crisis (Poncet D et al, 2008)PubMed. The presence of short telomeres and the altered expression of the telomeric and telomerase complex is characteristic of leukemia cells obtained from patients refractory to therapy with alkylating agents and/ or purine analogs and resistant to in vitro apoptosis induced by DNA damage (Brugat T et al, 2010a)PubMed. In these cells histone modifications have been identified that alter chromatin condensation at the level of telomeric sequences and cause the loss of the single-stranded cap at 3′ end. The appearance in these cells of chromosomal aberrations related to telomeres dysfunction, such as deletion of the telomeric sequences in one or both chromatids of a chromosome, were associated with the appearance of multiple cytogenetic abnormalities and complex karyotype (Brugat T et al, 2010bPubMed).

 

d) Conclusion

 

Telomeres physiology and pathology is an important field of study for the understanding of CLL biology. The role of telomere dynamics in disease progression and their prognostic significance has indicated telomerase as a potential therapeutic target. Inhibition of telomerase activity at an early stage of disease, when the cellular senescence checkpoint has not yet been impaired by critical telomere shortening, could have an important antineoplastic effect (Terrin L et al, 2007PubMed ; Ohyashiki JH et al, 2002PubMed ; Roos G et al, 2008PubMed). It was also demonstrated that telomerase activity is associated with functions such as promotion of cell survival and prevention of apoptosis induced by anticancer agents, independently from the activity of telomeres lengthening (Terrin L et al, 2007)PubMed. Ongoing studies exploring this therapeutic option deal with difficulties associated with the still incomplete understanding of the precise role of telomeres and telomerase in hematologic malignancies as well as with the possible detrimental effect that telomerase inhibitors might have on normal hematopoietic stem cells that constitutively express a certain level of telomerase activity (Ohyashiki JH et al, 2002PubMed).

 

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