Telomerase and telomere biology in hematological diseases: A new therapeutic target

Highlights

• Telomeres are structures confined at the ends of eukaryotic chromosomes.

• Telomerase neutralizes loss of telomeric sequences by adding telomere repeats.

• Telomere biology is frequently associated with human cancer.

• Telomerase mutations are risk factors for some hematological diseases.

• There is an increasing interest in targeting telomeres and telomerase in anti-cancer therapy.

Abstract

Telomeres are structures confined at the ends of eukaryotic chromosomes. With each cell division, telomeric repeats are lost because DNA polymerases are incapable to fully duplicate the very ends of linear chromosomes. Loss of repeats causes cell senescence, and apoptosis. Telomerase neutralizes loss of telomeric sequences by adding telomere repeats at the 3′ telomeric overhang.

Telomere biology is frequently associated with human cancer and dysfunctional telomeres have been proved to participate to genetic instability.

This review covers the information on telomerase expression and genetic alterations in the most relevant types of hematological diseases.

Telomere erosion hampers the capability of hematopoietic stem cells to effectively replicate, clinically resulting in bone marrow failure. Furthermore, telomerase mutations are genetic risk factors for the occurrence of some hematologic cancers. New discoveries in telomere structure and telomerase functions have led to an increasing interest in targeting telomeres and telomerase in anti-cancer therapy.

Introduction

Normal mammalian somatic cells in culture can replicate to a finite number of replications with the maximum number being referred to as the Hayflick limit, which can act as a molecular clock to check the replicative history of the cells [1]. Both in vitro and in vivo studies have shown negative correlation between telomere length and cellular aging [2], [3].

Telomeres are specialized DNA–protein structures that are confined at the ends of eukaryotic chromosomes. In humans, telomeric DNA is composed of tandem repeats of a DNA sequence (5′-TTAGGG-3′) that ends in a 3′ single-stranded overhang. With each cell division, telomeric repeats are lost because DNA polymerases are incapable to fully duplicate the very ends of linear chromosomes, and also because of C-strand degradation. Loss of repeats eventually causes critically short, dysfunctional telomeres unable to adequately cap chromosome ends, resulting in inadequate proliferative ability, cell senescence, and apoptosis. When telomere length (TL) reaches a certain limit, telomeres become dysfunctional and elicit a p53-dependent DNA damage checkpoint response which triggers either replicative senescence or apoptotic cell death [4], [5].

Telomeres also perform other roles, which comprise transcriptional silencing of genes located close to them, as well as ensuring right chromosome segregation during mitosis [6], [7], [8], [9], [10].

The length of telomeric DNA is a dynamic sum of shortening and lengthening movements. In contrast to the rest of the genome, semi-conservative DNA duplication is unable of replicating the entire telomere and, consequently, in the absence of a procedure to reestablish telomere length, approximately 50 base pairs of telomeric repeats are lost per generation.

In healthy subjects, the average length of telomeres ranges between 5 and 15 kb, and can differ among individuals, chromosomes, and chromosome arms, cells, and tissues. TL displays high interindividual variation at birth and afterward, and it is longer in women than in men and longer in African Americans than in whites of European descent. Telomere repair is upregulated by sex hormones and transcriptional factors like c-Myc. TL undergoes progressive attrition with age and is relatively short in aging-related diseases [11], [12], [13].

Telomere associated proteins are essential primarily for the solidity and regulation of telomere length, whereas some contribute even in the reparation of injured DNA. Shelterin is a significant protein complex that comprises six subunits (TRF1, TRF2, POT1, hRAP1, TIN2 and TPP1) and participates in forming a cap structure at the end of chromosomes [6]. Several additional proteins bind to TRF1 and TRF2 and thus form complexes that control the homeostasis of telomeres [14].

A higher-order telomere nucleoprotein complex aids dynamic change between the cap and loose structure. It presents two states that must be well regulated. The strongly packed loop structure

is the restrictive factor for the elongation of telomeres as it prevents the access of telomerase and other proteins participating in the elongation of telomeres.

In addition to shelterin, a trimeric complex consisting of conserved telomere maintenance component 1 (CTC1), suppressor of cdc13 1 (STN1) and telomeric pathway with STN1 (TEN1), contributes to telomere homeostasis. The role of the CTC1/STN1/TEN1 (CST) complex at the telomere is composite, functioning in the replication and processing of telomeres prior to and after telomerase action. The CST complex inhibits telomerase activity in vitro and knockdown of CST subunits results in telomerase-dependent telomere elongation in vivo. These data have led to the suggestion that CST reduces the extent to which a telomere-bound telomerase adds telomeric repeats. In addition, CTC1 stimulates the re-start of telomere lagging strand synthesis at stalled replication forks through interaction with polα primase. CTC1 conditionally null mice show a quick increase in 3′ single stranded telomeric DNA and subsequently catastrophic telomere sequence loss. This loss is accompanied by an increase of telomeric circles (t-circles) that are thought to rise when homologous recombination of telomeres is not sufficiency inhibited, and is most pronounced in rapidly dividing tissues, evoking a critical role for CTC1 in telomere replication. Even though initially viable, CTC1 null mice succumb within the first months of life due to hematopoietic stem cell reduction and bone marrow failure [15].

The classical replication mechanism cannot offer synthesis of a chromosomal end; that leads to telomere shortening after each round of cell division, damage of the genome, and senescence. To block those processes, cell uses different tools, the main being telomerase activation (Table 1).

In fact, in normal stem cells and germ lines, telomeres are maintained by a special ribonucleoprotein enzyme, called telomerase, which neutralize loss of telomeric sequences by adding telomere repeats at the 3′ telomeric overhang. The telomerase ribonucleoprotein complex actively maintains telomere length, slowing, but not totally preventing, telomere attrition [16], [17], [18], [19].

The actual telomerase reaction mechanism can be defined as a three-step action. The short telomere sequence at the 3′ matrix end of the chromosome binds to the RNA domain in the first step. Elongation, which is a template-directed addition of nucleotides, occurs in the second step; translocation, which enables repeated use of the same binding site, occurs in the last step [20], [21], [22], [23], [24], [25], [26], [27], [28].

Telomerase consists of a reverse transcriptase enzyme (TERT), an RNA template (TERC), and stabilizing proteins including dyskerin (encoded by DKC1) and TCAB1 [29], [30] (Fig. 1).

The catalytic subunit of telomerase hTERT is a polypeptide consisting of 1132 amino acids. The ectopic expression of hTERT enhances the replication life span of corneal epithelial cells and endothelial cells during unchanged differentiation without alterating their karyotypes or activating known oncogenes. While hTR is expressed in relatively identical amount in embryonic and somatic tissues, the expression of hTERT is precisely regulated and undetectable in many somatic cells. This indicates that the expression of hTERT is the limiting step in telomerase activation [8], [31], [32].

As far the synthesis and maturation of the telomerase RNA, its functions are not restricted to providing the template for telomeric repeat synthesis. Diverse parts of the molecule structure shape the telomerase catalytic center together with amino acids of TERT, participate in the catalytic reaction of the nucleotide incorporation, facilitate efficiency of the translocation process [33], [34], condition the connection of the different telomerase protein factors and complex assembly [35], [36], and play a central role in transport [37], [38] and regulation of telomerase activity [39].

Telomerase RNAs of different organisms differ significantly in terms of structure, sequence, and length. In vertebrates this structure is known as the core domain, which, together with the CR4/CR5 domain (trans-activating domain) and TERT permits reconstruction of the active complex. The other elements of TR high order structure are specific for particular classes of organisms [40], [41], [42], [43], [44], [45], [46]. For example, the presence of the Est1 binding hairpin in yeast TRs [45] or the presence of H/ACA scaRNA (small Cajal body RNA) domain in the 3′ region of vertebrate TRs [46].

The vertebrate one possesses an H/ACA box. The presence of this element changes radically TR biogenesis in higher organisms. It was shown that a small amount of TR localizes in the nucleolus. The necessity of the H/ACA domain in vivo and subcellular fractionation profile followed by the detection of TR transcript in different cellular organelles argue that one step of TR processing occurs in nucleolus, and after that TR is transported to the cytoplasm. It was suggested that the nucleolar telomerase complex possesses different properties in comparison with cytoplasmic or nuclear telomerase holoenzyme [47], [48], [49], [50], [51].

The study of telomeres and their relationship to human diseases take account of determination of telomere length. Several systems have been utilized to measure telomere length in human samples [52]. Southern blotting is considered to be the gold standard. In this technique, telomeric terminal restriction fragments (TRFs) are separated from heavily restriction-digested genomic DNA by gel electrophoresis and detected by hybridization with a telomeric probe, resulting in a report of mean TRF lengths. More usually, measurements based on telomere flow fluorescence in situ hybridization (FISH) or telomere quantitative polymerase chain reaction (Q-PCR) are described. In telomere flow FISH, peripheral blood leukocytes are subjected to flow cytometry following FISH using peptide nucleic acid probes specific for the telomeric repeat sequence. The fluorescence in different leukocyte populations is calculated and the absolute telomere length is measured based on the telomere length of a human reference sample determined by Southern blotting. The Q-PCR or its improved version determines the amount of telomeric DNA relative to a single-copy genomic DNA locus, referred to as the T/S ratio. This value most often represents an average of telomere lengths in all hematopoietic cells, though specific populations may be isolated for analysis through flow-sorting. Even though a more approximate measure, the benefits of the Q-PCR assay are in the small quantity of DNA required and the high-throughput design, together making this a useful technique in larger case-control studies where an absolute measurement is less important [53], [54], [55], [56].

Assays that indicate the biology of telomere disease are accessible commercially; analysis can be problematic. Prudent interpretation is recommended when comparing with controls, because telomere length varies with ethnicity and age [57], [58].