Pharmacological intervention strategies for affecting telomerase activity: future prospects to treat cancer and degenerative disease

Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.     

G-quadruplex preferentially forms at the very 3' end of vertebrate telomeric DNA

Human chromosome ends are protected with kilobases repeats of TTAGGG. Telomere DNA shortens at replication. This shortening in most tumor cells is compensated by telomerase that adds telomere repeats to the 3′ end of the G-rich telomere strand. Four TTAGGG repeats can fold into G-quadruplex that is a poor substrate for telomerase. This property has been suggested to regulate telomerase activity in vivo and telomerase inhibition via G-quadruplex stabilization is considered a therapeutic strategy against cancer. Theoretically G-quadruplex can form anywhere along the long G-rich strand. Where G-quadruplex forms determines whether the 3′ telomere end is accessible to telomerase and may have implications in other functions telomere plays. We investigated G-quadruplex formation at different positions by DMS footprinting and exonuclease hydrolysis. We show that G-quadruplex preferentially forms at the very 3′ end than at internal positions. This property provides a molecular basis for telomerase inhibition by G-quadruplex formation. Moreover, it may also regulate those processes that depend on the structure of the very 3′ telomere end, for instance, the alternative lengthening of telomere mechanism, telomere T-loop formation, telomere end protection and the replication of bulky telomere DNA. Therefore, targeting telomere G-quadruplex may influence more telomere functions than simply inhibiting telomerase.     

Targeting the telomere and shelterin complex for cancer therapy: current views and future perspectives

Aberrant telomere homeostasis is essential for cell immortality, enabling cells to evade telomere dependent senescence. Disruption of telomere structure and function in cancer cells is highly toxic as shown by detailed pre-clinical evaluation of telomerase inhibitors. Under telomerase inhibition, cells must divide sufficiently frequently to allow one or more telomeres to shorten to an unprotected length. Functioning telomeres are disguised from the DNA damage machinery by DNA remodelling and other activities of the telomere binding complex shelterin. Direct interference with shelterin has been shown to result in cell killing and small molecules directly targeting telomere DNA also have anti-tumour effects partially dependent on shelterin disruption. However, shelterin components have not generally been regarded as therapeutic targets in their own right. In this review, we explore the possibilities for therapeutic targeting of the shelterin complex.     

HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence

The telomere integrity is maintained via replication machinery, telomere associated proteins and telomerase. Many telomere associated proteins are regulated in a cell cycle-dependent manner. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a single-stranded oligonucleotide binding protein, is thought to play a pivotal role in telomere maintenance. Here, we identified hnRNP A1 as a novel substrate for vaccinia-related kinase 1 (VRK1), a cell cycle regulating kinase. Phosphorylation by VRK1 potentiates the binding of hnRNP A1 to telomeric ssDNA and telomerase RNA in vitro and enhances its function for telomerase reaction. VRK1 deficiency induces a shortening of telomeres with an abnormal telomere arrangement and activation of DNA-damage signaling in mouse male germ cells. Together, our data suggest that VRK1 is required for telomere maintenance via phosphorylation of hnRNP A1, which regulates proteins associated with the telomere and telomerase RNA.     

Generation of telomeric G strand overhangs involves both G and C strand cleavage

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.     

The yeast homolog of human PinX1 is involved in rRNA and small nucleolar RNA maturation,not in telomere elongation inhibition

In human cells, PinX1 protein has recently been shown to regulate telomere length by repressing the telomerase. In this work, we show that the putative yeast homolog of PinX1, encoded by the YGR280c open reading frame (ORF), is a new component of the ribosomal RNA processing machinery. The protein has a KK(E/D) C-terminal domain typical of nucleolar proteins and bears a putative RNA interacting domain widespread in eukaryotes called the G-patch. The protein was hence renamed Gno1p (G-patch nucleolar protein). GNO1 deletion results in a large growth defect due to the inhibition of the pre-ribosomal RNA processing first cleavage steps at sites A(0), A(1), and A(2). Furthermore, Gno1p is involved in the final 3'-end trimming of U18 and U24 small nucleolar RNAs. A mutational analysis showed that the G-patch of Gno1p is essential for both functions, whereas the KK(E/D) repeats are only required for U18 small nucleolar RNA maturation. We found that PinX1 complemented the gno1-Delta mutation, suggesting that it has a dual function in telomere length regulation and ribosomal RNA maturation in agreement with its telomeric and nucleolar localization in human cells. Conversely, we found that Gno1p does not exhibit the in vivo telomerase inhibitor activity of PinX1.     

Evolution of CST function in telomere maintenance

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack. In addition, telomeres facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.Key words: telomere, telomerase, telomere protein, CTC1, STN1, TEN1, OB-fold, arabidopsis, DNA polymerase alpha, RPA     

The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity

We have analyzed the de novo telomere synthesis catalyzed by the enzyme telomere terminal transferase (telomerase) from Tetrahymena. Oligonucleotides representing the G-rich strand of telomeric sequences from five different organisms specifically primed the addition of TTGGGG repeats in vitro, suggesting that primer recognition may involve a DNA structure unique to these oligonucleotides. The sequence at the 3' end of the oligonucleotide primer specified the first nucleotide added in the reaction. Furthermore, the telomerase was shown to be a ribonucleoprotein complex whose RNA and protein components were both essential for activity. After extensive purification of the enzyme by a series of five different chromatographic steps, a few small low abundance RNAs copurified with the activity.     

Processive and distributive extension of human telomeres by telomerase under homeostatic and nonequilibrium conditions

Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds ～60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.     

Interactions between telomerase and primase physically link the telomere and chromosome replication machinery

In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase alpha-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.     

Telomerase RNA localized in the replication band and spherical subnuclear organelles in hypotrichous ciliates

The intranuclear distribution of telomere DNA-binding protein and telomerase RNA in hypotrichous ciliates was revealed by indirect fluorescent antibody staining and in situ hybridization. The Oxytricha telomere protein colocalized with DNA, both being dispersed throughout the macronucleus except for numerous spherical foci that contained neither DNA nor the protein. Surprisingly, the telomerase RNA was concentrated in these foci; therefore, much of telomerase does not colocalize with telomeres. These foci persist through the cell cycle. They may represent sites of assembly, transport or stockpiling of telomerase and other ribonucleoproteins. During S phase, the macronuclear DNA replication machinery is organized into a disc-shaped structure called the replication band. Telomerase RNA is enriched in the replication band as judged by fluorescence intensity. We conclude that the localization of a subfraction of telomerase is coordinated with semiconservative DNA replication.     

The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres

Telomeres protect chromosome ends from being detected as lesions and from triggering DNA damage checkpoints. Paradoxically, telomere function depends on checkpoint proteins such as ATM and ATR, but a molecular model explaining this seemingly contradictory relationship has been missing so far. Here we show that the DNA damage machinery acts on telomeres in at least two independent steps. First, the ATR-dependent machinery is recruited to telomeres before telomere replication is completed, likely in response to single-stranded DNA resulting from replication fork stalling. Second, after replication, telomeres attract ATM and the homologous recombination (HR) machinery. In vivo and in vitro results suggest that the HR machinery is required for formation of a telomere-specific structure at chromosome ends after replication. Our results suggest that telomere ends need to be recognized as DNA damage to complete end replication and to acquire a structure that is essential for function.     

G-quadruplex formation at the 3' end of telomere DNA inhibits its extension by telomerase, polymerase and unwinding by helicase

Telomere G-quadruplex is emerging as a promising anti-cancer target due to its inhibition to telomerase, an enzyme expressed in more than 85% tumors. Telomerase-mediated telomere extension and some other reactions require a free 3' telomere end in single-stranded form. G-quadruplex formation near the 3' end of telomere DNA can leave a 3' single-stranded tail of various sizes. How these terminal structures affect reactions at telomere end is not clear. In this work, we studied the 3' tail size-dependence of telomere extension by either telomerase or the alternative lengthening of telomere (ALT) mechanism as well as telomere G-quadruplex unwinding. We show that these reactions require a minimal tail of 8, 12 and 6 nt, respectively. Since we have shown that G-quadruplex tends to form at the farthest 3' distal end of telomere DNA leaving a tail of no more than 5 nt, these results imply that G-quadruplex formation may play a role in regulating reactions at the telomere ends and, as a result, serve as effective drug target for intervening telomere function.     

有限延伸法检测端粒酶活性

人端粒酶是一种核蛋白体,通过其内含的RNA模板与端粒末端配对把重复端粒片段添加在端粒3＇末端|因此,端粒酶活性与细胞凋亡、衰老、永生化有密切关系,是癌症临床预测诊断的一个生物标签．现有的端粒酶活性检测方法,存在灵敏度低和不易定量等问题．本研究采用错配有限延伸法检测端粒酶活性：在人端粒酶延伸人工合成的游离端粒酶底物时,只加入dATP和dGTP,端粒酶只能把底物延伸4个脱氧核糖核苷酸AGGG．然后加入dNTP,让端粒酶延伸的产物和一条长的引物配对从而延伸出PCR模板|再加入引物进行热启动PCR．PCR后进行非变性PAGE (polyacrylamide gel electrophoresis),得到希望的唯一1条目标带．同时,用不同的端粒酶浓度梯度进行优化,发现有限延伸法检测端粒酶活性的下限达到250个HeLa细胞．