Preferential binding of a G-quadruplex ligand to human chromosome ends (original) (raw)
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Differential Effects of the G-Quadruplex Ligand 360A in Human Normal and Cancer Cells
DNA Repair and Human Health, 2011
Telomeres are essential for chromosome replication and genome integrity. The 3' singlestranded overhang of human telomere may adopt particular conformations such as T-loops and G-quadruplexes. Reactivated in most tumors, telomerase, a specific reverse transcriptase that elongates the telomeres, is thought to enable cancer cells to proliferate in an unlimited manner, thereby correcting the normal telomere erosion that occurs during cell division. The level of interest in G-quadruplex has increased due to their ability to inhibit telomerase activity. We have investigated chromosomal binding and the cellular effects induced by pyridine derivative G-quadruplex ligand in human normal and tumour cells. We show from our analysis that this G-quadruplex ligand preferentially binds the terminal regions of chromosomes in both normal and tumour cells. This compound also induces DNA damage signals in a strictly ATM-dependent manner, inhibits cell proliferation and induces apoptosis. We further observed by telo-FISH and Chromosome Orientation-FISH that this compound induces specific telomere aberrations either during or after replication and mainly consisting of sister telomere fusions and recombination events principally involving the lagging strand telomeres. We have also observed that ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia Related) reduce telomere instability independently of apoptosis suggesting its direct role in preventing inappropriate DNA repair at the telomeres. We have further demonstrated that, even at elevated concentrations, G-quadruplex ligand has limited effects on proliferation of normal cells, and does not induce apoptosis or telomere aberrations. Interestingly, we observed induction of reversible premature senescence in primary fibroblasts. Taken together, our results suggest that the protein composition and/or organization of the telomeres differ markedly between normal and cancer cells, and provide higher telomere stability to normal cells. 1.1 Structure of telomeres Telomeres are nucleoprotein structures located at the ends of chromosomes (Figure 1A). Human telomeric DNA contains double-stranded repeats of the motif TTAGGG (5-20 Kb) followed by a G-rich 3'-overhang (Makarov et al., 1997) (Figure 1B). In human chromosomes, the long terminal protrusions of single-stranded G-rich sequence have been reported to vary from 50 to more than 500 nucleotides (Makarov et al., 1997; Stewart et al., 2003). Telomeres have also been considered to be transcriptionally silent, but mammalian www.intechopen.com
Journal of Biological Chemistry, 2003
Ligands that stabilize the telomeric G-rich singlestranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. Ligand 12459, a potent G-quadruplex ligand that belongs to the triazine series, has been previously shown to induce both telomere shortening and apoptosis in the human A549 cell line as a function of its concentration and time exposure. We show here that A549 clones obtained after mutagenesis and selected for resistance to the short term effect of ligand 12459 frequently displayed hTERT transcript overexpression (2-6-fold). Overexpression of hTERT was also characterized in two resistant clones (JFD10 and JFD18) as an increase in telomerase activity, leading to an increase in telomere length. An increased frequency of anaphase bridges was also detected in JFD10 and JFD18, suggesting an alteration of telomere capping functions. Transfection of either hTERT or DN-hTERT cDNAs into A549 cells did not confer resistance or hypersensitivity to the short term effect of ligand 12459, indicating that telomerase expression is not the main determinant of the antiproliferative effect of ligand 12459. In contrast, transfection of DN-hTERT cDNA into resistant JFD18 cells restored sensitivity to apoptotic concentrations of ligand 12459, suggesting that telomerase does participate in the resistance to this G-quadruplex ligand. This work provides evidence that telomerase activity is not the main target for the 12459 G-quadruplex ligand but that hTERT functions contribute to the resistance phenotype to this class of agents.
Ligand Binding to Tandem G Quadruplexes from Human Telomeric DNA
ChemBioChem, 2008
Ligand-induced stabilization of intramolecular telomeric G quadruplexes produced in the single-stranded overhang of the human telomere has become an attractive strategy for the development of anticancer drugs. [1] Several distinct solution conformations of human telomeric G quadruplexes have been elucidated in the presence of sodium [2] and potassium [3] cations. The K +-form, hybrid-type G-quadruplex structure has been considered to be a physiologically relevant conformation of the human telomeric sequence, and thus, can be specifically targeted by G-quadruplex-interactive, small-molecule A C H T U N G T R E N N U N G drugs. [3a, b] Recently, a beads-on-a-string model was proposed for the telomeric overhang, in which every four consecutive Grich repeats adopt an individual G-quadruplex structure, and two G-quadruplex units are connected by one TTA linker. [3a, c, 4] Ligand binding to the G quadruplex has mostly been investigated on telomere sequences producing a single G quadruplex, but few studies of ligand binding to beads-on-a-string G quadruplexes have been reported. To gain insight into the beads-on-a-string model and the nature of ligand binding, we undertook the polymerase stop assay on human telomere sequences of three to eight repeats (Table S1 in the Supporting Information) with TMPyP4, a G-quadruplex-interactive ligand, [5] and sanguinarine (Scheme 1), a natural isoquinoline alkaloid. The results described in this paper confirm the beads-on-astring structure of telomeric overhang and suggest a mode of ligand binding between tandem G-quadruplex beads. These observations should be taken into account for structure-based design of anticancer drugs targeting human telomeric DNA. Sanguinarine is a natural isoquinoline alkaloid isolated from the North American herb bloodroot (Sanguinaria canadensis). It was approved by the FDA in 2003 to be added to oral cleansing products as an antibacterial agent. Sanguinarine also possesses potent anticancer activity. [6] We have previously reported its DNA-binding activity and distinct sequence selectivity to double-stranded DNA, [7] which was proposed to be one of the molecular mechanisms of its anticancer activity. The structural similarity of sanguinarine with berberine, another isoquinoline alkaloid possessing G-quadruplex-binding activity, [8] prompted us to speculate that sanguinarine is probably a G-quadruplex binder. In this communication, we report its binding to the A C H T U N G T R E N N U N G telomeric overhang using DNA polymerase stop assays. [9] DNA templates Tem-3 and Tem-4 (Table S1), which contains three and four human telomeric repeats dA C H T U N G T R E N N U N G (TTAGGG), respectively, were employed, and TMPyP4 was used as the reference compound in the assay. [5, 9a, 10] Neither TMPyP4 nor sanguinarine blocked DNA synthesis on Tem-3, because an intramolecular Gquadruplex structure could not form with three human telomeric repeats on Tem-3 (Figure 1). In contrast, both sanguinarine and TMPyP4 produced paused bands in DNA synthesis on Tem-4. The position of the paused bands was the same for the two ligands. For each ligand, a series of concentration-dependent paused bands appeared at the beginning of the G-quadruplex-forming site, that is, the first site of G-rich repeats in Tem-4 (from 3' to 5'). In the presence of 3 mm TMPyP4, the polymerase reaction was totally suppressed to give no elongation of the primer. The tight binding of sanguinarine and TMPyP4 to the K +-form hybrid-type G-quadruplex structure was clearly indicated from the large increase in the melting temperature (DT m
Molecules
Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashor...
The rational design of ligands targeting human telomeric DNA G‐quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G‐quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G‐quadruplex ligands (360A, Phen‐DC3 and pyridostatin) induce con‐ formational changes of telomeric DNA G‐quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K + (as determined by electrospray mass spectrometry), and hence presumably only two consecutive G‐quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel struc‐ tures, and L2H2‐6M(2)OTD, known not to disrupt the hybrid‐1 structure, did not show such K + removal. Instead, binding of the cyclic oxazole L2H2‐6M(2)OTD was accompanied by the uptake of one additional K +. Also contrasting with telomeric G‐quadruplexes, the parallel‐stranded Pu24‐myc G‐quadruplex, to which Phen‐DC3 is known to bind by end‐stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce confor‐ mational switching of the human telomeric G‐quadruplexes to an antiparallel structure, and that this conformational change is accompanied by removal of one inter‐quartet cation.
Reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action
Proceedings of the National Academy of Sciences, 2007
Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.
Binding properties of human telomeric quadruplex multimers: A new route for drug design
Biochimie, 2011
Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21e26 nt). However, the 3 0-terminal single-stranded human telomeric DNA is actually 100e200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences. In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG) 8 TT structure formed by two adjacent quadruplex units with the binding properties of the (AG 3 TT) 4 single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplexequadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.