A. De Cian - Academia.edu (original) (raw)

Papers by A. De Cian

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structur... more The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the stabilization and selectivity of two well-known quadruplex ligands (telomestatin and a cationic porphyrin) towards the human telomeric G-quadruplex species, with FRET. Both molecules strongly stabilize the G-quadruplex, but telomestatin appears much more selective, as shown by competition experiments with double-stranded DNA.

Genome research, 2014

The generation of genetically modified animals is important for both research and commercial purp... more The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and...

Journal of the American Chemical Society, 2007

Trying to control and regulate the expression of genes is emerging as a very appealing anticancer... more Trying to control and regulate the expression of genes is emerging as a very appealing anticancer strategy. Indeed, chemotherapy aiming at designing molecules able to interact with unusual structures of nucleic acids is currently subjected to a close examination. In that sense, quadruplex-DNA is a particularly attractive high-order structure since it appears to be putatively present in pivotal genomic regions such as telomeres, 2 promoters of oncogenes, and most growth control genes. 3 Convincing reports on the efficiency of quadruplex interacting molecules as therapeutically active agents are beginning to appear in the literature. 4 Thus, intensive investigations are currently oriented toward the design and development of new G-quadruplex ligands. Of particular interest are molecules with high quadruplex affinity that exhibit the ability to discriminate quadruplex-DNA from nucleus predominant duplex-DNA. Up to now, the leading G-quadruplex binder has been the natural product telomestatin. 5 Nevertheless, its total synthesis was achieved only very recently, and the complexity of the process renders its convenient exploitation difficult. Recently, some of us reported on bisquinolinium compounds that exhibit exceptional affinity and selectivity for quadruplex-forming oligonucleotides. 4c,7 The anti-proliferative activity of these compounds has been demonstrated, as well as their preferential binding to telomeric regions of human chromosomes, 7b thus providing new insights on quadruplex existence in vivo. These exciting results prompted us to develop the bisquinolinium family of compounds which was furthermore facilitated by a convenient and rapid synthetic access.

Biochimie, 2008

Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer... more Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer therapy. The objective of this review is to present the latest view on the mechanism(s) of action of telomerase inhibitors, with an emphasis on a specific class of telomere ligands called G-quadruplex ligands, and to discuss their potential use in oncology. Biochimie 90 (2008) 131e155 www.elsevier.com/locate/biochi 2.2. The shelterin complex and beyond The proteins that protect telomere extremities were identified during the last decade and make up a complex called the telosome [61] or shelterin [62]. This complex is composed of six proteins. Three of these bind directly to the telomeric 132 A. De Cian et al. / Biochimie 90 (2008) 131e155

Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zeb... more Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency and TALENs have also successfully been employed to knock-in DNA cassettes at defined loci via homologous recombination (HR). Here, we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double strand break (DSB) repair pathways. After coinjection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility to easily target DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss of function alleles. Due to its simplicity, flexibility and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rathe... more The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. General rules have been extended to longer DNA motifs or sequences containing modified bases such as 8-oxo or 7-deaza guanine. Results were compared with the canonical TG4T and TG5T tetramers: we demonstrate huge differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence.

Organic & Biomolecular Chemistry, 2007

A family of terpyridine metallo-organic complexes has been designed and its recognition propertie... more A family of terpyridine metallo-organic complexes has been designed and its recognition properties of G-quadruplex-DNA investigated. The series combines easy synthetic access and good affinity-selectivity ratio for quadruplex-DNA. Our study also highlights that the geometry of the metal center strongly governs the ability of the compounds to discriminate quadruplex from duplex-DNA.

Organic & Biomolecular Chemistry, 2006

A series of aminoglycoside-capped macrocyclic structures 9-12 has been prepared using intramolecu... more A series of aminoglycoside-capped macrocyclic structures 9-12 has been prepared using intramolecular bis-tethering of neomycin on three aromatic platforms (phenanthroline, acridine, quinacridine). Based on NMR and calculations studies, it was found that the cyclic compounds adopt a highly flexible structure without conformational restriction of the aminoglycoside moiety. FRET-melting stabilization measurements showed that the series displays moderate to high affinity for the G4-conformation of human telomeric repeats, this effect being correlated with the size of the aromatic moiety. In addition, a FRET competition assay evidenced the poor binding ability of all macrocycles for duplex DNA and a clear binding preference for loop-containing intramolecular G4 structures compared to tetramolecular parallel G4 DNA. Finally, TRAP experiments demonstrated that the best G4-binder (quinacridine 11) is also a potent and selective telomerase inhibitor with an IC 50 in the submicromolar range (200 nM).

Nucleic Acids Research, 2007

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a blo... more Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/ thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromoguanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5 0 end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Nucleic Acids Research, 2006

Repetitive 5 0 GGXGG DNA segments abound in, or near, regulatory regions of the genome and may fo... more Repetitive 5 0 GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5 0 GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallelstranded quadruplex formed by TG 4 T. We demonstrate large differences (up to 10 5 -fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG 4 T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG 4 T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.

Molecular Cancer Therapeutics, 2011

The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, ... more The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and gH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 mmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications. Mol Cancer Ther; 10(10); 1784-95. Ó2011 AACR.

Methods, 2007

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structur... more The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the Xuorescent tags, the incubation buVer, and the method chosen for T m calculation, complicating a direct comparison of the results obtained by diVerent laboratories.

Cytotechnology, 2004

The enzyme telomerase is involved in the replication of telomeres, specialized structures that ca... more The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3¢-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.

Biochimie, 2008

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simp... more We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex-over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplexand duplex-DNA matrices. The original protocol was tested using various quadruplex-and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays. that is not subjected to structural interconversion, the TBA (thrombin binding aptamer) . Finally, because quadruplex-vs. duplex-selectivity is a critical issue and because binding of both probe and ligand to duplex-DNA may be length-and sequence-dependent, we compare the results obtained with the 17-base pair duplex-DNA (dsl7) used in our initial work with a 26-bp duplex (ds26). G4-FID experiments reported herein were carried out with a set of 16 quadru-plex-ligands (from bisquinolinium to metallo-organic ligands) with two different QFOs (22AG , TBA ), in two different salt conditions (Na + , K + ) and with two different DNA control duplex-DNAs (dsl7 [31] and ds26 ).

Proceedings of the National Academy of Sciences, 2007

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of the... more 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.

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structur... more The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the stabilization and selectivity of two well-known quadruplex ligands (telomestatin and a cationic porphyrin) towards the human telomeric G-quadruplex species, with FRET. Both molecules strongly stabilize the G-quadruplex, but telomestatin appears much more selective, as shown by competition experiments with double-stranded DNA.

Genome research, 2014

The generation of genetically modified animals is important for both research and commercial purp... more The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and...

Journal of the American Chemical Society, 2007

Trying to control and regulate the expression of genes is emerging as a very appealing anticancer... more Trying to control and regulate the expression of genes is emerging as a very appealing anticancer strategy. Indeed, chemotherapy aiming at designing molecules able to interact with unusual structures of nucleic acids is currently subjected to a close examination. In that sense, quadruplex-DNA is a particularly attractive high-order structure since it appears to be putatively present in pivotal genomic regions such as telomeres, 2 promoters of oncogenes, and most growth control genes. 3 Convincing reports on the efficiency of quadruplex interacting molecules as therapeutically active agents are beginning to appear in the literature. 4 Thus, intensive investigations are currently oriented toward the design and development of new G-quadruplex ligands. Of particular interest are molecules with high quadruplex affinity that exhibit the ability to discriminate quadruplex-DNA from nucleus predominant duplex-DNA. Up to now, the leading G-quadruplex binder has been the natural product telomestatin. 5 Nevertheless, its total synthesis was achieved only very recently, and the complexity of the process renders its convenient exploitation difficult. Recently, some of us reported on bisquinolinium compounds that exhibit exceptional affinity and selectivity for quadruplex-forming oligonucleotides. 4c,7 The anti-proliferative activity of these compounds has been demonstrated, as well as their preferential binding to telomeric regions of human chromosomes, 7b thus providing new insights on quadruplex existence in vivo. These exciting results prompted us to develop the bisquinolinium family of compounds which was furthermore facilitated by a convenient and rapid synthetic access.

Biochimie, 2008

Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer... more Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer therapy. The objective of this review is to present the latest view on the mechanism(s) of action of telomerase inhibitors, with an emphasis on a specific class of telomere ligands called G-quadruplex ligands, and to discuss their potential use in oncology. Biochimie 90 (2008) 131e155 www.elsevier.com/locate/biochi 2.2. The shelterin complex and beyond The proteins that protect telomere extremities were identified during the last decade and make up a complex called the telosome [61] or shelterin [62]. This complex is composed of six proteins. Three of these bind directly to the telomeric 132 A. De Cian et al. / Biochimie 90 (2008) 131e155

Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zeb... more Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency and TALENs have also successfully been employed to knock-in DNA cassettes at defined loci via homologous recombination (HR). Here, we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double strand break (DSB) repair pathways. After coinjection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility to easily target DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss of function alleles. Due to its simplicity, flexibility and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rathe... more The melting of tetramolecular DNA or RNA quadruplexes is kinetically irreversible. However, rather than being a hindrance, this kinetic inertia allows us to study association and dissociation processes independently. General rules have been extended to longer DNA motifs or sequences containing modified bases such as 8-oxo or 7-deaza guanine. Results were compared with the canonical TG4T and TG5T tetramers: we demonstrate huge differences (up to 10(5)-fold) in the association constants of these quadruplexes depending on primary sequence.

Organic & Biomolecular Chemistry, 2007

A family of terpyridine metallo-organic complexes has been designed and its recognition propertie... more A family of terpyridine metallo-organic complexes has been designed and its recognition properties of G-quadruplex-DNA investigated. The series combines easy synthetic access and good affinity-selectivity ratio for quadruplex-DNA. Our study also highlights that the geometry of the metal center strongly governs the ability of the compounds to discriminate quadruplex from duplex-DNA.

Organic & Biomolecular Chemistry, 2006

A series of aminoglycoside-capped macrocyclic structures 9-12 has been prepared using intramolecu... more A series of aminoglycoside-capped macrocyclic structures 9-12 has been prepared using intramolecular bis-tethering of neomycin on three aromatic platforms (phenanthroline, acridine, quinacridine). Based on NMR and calculations studies, it was found that the cyclic compounds adopt a highly flexible structure without conformational restriction of the aminoglycoside moiety. FRET-melting stabilization measurements showed that the series displays moderate to high affinity for the G4-conformation of human telomeric repeats, this effect being correlated with the size of the aromatic moiety. In addition, a FRET competition assay evidenced the poor binding ability of all macrocycles for duplex DNA and a clear binding preference for loop-containing intramolecular G4 structures compared to tetramolecular parallel G4 DNA. Finally, TRAP experiments demonstrated that the best G4-binder (quinacridine 11) is also a potent and selective telomerase inhibitor with an IC 50 in the submicromolar range (200 nM).

Nucleic Acids Research, 2007

Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a blo... more Parallel tetramolecular quadruplexes may be formed with short oligodeoxynucleotides bearing a block of three or more guanines. We analyze the properties of sequence variants of parallel quadruplexes in which each guanine of the central block was systematically substituted with a different base. Twelve types of substitutions were assessed in more than 100 different sequences. We conducted a comparative kinetic analysis of all tetramers. Electrospray mass spectrometry was used to count the number of inner cations, which is an indicator of the number of effective tetrads. In general, the presence of a single substitution has a strong deleterious impact on quadruplex stability, resulting in reduced quadruplex lifetime/ thermal stability and in decreased association rate constants. We demonstrate extremely large differences in the association rate constants of these quadruplexes depending on modification position and type. These results demonstrate that most guanine substitutions are deleterious to tetramolecular quadruplex structure. Despite the presence of well-defined non-guanine base quartets in a number of NMR and X-ray structures, our data suggest that most non-guanine quartets do not participate favorably in structural stability, and that these quartets are formed only by virtue of the docking platform provided by neighboring G-quartets. Two notable exceptions were found with 8-bromoguanine (X) and 6-methyl-isoxanthopterin (P) substitutions, which accelerate quadruplex formation by a factor of 10 when present at the 5 0 end. The thermodynamic and kinetic data compiled here are highly valuable for the design of DNA quadruplex assemblies with tunable association/dissociation properties.

Nucleic Acids Research, 2006

Repetitive 5 0 GGXGG DNA segments abound in, or near, regulatory regions of the genome and may fo... more Repetitive 5 0 GGXGG DNA segments abound in, or near, regulatory regions of the genome and may form unusual structures called G-quadruplexes. Using NMR spectroscopy, we demonstrate that a family of 5 0 GCGGXGGY sequences adopts a folding topology containing double-chain reversals. The topology is composed of two bistranded quadruplex monomeric units linked by formation of G:C:G:C tetrads. We provide a complete thermodynamic and kinetic analysis of 13 different sequences using absorbance spectroscopy and DSC, and compare their kinetics with a canonical tetrameric parallelstranded quadruplex formed by TG 4 T. We demonstrate large differences (up to 10 5 -fold) in the association constants of these quadruplexes depending on primary sequence; the fastest samples exhibiting association rate equal or higher than the canonical TG 4 T quadruplex. In contrast, all sequences studied here unfold at a lower temperature than this quadruplex. Some sequences have thermodynamic stability comparable to the canonical TG 4 T tetramolecular quadruplex, but with faster association and dissociation. Sequence effects on the dissociation processes are discussed in light of structural data.

Molecular Cancer Therapeutics, 2011

The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, ... more The aim of this study was to test in vitro the efficacy of TAC, an original G-quadruplex ligand, as a potential radiosensitizing agent for glioblastoma multiforme (GBM). Two human radioresistant telomerase-positive GBM cell lines (SF763 and SF767) were analyzed, with and without TAC treatment, for telomere length, cell proliferation, apoptosis, cell-cycle distribution, gene expression, cytogenetic aberrations, clonogenic survival assay, 53BP1 immunofluorescence staining, and gH2AX phosphorylation. We found that low concentrations of TAC (0.5 and 1 mmol/L) inhibited the proliferation of GBM cells in a concentration-dependent manner after only 1 week of treatment, with minimal effects on cell cycle and apoptosis. TAC treatment had no visible effect on average telomere length but modified expression levels of telomere-related genes (hTERT, TRF1, and TRF2) and induced concentration-dependent DNA damage response and dicentric chromosomes. Survival curves analysis showed that exposure to nontoxic, subapoptotic concentrations of TAC enhanced radiation-induced killing of GBM cells. Analysis of DNA repair after irradiation revealed delayed repair kinetics in GBM cells treated with TAC. Furthermore, the combined treatment (TAC and radiation) significantly increased the frequency of chromosomal aberrations as compared with radiation alone. These findings provide the first evidence that exposure to a G4 ligand radiosensitizes human glioblastoma cells and suggest the prospect of future therapeutic applications. Mol Cancer Ther; 10(10); 1784-95. Ó2011 AACR.

Methods, 2007

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structur... more The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomeres and telomerase are relevant targets in oncology, and telomere ligands and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analysed the FRET method used to measure the stabilization and selectivity of quadruplex ligands towards the human telomeric G-quadruplex. The stabilization value depends on the nature of the Xuorescent tags, the incubation buVer, and the method chosen for T m calculation, complicating a direct comparison of the results obtained by diVerent laboratories.

Cytotechnology, 2004

The enzyme telomerase is involved in the replication of telomeres, specialized structures that ca... more The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3¢-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.

Biochimie, 2008

We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simp... more We report here the details of G4-FID (G-quadruplex fluorescent intercalator displacement), a simple method aiming at evaluating quadruplex-DNA binding affinity and quadruplex-over duplex-DNA selectivity of putative ligands. This assay is based on the loss of fluorescence upon displacement of thiazole orange from quadruplexand duplex-DNA matrices. The original protocol was tested using various quadruplex-and duplex-DNA targets, and with a wide panel of G-quadruplex ligands belonging to different families (i.e. from quinacridines to metallo-organic ligands) likely to display various binding modes. The reliability of the assay is further supported by comparisons with FRET-melting and ESI-MS assays. that is not subjected to structural interconversion, the TBA (thrombin binding aptamer) . Finally, because quadruplex-vs. duplex-selectivity is a critical issue and because binding of both probe and ligand to duplex-DNA may be length-and sequence-dependent, we compare the results obtained with the 17-base pair duplex-DNA (dsl7) used in our initial work with a 26-bp duplex (ds26). G4-FID experiments reported herein were carried out with a set of 16 quadru-plex-ligands (from bisquinolinium to metallo-organic ligands) with two different QFOs (22AG , TBA ), in two different salt conditions (Na + , K + ) and with two different DNA control duplex-DNAs (dsl7 [31] and ds26 ).

Proceedings of the National Academy of Sciences, 2007

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of the... more 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.