The Experts below are selected from a list of 351 Experts worldwide ranked by ideXlab platform
Kazuo Shinya - One of the best experts on this subject based on the ideXlab platform.
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synthesis of heptaoxazole macrocyclic analogues of Telomestatin and evaluation of their telomerase inhibitory activities
Bulletin of the Chemical Society of Japan, 2013Co-Authors: Kazuaki Shibata, Kazuo Shinya, Masahito Yoshida, Motoki Takagi, Takashi Takahashi, Takayuki DoiAbstract:We have demonstrated various synthetic routes to heptaoxazole macrocyclic analogues of Telomestatin and evaluated their inhibitory activities against telomerase. We synthesized three heptaoxazole m...
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atrx deficiency induces telomere dysfunction endocrine defects and reduced life span
Journal of Clinical Investigation, 2013Co-Authors: Ashley L Watson, Kazuo Shinya, Lauren A. Solomon, Frank Beier, Yan Jiang, Matthew Edwards, Nathalie G BerubeAbstract:Human ATRX mutations are associated with cognitive deficits, developmental abnormalities, and cancer. We show that the Atrx-null embryonic mouse brain accumulates replicative damage at telomeres and pericentromeric heterochromatin, which is exacerbated by loss of p53 and linked to ATM activation. ATRX-deficient neuroprogenitors exhibited higher incidence of telomere fusions and increased sensitivity to replication stress–inducing drugs. Treatment of Atrx-null neuroprogenitors with the G-quadruplex (G4) ligand Telomestatin increased DNA damage, indicating that ATRX likely aids in the replication of telomeric G4-DNA structures. Unexpectedly, mutant mice displayed reduced growth, shortened life span, lordokyphosis, cataracts, heart enlargement, and hypoglycemia, as well as reduction of mineral bone density, trabecular bone content, and subcutaneous fat. We show that a subset of these defects can be attributed to loss of ATRX in the embryonic anterior pituitary that resulted in low circulating levels of thyroxine and IGF-1. Our findings suggest that loss of ATRX increases DNA damage locally in the forebrain and anterior pituitary and causes tissue attrition and other systemic defects similar to those seen in aging.
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Telomestatin impairs glioma stem cell survival and growth through the disruption of telomeric g quadruplex and inhibition of the proto oncogene c myb
Clinical Cancer Research, 2012Co-Authors: Takeshi Miyazaki, Yang Pan, Kaushal Joshi, Deepti Purohit, Habibe Demir, Sarmistha Mazumder, Sachiko Okabe, Takao Yamori, Mariano S Viapiano, Kazuo ShinyaAbstract:Purpose: Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). Experimental Design: The effects of a G-quadruplex ligand, Telomestatin, were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSC), and normal fetal neural precursors in vitro and in vivo . The molecular targets of Telomestatin were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data were then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples. Results: Telomestatin impaired the maintenance of GSC stem cell state by inducing apoptosis in vitro and in vivo . The migration potential of GSCs was also impaired by Telomestatin treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to Telomestatin. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by Telomestatin in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb , as a novel molecular target of Telomestatin in GSCs, and pharmacodynamic analysis in Telomestatin-treated tumor-bearing mouse brains showed a reduction of c-Myb in tumors in vivo . Knockdown of c-Myb phenocopied Telomestatin-treated GSCs both in vitro and in vivo , and restoring c-Myb by overexpression partially rescued the phenotype. Finally, c-Myb expression was markedly elevated in surgical specimens of GBMs compared with normal tissues. Conclusions: These data indicate that Telomestatin potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBMs. Clin Cancer Res; 18(5); 1268–80. ©2012 AACR .
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design and synthesis of Telomestatin derivatives containing methyl oxazole and their g quadruplex stabilizing activities
ChemInform, 2011Co-Authors: Satoki Majima, Kazuo Shinya, Masayuki Tera, Keisuke Iida, Kazuo NagasawaAbstract:The macrocyclic hexaoxazole skeleton (I), containing bis- and tetramethyl oxazoles, is synthesized as a novel G-quadruplex ligand.
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abstract 3302 the effects of the g quadruplex ligand Telomestatin to human brain tumor stem cell survival and growth
Cancer Research, 2011Co-Authors: Takeshi Miyazaki, Kazuo Shinya, Yang Pan, Kaushal Joshi, Habibe Demir, Sachiko Okabe, Takao Yamori, Mariano S Viapiano, Hiroyuki Seimiya, Ichiro NakanoAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastoma multiforme (GBM) is the leading cause of death among primary brain tumors in adults and the current therapies have only palliative effect on prognosis of patients. Recently, stem cell-like cells in GBM (brain tumor stem-like cells; BTSC) have gained substantial attention as a potential therapeutic target. Hallmarks of BTSC include their self-renewal capacity and highly migratory potential. In this study, we demonstrate that treatment of patient-derived BTSC with a G-quadruplex ligand, Telomestatin (TMS), inhibits BTSC self-renewal and maintenance of their stem cell state, and induces their apoptosis in vitro and in vivo. In contrast, both normal precursors and non-stem tumor cells from the matched samples are relatively resistant to TMS treatment. Treatment with a lower dose of TMS specifically inhibits BTSC migration into normal brain. Immunofluorescence in situ hybridization with TMS-treated GBM cells displays both telomeric and non-telomeric DNA damage in BTSC but not in non-stem tumor cells. cDNA microarray analysis identifies a proto-oncogene, c-Myb, as a target of TMS in BTSC, and the pharmacodynamic analysis with TMS-treated tumor-bearing mouse brains demonstrates reduction of c-Myb expression in tumors. An elevated c-Myb expression is found in surgical specimens of GBM compared to normal brain tissues. Lastly, TMS treatment of BTSC-derived mouse intracranial tumors reduces tumor sizes in vivo without any noticeable apoptotic cells in the normal brain, and a combined treatment of TMS with radiation or temozolomide results in additive inhibitory effects on GBM sphere growth in vitro. Collectively, these data indicate a potential avenue toward BTSC-directed therapeutic strategy using TMS via telomeric DNA damage and inhibition of c-Myb, which may offer a novel therapeutic approach for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3302. doi:10.1158/1538-7445.AM2011-3302
Laurence H Hurley - One of the best experts on this subject based on the ideXlab platform.
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evidence for the presence of a guanine quadruplex forming region within a polypurine tract of the hypoxia inducible factor 1α promoter
Biochemistry, 2005Co-Authors: Richard De Armond, Daekyu Sun, Laurence H Hurley, Stacey Wood, Scot W EbbinghausAbstract:The promoter of the hypoxia inducible factor 1 alpha (HIF-1α) gene has a polypurine/polypyrimidine tract (−65 to −85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-1α expression. Oligonucleotides representing this region of the HIF-1α promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product Telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1α promoter that regulates basal HIF-1α expression.
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Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric g quadruplex structure
Journal of the American Chemical Society, 2005Co-Authors: Evonne M Rezler, Jeyaprakashnarayanan Seenisamy, Sridevi Bashyam, Mu Yong Kim, Elizabeth W White, David W Wilson, Laurence H HurleyAbstract:The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while Telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.
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facilitation of a structural transition in the polypurine polypyrimidine tract within the proximal promoter region of the human vegf gene by the presence of potassium and g quadruplex interactive agents
Nucleic Acids Research, 2005Co-Authors: Daekyu Sun, Kexiao Guo, Jadrian J Rusche, Laurence H HurleyAbstract:The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and Telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and Telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3′-side residue of the predicted G-quadruplex-forming region in the presence of KCl and Telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of Telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.
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the differential effects of tmpyp4 tmpyp2 se2sap and Telomestatin on c myc gene expression can be explained by their differential interactions with the g quadruplex in the c myc silencer element
Cancer Research, 2004Co-Authors: Jeyaprakashnarayanan Seenisamy, Cory L Grand, Thomas S Dexheimer, Evonne M Rezler, Sridevi Bashyam, Laurence H HurleyAbstract:2301 The purine-rich strand of DNA in the Nuclease Hypersensitive Element III1 (NHE III1) of c-MYC forms a parallel type of G-quadruplex with three G-tetrads and three loops, and acts as a transcriptional repressor element. This parallel type of G-quadruplex exists as an equilibrium mixture of at least four different loop G-quadruplex isomers. c-MYC transcription can be controlled by ligand-mediated G-quadruplex interaction. TMPyP4, Se2SAP and Telomestatin all demonstrate stabilization of the G-quadruplex structure in the NHE III1 of the c-MYC promoter in the order of Se2SAP>Telomestatin>>TMPyP4. In contrast, while TMPyP4 showed a greater effect on lowering of c-MYC expression than Se2SAP, Telomestatin did not show any suppression of c-MYC transcription. TMPyP2 did not show any c-MYC down regulation because of its inability to interact with the G-quadruplex structure. Our previous studies suggested that the c-MYC transcriptional activator NM23-H2 recognizes and unfolds the parallel G-quadruplex structure in the silencer element of the c-MYC promoter region for subsequent transcriptional activation by hnRNPK and CNBP. Subsequent experiments on the inhibition of alkali induced cleavage of the purine-rich strand by NM23-H2 using the same compounds also correlated well with their differential effects on c-MYC expression. CD studies showed the induced structural changes in the parallel c-MYC G-quadruplex with Se2SAP and TMPyP4, while Telomestatin and TMPyP2 did not show any structural changes. Photocleavage experiments were conducted to identify the induced structural changes with TMPyP4 and Se2SAP. Se2SAP converts one loop of the parallel c-MYC G-quadruplex to the antiparallel direction while maintaining the other two loops in the parallel direction and forms a mixed parallel/antiparallel type of G-quadruplex. TMPyP4 converts all three loops to the antiparallel direction, forming a chair type of G-quadruplex. Based upon these results we can now fully rationalize the differential effects of these drugs on c-MYC expression, and most importantly, we have identified the chair type of G-quadruplex in the NHE III1 as the inactive form for c-MYC transcription activation.
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telomerase inhibition and cell growth arrest after Telomestatin treatment in multiple myeloma
Clinical Cancer Research, 2004Co-Authors: Masood A Shammas, Laurence H Hurley, Robert Shmookler J Reis, Hemanta Koley, Kenneth C Anderson, Nikhil C MunshiAbstract:Purpose: The aim of this study was to test the efficacy of Telomestatin, an intramolecular G-quadruplex intercalating drug with specificity for telomeric sequences, as a potential therapeutic agent for multiple myeloma. Experimental Design: We treated ARD, ARP, and MM1S myeloma cells with various concentrations of Telomestatin for 7 days and evaluated for telomerase activity. Myeloma cells were treated with the minimal effective Telomestatin concentration for 3–5 weeks. Every 7 th day the fraction of live cells was determined by trypan blue exclusion, aliquots of cells were removed for various molecular assays, and the remaining cells were replated at the same cell number and at the same concentration of Telomestatin. Telomere length, apoptosis, and gene expression changes were monitored as described in detail in “Materials and Methods.” Results: Telomestatin treatment led to inhibition of telomerase activity, reduction in telomere length, and apoptotic cell death in ARD, MM1S, and ARP myeloma cells. Gene expression profile after 1 and 7 days of Telomestatin treatment revealed ≥2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. No changes were seen in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination. Conclusions: These results demonstrate that Telomestatin exerts its antiproliferative and proapoptotic effects in myeloma cells via inhibition of telomerase and subsequent reduction in telomere length. We conclude that telomerase is an important potential therapeutic target for multiple myeloma therapy, and G-quadruplex interacting agents with specificity for binding to telomeric sequences can be important agents for additional evaluation.
Jean -françois Riou - One of the best experts on this subject based on the ideXlab platform.
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the g quadruplex ligand Telomestatin impairs binding of topoisomerase iiiα to g quadruplex forming oligonucleotides and uncaps telomeres in alt cells
PLOS ONE, 2009Co-Authors: Nassima Temimesmaali, Chantal Trentesaux, Jean -françois Riou, Kazuo Shinya, Lionel Guittat, Assitan SidibeAbstract:In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIα (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by Telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, Telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in Telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.
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The G-quadruplex ligand Telomestatin impairs binding of topoisomerase IIIalpha to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.
Public Library of Science (PLoS), 2009Co-Authors: Nassima Temime-smaali, Chantal Trentesaux, Kazuo Shin-ya, Lionel Guittat, Assitan Sidibe, Jean -françois RiouAbstract:In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIalpha (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by Telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, Telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in Telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed
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Telomestatin induced telomere uncapping is modulated by pot1 through g overhang extension in ht1080 human tumor cells
Journal of Biological Chemistry, 2006Co-Authors: Dennis Gomez, Chantal Trentesaux, Thomas Wenner, Bertrand Brassart, Celine Douarre, Victoria El Khoury, Hamid Morjani, Mariefrancoise Odonohue, Kazuo Shinya, Jean -françois RiouAbstract:Abstract Telomestatin is a potent G-quadruplex ligand that interacts with the 3′ telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by Telomestatin. We show here that the treatment of HT1080 human tumor cells by Telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the γ-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for Telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to Telomestatin treatment characterized by a protection to the Telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased Telomestatin concentration. Altogether these results suggest that Telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.
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interaction of Telomestatin with the telomeric single strand overhang
Journal of Biological Chemistry, 2004Co-Authors: Dennis Gomez, Kazuo Shinya, Rajaa Paterski, Thibault Lemarteleur, Jeanlouis Mergny, Jean -françois RiouAbstract:The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicative senescence. The repeated sequence forming a G-overhang is able to adopt a peculiar four-stranded DNA structure in vitro called a G-quadruplex, which is a poor substrate for telomerase. Small molecule ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and a delayed growth arrest. Here we show that the G-quadruplex ligand Telomestatin has a dramatic effect on the conformation of intracellular G-overhangs. Competition experiments indicate that Telomestatin strongly binds in vitro and in vivo to the telomeric overhang and impairs its single-stranded conformation. Long-term treatment of cells with Telomestatin greatly reduces the G-overhang size, as evidenced by specific hybridization or telomeric oligonucleotide ligation assay experiments, with a concomitant delayed loss of cell viability. In vivo protection experiments using dimethyl sulfate also indicate that Telomestatin treatment alters the dimethyl sulfate effect on G-overhangs, a result compatible with the formation of a local quadruplex structure at telomeric overhang. Altogether these experiments strongly support the hypothesis that the telomeric G-overhang is an intracellular target for the action of Telomestatin.
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telomerase downregulation induced by the g quadruplex ligand 12459 in a549 cells is mediated by htert rna alternative splicing
Nucleic Acids Research, 2004Co-Authors: Dennis Gomez, Thibault Lemarteleur, Jeanlouis Mergny, Laurent Lacroix, Patrick Mailliet, Jean -françois RiouAbstract:Ligand 12459, a potent G-quadruplex-interacting agent that belongs to the triazine series, was previously shown to downregulate telomerase activity in the human A549 lung carcinoma cell line. We show here that the downregulation of telomerase activity is caused by an alteration of the hTERT splicing pattern induced by 12459, i.e. an almost complete disappearance of the active (+α,+β) transcript and an over-expression of the inactive –β transcript. Spliced intron 6 forming the –β hTERT transcript contained several tracks of G-rich sequences able to form G-quadruplexes. By using a specific PCR-stop assay, we show that 12459 is able to stabilize the formation of these G-quadruplex structures. A549 cell line clones selected for resistance to 12459 have been analyzed for their hTERT splicing pattern. Resistant clones are able to maintain the active hTERT transcript under 12459 treatment, suggesting the appearance of mechanisms able to bypass the 12459-induced splicing alterations. In contrast to 12459, Telomestatin and BRACO19, two other G-quadruplex-interacting agents, have no effect on the hTERT splicing pattern in A549 cells, are cytotoxic against the A549-resistant clones and display a lower efficiency to stabilize hTERT G-quadruplexes. These results lead us to propose that 12459 impairs the splicing machinery of hTERT through stabilization of quadruplexes located in the hTERT intron 6. Differences of selectivity between 12459, BRACO19 and Telomestatin for these hTERT quadruplexes may be important to explain their respective activity and inactivity against hTERT splicing.
Dennis Gomez - One of the best experts on this subject based on the ideXlab platform.
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Telomestatin induced telomere uncapping is modulated by pot1 through g overhang extension in ht1080 human tumor cells
Journal of Biological Chemistry, 2006Co-Authors: Dennis Gomez, Chantal Trentesaux, Thomas Wenner, Bertrand Brassart, Celine Douarre, Victoria El Khoury, Hamid Morjani, Mariefrancoise Odonohue, Kazuo Shinya, Jean -françois RiouAbstract:Abstract Telomestatin is a potent G-quadruplex ligand that interacts with the 3′ telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by Telomestatin. We show here that the treatment of HT1080 human tumor cells by Telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the γ-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for Telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to Telomestatin treatment characterized by a protection to the Telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased Telomestatin concentration. Altogether these results suggest that Telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.
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The G-quadruplex Ligand Telomestatin Inhibits POT1 Binding to Telomeric Sequences In vitro and Induces GFP-POT1 Dissociation from Telomeres in Human Cells
Cancer Research, 2006Co-Authors: Dennis Gomez, Thomas Wenner, Celine Douarre, Marie-françoise O'donohue, Jérome Macadré, Pascale Koebel, Marie-josèphe Giraud-panis, Hervé Kaplan, Alain Kolkes, Kazuo Shin-yaAbstract:Telomestatin is a potent G-quadruplex ligand that specifically interacts with the 3' telomeric overhang, leading to its degradation and that induces a delayed senescence and apoptosis of cancer cells. Protection of Telomere 1 (POT1) was recently identified as a specific single-stranded telomere-binding protein involved in telomere capping and T-loop maintenance. We showed here that a Telomestatin treatment inhibits POT1 binding to the telomeric overhang in vitro. The treatment of human EcR293 cells by Telomestatin induces a dramatic and rapid delocalization of POT1 from its normal telomere sites but does not affect the telomere localization of the double-stranded telomere-binding protein TRF2. Thus, we propose that G-quadruplex stabilization at telomeric G-overhang inactivates POT1 telomeric function, generating a telomere dysfunction in which chromosome ends are no longer properly protected.
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interaction of Telomestatin with the telomeric single strand overhang
Journal of Biological Chemistry, 2004Co-Authors: Dennis Gomez, Kazuo Shinya, Rajaa Paterski, Thibault Lemarteleur, Jeanlouis Mergny, Jean -françois RiouAbstract:The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicative senescence. The repeated sequence forming a G-overhang is able to adopt a peculiar four-stranded DNA structure in vitro called a G-quadruplex, which is a poor substrate for telomerase. Small molecule ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and a delayed growth arrest. Here we show that the G-quadruplex ligand Telomestatin has a dramatic effect on the conformation of intracellular G-overhangs. Competition experiments indicate that Telomestatin strongly binds in vitro and in vivo to the telomeric overhang and impairs its single-stranded conformation. Long-term treatment of cells with Telomestatin greatly reduces the G-overhang size, as evidenced by specific hybridization or telomeric oligonucleotide ligation assay experiments, with a concomitant delayed loss of cell viability. In vivo protection experiments using dimethyl sulfate also indicate that Telomestatin treatment alters the dimethyl sulfate effect on G-overhangs, a result compatible with the formation of a local quadruplex structure at telomeric overhang. Altogether these experiments strongly support the hypothesis that the telomeric G-overhang is an intracellular target for the action of Telomestatin.
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telomerase downregulation induced by the g quadruplex ligand 12459 in a549 cells is mediated by htert rna alternative splicing
Nucleic Acids Research, 2004Co-Authors: Dennis Gomez, Thibault Lemarteleur, Jeanlouis Mergny, Laurent Lacroix, Patrick Mailliet, Jean -françois RiouAbstract:Ligand 12459, a potent G-quadruplex-interacting agent that belongs to the triazine series, was previously shown to downregulate telomerase activity in the human A549 lung carcinoma cell line. We show here that the downregulation of telomerase activity is caused by an alteration of the hTERT splicing pattern induced by 12459, i.e. an almost complete disappearance of the active (+α,+β) transcript and an over-expression of the inactive –β transcript. Spliced intron 6 forming the –β hTERT transcript contained several tracks of G-rich sequences able to form G-quadruplexes. By using a specific PCR-stop assay, we show that 12459 is able to stabilize the formation of these G-quadruplex structures. A549 cell line clones selected for resistance to 12459 have been analyzed for their hTERT splicing pattern. Resistant clones are able to maintain the active hTERT transcript under 12459 treatment, suggesting the appearance of mechanisms able to bypass the 12459-induced splicing alterations. In contrast to 12459, Telomestatin and BRACO19, two other G-quadruplex-interacting agents, have no effect on the hTERT splicing pattern in A549 cells, are cytotoxic against the A549-resistant clones and display a lower efficiency to stabilize hTERT G-quadruplexes. These results lead us to propose that 12459 impairs the splicing machinery of hTERT through stabilization of quadruplexes located in the hTERT intron 6. Differences of selectivity between 12459, BRACO19 and Telomestatin for these hTERT quadruplexes may be important to explain their respective activity and inactivity against hTERT splicing.
Robert M Brosh - One of the best experts on this subject based on the ideXlab platform.
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the q motif of fanconi anemia group j protein fancj dna helicase regulates its dimerization dna binding and dna repair function
Journal of Biological Chemistry, 2012Co-Authors: Joshua A Sommers, Jason A Loiland, Hiroyuki Kitao, Jochen Kuper, Caroline Kisker, Robert M BroshAbstract:The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or Telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo.
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inhibition of helicase activity by a small molecule impairs werner syndrome helicase wrn function in the cellular response to dna damage or replication stress
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Monika Aggarwal, Joshua A Sommers, Robert H Shoemaker, Robert M BroshAbstract:Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex–binding compound Telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response.
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Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes
American Society of Hematology, 2010Co-Authors: Joshua A Sommers, Kazuo Shin-ya, Hiroyuki Kitao, Avvaru N. Suhasini, Thomas Leonard, Julianna S. Deakyne, Alexander V. Mazin, Robert M BroshAbstract:Abstract Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or Telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair.
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fancj helicase defective in fanconia anemia and breast cancer unwinds g quadruplex dna to defend genomic stability
Molecular and Cellular Biology, 2008Co-Authors: Kazuo Shinya, Robert M BroshAbstract:FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound Telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.
