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• W2009139426 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. 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. Telomeres play an important role in structural chromosome integrity. They cap and protect their extremities from illegitimate recombination, degradation, and end-to-end fusion (1Blackburn E.H. Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1750) Google Scholar). Telomere replication is sustained in proliferative somatic cells and in most cancer cells by telomerase, a ribonucleoprotein complex that elongates the chromosome ends to compensate losses occurring at each cell division, because of the inability of polymerase to fully replicate telomeric extremities (2McEachern M.J. Krauskopf A. Blackburn E.H. Annu. Rev. Genet. 2000; 34: 331-358Crossref PubMed Scopus (603) Google Scholar). In somatic cells, the absence of telomerase provokes a progressive shortening of telomeric DNA that ultimately leads to replicative senescence, once a critical telomere length has been reached (3Shay J.W. Wright W.E. Cancer Cell. 2002; 2: 257-265Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Numerous observations, notably that inhibition of telomerase activity limits tumor cell growth (4Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1971) Google Scholar), have led to the proposal that telomere and telomerase are potential targets for cancer chemotherapy (3Shay J.W. Wright W.E. Cancer Cell. 2002; 2: 257-265Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 5Neidle S. Parkinson G. Nat. Rev. Drug Discov. 2002; 1: 383-393Crossref PubMed Scopus (597) Google Scholar).In human, telomeres consist of the repetition of the G-rich duplex sequence 5′-TTAGGG-3′. A G-rich 3′-strand extends beyond the duplex to form a 130-210-base overhang (G-overhang) (6Makarov V.L. Hirose Y. Langmore J.P. Cell. 1997; 88: 657-666Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, 7Wright W.E. Tesmer V.M. Huffman K.E. Levene S.D. Shay J.W. Genes Dev. 1997; 11: 2801-2809Crossref PubMed Scopus (583) Google Scholar). Telomeres may be structurally organized in different conformations together with several telomere-associated proteins, such as TRF1, TRF2, and POT1 (8Smogorzewska A. De Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (657) Google Scholar). The G-overhang is either accessible for telomerase extension in an open state or inaccessible in a capped (or closed) conformation that involves the formation of a putative t-loop structure (8Smogorzewska A. De Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (657) Google Scholar). Although the t-loop structure has not been defined in detail, it may be created by the invasion of the G-overhang into the duplex part of the telomere (9Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1904) Google Scholar). The t-loop structure is induced in vitro by the binding of TRF2 in the vicinity of the telomeric G-overhang (10Stansel R.M. de Lange T. Griffith J.D. EMBO J. 2001; 20: 5532-5540Crossref PubMed Scopus (404) Google Scholar).Telomeric proteins stabilize the telomere by protecting the single-stranded G-overhang from degradation (8Smogorzewska A. De Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (657) Google Scholar). Uncapping of the telomere ends leads to telomeric dysfunction characterized by end-to-end fusion, inappropriate recombination, anaphase bridges, and G-overhang degradation that either lead to apoptosis or senescence (11Blackburn E.H. Chan S. Chang J. Fulton T.B. Krauskopf A. McEachern M. Prescott J. Roy J. Smith C. Wang H. Cold Spring Harbor Symp. Quant. Biol. 2000; 65: 253-263Crossref PubMed Scopus (55) Google Scholar, 13Karlseder J. Smogorzewska A. de Lange T. Science. 2002; 295: 2446-2449Crossref PubMed Scopus (656) Google Scholar).A dominant negative mutant of TRF2, TRF2ΔBΔM, displaces TRF2 and its interacting factors off the telomeres and causes a loss of telomeric overhangs, apoptosis, senescence, and chromosome abnormalities (8Smogorzewska A. De Lange T. Annu. Rev. Biochem. 2004; 73: 177-208Crossref PubMed Scopus (657) Google Scholar). POT1 (protection of telomere 1) binds specifically to the single-stranded G-overhang (14Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (796) Google Scholar) and has been described as a regulator of telomere length (15Colgin L.M. Baran K. Baumann P. Cech T.R. Reddel R.R. Curr. Biol. 2003; 13: 942-946Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 16Loayza D. de Lange T. Nature. 2003; 25: 1013-1018Crossref Scopus (537) Google Scholar). POT1 has been found associated with the double-stranded telomeric DNA protein TRF1 and TRF2 through the bridging proteins PTOP/TINT1/PIP1 and TIN2 (17de Lange T. Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2240) Google Scholar). Suppression of POT1 function by RNA interference in human cells leads to the loss of the telomeric single-stranded overhang, induced apoptosis, senescence, and chromosomal instability in human cells (18Veldman T. Etheridge K.T. Counter C.M. Curr. Biol. 2004; 14: 2264-2270Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 19Yang Q. Zheng Y.L. Harris C.C. Mol. Cell. Biol. 2005; 25: 1070-1080Crossref PubMed Scopus (135) Google Scholar).Because of the repetition of guanines, the G-overhang is prone to form a four-stranded G-quadruplex structure that has been shown to inhibit telomerase activity in vitro (20Zahler A.M. Williamson J.R. Cech T.R. Prescott D.M. Nature. 1991; 350: 718-720Crossref PubMed Scopus (990) Google Scholar). Small molecules that stabilize the G-quadruplex are effective as telomerase inhibitors (21Sun D. Thompson B. Cathers B.E. Salazar M. Kerwin S.M. Trent J.O. Jenkins T.C. Neidle S. Hurley L.H. J. Med. Chem. 1997; 40: 2113-2116Crossref PubMed Scopus (714) Google Scholar, 24Koeppel F. Riou J.F. Laoui A. Mailliet P. Arimondo P.B. Labit D. Petitgenet O. Helene C. Mergny J.L. Nucleic Acids Res. 2001; 29: 1087-1096Crossref PubMed Scopus (211) Google Scholar), and several series have been reported to date to induce replicative senescence after long term exposure to tumor cell cultures (25Duan W. Rangan A. Vankayalapati H. Kim M.Y. Zeng Q. Sun D. Han H. Fedoroff O.Y. Nishioka D. Rha S.Y. Izbicka E. Von Hoff D.D. Hurley L.H. Mol. Cancer Ther. 2001; 1: 103-120PubMed Google Scholar, 29Tauchi T. Shin-Ya K. Sashida G. Sumi M. Nakajima A. Shimamoto T. Ohyashiki J.H. Ohyashiki K. Oncogene. 2003; 22: 5338-5347Crossref PubMed Scopus (180) Google Scholar). Among them, the natural product telomestatin is one of the most active and selective telomeric G-quadruplex ligands (30Rosu F. Gabelica V. Shin-ya K. De Pauw E. Chem. Commun. (Camb.). 2003; 21: 2702-2703Crossref PubMed Scopus (87) Google Scholar, 32Kim M.Y. Vankayalapati H. Shin-Ya K. Wierzba K. Hurley L.H. J. Am. Chem. Soc. 2002; 124: 2098-2099Crossref PubMed Scopus (468) Google Scholar). We have shown recently that telomestatin impairs the conformation and the length of the telomeric G-overhang, an effect that is thought to be more relevant than double-stranded telomere erosion as a marker for its cellular activity (33Gomez D. Paterski R. Lemarteleur T. Shin-Ya K. Mergny J.L. Riou J.F. J. Biol. Chem. 2004; 279: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Telomestatin also inhibits POT1 to the telomeric G-overhang in vitro and induces GFP-POT1 dissociation from telomeres in EcR293 cells (34Gomez D. O'Donohue M.F. Wenner T. Douarre C. Macadré J. Koebel P. Giraud-Panis M.J. Kaplan H. Kolkes A. Shin-Ya K. Riou J.F. Cancer Res. 2006; 66: 6908-6912Crossref PubMed Scopus (177) Google Scholar). In tumor cells, telomestatin was shown to completely dissociate TRF2 from telomeres, a result thought to be the consequence of the t-loop disruption (35Tahara H. Shin-Ya K. Seimiya H. Yamada H. Tsuruo T. Ide T. Oncogene. 2006; 25: 1955-1966Crossref PubMed Scopus (195) Google Scholar).In this study we have investigated the effect of telomestatin treatment in the tumor cell line HT1080. Our results indicate that G-quadruplex stabilization provokes the rapid degradation of both G-overhang and telomere together with the delocalization of GFP-POT1 and TRF2 from telomeres. DNA damage response is induced by telomestatin, which is partially localized at telomeres. The expression of GFP-POT1 in HT1080 induces the lengthening of the G-overhang and partially protects cells from telomestatin-induced G-overhang degradation and senescence induction. These data confirm the importance of POT1 and G-overhang in the action of telomestatin but suggest the presence of additional DNA sites of action.EXPERIMENTAL PROCEDURESOligonucleotides and Compounds—All oligonucleotides were synthesized and purified by Eurogentec (Seraing, Belgium). Telomestatin was purified according to Ref. 31Shin-ya K. Wierzba K. Matsuo K. Ohtani T. Yamada Y. Furihata K. Hayakawa Y. Seto H. J. Am. Chem. Soc. 2001; 123: 1262-1263Crossref PubMed Scopus (468) Google Scholar. Other compounds were commercially available (Sigma). Telomestatin was prepared at 5 mm in MeOH/Me2SO (50:50). Further dilutions were made in water.Plasmids—Full-length hPOT1 was cloned into the pET22b expression vector by PCR using the Marathon testis cDNA library (Clontech). The cDNA was completely sequenced and corresponded to the sequence previously released (14Baumann P. Cech T.R. Science. 2001; 292: 1171-1175Crossref PubMed Scopus (796) Google Scholar). This construct contained an N-terminal T7 sequence allowing its coupled transcription/transcription. The GFP-POT1 plasmid was constructed by insertion of the POT1 cDNA after PCR amplification from pET22bPOT1 vector at BamHI-XbaI of the pEGFP-C1 plasmid (Clontech). The ΔOB-POT1 and TRF2 sequences were cloned by PCR from POT1 and TRF2 cDNAs (a gift from E. Gilson, ENS, Lyon, France), using pfu polymerase and inserted in the cloning sites of a pCDNA3 vector.Cell Culture and Transfection—HT1080 was obtained from the ATCC. Cells were grown in DMEM 5The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation. with 100 units of penicillin and 0.1 mg of streptomycin per ml and 10% fetal bovine serum (Invitrogen). 70-80% confluence cells were transfected with 5 μg of plasmid in Lipofectamine 2000 complex in fetal bovine serum and antibiotic-free DMEM according to the manufacturer (Invitrogen). The media were replaced after 24 h, and the cells were grown in DMEM with 100 units of penicillin and 0.1 mg of streptomycin/ml containing 400 μg/ml of geneticin. After 15 days of geneticin selection, GFP-positive cells were sorted by fluorescence-activated cell sorter.For long term cell growth studies, transfected cells were seeded at 15 × 103 cells/ml into a 25-cm2 tissue culture flask, in the presence or the absence of telomestatin (2 μm), cultured for 4 days, then trypsinized, and counted. At each passage, 15 × 103 cells/ml were replated into a new culture flask with fresh medium containing drug solution. Results were expressed as the cumulated population doubling as a function of the time of culture as described previously (36Gomez D. Aouali N. Renaud A. Douarre C. Shin-Ya K. Tazi J. Martinez S. Trentesaux C. Morjani H. Riou J.F. Cancer Res. 2003; 63: 6149-6153PubMed Google Scholar).Immunofluorescence—For immunofluorescence microscopy, HT1080-GFP-POT1 cells plated on glass coverslips were permeabilized in 0.5% Triton X-100/PBS and fixed with 3% paraformaldehyde. Cells were then washed twice in PBS and treated with permeabilization buffer (20 mm Tris-HCl (pH 8.0), 50 mm NaCl, 3 mm MgCl2, 300 mm sucrose, and 0.5% Triton X-100), washed twice with PBS followed by antibody staining with 1 ng/μl TRF2 4A794 mouse monoclonal antibody (Upstate Biotechnology, Inc.) or TRF2 H-300 (sc-9142) rabbit polyclonal antibody (Santa Cruz Biotechnology), and/or 2 ng/μl antiphospho-γH2AX (Ser-139) (Upstate Biotechnology) in 0.5% Triton X-100/PBS. The nuclear DNA was stained with 1 μm Hoechst. Secondary antibodies raised against mouse were labeled with Alexa 568 (Molecular Probes), and those raised against rabbit were labeled with Alexa 488 (Molecular Probes).We obtained images of fixed cells using a 100× (NA 1.4) plan apochromat objective mounted on a piezo translator (Physik Instrumente, Karlsruhe, Germany) and imaged with a Cool-snap HQ camera controlled by Metamorph software (Roper Scientific, Duluth, GA). Appropriate excitation and emission filters placed in two filter wheels driven by a Lambda 10-2 controller (Sutter Instruments, Novato, CA) were combined to specific double or triple band dichroic filters (Chroma Technology, Rockingham, VT). Stacks of 60-100 images (12-bit grayscale) were acquired with a z-step of 0.12 μm with a low illumination intensity to avoid photo-bleaching. For data processing, experimental point spread functions were obtained from infra-resolution fluorescent microspheres emitting at specific wavelengths (Molecular Probes), whose stacks were acquired in the same sampling conditions as those used for the volumes to be analyzed. Deconvolution was performed with Metamorph software on a 2.4-GHz Dell computer equipped with a GeForce4 Ti 4800 Se Winfast A280 video card (Leadtek Research Inc., Almere, The Netherlands).G-overhang Assays—The nondenaturing hybridization assay to detect the 3′ telomere G-overhang was performed as described previously on 2.5-μg aliquots of undigested genomic DNA using a labeled 5′-CCCTAACCCTAACCCTAACCC-3′ oligonucleotide (21C) (33Gomez D. Paterski R. Lemarteleur T. Shin-Ya K. Mergny J.L. Riou J.F. J. Biol. Chem. 2004; 279: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The procedure allows detection of the amount of single strand overhang available for hybridization. Experiments were performed either on genomic DNA from treated cells or on purified genomic DNA treated in vitro with telomestatin, as indicated in the text.TRF Analysis—Aliquots of 5 μg of undigested genomic DNA were hybridized at 37 °C overnight with 0.5 pmol of [γ-32P]ATP-labeled (5′-(CCCTAA)3CCC-3′) oligonucleotide in hybridization buffer (10 mm Tris-HCl (pH 7.9), 50 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol) in the presence of RsaI and HinfI restriction enzymes in a volume of 20 μl. Reaction was stopped with 2 μl of 1%SDS, 1 mg/ml proteinase K and incubated for 30 min at 50 °C. Hybridized samples were size-fractionated on 0.8% agarose gels in 1× TBE buffer. The gels were stained with ethidium bromide, washed, and dried on Whatman filter paper. Ethidium fluorescence and radioactivity were scanned in a PhosphorImager (Typhoon 9210, Amersham Biosciences). Telomeric smears were revealed by exposure in a PhosphorImager (Typhoon 9210, Amersham Biosciences), and the mean length of the TRF corresponds to the peak of the integration curve from three separate experiments.Chromatin Immunoprecipitations (ChIP)—ChIP was performed according to the manufacturer's procedure (Upstate Biotechnology) using TRF2 antibody (H-300; Santa Cruz Biotechnology). Telomeric sequences in immunoprecipitates were evidenced by PCR amplification according to a method described previously (37Cawthon R.M. Nucleic Acids Res. 2002; 30: e47Crossref PubMed Scopus (2471) Google Scholar). The final telomere primer concentrations were 270 nm (tel1) and 900 nm (tel2), and PCR amplification was subjected to 35 cycles of 95 °C for 15 s, 54 °C for 2 min. The primer sequences were as follows: tel1 5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′ and tel25′-TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3RESULTSTelomestatin Induces a Decrease of the Telomeric G-overhang in HT1080 Cells—Recent studies have indicated that the telomeric G-overhang represents one of the direct targets of quadruplex ligands (33Gomez D. Paterski R. Lemarteleur T. Shin-Ya K. Mergny J.L. Riou J.F. J. Biol. Chem. 2004; 279: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 38Douarre C. Gomez D. Morjani H. Zahm J.M. O'Donohue M.F. Eddabra L. Mailliet P. Riou J.F. Trentesaux C. Nucleic Acids Res. 2005; 33: 2192-2203Crossref PubMed Scopus (48) Google Scholar). We analyzed the effect of telomestatin on the telomeric G-overhangs from HT1080 cells. As shown before, hybridization of a telomeric C-rich probe (21C) under nondenaturing conditions allowed the measurement of the relative single-stranded G-overhang signal in undigested genomic DNA samples (33Gomez D. Paterski R. Lemarteleur T. Shin-Ya K. Mergny J.L. Riou J.F. J. Biol. Chem. 2004; 279: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar).Treatment of HT1080 cells with 1, 2, and 5 μm telomestatin for 48 h induces a dose-dependent decrease of the G-overhang signal, which represents 32, 15, and 10% of the untreated control, respectively (Fig. 1, A and C).Previous results with telomestatin in A549 cells (33Gomez D. Paterski R. Lemarteleur T. Shin-Ya K. Mergny J.L. Riou J.F. J. Biol. Chem. 2004; 279: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) indicate that the apparent decrease in G-overhang signal may result from the stabilization of the quadruplex, making it less prone to hybridization to its complementary C-rich probe. To exclude this possibility, we performed the following experiment: telomestatin (1-5 μm) was added to purified DNA just prior to the hybridization reaction. This results in a nearly complete inhibition of the G-overhang signal (Fig. 1, B and C). The inhibition is almost completely reversed in the presence of another G-quadruplex competitor (Pu22myc) that traps the ligands, leaving the overhang free for hybridization with 21C probe (Fig. 1, B and C).To determine the real degradation of the G-overhang induced by telomestatin in HT1080 cells, in this study we used the competition with Pu22myc on DNA samples from telomestatin-treated HT1080 cells. Results indicated a limited reversion (∼15%) of the G-overhang signal decrease (Fig. 1, A and C, compare with reactions in the absence of Pu22myc). Therefore, we concluded that the G-overhang signal loss in HT1080-treated cells mainly corresponds to an effective degradation of the telomeric G-overhang in vivo.Treatment with Telomestatin Delocalizes Telomeric POT1 in Human Cell Lines—To examine the effects of telomestatin treatment on the binding of POT1 to telomeres in cultured cells, we have designed a GFP-POT1 vector that was transfected in HT1080 cells. As reported previously (15Colgin L.M. Baran K. Baumann P. Cech T.R. Reddel R.R. Curr. Biol. 2003; 13: 942-946Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 34Gomez D. O'Donohue M.F. Wenner T. Douarre C. Macadré J. Koebel P. Giraud-Panis M.J. Kaplan H. Kolkes A. Shin-Ya K. Riou J.F. Cancer Res. 2006; 66: 6908-6912Crossref PubMed Scopus (177) Google Scholar), GFP-POT1 overexpression in telomerase-positive cells results in telomere length elongation (supplemental Fig. S1), suggesting that the N-terminal fusion with GFP does not alter the functional property of the fusion protein to transduce telomere extension.To localize GFP-POT1 in HT1080 cells, a co-localization experiment has been performed on fixed cells by confocal microscopy using a TRF2 antibody. As shown in Fig. 2, GFP-POT1 colocalizes with almost all the TRF2 dots, suggesting that GFP-POT1 protein is present at telomeres in HT1080 cells. Thus, cells expressing GFP-POT1 fusion protein may be used as a model to investigate the effect of telomestatin on POT1 localization. HT1080 cells expressing GFP-POT1 have been treated for 48 h with 2 μm telomestatin (Fig. 2), a concentration and time exposure with the ligand at which most of the cells are still viable, because the IC50 values for 2 and 4 days of treatment were equal to 5 and 1.5 μm, respectively. Microscopic examination of treated cells indicated a dramatic change in the nuclear organization of GFP-POT1. Telomestatin strongly reduced the GFP-POT1 punctated signal associated with telomeres to nearly undetectable levels, as compared with untreated controls (Fig. 2).FIGURE 2Telomestatin alters the GFP-POT1 and TRF2 localization at telomeres in HT1080 cells. Effect of telomestatin (2 μm) on GFP-POT1 in HT1080 cells treated for 48 h. Fluorescence for GFP-POT1 (green), TRF2 (red), and Hoechst (blue) was determined on fixed cells. GFP-POT1 and TRF2 signals colocalize in control untreated cells. Telomestatin treatment induces a strong decrease of the telomeric sites of GFP-POT1 fluorescence and also induces a decrease of the TRF2 fluorescence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The dose-dependent effect of telomestatin was also studied in HT1080GFP-POT1 cells after 48 h of treatment (supplemental Fig. S2). At 0.5 μm telomestatin, no obvious decrease or modification of the GFP-POT signal was detectable in >90% of the cells. At 1 μm, a decrease of telomeric GFP-POT1 fluorescence was observed in 20-30% of the cells. The main effect of the ligand, i.e. a decrease of the telomeric GFP-POT1 fluorescence, was detectable at 2 μm in about 50-60% of the cells. In addition, a significant fraction of the cells (25-30%) presented a nucleolar localization of GFP-POT1. At 5 μm, a telomestatin concentration that significantly impaired the growth of the cells and induced apoptosis (supplemental Fig. S3), nearly all surviving cells presented a complete loss of the GFP-POT1 telomeric signal and a strong nucleolar accumulation, as well as a marked cytoplasmic GFP-POT1 accumulation (supplemental Fig. S2).Telomestatin Impairs TRF2 Binding at Telomeres and Decreases Telomere Length in HT1080 Cells—To examine whether the delocalization of POT1 is a consequence of a general effect on the telomere structure, we have determined the effect of telomestatin on TRF2 localization. Telomestatin treatment of HT1080 cells (2 μm, 48 h) induced a noticeable decrease of the TRF2 signal at telomeres (Fig. 2) that paralleled the effect of telomestatin on GFP-POT1.The telomestatin effect was also evaluated by ChIP experiments using TRF2 antibodies. In these experiments, the immunoprecipitated telomere sequences were evaluated by specific PCR amplification, as described previously (37Cawthon R.M. Nucleic Acids Res. 2002; 30: e47Crossref PubMed Scopus (2471) Google Scholar). ChIP experiments indicate that telomestatin (2 μm, 48 h) provokes the removal of an important fraction of TRF2 from telomeric sequences, in agreement with the immunofluorescence results (supplemental Fig. S4).The effect of the ligand on TRF2 suggests that either the double-stranded telomeric repeats or the t-loop conformations have been altered. We have thus determined the effect of telomestatin to decrease the length of the double-stranded telomere. Interestingly, the exposure of HT1080 cells to telomestatin (2 μm) induces a rapid telomere shortening detectable after short term treatment (supplemental Fig. S5A). The TRF decrease corresponds to 300 and 600 bases after 4 and 8 days, respectively (supplemental Fig. S5B). These results indicate that telomestatin also induces a dramatic and rapid alteration of the double-stranded telomere repeats and TRF2 binding to telomeres in addition to the G-overhang degradation and the GFP-POT1 delocalization.Telomestatin Induces an Early DNA Damage Response at Telomeres—Telomere-initiated senescence or dysfunctional telomeres have been shown to be associated with a DNA damage response involving factors such as 53BP1 and γH2AX (17de Lange T. Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2240) Google Scholar, 39Celli G.B. de Lange T. Nat. Cell Biol. 2005; 7: 712-718Crossref PubMed Scopus (461) Google Scholar). The rapid effect of telomestatin to trigger telomere degradation together with POT1 and TRF2 removal may suggest the induction of a DNA damage response at telomeres. To study such DNA damage, we used γH2AX immunofluorescence after short term treatment with the ligand. As shown in Fig. 3A, telomestatin treatment induces a marked DNA damage response evidenced by a strong increase in the γH2AX foci. The effect started at 0.5 μm telomestatin and reaches nearly all cells in the presence of 2 μm telomestatin (Fig. 3, A and B). We have also determined the colocalization of γH2AX foci in HT1080GFP-POT1 with telomeric GFP-POT1 under telomestatin treatment. As shown in Fig. 3B, treatment with telomestatin 0.5 μm mainly triggers a DNA damage response outside from the telomeric foci. Only a fraction of the total γH2AX foci colocalizes with GFP-POT1 in treated cells (indicated by arrowheads in Fig. 3B). The γH2AX and GFP-POT1 colocalization is significantly increased by 2.5-fold (p < 0.01), as compared with controls, in cells treated with 0.5, 1, or 2 μm telomestatin, where the GFP-POT1 telomeric signal is still detectable (Fig. 3C). However, in cells treated with 5 μm telomestatin, the analysis was not possible, because of the complete delocalization of the GFP-POT1 protein (see supplemental Fig. S2). These results have been confirmed by Telo-fluorescence in situ hybridization experiments using a telomeric fluorescein isothiocyanate-peptidic nucleic acid probe to determine the localization of γH2AX foci in telomestatin-treated HT1080 cells (supplemental Fig. S6). Analysis of HT1080 cells treated with telomestatin also showed that in some metaphases the γH2AX response is observed at the extremities of chromosomes, in agreement with a response at telomeres (Fig. 3D).FIGURE 3DNA damage response at telomere after telomestatin treatment in HT1080 cells. A, telomestatin induces a DNA damage response in HT1080 cells. Immunofluorescence for γH2AX (red) and Hoechst fluorescence (blue) in untreated (control) or cells treated for 48 h with 2 μm telomestatin (+ Telo). B, merge fluorescence for γH2AX (red), GFP-POT1 (green), and Hoechst (blue) in untreated (control) or cells treated for 48 h with 0.5 μm telomestatin (+ Telo). Telomestatin induced a partial colocalization of γH2AX and GFP-POT1 foci, as compared with untreated cells. Co-localized sites of DNA damage at telomere are indicated by arrowheads. C, number of γH2AX-GFPPOT1 colocalizing foci in HT1080 cells treated by telomestatin (0.5-2 μm). Telomestatin significantly induce" @default.
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