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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

¹ Laboratoire d'Onco-Pharmacologie, JE 2428; ² Centre National de la Recherche Scientifique UMR 6142; ³ Institut Fédératif de Recherche 53, Université de Reims Champagne-Ardenne; ⁴ Institut Jean-Godinot, Reims, France; ⁵ Laboratoire de Biologie Moléculaire de la Cellule, Centre National de la Recherche Scientifique UMR 5161, Ecole Normale Supérieure de Lyon, Lyon, France; and ⁶ Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan

Requests for reprints: Jean-François Riou, Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51096 Reims, France. Phone: 33-3-26-91-80-13; Fax: 33-3-26-91-89-26; E-mail: jf.riou{at}univ-reims.fr.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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. (Cancer Res 2006; 66(14): 6908-12)

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
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- to 210-base overhang (G-overhang; ref. 1). Telomeres may be structurally organized in different conformations together with several telomere-associated proteins, such as TRF1, TRF2, and POT1 (2). 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 (2).

Telomeric proteins stabilize the telomere by protecting the single-stranded G-overhang from degradation (2). Protection of Telomere 1 (POT1) binds specifically to the single-stranded G-overhang (3) and has been described as a regulator of telomere length (4, 5). POT1 has been found associated with the double-stranded telomeric DNA proteins TRF1 and TRF2 through the bridging proteins PTOP/TINT1/PIP1 and TIN2 (6). 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 (7, 8).

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 (9). Small molecules that stabilize the G-quadruplex are effective as telomerase inhibitors, and several series have been reported to date to induce replicative senescence after long-term exposure to tumor cell cultures (9). Among them, the natural product telomestatin is one of the most active and selective telomeric G-quadruplex ligand (10). It has been recently shown 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 (11, 12).

Because telomestatin causes cellular effects analogous to those due to dysfunctional telomeric proteins, and because POT1 regulates in vitro the G-quadruplex conformation at telomeres (13), we have investigated the effect of a telomestatin treatment on POT1 binding in vitro and in human cells using a green fluorescent protein (GFP) fusion protein. Our results indicate that G-quadruplex stabilization impairs POT1 binding to telomere sequences and provokes the rapid delocalization of GFP-POT1 from telomeres in human cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Plasmids. Full-length hPOT1 was cloned into the pET22b expression vector by PCR using the Marathon testis cDNA library (Clontech, Palo Alto, CA). The cDNA was completely sequenced and corresponded to the sequence previously released (3). This construct contained an NH₂-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).

Electrophoretic mobility gel shift assay. POT1 protein was prepared with the TNT Coupled Transcription/Translation system (Promega, Charbonnières, France) using the pET22bPOT1 vector. Purified TRF2 protein was obtained from an Escherichia coli strain expressing TRF2 fused to a NH₂-terminal Tag containing six histidines. Purification of the protein was done by affinity chromatography through a nickel-containing resin (Ni-NTA agarose from Qiagen, Courtaboeuf, France; details on cloning and purification will be published elsewhere). Oligonucleotides were labeled at the 5' end with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [-³²P]ATP (3,000 Ci/mmol, Amersham Bioscience Europe, Orsay, France) and gel purified.

The binding reactions were done in 20 µL of the following buffer: 50 mmol/L Tris HCl (pH 8), 100 mmol/L KCl, 2 mmol/L MgCl₂, 10% glycerol, 0.1% NP40, 20 nmol/L labeled TEL1 probe in the presence of POT1 (2 µg) or TRF2 (50 ng). For POT1 reactions, the SACC1 oligonucleotide (250 nmol/L) and salmon sperm DNA (50 µg/mL) were also added as competitors. For TRF2 reactions, the CXext oligonucleotide (250 nmol/L) that hybridized to the TEL1 overhang was also added. Telomestatin was added at room temperature 15 minutes before the protein. Then, the protein was added, and binding was done for 15 minutes at room temperature. Reaction products were separated by electrophoresis in 6% nondenaturing polyacrylamide gels in 0.25x Tris borate EDTA buffer. The gels were run at 180 V for 1.5 hours, dried on Whatman DE81 paper at 80°C, and visualized by a Phosphorimager (Typhoon 9210, Amersham).

Oligonucleotides used were as follows: TEL1, 5'-TAACCCTAACCCTAAGCGAATTCGTCATGCGAATTCGCTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG-3'; CXext, 5'-GTGCCTTACCCTCTACCCTTACCCTAA-3'; SACC1, 5'-ACTGTCGTACTTGATATGTGGGTGTGTGTGGG-3'; TR2, 5'-TTAGGGTTAGGG-3'.

Cell culture and transfection. EcR293 telomerase-positive cells (14) were grown in DMEM with 100 units of penicillin and 0.1 mg of streptomycin per milliliter and 10% fetal bovine serum (FBS; Invitrogen, Cergy Pontoise, France); 70% to 80% confluence cells were transfected with 5 µg of plasmid in LipofectAMINE 2000 complex in FBS and antibiotic-free DMEM according to the manufacturer (Invitrogen). The medium was replaced after 24 hours, and the cells were grown in DMEM with 100 units of penicillin and 0.1 mg of streptomycin per milliliter containing 400 µg/mL of geneticin. After 15 days of geneticin selection, GFP-positive cells were sorted by fluorescence-activated cell sorting.

Immunofluorescence. For immunofluorescence microscopy, EcR293-GFPPOT1 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 mmol/L Tris-HCl (pH 8), 50 mmol/L NaCl, 3 mmol/L MgCl₂, 300 mmol/L sucrose, and 0.5% Triton X-100] and washed twice with PBS followed by antibody staining with 1 ng/µL for TRF2 4A794 mouse monoclonal (Chemicon-Upstate, Hampshire, United Kingdom) in 0.5% Triton X-100/PBS. The nuclear DNA was stained with 1 µmol/L Hoechst. Secondary antibodies raised against mouse were labeled with Alexa 568 (Molecular Probes, Eugene, OR).

For experiments on living cells, EcR293GFP-POT1 cells were plated on glass coverlips in complete media supplemented with 0.1 µmol/L Hoechst 33342. GFP and Hoechst fluorescence were recorded on a heated stage (37°C) and CO₂ chamber of a Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany).

Chromatin immunoprecipitations. Chromatin immunoprecipitation was done according to manufacturer procedure (Upstate Biotechnology) using, either TRF2 antibody (H-300, Santa Cruz Biotechnology, Santa Cruz, CA) or with a telomere antibody that recognizes the single- and double-stranded telomere repeats in vitro (19C7, a generous gift from Dr E. Mandine, Sanofi-Aventis, Vitry sur Seine, France). Telomeric sequences in immunoprecipitates were evidenced by PCR amplification according to a previously described method (15). The final telomere primer concentrations were 270 nmol/L (tel1) and 900 nmol/L (tel2), and PCR amplification was subjected to 35 cycles of 95°C for 15 seconds and 54°C for 2 minutes.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
G-quadruplex formation inhibits POT1 binding to telomeric sequences in vitro. To study the effect of telomestatin on POT1 binding in vitro, a short hairpin oligonucleotide (i.e., TEL1), which reconstitutes the double-stranded telomere with a short 3'overhang, has been used. In the presence of translated POT1, two specific POT1-TEL1 complexes are evidenced by electrophoretic mobility shift assay (EMSA; Fig. 1A , see * and ** bands). These two complexes correspond to the binding of POT1 at the single-stranded extension, as the addition of oligonucleotides (a) CXext that hybridizes to the single-stranded extension and (b) TR2 that titrates POT1 (16) inhibit the formation of these complexes.

View larger version (17K): [in this window] [in a new window]   Figure 1. Telomestatin inhibits POT1 but not TRF2 binding to telomeric sequences. A, EMSA assay of in vitro translated hPOT1 protein (2 µg) on TEL1 oligonucleotide. POT1 forms two specific complexes (* and **) with TEL1 compared with control vector (pET22b). Competitions with CXext or TR2 oligonucleotides (250 nmol/L) abolish the formation of the complexes, indicating that POT1 binds the TEL1 overhang. The addition of telomestatin (2 or 5 µmol/L) decreases the formation of the POT1/TEL1 complexes. Bottom, underexposed and allowed to follow the migration of the TEL1 probe. Telomestatin also binds the TEL1 3' overhang, as evidenced by a faster electrophoretic mobility (see TEL1+ arrow). B, quantification of the telomestatin effect on POT1-TEL1 upper (*) and lower (**) complexes. Results are relative to untreated control. Points, mean of three independent experiments; bars, SD. C, direct treatment of TEL1 oligonucleotide by telomestatin (2-10 µmol/L) results in the formation of the faster mobility oligonucleotide TEL1+. Bottom, relative percentage of TEL1+ compared with total TEL1. D, EMSA assay of purified TRF2 protein (50 ng) on TEL1 oligonucleotide in the presence of CXext oligonucleotide hybridized to the TEL1 overhang. TRF2 forms a specific complex with TEL1 (*), which is not affected by telomestatin (1.25-10 µmol/L).

The telomestatin treatment also induces in a dose-dependent manner a faster electrophoretic mobility for TEL1 oligonucleotide (Fig. 1A, see TEL1⁺ at bottom), with a maximal mobility at 10 µmol/L telomestatin, where TEL1⁺ represents 87% of the total TEL1 oligonucleotide (Fig. 1C).

Interestingly, this effect on TEL1⁺ correlates with the binding inhibition of POT1 and may result from the interaction of telomestatin with the TEL1 overhang fold in a G-quadruplex structure, in agreement with previous findings (11, 17).

To further establish the selectivity of the telomestatin interaction with telomeric DNA, we also examined its effect on the binding of purified TRF2 to the TEL1 oligonucleotide by EMSA. In the absence of telomestatin, we observe the formation of a specific TRF2-TEL1 complex, as already described (18). In the presence of telomestatin (1.25-10 µmol/L), we did not observe any significant effect of telomestatin (up to 10 µmol/L) on the TRF2-TEL1 complex (Fig. 1D).

These results suggest that telomestatin is able to inhibit in vitro the binding of POT1 to a short telomeric G-overhang but is not able to inhibit the interaction of telomeric proteins, such as TRF2, with the double-stranded telomeric DNA.

GFP-POT1 is localized at telomeres in EcR293 cells. 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 EcR293 cells. Western blot analysis using a GFP antibody indicated that EcR293-GFPPOT1 cells stably express the fusion protein (Fig. 2A ). As previously reported, POT1 overexpression in telomerase-positive cells results in telomere length elongation (4). Similar findings are observed in EcR293 cells transfected with either GFP-POT1 or POT1 (Fig. 2B), suggesting that the NH₂-terminal fusion with GFP does not alter the functional property of the fusion protein to transduce telomere extension.

View larger version (19K): [in this window] [in a new window]   Figure 2. Transfection of GFP-POT1 induces a telomere lengthening and colocalizes with TRF2 in human cell lines. A, immunoblot analysis to detect GFP in EcR293 cells stably transfected by GFP-POT1 or by control GFP vector. B, overexpression of POT1 and GFP-POT1 induced a telomere lengthening in EcR293 cells. TRF analysis of EcR293 control cells and cells transfected with POT1 or GFP-POT1, as indicated. The TRF analysis was done 2 months after the selection of transfected cells.

View larger version (34K): [in this window] [in a new window]   Figure 3. Telomestatin alters the GFP-POT1 localization at telomeres in EcR293 cells. A, effect of telomestatin (2 µmol/L) on GFP-POT1 in EcR293 cells treated for 48 hours. Fluorescence for GFP-POT1 (green), TRF2 (red), and Hoechst (blue) were determined on fixed cells. GFP-POT1 colocalizes with TRF2 signal in control EcR293 cells. Telomestatin treatment induced a marked decrease of the normal telomeric sites of GFP-POT1 fluorescence and induced a nucleolar accumulation of GFP-POT1 (arrowheads) in EcR293 cells but does not modify the TRF2 localization at telomeres. B, effect of telomestatin (2 µmol/L) in living EcR293 cells for 24 to 72 hours (as indicated). Fluorescence for GFP-POT1 (green) and Hoechst (blue) were merged. Cytoplasmic foci of GFP-POT1 are indicated by arrows. Diffuse GFP-POT1 aggregates inside the nucleus or the nucleoli are indicated by arrowheads. C, doxorubicin (DOX) and etoposide (VP16) do not affect the GFP-POT1 localization in living EcR293 cells treated for 48 hours. The drug concentration used was 10 nmol/L, corresponding to the IC50 at 96 hours, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Fluorescence for GFP-POT1 (green) and Hoechst (blue) were merged.

Microscopic examination of living cells confirms these results obtained with fixed cells (Fig. 3B) but also shows that the telomestatin treatment provokes the formation of additional cytoplasmic GFP-POT1 foci that were not or poorly observed in fixed cells. These foci may correspond to proteasome-dependent degradation of GFP-POT1.

All together, our results indicate that telomestatin induces a reduction of the GFP-POT1 telomeric foci in EcR293 cells at noncytotoxic concentrations and in the range to those used to inhibit POT1 binding at telomeric overhang in vitro.

To further establish that POT1 delocalization is specifically due to telomestatin treatment, we also used several anticancer agents (doxorubicin, etoposide, and vinblastin) whose mechanisms of action are not related to G-quadruplex stabilization. At drug concentrations corresponding to the IC₅₀ on EcR293GFP-POT1 cells, we did not observe any effect on the telomere localization of POT1 in viable cells for doxorubicin (10 nmol/L) and etoposide (10 nmol/L; Fig. 3C) and for vinblastin (data not shown). In contrast, cells undergoing apoptosis present a strong decrease of telomeric POT1 staining (data not shown), in agreement with the degradation of telomeric sequences observed during the late stages of apoptosis (20). Therefore, we conclude that POT1 delocalization by telomestatin is an early event occurring before any evidence for apoptosis or cell growth arrest.

Telomestatin does not impair TRF2 binding at telomeres in EcR293 cells. To examine whether the delocalization of POT1 is a consequence of a general effect on the telomere structure, we have determined whether the telomeric localization of TRF2 is also modified by telomestatin. As shown in Fig. 4A (see also Fig. 3A), the telomere localization of TRF2 is not or barely altered by telomestatin treatment in conditions that provokes the GFP-POT1 delocalization in EcR293 cells. The selectivity of the telomestatin effect was also evaluated by chromatin immunoprecipitation experiments using TRF2. In these experiments, the immunoprecipitated telomere sequences were evaluated by specific PCR amplification, as described previously (15). As shown in Fig. 4B, telomestatin treatment of EcR293GFP-POT1 cells to 48 hours and up to 96 hours does not modify the telomere immunoprecipitation by TRF2, in agreement with the immunofluorescence results. Chromatin immunoprecipitation with telomere antibodies were used as internal controls and did not present variation under telomestatin treatment (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   Figure 4. Telomestatin does not alter TRF2 binding to telomere in EcR293 cells. A, effect of telomestatin (2 µmol/L) on GFP-POT1 in EcR293 cells treated for 48 hours. Fluorescence for GFP-POT1 (green), TRF2 (red), and Hoechst (blue) were determined on fixed cells. Telomestatin treatment induced a marked decrease of the normal telomeric sites of GFP-POT1 fluorescence but does not modify the TRF2 localization at telomeres. B, chromatin immunoprecipitation of EcR293 treated by telomestatin (2 µmol/L) for 48 or 96 hours with TRF2 antibodies or telomere antibodies as internal standard. Immunoprecipitated telomere sequences were evidenced by PCR amplification (see Materials and Methods). PCR telomere fragments migrated as a smear indicated by bracket. Primer dimers forming in the blank PCR migrated as a distinct single band.

Telomestatin was also reported to induce alterations of the telomeric G-overhang conformation in different tumor cell lines that leads to a degradation of the G-overhang (11, 12). Indeed, telomestatin also provokes a decrease of the G-overhang signal, in EcR293 cells treated for 48 hours at 2 µmol/L (results not shown). This may indicate that the G-overhang alteration preferentially impairs the telomere localization of POT1 in EcR293 cells compared with TRF2.

Experiments were also done on the HT1080 tumor cell line. Preliminary results indicated that both GFP-POT1 and TRF2 binding to telomere were sensitive to telomestatin treatment (12).⁷ The kinetic of their dissociation, compared with the G-overhang erosion, is currently under investigation and will give interesting clues for the importance of these proteins in the mechanism of action of telomestatin.

In conclusion, our results show that the G-quadruplex ligand telomestatin impairs the binding of POT1 to single-stranded telomeric sequences in vitro and alters the nuclear localization of GFP-POT1, suggesting that major modifications of the telomeric end structure are induced by this ligand for which POT1 is a highly sensitive nuclear marker.

	Acknowledgments

 
Grant support: Association pour la Recherche contre le Cancer grant 3644 and Ligue Nationale Contre le Cancer (Equipe labellisée 2006).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. E. Gilson, J.L. Mergny, A. Londono-Vallejo, C. Trentesaux, and H. Morjani for helpful discussions.

	Footnotes

 
Note: D. Gomez is currently at the Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique UMR 5089, 205 route de Narbonne, 31077 Toulouse, France.

⁷ D. Gomez, T. Wenner, unpublished results.

Received 5/ 1/06. Revised 5/30/06. Accepted 6/ 2/06.

	References

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