Telomere Targeting with a New G4 Ligand Enhances
Radiation-Induced Killing of Human Glioblastoma Cells
Patrick Merle3,4, Bertrand Evrard1,3,5, Anne Petitjean8,9, Jean-Marie Lehn8,Marie-Paule Teulade-Fichou10, Emmanuel Chautard3,7, Anne De Cian11, Lionel Guittat11, Phong Lan Thao Tran12, Jean-Louis Mergny11,12, Pierre Verrelle3,7, and Andreï Tchirkov2,3,6,7
Abstract
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. 2011 AACR.
Introduction
Glioblastoma multiforme (GBM) is the most common human primary brain tumor with a very poor prognosis. GBM patients are treated by neurosurgery followed by radiotherapy with concomitant administration of temo-zolomide (1). This treatment is not curative, and the tumor
almost always reappears inside the irradiated tumor volume. Increasing the sensitivity of tumor cells to radio-therapy would improve outcome in patients with GBM. Telomeres are nucleoprotein structures at the end of chromosomes that stabilize and protect them from nucleotide degradation and end-to-end fusion. Telome-rase is a ribonucleoprotein complex, with reverse tran-scriptase activity maintaining telomere length by adding hexameric GGTTAG repeats to the chromosomal ends (2). Telomerase activity is regulated at the expression level of the human telomerase reverse transcriptase (hTERT) gene. In humans, telomerase is expressed in germinal and stem cells but is repressed in most somatic cells. In contrast, telomerase is active in more than 85% of cancers and allows unlimited proliferation of tumor cells (3). hTERT expression is strongly associated with GBM (4), and patients with highhTERT levels have very short survival (5). Suppression ofhTERT in GBM cells induces senescence and inhibits proliferation (6). The telomerase antagonist, imetelstat, can inhibit GBM growth, showing an increased efficacy in combination with radiation and temozolomide (7). In addition, telomeric dysfunction due to genetic deficiency of telomerase activity increases radiosensitivity and decreases the capacity of DNA repair (8). Thus, there is a pathogenetic link between telomere, telomerase, and radiosensitivity.
Authors' Affiliations:1
Laboratoire d’Immunologie,2
Histologie Embryo-logie Cytogenetique, and 3EA 3846, Clermont Universite, Universite
d’Auvergne; 4
Service de Pneumologie, 5Laboratoire d’Immunologie,
and 6Service de Cytogenetique Medicale, CHU Clermont-Ferrand; 7Departement de Radiotherapie, Centre Jean Perrin, Clermont-Ferrand; 8ISIS, Universite de Strasbourg, Strasbourg, France; 9
Department of Chemistry, Queen's University, Kingston, Ontario, Canada;10Institut Cu-rie, UMR 176, Centre Universitaire, Orsay;11INSERM U565, Museum
National d’Histoire Naturelle, CNRS UMR 7196, Paris; and12
INSERM U869, Institut Europeen de Chimie Biologie, Universite de Bordeaux, Pessac, France
Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
P. Merle and B. Evrard contributed equally to this work.
Corresponding Author: Andreï Tchirkov, Departement de Radiotherapie, Centre Jean Perrin, 58 rue Montalembert, BP 392, 63011 Clermont-Ferrand, Cedex 1, France. Phone: 81-42; Fax: 33-4-73-27-81-25; E-mail: atchirkov@chu-clermontferrand.fr
doi: 10.1158/1535-7163.MCT-10-0664
Targeting telomeres and/or telomerase using G-quadruplex (G4) ligands is a promising strategy for cancer treatment (9). G4 ligands bind to a family of DNA secondary structures called G-quadruplexes, which result from the cation-stabilized stacking of several G-quartets (typically 2–5), each quartet being composed of 4 coplanar guanines. Quadruplexes can be very stable under physiologic conditions. Any genomic sequence containing a repetitive motif with several consecutive guanines can in principle form a quadru-plex (10). This is the case for the telomeric TTAGGG motif, which is single stranded at chromosomal ends. Thus, G4 ligands interact with telomeres (11), induce telomere uncapping, and, indirectly, affect telomerase function by preventing telomerase binding on its target (12). Some of these ligands induce proliferation arrest and apoptosis in GBM cell lines (13).
Here, we assessed the ability of an original G4 ligand to inhibit tumor cell proliferation and to enhance the sensitivity to ionizing radiation of 2 human telomerase-positive GBM cell lines.
Materials and Methods Cell lines
Human GBM cell lines, SF763 and SF767, were obtained from the Brain Tumor Research Center Tissue Bank of the University of California. Cell lines were Mycoplasma free (Mycoplasma Detection Kit; InvivoGen). No authentication of cell lines was done by the authors. Normal skin fibroblasts were obtained from a healthy 30-year-old female donor. Cultures were carried out in Dulbecco’s Modified Eagle’s Media sup-plemented with 10% of FBS, 1% of nonessential amino acids, and 1% of pyruvate of sodium and gentamicin (Gibco Invitrogen).
Chemical compounds
TAC consists of N-methylated triflate derivatives of 4,6-bis-(6-(acrid-9-yl)-pyridin-2-yl)-pyrimidine (U.S. patent 20080119492; see Supplementary Methods for synthesis details). Mass spectrometric analyses (liquid chromatography/mass spectrometry and electrospray-mass spectrometry; Supplementary Fig. S1–3) indicated that TAC is a 2:1 mixture of bis-methylated and tris-methylated products. Because of the small amount of material available, the 2 compounds were not separated and the 2:1 mixture was used as such for biological experiments. TAC was dissolved in dimethyl sulfoxide at a concentration of 2 mmol/L to produce aliquots of stock solution. The aliquots were stored at 20C and diluted in culture medium immediately before use to obtain a working solution at 100 mmol/L.
Equilibrium dialysis and fluorescence resonance energy transfer melting assays
Competitive dialysis is an effective method to compare the binding of a compound to different nucleic acid
structures. Experiments were carried out as previously described on a panel of single strands, duplexes, i-motif, and quadruplexes (14). Binding was quantified by the absorbance and fluorescence signals of TAC.
Fluorescence resonance energy transfer (FRET) melting assays allow a semiquantitative assessment of quadru-plex stabilization. The F21T oligonucleotide was double labeled with fluorescein or carboxytetramethylrhoda-mine (TAMRA), and apparent melting temperatures were determined in sodium and potassium conditions, with or without added ligand, at various concentrations (micromolar range) as previously described (15). Proliferation assessment
Cells were grown for 14 days with an intermediate passage after 7 days of culture. After harvesting, trypan blue–negative viable cells were counted. The number of population doublings (PD) after 7 and 14 days was calculated as follows: PD ¼ log2(Nf/N0), where Nf is the final cell number and N0 is the initial number of seeded cells.
Cell-cycle analysis
Cell membranes were lyzed by immersion in liquid nitrogen. The cell pellet was then resuspended in 500 mL of ribonuclease A (Sigma) and 500 mL of propidium iodide (PI; Sigma) for 20 minutes at room temperature. Cell-cycle distribution and sub-G0–G1 fraction were determined by flow cytometry (EPICS XL; Beckman Coulter) using CellQuest software.
Apoptosis assessment
The Annexin V kit of Caltag Laboratories, Inc., was used to assess apoptosis. Cells (1 105) were double labeled with PI and anti-human Annexin V antibody and then analyzed by flow cytometry (EPICS XL). Early apoptotic cells were scored as the percentage of Annexin V–positive/PI-negative cells, and late apoptotic and necrotic cells were scored as the percentage of cells staining positive for both Annexin V and PI.
Quantitative real-time reverse transcriptase PCR Total RNA was extracted from tumor cells using TRIzol reagent (Invitrogen). One microgram of RNA was reverse transcribed with SuperScript II enzyme (Invitrogen). Quantitative real-time reverse transcrip-tase PCR (qRT-PCR) was carried out with the Light-Cycler system (Roche Diagnostics). The primers used for amplification ofhTERT, TRF1, and TRF2 (telomeric repeat binding factors 1 and 2) have been described elsewhere (16, 17). In addition, the amount of ABL transcripts was quantified in all samples as an internal control to normalize the results (18).
Cytogenetics
Cells were harvested after colcemide exposure (0.01 mg/mL) for 20 hours. Chromosomal slides were prepared and stained according to standard methods.
The frequency of chromosomal aberrations was deter-mined by the analysis of 100 metaphases.
Cell irradiation
Photon irradiations were conducted at room temperature with a linear accelerator PHILIPS SL 75-5 (in X-ray production mode) at a dose rate of 3 Gy/min.
Clonogenic survival assay
Clonogenic cell survival was determined by a standard colony-forming assay. Cells (1,500) were plated in culture flasks and were allowed to attach for 24 hours. The following day, flasks were irradiated with 0 to 8 Gy. Cells were reincubated for 10 days, then fixed with methanol, and stained with Giemsa. Colonies containing more than 50 cells were scored. Surviving fractions at each dose were calculated relative to the plating effi-ciency of nonirradiated control cells.
53BP1 immunofluorescence staining
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. For immunos-taining, cells were incubated with a mouse monoclonal anti-53BP1 (p53-binding protein 1) antibody (Santa Cruz Biotechnology) overnight at 4C. Cells were then exposed to the secondary fluorescein isothiocyanate– labeled rabbit anti-mouse antibody (Santa Cruz Bio-technology) for 1 hour. The nuclei were stained by 40,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were analyzed with a Zeiss confocal laser scan-ning microscope.
Determination of gH2AX phosphorylation by flow cytometry
The level of gH2AX phosphorylation was deter-mined by flow cytometry using H2AX Phosphorylation Assay Kit (Upstate) according to the manufacturer’s instructions. Fluorescence intensity, in arbitrary units, was expressed relative to the nonirradiated control. For each experiment, all the compared samples were ana-lyzed the same day.
Statistical analysis
Standard errors were calculated for each mean, and statistical differences between groups were determined by Student’s t test, Kruskal–Wallis H test, or ANOVA using SEM software (19). Survival data were fitted according to the linear quadratic model using Kaleida-Graph software (4.0).
Results
Chemical properties of TAC and its interaction with quadruplex (telomeric) DNA
The major component (70%) of TAC (TAC-Me2) comprises 2 acridinium moieties joined by a heterotriaryl spacer containing 2 pyridine rings and 1 pyrimidine ring in alternation (abbreviated as py-pym-py; Fig. 1A). The minor component (TAC-Me3, 30%; Supplementary Fig. S4) has a third methylated site that cannot be precisely attributed (methylated pyridine or pyrimidine). Hence, throughout the present study, all discussion of the data refers to the bis-methylated compound. Owing to the propensity of the py-pym-py spacer to fold in a U-shaped
D
C
1 0.8 0.6 0.4 0.2 0 poly(dA-dT) poly(dC-dG) ctDNA ds26 poly(dC) 22CT 22AG 24G20 poly(dT) poly(dA) poly(rU) poly(rA) Relative bindingNucleic acid structure
Single strands Double strands i-motifs G4
B
A
N N N N N N 0 5 10 15 20 5 4 3 2 1 0TAC concentration (µmol/L)
Δ
Tm
(ºC)
Figure 1.Chemical structure, selectivity, and activity of the G4-interactive molecule TAC. A, structure of TAC-Me2 major component. B, speculative U-shaped conformation of TAC-Me2. C, nucleic acid binding by TAC was tested by equilibrium dialysis on a variety of nucleic acid structures (duplexes,
quadruplexes, i-motif, and single strands). D, FRET melting stabilization assays with the F21T telomeric sequence
[50-fluorescein-(GGGTTA)3
GGG-TAMRA] as a function of TAC concentration in sodium (open squares) and potassium (filled circles) conditions.
conformation, TAC-Me2 may possibly adopt a tweezer-type conformation in water (Fig. 1B), similar to that observ-ed for the unchargobserv-ed and monomethylatobserv-ed analogues in the solid state and in organic solvents (20). Interestingly, tweezer-type molecules holding 2 potentially intercalating units 7 A apart have a moderate affinity for duplex DNA based on the neighbor exclusion principle (21, 22), which excludes bis-intercalation between contiguous base pairs. In contrast, such compounds can display strong binding affinity for non-B DNA architectures that can provide less sterically constrained binding sites (23). In particular, G4 DNA can accommodate a large diversity of small molecules due to the presence of 3 loops and 4 grooves in the vicinity of the guanines quartets. Although the first generations of G4 ligands were mainly restricted to planar aromatic molecules (24–26), several recent studies reported the design of second-generation quadruplex binders that exhibit a more complex 3-dimen-sional topology (27–31). For instance, molecular modeling studies have suggested that ligands possessing 2 interca-lative aromatic units can establish a dual quartet–loop interaction (32). It was thus of great interest to assess the ability of TAC to bind the quadruplex form of human telomeric DNA, as it is now suggested that this unusual DNA structure affects cancer cell viability. A monomeric acridinium salt CH12-4 was prepared and used as control (Supplementary Fig. S5). This molecule has a planar con-formation and most likely behaves in the same manner as a classical duplex DNA intercalator.
Two complementary methods were used to show that TAC is a quadruplex ligand. Competitive dialysis experi-ments (Fig. 1D) indicate that this compound has a pref-erence for quadruplex structures. These experiments compare the amount of dye bound to a variety of struc-tures (typically 12–20), each trapped in a small dialysis chamber immerged in a large bath containing 1 mmol/L TAC. Binding to the 2 quadruplex samples [22AG and 24G20, corresponding to the human telomeric motif [A(GGGTTA)3GGG] and T2G20T2sequence, respectively] was stronger than that for duplexes and DNA and RNA single strands [poly(rA), poly(rU), poly(dT), and poly(dU)] or i-motifs (formed with C-rich sequences). Binding to quadruplexes was not exclusive, as lower but significant binding was also observed for double strands. FRET melting experiments were carried out with a fluorescently labeled oligonucleotide F21T mimicking 3.5 repeats of the human telomeric motif fluorescein-d-(GGGTTA)3GGG-TAMRA. Adding increasing concen-trations of TAC led to an increase in the apparent melting temperature of F21T. Figure 1D shows that stabilization reachedþ20C at 5 mmol/L ligand concentration in KCl (circles) but was systematically lower in NaCl (squares), as observed for many other ligands.
Finally, TAC was active in the telomere repeat ampli-fication protocol (TRAP) assay with an apparent IC50 value of 0.15 mmol/L (data not shown). However, as previously reported, this is the result of inhibiting the amplification of G-rich sequences rather than true
telomerase inhibition (33). Under the conditions used here, the TRAP assay should be considered as a useful technique to confirm quadruplex recognition rather than one to test its activity against telomerase.
Short-term treatment with G4 ligand TAC inhibits GBM cell growth
SF763 and SF767 cell lines were maintained in culture for 14 days in the presence of 0.5, 1.0, and 2.0 mmol/L of TAC. This treatment inhibited the growth of GBM cells in a concentration-dependent way (ANOVA; P < 107; Fig. 2A), with a more marked effect on SF763 cells. Analysis of cellular viability indicated that the treatments were not toxic (<10% Trypan blue–positive cells).
In parallel, we tested the same concentrations of TAC on control normal human fibroblasts. No changes in proliferation were observed (Supplementary Fig. S6), in line with previous studies with other G4 ligands showing that they did not affect the proliferation and viability of normal human telomerase-negative cells like fibroblasts (34) or astrocytes (13). However, the toxicity of TAC to telomerase-dependent normal human cells as hematopoietic cells or other rapidly proliferating cells remains to be explored. In addition, the control com-pound CH12-4, a classical duplex DNA intercalator, did not inhibit GBM cell proliferation (Supplementary Fig. S7). Taken together, these results indicate the speci-ficity of TAC effects on tumor cell proliferation.
We next analyzed the distribution of cells in different phases of the cell cycle by flow cytometry (Fig. 2B). As compared with untreated controls, the distribution was not significantly modified in SF763 and SF767 cells trea-ted with TAC for 14 days, with only a moderate cell accumulation in the G2–M phase.
To determine whether inhibition of tumor cell growth was related to apoptosis induction, the fraction of early apoptotic cells was evaluated by flow cytometry after 14 days of treatment (0.5–2 mmol/L). The apoptotic frac-tions were low (<10%) and closely similar in controls and in cells treated with 0.5 and 1 mmol/L TAC. In contrast, the early apoptotic fraction increased to 32% and 23% (Student’st test; P < 103) in SF763 and SF767 cells treated with 2 mmol/L of TAC, respectively (Fig. 2C). There was no significant increase in the fraction of late apoptotic and necrotic cells, which is in agreement with the low percentage of cells stained with Trypan blue. In addition, sub-G0–G1analysis also detected a significant increase in apoptosis in both cell lines treated with 2 mmol/L TAC (Supplementary Fig. S8).
In the absence of significant changes in cell-cycle dis-tribution and apoptosis rate after treatment with low con-centration of TAC, the observed proliferation inhibition may be related to an increase in the cell-cycle duration. To address this point, we estimated the length of cell doubl-ing time in the exponential phase of cell growth, which roughly equals the cell-cycle duration. This estimation was done after 10 days of culturing, with an intermediate passage of cultures after 5 days. The treatment with
1 mmol/L TAC significantly increased the duration of the cell cycle from 23.2 1.3 to 45.4 2.4 hours in SF763 cells (Student’st test; P ¼ 9 104) and from 22.1 0.9 to 34.0 0.9 hours in SF767 cells (P ¼ 7 104).
TAC triggershTERT overexpression and DNA damage but does not induce telomere shortening
SF763 and SF767 cell lines have relatively short telo-meres, with an average length for the telomeric restric-tion fragments of 4.4 and 3.3 kb, respectively. The treatment with 1 mmol/L TAC did not induce any significant reduction of total or 30-overhang telomeric DNA, as radioactive signal corresponding to 30-telomeric single-stranded DNA was roughly the same before and after 14 days of treatment for similar DNA quantities (Supplementary Fig. S9).
The positive telomerase status of the GBM cell lines was confirmed with qRT-PCR, which revealed hTERT expression. We then evaluated expression levels of the
hTERT gene as well as TRF1 and TRF2 genes, which encode the core members of the shelterin telomere pro-tection complex, in cells treated with TAC for 14 days (Fig. 3A). In comparison with untreated controls, the expression of hTERT was greatly increased (3-fold for SF763 and 10-fold for SF767 cells) after TAC treatment (ANOVA, P ¼ 2.5 107andP ¼ 1.7 107, respec-tively). The treatment also led to a 2-fold increase in TRF2 expression (SF763, P ¼ 6.6 104; SF767, P ¼ 4.4 105) whereas the expression ofTRF1 gene was decreased (SF763,P ¼ 1.6 103; SF767,P ¼ 6.0 106). To study the induction of DNA double-strand breaks (DSB), we counted the number of phosphorylated 53BP1 foci by in situ immunofluorescence staining. G4 ligands have been reported to induce DNA damage after only a few hours of treatment (34). Indeed, we found that 24-hour treatment with TAC significantly increased the number of 53BP1 foci both in SF763 cells exposed to 0.5 mmol/L TAC (P ¼ 4 104,H test), 0 2 4 6 8 10 12 14 7 0 Days Population doublings NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TAC 0 2 4 6 8 10 12 14 7 0 Days Population doublings NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TAC
A
B
C
SF767 SF763 0 10 20 30 40 % of apoptotic cells*
0 10 20 30 40 2 µmol/L TAC 1 µmol/L TAC 0.5 µmol/L TAC NT 2 µmol/L TAC 1 µmol/L TAC 0.5 µmol/L TAC NT % of apoptotic cells*
0 10 20 30 40 50 60 70 80 G2–M S G0–G1 % of cells NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TAC 0 10 20 30 40 50 60 70 80 G2–M S G0–G1 % of cells NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TACFigure 2.Cellular effects of TAC in SF763 and SF767 cell lines treated continuously with TAC (0.5, 1, and 2mmol/L) for 14 days as compared with nontreated (NT) cells. Mean SE values of 3 independent experiments each carried out in triplicate are shown. A, proliferation assessment: population doubling of the cell lines. TAC induced a concentration-dependent proliferation inhibition in both cell lines. B, cell-cycle analysis: distribution of cells in the different cell-cycle phases. Cells exposed to TAC showed few modifications in this distribution, with only a moderate accumulation of cells in G2–M phase. C, flow cytometric
assessment of apoptosis: apoptotic cells were scored as the percentage of Annexin V positive/ PI negative cells. Cells treated with 2mmol/L TAC, but not cells exposed to a lower concentration, showed an increased apoptotic fraction. *, statistically significant difference (P values are given in the text).
1 mmol/L TAC (P ¼ 7 105), and 2 mmol/L TAC (P < 107) and in SF767 cells exposed to 1 mmol/L TAC (P ¼ 0.04) and 2 mmol/L TAC (P ¼ 9 106; Fig. 3B and C). This effect was more pronounced in SF763 cells (3-fold increase at 2 mmol/L) than in SF767 cells (1.5-fold increase at 2 mmol/L).
To assess the induction of chromosomal aberrations, we evaluated untreated and treated (0.5 mmol/L TAC, 14 days) cultures for the presence of dicentric chromo-somes. Average frequencies of dicentrics per cell were 0.11 in SF763 and 0.06 in SF767 cells, as compared with 0.02 and 0 for untreated controls, respectively. Thus, the treatment induced dicentric chromosomes, which can result from end-to-end chromosome fusions and repre-sent signatures of telomere dysfunction.
These observations indicated that short-term TAC treatment of GBM cells modified the level of hTERT, TRF1, and TRF2 gene expression without a visible effect on average telomere length. The treatment induced concentration-dependent DNA damage and dicentric chromosomes.
TAC enhances radiation-induced killing of GBM cells
The intrinsic radiosensitivity of SF763 and SF767 cells was first studied in triplicate experiments by clonogenic
survival assay after increasing doses of radiation (0–8 Gy). Both GBM lines were found to be radioresistant, as previously observed (35). The survival fraction at 2 Gy was higher for SF763 cells (86%) than for SF767 cells (65%), indicating an even stronger resistance of SF763 cells to radiation.
Subapoptotic, nontoxic concentrations of TAC (0.5 and 1 mmol/L) were selected for combination experiments. Of note, these concentrations did not reduce plating efficiency (data not shown). SF763 and SF767 cells were treated with TAC for 14 days, the time point where TAC had shown an appreciable antiproliferative effect in both cell lines. These cells were then irradiated and clonogenic survival assay was conducted. During incubation, TAC was present in the culture medium. The comparison between sur-vival curves obtained after irradiation alone and after the combined treatment showed a strong concentra-tion-dependent enhancement of radiation sensitivity after TAC treatment (Fig. 4; ANOVA,P < 107), show-ing a synergistic effect in both cell lines. Survival (S) data after irradiation dose (D) were fitted according to the linear quadratic model as follows: S(D)/S(0) ¼ exp(aD bD2
). The a and b parameters were used to calculate the dose enhancement factor at 10% sur-vival (DEF10). The DEF10values for 0.5 and 1 mmol/L
Figure 3.TAC effects at RNA and DNA levels. A, qRT-PCR results for the expression ofhTERT and telomere-related genesTRF1 and TRF2. In both cell lines, the treatment with TAC led to an increase in the expression of hTERT and TRF2 genes whereas the expression ofTRF1 was decreased after TAC treatment. B and C, DNA breaks in GBM cells, nontreated (NT) or treated with TAC, detected with 53BP1 immunofluorescence. Immunofluorescence staining of SF763 cells was carried out after treatment for 24 hours with TAC. Nuclei were stained with DAPI (blue) and 53BP1 (green). TAC increased the number of 53BP1 foci per cell in a concentration-dependent manner (B). This increase in the number of 53BP1 foci per cell was significant in SF763 cells exposed to 0.5, 1, and 2mmol/L TAC and for SF767 cells exposed to 1 and 2mmol/L TAC (C). *, statistically significant difference (P values are given in the text).
B
C
Nontreated 1 µmol/L TAC 0.5 µmol/L TAC 2 µmol/L TAC 0 100 200 300 400 500 TRF2 TRF1 hTERTNormalized mRNA expression
*
*
*
0 100 200 300 400 500 TRF2 TRF1 hTERTNormalized mRNA expression
NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TAC NT 0.5 µmol/L TAC 1 µmol/L TAC 2 µmol/L TAC
*
*
*
SF767 SF763A
0 1 2 3 4 5 Number of 53BP1foci per cell
* * 0 1 2 3 4 5 2 µmol/L TAC 1 µmol/L TAC 0.5 µmol/L TAC NT 2 µmol/L TAC 1 µmol/L TAC 0.5 µmol/L TAC NT Number of 53BP1
foci per cell
*
* *
SF 763
TAC were 1.68 and 2.32 in SF763 cells and 1.96 and 2.92 in SF767 cells, respectively.
TAC modifies the repair of DNA DSBs induced by radiation
To investigate possible radiosensitization mechan-isms of TAC, we assessed the induction and repair of DSB after irradiation through the study of phosphory-lated 53BP1 and gH2AX foci that colocalize with DNA DSB.
Despite extensive analysis (at least 100 cells), we did not detect any consistent increase in DNA break induc-tion after combined TAC (0.5 mmol/L) and radiainduc-tion (2 Gy) treatment by in situ immunofluorescence ana-lysis for 53BP1. Initial levels of foci at 0.5 hours after irradiation were around 30 per cell. The repair was apparently efficient after this dose, as the numbers of residual foci 24 hours following irradiation were very low (<3 foci per cell).
Because the radiosensitizing effect of TAC was stron-ger at higher radiation doses, we applied a different approach with flow cytometric analysis of phosphorylat-ed gH2AX (36), which enablphosphorylat-ed us to use the radiation dose of 6 Gy. We measured the initial peak at 0.5 hours and signal reduction at 3 and 24 hours after irradiation. No significant difference was noted at 0.5 and 3 hours between treated (0.5 mmol/L) and nontreated cells. In contrast, a higher residual level in the presence of TAC was observed at 24 hours (P ¼ 0.016 for SF763 andP ¼ 0.04 for SF767; Fig. 5A), indicating a significant deceleration of DNA repair.
These results suggest that TAC affected DNA repair after irradiation. To further investigate this hypothesis, we analyzed the frequency of radiation-induced chromo-somal aberrations after irradiation with 4 Gy. The treat-ment with 0.5 mmol/L TAC for 14 days significantly increased the frequency of both chromosome-type and chromatid-type aberrations (Table 1). Of note, the highest increase was observed in the frequency of dicentric chro-mosomes (Fig. 5B) after subtraction of the background incidence of dicentrics induced by TAC alone.
In addition, we conducted cell-cycle analysis at 48 hours after irradiation with 6 Gy in untreated cul-tures and in culcul-tures treated with 1 mmol/L of TAC for 14 days. Both GBM cell lines treated with TAC exhibited a significant increase in percentage of cells in the G2–M phase (P < 0.01), reflecting a more important radiation-induced G2-phase block (Fig. 5C).
To address the issue of apoptosis induction after TAC and radiation, we conducted Annexin V/PI staining and flow cytometric analysis of cells harvested 3, 6, 24, 48, and 72 hours after treatments with either radiation (6 Gy) alone or radiation (6 Gy) in combination with 1 mmol/L TAC. During this time course, both early and late apopto-sis/necrosis was slightly but not significantly induced by the treatments (data not shown).
Discussion
TAC is a quadruplex ligand active on telomeric DNA in vitro. Telomere damage and DNA damage response induced by G4 ligands such as RHPS4, BRACO19, and
SF767 SF763 0.01 0.1 1 10 100 8 6 4 2 0
Radiation dose (Gy)
Surv iv al fraction (% ) NT 0.5 µmol/L TAC 1 µmol/L TAC P < 10-7
Radiation dose (Gy) 0.01 0.1 1 10 100 8 6 4 2 0 Surv iv al fraction (% ) NT 0.5 µmol/L TAC 1 µmol/L TAC P < 10-7 β α NT 0.034 0.017 0.5 µmol/L TAC 0.216 0.024 1 µmol/L TAC 0.043 0.101 β α NT 0.166 0.023 0.5 µmol/L TAC 0.464 0.050 1 µmol/L TAC 0.578 0.158 S(D)/S(0) = exp (–αD – βD2) S(D)/S(0) = exp (–αD – βD2)
Figure 4.TAC radiosensitizes GBM cells. Clonogenic survival curves of SF763 and SF767 cells, nontreated (NT) or treated continuously for 14 days with 0.5 and 1mmol/L TAC, after X-ray irradiation with doses ranging from 0 to 8 Gy. Mean SE values of 3 independent experiments, each carried out in triplicate are shown. Survival (S) data after a radiation dose (D) were fit according to the linear quadratic model. The linear parametera and the quadratic parameterb are given for each experimental condition. Radiation-induced killing of TAC-treated cells was significantly enhanced in a concentration-dependent manner (ANOVA;P < 107).
telomestatin (37–39) lead to a short-term inhibition of proliferation and rapid apoptosis of tumor cells. In line with these reports, 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. The effects of these low con-centrations on cell-cycle distribution and apoptosis were minimal, but the treatment apparently increased the cell-cycle length, which can explain the decreased cell proliferation rate. However, TAC could also induce rapid and substantial apoptosis (up to 32% of cells) at a higher concentration (2 mmol/L). Antitumor properties of several G4 ligands (pyridine dicarboxamide derivatives) have already been tested on GBM cell lines by Pennarun
and colleagues (13). They showed an early antiprolifera-tive effect and a massive apoptosis (35% of cells) in cultures treated with a G4 ligand at 5 mmol/L concen-tration. Tumor cell apoptosis could be the linking factor between telomere instability, DNA damage response, and decreased proliferation.
G4 ligands were initially evaluated as telomerase inhi-bitors, but their antiproliferative effect may occur quick-ly, before any telomere shortening takes place (40). This short-term effect cannot be explained by telomerase inhibition, which would lead to a gradual shortening of telomeres after a certain number of cell divisions. The observations that G4 ligands delocalize telomeric pro-teins from telomeres (34, 41), activate DNA damage
B
A
SF767 SF763 0 1 2 3 4 5 24 h 3 h 0.5 hTime after irradiation
H2AX MFI (arbitrary units)
NT + 6 Gy 0.5 µmol/L TAC + 6 Gy
*
0 1 2 3 4 5 24 h 3 h 0.5 hTime after irradiation
H2AX MFI (arbitrary units)
NT + 6 Gy 0.5 µmol/L TAC + 6 Gy
*
SF767 SF763C
0 10 20 30 40 50 60 70 80 G2–M S G0–G1 % of cells NT + 6 Gy 1 µmol/L TAC + 6 Gy*
*
0 10 20 30 40 50 60 70 80 G2–M S G0–G1 % of cells NT + 6 Gy 1 µmol/L TAC + 6 Gy*
*
Figure 5.Delayed repair of radiation-induced DNA breaks, induction of chromosome aberrations, and G2–M accumulation in GBM cells after combined
treatment with TAC and irradiation. A, kinetics of DNA repair analyzed bygH2AX phosphorylation by flow cytometry in SF763 and SF767 cells treated with 0.5mmol/L TAC for 14 days and irradiated with 6 Gy. Mean SE values of 3 independent experiments each carried out in triplicate are shown. Results of mean fluorescence intensity (MFI) are expressed in arbitrary units. At 0.5 and 3 hours, no difference was observed between nontreated (NT) cells and cells treated with TAC. At 24 hours, cells treated with TAC showed a significant increase in the expression of phosphorylatedgH2AX compared with NT controls for SF763 and SF767 cell lines. B, dicentric chromosomes (arrows) in SF763 cell line treated with radiation and 0.5mmol/L TAC. C, cell-cycle distribution at 48 hours after 6 Gy irradiation in SF763 and SF767 cells, nontreated or treated with 1mmol/L TAC for 14 days. Significant increase in G2–M fraction in treated cells was observed for both cell lines. Mean SE values of 3 independent experiments each carried out in triplicate are shown.
response at telomeres, and induce chromosomal end-to-end fusions (38) strongly suggest that their direct target is the telomere and not the telomerase enzyme.
In agreement with these reports, we found that TAC caused DNA damage without significant telomere short-ening. This damage was, in particular, revealed by an increase in chromosomal fusions in both GBM cell lines. Pennarun and colleagues (13) also found in GBM cells treated with G4 ligands a very clear increase in telomeric fusions in metaphase and anaphasic bridges, evidence of telomeric instability not related to telomere attrition.
The chromosome end fusions are formed when telo-meres are dysfunctional and are recognized as DSBs by repair machinery, which joins chromosomal ends. Dysfunctional (uncapped) telomeres, resulting from quadruplex DNA stabilization by G4 ligand, associate directly with many DNA damage response proteins. This DNA damage response may be evidenced by the appearance of phosphorylated 53BP1 or gH2AX foci in cells exposed to G4 ligands, of which about 70% are located at telomeres (34). In the present study, we found a rapid and significant increase in the number of 53BP1 foci after TAC treatment.
The structure and function of telomeres are controlled both by telomerase and by the shelterin complex. Pre-vious studies showed that tumor cells overexpressing hTERT or TRF2, a shelterin complex component, become resistant to treatments with G4 ligands (34, 42). These changes were considered to represent compensatory defense mechanisms activated in response to G4 ligand treatment, which is consistent with the view that telomere is a direct target of G4 ligands (34, 42). In our study, the effect of TAC on telomeres was also evidenced by
changes in the expression of telomere-related genes in treated cells. In particular, we observed a strong increase in the expression of hTERT, which might counteract the deleterious effects of the stabilization of G4 DNA by a ligand through the extension of the single-stranded G-rich telomeric portion, which is termed 30 overhang (42). We also found increased TRF2 expression, which could facilitate the formation of a loop structure (t-loop), masking the 30 overhang against G4 ligand binding, and/or the recruitment and stimulation of RecQ heli-cases, which resolve G4 DNA structures (32). In addition, we observed a concomitant decrease in the expression of TRF1, which may contribute to hTERT-dependent telo-mere elongation.TRF1 controls the access of telomerase to telomeric DNA, and its downregulation was shown to result in the elongation of telomeres (43).
Thus, our results indicate that TAC targets telomeres, thereby increasing telomeric instability in tumor cells, and this inhibits tumor growth and, at higher doses, leads to tumor cell apoptosis. As telomeric dysfunction was reported to increase radiosensitivity and to decrease DNA repair capacity (8), we then investigated the impact of telomeric instability induced by TAC on the radio-sensitization of GBM cells.
Applying low concentrations of TAC, which itself did not induce apoptosis or cell-cycle changes, we found a strong concentration-dependent radiosensitizing effect on GBM cell lines within a wide dose range, including the dose corresponding to the daily therapeutic radiation dose (2 Gy) administered to GBM patients in clinical practice. This result is significant, because the adminis-tration of temozolomide, the new standard chemothera-py (1) given concomitantly with radiotherachemothera-py, is based on Table 1. Chromosome aberration frequencies in GBM cell lines after irradiation and after combined treatment with radiation and G4 ligand TAC
Frequency of chromosomal aberrations Cells analyzed Acentrics per cell (95% CI) Dicentrics per cell (95% CI) Chromatid fragments per cell (95% CI) Chromatid exchanges per cell (95% CI) SF763 cells Irradiated (4 Gy) 100 0.34 (0.21–0.47) 0.32 (0.19–0.44) 1.12 (0.89–1.34) 0.31 (0.19–0.43) Irradiated (4 Gy) and treated
with 0.5mmol/L TAC
100 0.85 (0.62–1.08) 1.02 (0.83–1.21)a 1.72 (1.45
–1.99) 0.81 (0.58–1.04)
P (H test) 0.011 <106 0.0035 0.023
SF767 cells
Irradiated (4 Gy) 100 0.46 (0.32–0.59) 0.38 (0.24–0.52) 0.86 (0.63–1.09) 0.24 (0.11–0.37) Irradiated (4 Gy) and treated
with 0.5mmol/L TAC
100 0.86 (0.64–1.07) 0.80 (0.62–0.98)a 1.42 (1.12–1.72) 0.58 (0.39–0.77)
P (H test) 0.043 0.00076 0.0093 0.0068
a
The background frequencies of dicentric chromosomes induced by TAC alone in nonirradiated cells (0.09 and 0.06 for SF763 and SF767 cells, respectively) were subtracted.
preclinical data (44, 45), which suggested that a combi-nation of temozolomide with radiation was essentially additive.
Radiosensitivity in an animal model with dysfunc-tional telomeres correlated with a deceleration of DNA repair kinetics and persistence of chromosomal aberra-tions (8). We investigated whether TAC induced these effects in GBM cell lines. There is a correspondence between the formation and subsequent loss of phos-phorylated gH2AX or 53BP1 foci and the recognition and subsequent repair of DNA damage. On the basis of this, MacPhail and colleagues (36) and Olive and Banath (46) proposed that the half-loss time of phosphoryl-ated gH2AX and the residual rate could be significant factors in the evaluation of cell response to ionizing radiation, with more rapid loss and less retention asso-ciated with more radioresistant cell lines. In our study, analysis of DNA repair after irradiation by a similar approach revealed delayed repair kinetics in GBM cells treated with TAC. The persistence of significant amounts of residual phosphorylated gH2AX was ob-served in both cell lines, indicating that the GBM cells had become more sensitive to radiation.
An increase in radiosensitivity was also correlated with a higher frequency of radiation-induced chromosomal aberrations after combined treatment with radiation and TAC as compared with radiation alone. Dysfunctional telomeres, which act as DNA breaks, might join radia-tion-induced DNA DSB, thus increasing radiation sensi-tivity (47). As TAC induces telomere dysfunction, we suggest that this mechanism was involved in the radio-sensitization of GBM cells observed after TAC treatment. Notably, the highest increase was seen in the frequency of dicentric chromosomes, which could be due to DSB– telomere joinings.
Cells exposed to ionizing radiation show delay or arrest in the cell-cycle progression. In particular, G2-phase block prevents G2cells with damaged DNA entering mitosis. The DNA damage can be reduced by means of DNA repair, but heavily damaged cells are unable to reach mitosis and are arrested in the G2phase. G2–M accumulation after irradiation in GBM cells trea-ted with TAC compared with untreatrea-ted cells may reflect the presence of cell population with increased DNA damage. This cell-cycle arrest can explain, at least in part, the radiosensitizing effect of TAC at the cellular level.
We did not observe any significant apoptosis induc-tion up to 72 hours after treatment with radiainduc-tion and TAC. This result may be explained by the fact that the cells were blocked in the G2–M phase after radiation, which was particularly marked after the combination of radiation and TAC. This blockage delays cell death, which could occur much later, after 1 or more cell divisions (48).
Here, we showed efficacy of TAC combined with radiation on GBM cells in vitro. Next step will be to test the radiosensitizing effect of intratumoral injections
of TACin vivo, using subcutaneous human GBM xeno-grafts in animals. Because one of the major problems in brain tumor therapy remains the difficulty of drug delivery across the blood–brain barrier, it will also be essential to evaluate in an orthotopic model whether TAC is capable of crossing the blood–brain barrier to reach and radiosensitize GBM tumors. Another possi-bility can be the direct injection of G4 ligand into the tumor-infiltrated brain parenchyma under positive pressure using convection-enhanced delivery. Con-versely to conventional diffusion, convection-enhanced delivery creates a homogenous drug concentration extending over several centimeters in diameter, allow-ing to target brain tumor tissue as well as the area surrounding the tumor, which may contain tumor cells. This approach is successfully used to test novel antitu-mor agents in animal glioma models and in clinical trials with acceptable safety (49).
In conclusion, we showed that TAC, an original G4 ligand, could enhance the sensitivity to ionizing radia-tion of 2 human hTERT-positive GBM cell lines. A possible mechanism of radiosensitization is related to delayed DNA break repair after irradiation, which increases the number of chromosomal abnormalities such as dicentrics, leading to cell death. Our work thus provides the first evidence that exposure to a G4 ligand radiosensitizes radioresistant human GBM cells. Radiosensitizing effects will be investigated with other G4 ligands. Many elements concerning molecular mechanisms of altering tumor growth with TAC have to be elucidated. In particular, it will be interesting to study the effects of TAC on G4 ligands located outside telomeric regions, in promoters of human genes, which are involved in cell proliferation (50). Further studies in animal models using human GBM tumor xenografts are necessary to explore the therapeu-tic potential of this radiosensitizing approach.
Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments
The authors thank M. Bombled and D. Monchaud for liquid chroma-tography/mass spectrometry analysis of chemical compounds, N. Saettel for help in the drawings, and Y. Communal, R. Veyrat-Masson, and J. Chassagne for help in flow cytometric analysis. The authors are also grateful to Aurore Guedin (U869).
Grant Support
This work was supported by grants from the Region Aquitaine, Fondation pour la Recherche Medicale, and ANR (G4-TOOLBOX, ANR-Blan-09-355) to J.-L. Mergny.
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.
Received July 13, 2010; revised July 22, 2011; accepted August 1, 2011; published online October 10, 2011.
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