Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells

Article abstract of DOI:10.1016/j.biochi.2012.07.003

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.

Full text of DOI:10.1016/j.biochi.2012.07.003

Biochimie 94 (2012) 2559e2568
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journal homepage: www.elsevier.com/locate/biochi
Research paper
Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide
series on the terminal sequence of XpYp telomere in HT1080 cells
Assitan Sidibe a, Florian Hamon b, Eric Largy b, Dennis Gomez c, Marie-Paule Teulade-Fichou b,
,
Chantal Trentesaux a, Jean-François Riou a
*
a Structure des Acides Nucléiques, Télomères et Evolution, Inserm U565, CNRS UMR 7196, Muséum National d’Histoire Naturelle, 43 rue Cuvier, 75231 Paris cedex 05, France
b Synthèse et Vectorisation de Biomolécules, CNRS UMR 176, Institut Curie, Bat 110-112, Université Paris-Sud, 91405 Orsay, France
c Radiology and DNA repair group, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during
its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to
disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric
dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical
synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that
belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for
biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity
for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080
tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom
seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo
derivative (360AeBr) was selected for the cellular assays. Since POT1 participates to the fine tuning of the
C-strand end resection during telomere replication, we investigated the effect of 360AeBr to alter the
terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated
for up to 24 days with 360AeBr presented some minor but significant variations of C-strand terminal
nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these
minor modifications of the telomeric C-strand resection induced by 360AeBr in HT1080 cells are
discussed.
Received 29 May 2012
Accepted 5 July 2012
Available online 14 July 2012
Keywords:
G-Quadruplex
Telomere
Pyridine dicarboxamide halogenated
derivatives
POT1
C-STELA
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
essential for the formation of the t-loop, a potential protective
structure at telomere, in which the G-overhang invades the telo-
The nucleoprotein structure at the end of telomeres is essential
to maintain chromosome stability and to avoid end of chromo-
somes to be recognized as a double-strand break by the DNA repair
machinery [1]. Human telomeres are composed of tandem repeats
of the sequence TTAGGGn and a single-stranded 30 extension (G-
overhang) associated with six proteins from the shelterin complex
(TRF1, TRF2, hRAP1, TIN2, TPP1 and POT1) that play important
functions in the protection of telomere stability and during its
replication [2,3]. Telomeric G-overhang plays a complex role in
meric duplex to form a D-loop [6]. Ends of telomeres are generated
during replication through a complex mechanism involving G-
overhang extension, C-strand resection and C-strand fill-in [7].
Interestingly, the final nucleotide at the 50 end of the C-strand
remains precisely defined, suggesting a fine tuning of the C-strand
resection and corresponds to 30-ATC-50 (80%) [8]. The nuclease
responsible for the C-strand resection is likely to be recruited by the
shelterin protein POT1, since the depletion of POT1 by siRNA leads
to the randomization of the terminal nucleotide sequence at C-
strand [9,10]. The artemis-like nuclease Apollo (hSNM1B) previ-
ously found to be associated to the shelterin complex was also
found to be implicated in the G-overhang maintenance specifically
at telomeres generated by leading strand synthesis and is a poten-
tial candidate for the resection enzyme [11].
telomere maintenance. G-overhang provides
a template for
extension by telomerase or alternative mechanisms of telomere
extension based on recombination [4,5]. In addition, G-overhang is
Evidences are accumulating that non-canonical four-stranded
structures called G-quadruplexes can form among telomere repeats
* Corresponding author. Tel.: þ33 1 40 79 36 98; fax: þ33 1 40 79 37 05.
E-mail address: riou@mnhn.fr (J.-F. Riou).
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2012.07.003

2560
A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
during lagging strand DNA replication and at the 30 telomeric G-
overhang [12,13]. G-quadruplexes are well characterized confor-
mations of sequences containing several repeats of guanines that
arrange in guanine tetrads, stabilized by Hoogsteen hydrogen
bounding and coordinated by central cations [12,14]. Human telo-
meric sequence can form intramolecular G-quadruplexes showing
an antiparallel conformation in sodium containing solutions or
a parallel “propeller” structure in potassium containing dehydrated
solutions or in crystal [15,16]. Although their existence in
mammalian cells is based on indirect evidences [13], it has been
demonstrated using specific antibodies that G-quadruplexes are
formed in the macronuclei from ciliates [17]. Further bioinformatic
analysis of the human genome indicated the presence of thousands
of potential G-quadruplex-forming sequences (PQS) with a specific
enrichment in regulatory regions such as gene promoters, UTRs and
splice sequences [18], suggesting a role for G-quadruplexes in the
regulation of gene expression.
A large number of small molecule ligands have been synthesized
during the last decade to bind and to stabilize G-quadruplexes [19].
Some of them present remarkable selective binding properties to
G-quadruplex relative to duplex DNA [20]. G-quadruplex ligands
were shown to induce a delayed growth arrest or apoptosis in both
telomerase positive and ALT tumor cell lines associated with
alterations of the telomere stability [12,19,21]. All G-quadruplex
ligands were shown to interfere more or less with the binding of
essential shelterin components, such as POT1 and TRF2, leading to
a DNA damage response at telomere (TIFs) and G-overhang short-
ening [22e32].
telomeric G-quadruplex and delayed growth inhibitory properties,
but triggers mild modifications of the telomeric C-strand in
agreement with a partial displacement of POT1, whose significance
is discussed.
2. Materials and methods
2.1. Synthesis
1H and 13C spectra were recorded at 25 ꢀC on a Bruker Avance
300 using TMS as internal standard. Deuterated CDCl3 and DMSO-
d6 were purchased from SDS. The following abbreviations are used:
singlet (s), doublet (d), triplet (t) and multiplet (m). Low resolution
mass spectrometry (ESI-MS) was recorded on a micromass ZQ 2000
(waters). High resolution mass spectrometry (ESI-MS) were
provided by the I.C.S.N. (Institut de Chimie des Substances
Naturelles, Gif-sur-Yvette). TLC analysis was carried out on silica gel
(Merck 60F-254) with visualization at 254 and 366 nm. Melting
points were taken on a Kofler melting point apparatus and are
uncorrected. Preparative flash chromatography was carried out
with Merck silica gel (Si 60, 40e63 mm). Reagents and chemicals
were purchased from Sigma-aldrich, Acros or Alfa-aesar unless
otherwise stated. Solvents were purchased from SDS.
2.2. FRET experiments
Quantitative FRET experiments were obtained on a Stratagene
MX3000P (La Jolla, CA) quantitative PCR apparatus using 0.2 mM
G-quadruplex ligands also induce telomere-independent DNA
damage foci or binding sites that might be related to the presence of
G-quadruplexes in other regions of the genome [25,33], as evi-
denced recently for pyridostatin [34]. These effects correspond to
the lack of selectivity reported for all G-quadruplex ligands between
telomeric over the others G-quadruplexes [35,36] that may be
F21T (50-FAM-GGGTTAGGGTTAGGGTTAGGG-Tamra-30). Melting
profiles were recorded in a 10 mM lithium cacodylate (pH 7.2)
buffer with 100 mM NaCl or 10 mM KCl þ 90 mM LiCl. Excitation
wavelength was 496 nm and emission was recorded at 516 nm. For
competition experiments, various concentrations of the double-
stranded ds26 competitor (50-CAATCGGATCGAATTCGATCCGATTG-
30) were added prior to the melting experiment.
explained by their predominant binding mode through aromatic
p
stacking to one of the external G4 quartet [16]. In some cases, minor
chemical modifications of G4-ligands were reported to affect both
their biophysical and biological properties. As an example, compare
ligands 360A, 307A and 832A from the 2,6 pyridine dicarboxamide
series for G4-FID displacement assayand growth inhibition in tumor
cell lines in [31,36]. Indeed, compounds presenting a close G-
quadruplex binding profile (360A and Phen-DC3) displayed
remarkable differences at the level of transcriptional changes in
quadruplex-containing genes[36,37]. Altogether these observations
suggest that minor modifications of the G-quadruplex ligand
structure and of the G-quadruplex binding profile might have an
important impact on their overall biological activity.
Inthisstudy, wehaveinvestigatedtheimpactoftheintroductionof
a halogen atom (Cl, Br, I) on the central pyridine moiety. The intro-
duction of halogen atoms on biologically active compound is classi-
cally used in drug design and is known to improve membrane
permeability (cellular and blood brain barrier). In addition steric and
electronic effects of halogen atoms may improve binding to molecular
targets [38]. Thus both improvements of 360A activity could be ex-
pectedbothintermsofquadruplexrecognitionandintermsofcellular
effects.
2.3. HT-G4-FID
HT-G4-FID measurements were performed as described previ-
ously [39], on a FLUOstar Omega microplate reader (BMG Labtech)
with a 96-well black quartz microplate (Hellma). TO (thiazole
orange) and cacodylic acid were purchased from Aldrich and used
without further purification. Stock solutions of TO and ligands
(2 mM in DMSO) were used. 22AG oligonucleotide (corresponding
to the human telomeric repeat; [50-AG3(T2AG3)3-30]), purified by
reverse phase HPLC, was purchased from Eurogentec (Belgium).
The percentage of displacement is calculated from the fluores-
cence intensity (F), using: TOD (%) ¼ 100 ꢁ [(F/F0) ꢂ 100], F0 being
the fluorescence from the fluorescent probe bound to DNA without
added ligand. The TOD is then plotted as a function of the
concentration of added ligand. The ligand affinity was evaluated by
the concentration of ligand required to decrease the fluorescence of
the probe by 50%, noted DC50, and determined after non-linear
fitting of the displacement curve.
2.4. EMSA experiments
The three chloro, bromo and iodo derivatives (360AeCl,
360AeBr, 360AeI) were evaluated for their quadruplex interac-
tion in vitro using biophysical and biochemical assays. In addition,
because all G-quadruplex ligands were reported to alter the binding
of POT1 at telomere and that this shelterin protein controlled the C-
strand resection processes, we have also studied the alterations of
the C-strand nucleotide composition at XpYp telomere in HT1080
cells using C-STELA with the bromo derivative (360AeBr). We
report that 360AeBr retains the potent affinity and selectivity for
Topo III
procedure [40]. Electrophoretic mobility shift assays using Topo III
was performed in 10 L of the following solution: 50 mM HEPES
(pH 7.9), 100 mM NaCl, 0.1 mM EDTA, 4% w/v sucrose, 2% v/v
glycerol, 0.1 mg/mL BSA, 0.02% w/v bromophenol blue, 20 nM
labeled 22AG (50-AGGGTTAGGGTTAGGGTTAGGG-30) and Topo III
(100 nM). The indicated concentrations of ligands were added in
a was purified according to a previously published
m
a volume of 1
mL. The reaction mixture was incubated at room

A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
2561
temperature for 30 min. Each individual mixture was separated
immediately by electrophoresis on 1% agarose gels in 0.5ꢂ Tris-
Borate-EDTA buffer. The gels were run at 80 V for 45 min, dried
on Whatman DE81 paper, and visualized by a phosporimager
(Typhoon 9210, Amersham). Data was analyzed using ImageQuant
software (Amersham) and results were expressed relative to the
fraction of DNA bound to Topo III in the absence of treatment,
defined as 100%. Values correspond to the mean value of three
independent experiments ꢃ SD.
For each ligation, EcoR1 digested DNA (80 ng) was incubated
overnight at 37 ꢀC in 20
L of reaction mix (1ꢂ ligase buffer, 10 U of
T4 ligase and 10ꢁ3
M of each C-telorette). PCR amplification
reactions were performed with 2 ng of ligated DNA, 1 M primers
m
m
m
[XpYpE2 (forward primer subtelomeric): 50-TTGTCTCAGGGTCC-
TAGTG-30; C-teltail (reverse primer): 50-TGCTCCGTGCATCTGG-
CATC-30], 1U of Failsafe enzyme mix in 1ꢂ Failsafe buffer H
(Epicenter) in a final volume of 25
58 ꢀC for 20 s, and 72 ꢀC for 10 min.
m
L for 26 cycles of 95 ꢀC for 15 s,
Amplification products were resolved on a 0.8% agarose gel,
denatured, transferred onto a positively charged nylon membrane
(Hybond Nþ), fixed with UV, and hybridized with the telomeric
2.5. Cell culture and drug treatment
probe 24CA end-labeled with g[
32P]-ATP with T4 kinase. Telomeric
Human HT1080 fibrosarcoma cell line was obtained from the
AmericanType Culture Collection (Rockville, MD). Cells were cultured
in DMEM with glutamax, supplemented with 10% fetal calf serum at
37 ꢀC in an atmosphere containing 5% CO2. For growth inhibition
experiments, HT1080 cells were seeded at 50,000/mL in 75 cm2
culture dish and after 72 h of treatment, cells were trypsinised,
counted and results expressed relative to cells treated with DMSO,
defined as 100%, represent the mean value of two independent
experiments.Othercelllines,KB,A549, MCF7,HCT116andMRC5, were
fragments were exposed on a screen and revealed with a phospor-
imager (Typhoon 9210, Amersham). Data were analyzed using
ImageQuant software (Amersham) for radioactivity on the whole
lane or for the number of telomere (as indicated in the text) on at
least three independent ligations and statistical analysis was per-
formed using student test.
2.7. SiRNA experiments
assayed for growth inhibition at 1 and 10 mM for 72 h in the cellular
platform from the Institut de Chimie des Substance Naturelles (CNRS
UPR 2301, Gif-sur-Yvette, France), each point in triplicate. For cell
senescence experiments, HT1080 cells were seeded at 40,000/mL in
75 cm2 culture dish. After 4 days of treatment, cells were trypsined,
counted and replated at 40,000/mL. At each passage, population
doubling (PD) from untreated or ligand-treated cultures were calcu-
lated according to the formula: PD ¼ Log Cell density Day 4 ꢁ Log
40000/Log 2.
For siRNA experiments, HT1080 cells were plated in 6-well
culture plates at 1 ꢂ 105 cells/well in 2 mL DMEM medium
without antibiotics. After 12 h, 100 nM siRNA POT1ex7 were
transfected for 4 h, using Lipofectamin 2000 (Invitrogen). A second
transfection was performed 24 h later with 100 nM siRNA
POT1ex18. 2 days after the last transfection, treated cells were
processed for Western-blotting with POT1 antibody or for C-STELA
experiments. The siRNA used here are:
siRNA POT1ex7: 50-GGGUGGUACAAUUGUCAAU-30,
siRNA POT1ex18: 50-GUACUAGAAGCCUAUCTCA-30,
2.6. Single telomere length analysis
siRNA control: 50-UGCGCUACGAUGGACGAUG-30.
The C-STELA assay was performed as described previously
[8,41]. Each DNA sample was prepared in parallel from HT1080
treated or untreated cells using QIAamp DNA mini kit (Qiagen) and
ligation reactions were perfomed with individual C-telorettes:
C-telorette 1: 50-TGCTCCGTGCATCTGGCATCCCCTAAC-30;
C-telorette 2: 50-TGCTCCGTGCATCTGGCATCTAACCCT-30;
C-telorette 3: 50-TGCTCCGTGCATCTGGCATCCCTAACC-30;
C-telorette 4: 50-TGCTCCGTGCATCTGGCATCCTAACCC-30;
C-telorette 5: 50-TGCTCCGTGCATCTGGCATCAACCCTA-30;
C-telorette 6: 50-TGCTCCGTGCATCTGGCATCACCCTAA-30.
2.8. Antibodies and western blot analysis
Cells were washed with ice-cold PBS and lysed in buffer
(TriseHCl pH 7.4 50 mM, NP40 1%, NaCl 150 mM, EDTA 1 mM, PMSF
1 mM) including a protease inhibitor cocktail at 1
complete protease, Roche Diagnostics). After 30 min on ice, lysates
were cleared by centrifugation. Protein concentration was quanti-
fied with the Bio-Rad protein assay. Cell lysates containing equal
mg/mL (Mini
amounts of total protein (25
mg) were resolved on a 10% SDS-
l
l
l
l
l
l-
l-
l-
l
l
-
l-
l
l
Fig. 1. Chemical synthesis of halogenated 360A derivatives (360AeX). Synthesis: a) 1) Ph-POCl2, 120 ꢀC, 2 h, 2) MeOH, 0 ꢀC, 1 h; b) 1) PBr5, 90 ꢀC, 6 h; 2) absolute EtOH, 0 ꢀC; c) aq.
LiOH, THF, 3 h, RT or aq. NaOH, reflux, 1 h then HCl; d) 3-aminoquinoline, EDCi, HOBt, DMF (CH2Cl2), 16 h, RT; e) CH3I, DMF (MeOH), 50 ꢀC, see also Supplementary information for
more detailed procedures.

2562
A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
polyacrylamide gel by electrophoresis, transferred to PVDF-
membrane (Macherey-Nalgel) by electro-blotting in 25 mM
TriseHCl pH 8.3, 192 mM glycine, 20% ethanol. Membranes were
blocked for 3 h at room temperature in 10 mM TriseHCl pH 7.5
containing 0.15 M NaCl, 0.1% Tween 20 and 5% nonfat dry milk.
Primary and secondary immunodetection, as well as washes, were
performed in the same buffer using 5% dry milk. Western blot
analysis was accomplished according to standard procedure using
SuperSignal West Pico chemiluminescent substrate (Pierce). The
independently of the cation present in the medium. In consistency
with theFRET data, thechloro compound was found significantlyless
active in Kþ as compared to the two other derivatives. However the
same is observed in Naþ which was not the case in the FRET-melting
experiment. Altogether the low DC50 obtained sorts the bromo and
iodo compounds among the most potent G4-ligands evaluated so far
in this assay [39]. Altogether the data collected from the two assays
indicate that the sterically demanding bromo and iodo substituents
have no negative influence on the binding strength of the quad-
ruplex/ligand interaction. On the opposite, the introduction of
halogen atom seems to afford a slight advantage considering that all
following primary antibodies were used: Monoclonal anti-b actin
clone AC-15 (Sigma) (1:5000), anti-cleaved PARP asp 214 (Cell
signaling) (1:1000), anti P21 (BD Pharmingen) (1:1000), anti POT1
#21382 (Abcam) (1:1000).
DT1/2 values are higher and most DC50 values are lower thereby
suggesting a stronger association to G-quadruplex as compared to
that of 360A (Fig. 2A).
3. Results
The different behavior of the chloro derivative is difficult to
rationalize on the ground of electronegativity and steric consider-
ations, this will be studied further in the light of recently identified
halogen bonds in drugetarget complexes [38]. For this reason and
owing to the poor metabolic stability of iodo compounds only the
bromo derivative was used for the biochemical and cellular assays.
3.1. Synthesis of the three halogenated 360A derivatives
Halogenated 360A derivatives (360AeX) were prepared
following a common synthetic pathway (Fig. 1). First, chloration of
chelidamic acid was carried out in phenylphosphonic dichloride
followed by methanolysis at 0 ꢀC affording the corresponding
dimethyl ester 1 with a 68% yield over the two steps. The corre-
sponding iodo compound 3 was obtained by halogenehalogen
exchange (Finkelstein reaction, 88% yield) [42]. Similarly, bromi-
nation of chelidamic acid in phosphorus pentabromide was carried
out followed by ethanolysis at 0 ꢀC leading to diester 2 with 47%
yield over the 2 steps. Hydrolysis of three halogeno-derivatives was
conducted either with LiOH in aqueous THF or with an aqueous
solution of NaOH with yield ranging from 42% to 98%. The corre-
sponding diacids 4, 5 and 6 were then coupled to 3-aminoquinoline
in presence of EDCI and HOBt in DMF with yield comprised
between 30 and 81%. Finally methylation of 7, 8 and 9 by iodo-
methane in a solvent mixture afforded 360AeCl, 360AeBr and
360AeI with 99%, 44% and 99% yield respectively. The overall yields
for the synthesis of the three 360AeX compounds are 42, 16% and
7% over 5, 5 and 6 steps, respectively.
K+
30
A
25
+ ds26
0
20
3 µM
Δ
T1/2 ( C)
15
10
5
°
10 µM
0
360A 360A-Cl 360A-Br 360A-I
Na+
30
25
20
15
10
5
Δ
T1/2 ( C)
°
3.2. FRET, HT-G4-FID and EMSA analysis
The ability of 360A-halogenated derivatives to stabilize the telo-
meric G-quadruplex DNA (F21T) was evaluated in a FRET-melting
temperature assay, in comparison with 360A. Melting temperature
0
360A 360A-Cl 360A-Br 360A-I
shifts were compared at a drug concentration of 1
mM and using
0.2
m
M F21T and the experiment was conducted both in Naþ and Kþ
360A-Cl ( ),360A-Br ( ),
360A-I ( ) and 360A ( )
rich buffer, results are summarized in Fig. 2A. All compounds show
100
B
very high affinity for the human telomeric G-quadruplex sequence
F21T, leading to increases in melting temperature (D
T1/2) of 23e27 ꢀC
80
60
40
20
0
in Naþ (NaCl 100 mM) and of up to 25e28 ꢀC in Kþ (KCl 10 mM, LiCl
90 mM). The chloro derivative 360AeCl represents the exception
with a strikingly low binding in Kþ conditions whereas it is strongly
active in Naþ conditions (Fig. 2A). The selectivityof the ligands for the
G-quadruplex structure subsequently was evaluated in a competitive
Na+
K+
Conditions
360A
360A-Cl
360A-Br
360A-I
0.45
0.84
0.40
0.42
0.55
0.86
0.53
0.68
FRET-melting experiment, in which different amounts (3 and 10
of duplex DNA (ds26) were added to the telomeric sequence F21T
(0.2 M). In the presence of 10 M of ds26 competitor (i.e., a 50-fold
mM)
m
m
molar excess vs F21T), the strong stabilization induced by both
ligands was not significantly lowered (from 0 to 2 ꢀC), thereby sug-
gesting ahighaffinityandselectivityfortelomericG-quadruplexover
duplex DNA, as previously reported for this series [31,36]. Finally, the
high binding affinity of the three halogeno compounds was
confirmed using the HT-G4-FID assay (Fluorescent Intercalator
Displacement assay) (Fig. 2B). DC50 values (concentration of ligand
displacing 50% of the fluorophore) similar to that of 360A (around
0.0
0.5
1.0
1.5
2.0
2.5
Ligand concentration (µM)
Fig. 2. FRET melting and G4-FID assays of halogenated 360A derivatives. (A) F21T
(0.2 mM) FRET-based D mM)
T1/2 values (ꢀC) for 360A, 360AeCl, 360AeI and 360AeBr (1
at the indicated ionic conditions (Kþ or Naþ) in the presence or absence of ds26
competitor. (B) HT-G4-FID assay using the human telomeric sequence 22AG with
different concentrations of 360A derivatives, as indicated. Results are expressed in
percent of TO Displacement (TOD; %). DC50 values (
indicated in the table.
m
M) for Kþ or Naþ conditions are
0.40e0.45
mM) were found for the bromo and iodo compounds

A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
2563
The inhibition of Topo III
22AG by the ligands 360A and 360AeBr were examined in vitro
using EMSA assays [43]. Both compounds inhibit the binding of
a
binding to a telomeric DNA sequence
360AeBr were inactive against the normal cell line MRC5 and
presented weak growth inhibitory properties (ranging from 5 to
25%) against KB, A549, MCF7 and HCT116 tumor cell lines, as ex-
pected for a specific G-quadruplex ligand. Interestingly, HT1080
tumor cell line was found to be more sensitive to 360AeBr than
360A. These data suggested that the introduction of the Bromine
moiety at position 4 of the pyridine has at least a neutral or a slight
advantage to improve the cellular activity.
Topo III
equal to 1.2 and 0.7
a
at low micromolar concentrations (Fig. 3), with IC50’s
M for 360A and 360AeBr, respectively. This
m
result suits well with the G-quadruplex stabilizing properties of
these ligands, by analogy with other compounds previously
examined in this assay [43,44]. All together, the FRET and EMSA
in vitro results suggest that 360AeBr derivative presents a slightly
higher interaction with telomeric G-quadruplex as compared to the
benchmark compound 360A and retains the exceptionally high
selectivity of the latter toward duplex DNA.
In order to examine the long-term effects of 360eBr, HT1080
and A549 cells were treated with 10 mM of ligand for 24 and 21
days, respectively. Treated cells were replated every 3 or 4 days
(with fresh compound added at each replating) and the cumulated
population doubling was measured (Fig. 5A). Results show that
360AeBr induced a decrease of the population doubling starting
between days 4 and 8 on both cell lines and corresponding to
a delayed growth inhibition. After 24 or 21 days of treatment, the
population doubling in HT1080 and A549 cells was reduced to 42%
and 37% of controls, respectively. Western blot experiments, using
cleaved-PARP and P21 antibodies, were performed on A549 cells
during the time-course of 360AeBr treatment. Results presented in
Fig. 5B indicated that 360AeBr induces a significant increase of P21
expression but does not trigger any expression of cleaved-PARP, as
3.3. Growth inhibitory properties on cell lines
Pyridine dicarboxamide derivative 360A was reported to induce
a delayed growth inhibition in different tumor cell line models,
associated with the induction of telomeric instability, as shown by
telomere-end fusion and G-overhang shortening [31,45]. To deter-
mine whether the substitution with a bromine influences cellular
growth inhibitory properties of the series, short-term anti-
proliferative effect (72 h) was examined against different cell lines,
compared to camptothecin treatment (0.5 mM for 24 h). Thus, these
using 10 mM of compound (Fig. 4). Results indicated that 360A and
results are in agreement with a delayed growth inhibition associ-
ated with the activation of P21, a figure already reported for
different G-quadruplex ligands [32,46,47].
360A
360A-Br
A
0.3
+
1
+
3
+
10
+
0.3
+
1
+
3
+
10
+
Topo III:
22AG -
-
+
+
3.4. Effect of 360AeBr on the C-strand terminal nucleotide
composition of XpYp telomere
G-quadruplex ligands, such as telomestatin, RHPS4, pyridostatin
and BRACO-19 have been reported to trigger POT1 removal from
telomeres [22,26e28,30]. Interestingly, depletion of POT1 by shRNA
360A
360A-Br
120
100
80
60
40
20
0
360A
360A-Br
B
100
90
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
Concentration (μM)
Fig. 3. Inhibition of Topo III binding to the telomeric sequence 22AG by 360A and
360AeBr. (A) Representative bandshift assay using purified Topo III (100 nM) and
[32]P-labeled 22AG (20 nM) in the presence of the indicated concentrations (
mM) of
360A or 360AeBr on the top of the panel. Control 22AG alone is indicated by (ꢁ).
(B) Quantification of the bandshift experiments with 360A (closed circles) or 360AeBr
(open circles). Results correspond to the mean ꢃ SD of three determinations and were
expressed relative to the fraction of Topo III bound to 22AG, defined as 100%.
Fig. 4. Antiproliferative activities of 360A and 360AeBr against a panel of human cell
lines. Cells were treated with 10
M of compounds for 72 h and results (mean ꢃ S.E.)
expressed in percent of untreated controls, defined as 100%.
m

2564
A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
A
HT1080
+ 360A-Br
A549
+ 360A-Br
30
20
10
0
30
20
10
0
0
4
8
12
16
20
24
0
4
8
12
16
20
24
Days
Days
days
4
11
10
21
10 CPT
B
360A-Br
0
10
0
0
Cleaved PARP -
Actin -
days
7
11
3
360A-Br
P21 -
0
3
10
0
10 CPT
Actin -
Fig. 5. 360AeBr induced a delayed growth inhibition in human tumor cell lines without apoptosis. (A) Long-term proliferation inhibition of A549 and HT1080 cells induced by
360AeBr (10 M). Cells were treated for the indicated times (21 or 24 days) and results represent the cumulated population doubling measured at each replating in control
untreated cells or 360AeBr-treated cells. (B) Western blot analysis of cleaved-PARP and P21 protein expression in A549 cells untreated or treated with 360AeBr (3 or 10 M) for the
-actin was used as a control for protein
m
m
indicated times (3, 11 or 21 days). Camptothecin (CPT, 0.5
loading.
mM for 24 h) was used as a positive control for cleaved-PARP and P21 expression. b
or siRNA was found to alter the C-strand resection leading to the
randomization of the terminal telomeric sequence [9,10]. To
address whether cell treatment by 360AeBr induces a modification
of the terminal nucleotide composition, we have investigated by C-
STELA analysis the composition of the terminal nucleotide at the C-
strand of XpYp telomere during the long-term treatment of HT1080
cells. In this assay, C-telorettes #1 to #6, representing the six
possible ends of the 50 C-strand telomeric sequence were individ-
ually ligated to DNA samples extracted from HT1080-treated cells,
using the telomeric overhang as a template for annealing [8]. Each
ligation product is amplified with a sequence (C-teltail) common to
all C-telorettes and a primer that anneal to a XpYp subtelomeric site
present at 450 bases from the start of the telomere repeats [41,48].
Individual telomeres, corresponding to discrete bands are revealed
by southern blot hybridization and are proportional to the number
of ligated telomeres. In our hands, the C-STELA assay showed the
expected predominance of the ATC-50 in untreated HT1080 cells
since C-telorette #3 corresponded to 64% of the ligated and
amplified telomeres (Fig. 6). We also found, that C-telorettes #2 and
#4 corresponding to CAA-50 and AAT-50 ends, and immediately
adjacent to ATC-50, are ligated with a non-negligible percentage,
equal to 20 and 12.5%, respectively.
C-STELA analysis of HT1080 cells treated by 360AeBr was per-
formed on DNA samples harvested at each passages (days 4, 8, 12,
16, 20 and 24 days) and representative gels of the C-telorette profile
indicated that the overall ATC-50 predominance is conserved under
drug treatment, as compared to untreated HT1080 cells (Fig. 7A).
Quantification of the experiments only revealed a non-significant
change of the C-telorette ligation profile, when all treated DNA
samples (day 4 to day 24) were compared to the overall untreated
controls (Fig. 7B). However, a significant decrease to 8.5% (p
<0.005) and increase to 20.4% (p <0.05) in the proportion of the
ligation of C-telorette #2 and #4 corresponding to the CAA-50 and
AAT-50 telomere ends, respectively were found during the first part
of the cell treatment (days 4e12) (Fig. 7C, see also Fig. S1) where the
growth inhibition is the most pronounced (compare days 4e12 and

A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
2565
A
B
Fig. 6. Terminal nucleotide composition at telomeric C-strand in HT1080 cells. (A) Individual telomere products generated by the C-STELA assay in HT1080 cells (see Materials and
methods). Each C-telorette was used in an independent assay (duplicate ligation in this example) and run in independent lanes. C-telorette number (#1e6) is indicated at the top of
the gel. The hybridizing part of the telorette is underlined. (B) Schematic representation of XpYp telomere 30 G-overhang and 50 C-strand, with the terminal nucleotide composition
corresponding to each individual C-telorette at C-strand. The relative percentage (mean) generated by each individual C-telorette is indicated on the right of the panel and
corresponds to the quantification of 15 independent ligation assays. The total telomere signal generated by the six C-telorettes in each assay and quantified by ImageQuant (on the
whole lane) is defined as 100%. See also Fig. 7, for the S.D. values of the assay. Individual telomeres generated by C-telorettes #2, 3 and 4 contributed to 96% of the total signal in
untreated HT1080 cells.
16e24 in Fig. 5A). In contrast, during the second part of the treat-
ment (days 16e24), the C-telorette ligation profile becomes again
similar in proportions to untreated controls. It must be noticed that
a reproducible decrease of the efficiency of telomere ligation and/or
PCR amplification reaction was also observed during this stage (see
days 16e24 in Fig. 7B). Indeed, the mean telomere number in
untreated and treated cells (days 20e24) is equal to 38.8 and 23.3 (p
<0.05), respectively (Fig. 7D). Such effect was also found after long-
term treatment of HT1080 cells with another PDC derivative (360A-
biot) [49](A. Sidibe, data not shown) and may be due to the accu-
mulation of DNA lesions occurring at telomere, such as telomere-
end fusions, telomere doublets or deletions that were detected by
this class of ligand (360A) in different tumor cell lines [45,50].
In order to determine the sensitivity of the C-STELA assay to
detect the randomization of the telomeric C-strand, we have also
evaluated the effect of POT1 depletion using siRNA directed against
POT1. HT1080 cells were transfected with siRNA control or with
two different siRNA directed against POT1 that were previously
reported [10]. Western blot experiments indicated that siRNA
directed against exon 18 of POT1 gene (siPOT1ex18) induced
a significant decrease of POT1 (50%), but not siRNA directed against
exon 7 (siPOT1ex7) in HT1080 cells, 72 h after transfection (Fig. 8A).
In parallel, C-STELA analysis of HT1080 cells depleted for POT1
protein by siRNA POT1ex18 was performed and compared to
HT1080 cells treated with siRNA control (Fig. 8B). Results from
triplicate ligations on HT1080 treated with siRNA control showed
a more marked ATC-50 predominance in this experiment since
products from telorette #3 corresponded to 83% and the absence of
products from telorette #4 (Fig. 8B and C). We attribute these
variations to differences in the quality of the DNA preparations and/
or efficiency in the ligation reaction (see Discussion). After treat-
ment with siRNA POT1ex18, a partial randomization of the terminal
nucleotide sequence was observed, as revealed by a decrease of the
products from telorettes #2 and 3 and the increase of those from
telorettes #4 and 6, suggesting that the partial loss of POT1 is
sufficient to trigger a detectable alteration of the C-telorette liga-
tion profile.
Interestingly, we observe a similar alteration of C-telorette
profile between 360AeBr treatment (days 4e12) and POT1 deple-
tion (compare the relative decrease of C-telorette #2 and the
relative increase of C-telorette #4 in Figs. 7C and 8C), suggesting
that in our experimental conditions the effect of the ligand may
correspond to a partial depletion of POT1.
4. Discussion
The 360AeBr PDC derivative reported in this paper shows high
affinity for telomeric G-quadruplex DNA, as evidenced by different
techniques such as FRET-melting, G4-FID and EMSA assays and
retains the high selectivity toward duplex DNA of 360A. In agree-
ment, the substitution at position 4 of the pyridine by a 2-
aminoethoxy (pyridostatin) or by a biotin (PDC-biotin) were also
shown to maintain the affinity and the selectivity toward telomeric
G-quadruplex in this series [34,49].
Although the panel of cell line used here is limited, our results
indicated that 360A and 360AeBr could not be discriminated
through their cellular activity at short-term (72 h). Both
compounds were found inactive against the normal cell line MRC5,
suggesting an in vitro selective activity for tumor cells as already
reported for other selective G-quadruplex ligands such as telo-
mestatin [51], 307A [31] and pyridostatin [52].
In contrast to its close analog 307A in glioma cells lines [31] or to
the triazine derivative 12459 in A549 cells [47], we do not observe
any apoptotic process induced by 360AeBr in A549 or HT1080 cells,
but rather a delayed growth inhibition associated with the activa-
tion of P21. Interestingly, pyridostatin and different analogs also

2566
A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
B
A
D
C
Fig. 7. Effect of 360AeBr on the terminal nucleotide composition at telomeric C-strand in HT1080 cells. (A) Representative experiments of the C-STELA assay from HT1080 cells
untreated or treated with 360AeBr (10
mM) harvested at the indicated days. C-telorette number (1e6) is indicated. (B) Quantification of the relative percentage (mean ꢃ SD)
generated by each individual C-telorettes in HT1080 cells untreated or treated by 360AeBr. In each lane, the telomere signal generated was quantified and expressed in percentage,
the total telomere signal generated by the six telorettes in each ligation defined as 100%. Untreated controls corresponded to 15 independent ligations and treated to 18 independent
ligations. P values after statistical analysis by student test are indicated. (C) Same analysis that differentiates days 4e12 and 16e24 of the treatment by 360AeBr, as indicated. Ns
corresponded to p >0.05. (D) Total telomere number generated by the six C-telorettes in each ligation. Results corresponded to the mean ꢃ SD for untreated HT1080 (HT1080,
n ¼ 15), HT1080 treated with 360AeBr (days 4e16, n ¼ 12 and days 20e24, n ¼ 6).
induced a senescence-like phenotype in HT1080 cells during long-
term treatments but did not induce an elevated level of cell death
[52]. Both pyridostatin and 360A trigger a partial delocalization of
POT1 from telomeres in this cell line ([26] and result not shown).
This suggests a common phenotypic action of PDC ligands in
HT1080 cells.
Thus, we used 360AeBr, as a representative member of the PDC
series, to further investigate its effect to randomize the telomeric C-
strand by using the C-STELA assay in HT1080 cells. Since depletion
of POT1 by shRNA was shown to alter the C-strand resection,
leading to the randomization of the C-strand, we expected to obtain
an easily detectable effect on C-STELA, after 360AeBr treatment.
We first noticed some variations in the C-STELA assay profile in
HT1080 cells, compared to the original report on BJ cells [8], and
concerning the presence of a significant amount of AAT-50 and
CAA-50 ends in untreated HT1080 cells. These variations were also
observed for other cells lines in this report and in ulterior studies
[8e10]. Comparison between different cell lines confirmed the
predominance of ATC-50, but in proportion varying from 35 to 50%
(see Fig. S1 in [8]) or equal to 100% [10]. In these studies, AAT-50
ends were found present, varying from 10 to 20%, in agreement
with our finding. It is possible that these differences result from
minor differences in the experimental protocol for C-STELA (see
Table S1), or from the used cell line. However, we also observed
a variation between two series of experiments using HT1080 cells
concerning the detection of AAT-50 ends (compare telorette #4 in
Figs. 7 and 8). Since these DNA samples were prepared at different
dates, these variations may be due to differences in the quality of
the DNA preparation and/or efficiency in the ligation reaction. In
order to limit these reproducibility issues, DNA extraction for all

A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
2567
detectable). Similar results were obtained by measuring the
number of telomere amplified instead of quantifying the radioac-
tivity in each individual lane (Fig. S2). At first instance, these results
suggest that 360AeBr treatment does not induce the complete
randomizing effect reported when POT1 is nearly completely
depleted [10]. In our experimental conditions, we found that
siRNA-mediated depletion of POT1 by 50% only triggers a partial
but detectable randomization of the C-strand resection. Partial
randomization was also found in HeLa cells partially depleted for
POT1 (46%) using the same siRNA sequence [9]. Interestingly, these
partial depletion of POT1 trigger the same relative variations in
CAA-50 and AAT-50 ends than that observed after 360AeBr treat-
ment. We conclude that the effect of the ligand might correspond to
a partial depletion of POT1.
A
POT1 -
*
Actin-
1
Telorette#
SiControl
2
3
4
5
6
B
A possible explanation for this limited action of the ligand may
reside in the different mode of replication of telomeres between
leading and lagging strand. Recent observations suggested that C-
strand resection and G-overhang formation are uncoupled between
leading and lagging strands at the end of telomere replication [7].
An additional step of C-strand fill-in by Pola recruited by the CST
complex is necessary at lagging strand that requires the displace-
ment of POT1 [7,53]. It is therefore possible that the recruitment of
the nuclease responsible for the resection is directed by a fraction of
POT1 interacting with TIN2eTRF1/2 that does not bind G-strand
and not by the one interacting with G-strand. Since a G-quadruplex
ligand is expected to block only the fraction of POT1 interacting
with telomeric DNA, this might explain why a nearly complete
depletion of POT1 would be necessary to observe the complete
randomization of telomeric ends.
SiPOT1ex18
On the other hand, we also observed that the variation of C-
strand 50-ends induced by 360AeBr is a transient event detectable
between days 4e12 and not observed later. A decrease in the
360AeBr growth inhibitory efficiency was also found between days
16e24 (see Fig. 5A), suggesting that the cell line may adapt to the
treatment. In agreement, A549 cells induced to resistance to 360A
presented increased expression of POT1 and TRF2 (C. Trentesaux,
unpublished results).
Because of the minor modification induced by 360AeBr, further
works using additional cell lines models and/or different chemical
series of G-quadruplex ligands are therefore needed to confirm
these data.
SiControl
SiPOT1
C
100
90
80
70
60
50
40
30
20
10
Finally, since the C-STELA analysis only explore the behavior of
XpYp telomere, it would be also interesting to address whether this
situation could be generalized at other chromosome ends where
STELA analysis is possible [54], or using the Universal STELA that
bypass the need to design chromosome proximal primers in sub-
telomeric regions [55].
0
1
2
3
4
5
6
C-telorette number
Fig. 8. Effect of POT1 depletion by siRNA on the terminal nucleotide composition at
telomeric C-strand in HT1080 cells. (A) Western blot analysis of POT1 protein
expression in HT1080 cells untreated or transfected with 100 nM siRNAs (siRNA
control, siRNA POT1ex7, siRNA POT1ex18, as indicated) for 72 h. The asterisk indicated
a non-specific band revealed by the POT1 antibody. b-actin was used as a control for
Acknowledgments
protein loading. (B) C-STELA assay from HT1080 cells treated with siRNA control
(100 nM) or siRNA POT1ex18 (100 nM), as indicated. C-telorette number (1e6) is
indicated and each ligation was performed in triplicate. (C) Quantification of the
experiment. In each lane, the telomere signal generated was quantified and expressed
in percentage (mean ꢃ SD, n ¼ 3), with the total telomere signal generated by the six
C-telorettes in each ligation defined as 100%.
We thank Nathalie Rousselet and Aurore Guédin for technical
assistance, Gregory Méchin for help in cell culture, Dr A. Londono-
Vallejo and Dr L. Guittat for helpful discussions. This work was
supported by a grant from the “ Ligue Nationale contre le Cancer,
Equipe Labellisée 2010” to J.F. Riou and the Agence Nationale de la
Recherche (ANR-09-BLAN-0355) to J.F. Riou and M.P. Teulade-
Fichou. A. Sidibe was supported by a PhD fellowship from the
“Ligue Nationale contre le Cancer”. E. Largy was supported by
a joint fellowship from Centre National de la Recherche Scientifique
(CNRS) and Institut Curie.
samples in a study and subsequent ligations should be performed
in parallel, as it was done for 360AeBr or siRNA treatment. In these
conditions, the assay in triplicate gives an acceptable reproduc-
ibility (compare HT1080 controls between days 4e12 and days
16e24 in Fig. 7C).
The only statistically significant variation in the C-telorette
profile after 360AeBr treatment corresponded to a decrease of
CCA-50 and an increase of AAT-50 ends, but without obvious change
in the ATC-50 predominance (see also Fig. S1 for quantifications at
each passage where a trend for some significant variations is
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.biochi.2012.07.003.

2568
A. Sidibe et al. / Biochimie 94 (2012) 2559e2568
[31] G. Pennarun, C. Granotier, L.R. Gauthier, D. Gomez, F. Hoffschir, E. Mandine,
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