![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Laboratoire d'Onco-Pharmacologie, JE 2428, Unité de Formation et de Recherche de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France (B.B., D.G., R.P., N.T.-S., C.T., J.-F.R.); Institut National de la Santé et de la Recherche Médicale, U565, Acides Nucléiques: Dynamique, Ciblage et Fonctions Biologiques, Paris, France (A.D.C., J.-L.M.); Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 5153, Muséum National d'Histoire Naturelle USM503, Département de "Régulations, Développement et Diversité Moléculaire", Laboratoire des Régulations et Dynamique des Génomes, Paris, France (A.D.C., J.-L.M.); and Institut de Chimie des Substances Naturelles, Unité Propre de Recherche CNRS 2301, Gif sur Yvette, France (A.M., K.-H.Q., F.G.)
Received March 29, 2007; accepted June 20, 2007
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Telomere replication is sustained in proliferative somatic cells and in most cancer cells by telomerase, a ribonucleoprotein complex that elongates the chromosome ends to compensate losses occurring at each cell division, because of the inability of polymerase to fully replicate telomeric extremities (McEachern et al., 2000). In somatic cells, the absence of telomerase provokes a progressive shortening of the telomeric DNA at each round of division that ultimately leads to replicative senescence once a critical telomere length has been reached (Shay and Wright, 2002). Numerous observations, notably that inhibition of telomerase activity limits tumor cell growth (Hahn et al., 1999), have led to the proposal that telomere and telomerase are potential targets for cancer chemotherapy (Lavelle et al., 2000; Neidle and Parkinson, 2002; Shay and Wright, 2002).
In humans, the telomere is composed of tandem repeats of the G-rich duplex sequence 5'-TTAGGG-3', with the G-rich 3' strand extending beyond the duplex to form a 130- to 210-base overhang, called the G-overhang (Makarov et al., 1997; Wright et al., 1997). Telomeres are believed to exist in different conformations together with several telomere-associated proteins, such as telomere repeat factors (TRF1, TRF2) and POT1 (Smogorzewska and de Lange, 2004). The G-overhang is accessible for telomerase extension in the open state or inaccessible in a capped (or closed) conformation that involves the formation of a telomeric loop motif (Smogorzewska and de Lange, 2004). Although the telomeric loop structure has not been defined in detail, it may be created by the invasion of the G-overhang into the duplex region of the telomere (Griffith et al., 1999). Uncapping of the telomere ends leads to telomeric dysfunction characterized by end-to-end fusion, inappropriate recombination, anaphase bridges, and G-overhang degradation that may lead to either apoptosis or senescence (Blackburn et al., 2000; Duan et al., 2001; Karlseder et al., 2002; Li et al., 2003).
Because of the repetition of guanines, the G-overhang is prone to formation of a four-stranded G-quadruplex structure that has been shown to inhibit telomerase activity in vitro (Mergny et al., 2002; Davies, 2004). Small molecules that stabilize G-quadruplexes are effective as telomerase inhibitors, and several series of compounds have been identified using techniques such as temperature melting fluorescence assays on oligonucleotides (Mergny et al., 2001), electrophoresis analysis of quadruplex formation (Koeppel et al., 2001), electrospray ionization mass spectrometry (Rosu et al., 2003a), and the telomeric repeat amplification protocol that measures telomerase activity in cell extracts (Gomez et al., 2002) (for review, see Guittat et al., 2004). The ligands that stabilize G-quadruplex structures include cationic porphyrins (Han et al., 1999, 2001; Dixon et al., 2007), perylenes (Fedoroff et al., 2000), amidoanthracene-9,10-diones (Perry et al., 1998), 2,7-disubstituted amidofluorenones (Perry et al., 1999), acridines (Read et al., 1999; Harrison et al., 2003), ethidium derivatives (Koeppel et al., 2001; Rosu et al., 2003a), disubstituted triazines (Riou et al., 2002), fluoroquinoanthroxazines (Kim et al., 2003a), indoloquinolines (Caprio et al., 2000), dibenzophenanthrolines (Mergny et al., 2001), bisquinacridines (Teulade-Fichou et al., 2003), pentacyclic acridinium (Gowan et al., 2001), telomestatin (Shin-ya et al., 2001), and the recently discovered bisquinolinium derivatives (Lemarteleur et al., 2004; Pennarun et al., 2005; De Cian et al., 2007) (for review, see Kerwin, 2000; Cuesta et al., 2003; Guittat et al., 2004; Pendino et al., 2006). Because of the peculiar features of the quadruplex structure, compared with classic double-stranded B-DNA, a selective recognition of telomeric G-quadruplex by small-molecule ligands should be possible (Neidle and Parkinson, 2002; Parkinson et al., 2002; Clark et al., 2003; Ambrus et al., 2006). Some partial selectivity for G-quadruplex relative to duplex DNA was obtained with triazine (Riou et al., 2002) and with ethidium derivatives (Rosu et al., 2003a), and selectivity was significantly enhanced with the natural product telomestatin (Kim et al., 2002, 2003b; Rosu et al., 2003b), with a new series of 2,6-pyridin-dicarboxamide derivatives (Pennarun et al., 2005), and with a porphyrin derivative (Dixon et al., 2007).
To test the paradigm of the lifespan control by telomerase activity and telomere length, G-quadruplex ligands were evaluated in cells. This paradigm is at least partially true because a functional telomerase inhibition was observed in cell lines treated for several weeks with a subtoxic dosage of a compound that provokes a telomere shortening, and this shortening was correlated with the induction of senescence (large morphology of cells and SA-
-galactosidase activity) (Riou et al., 2002). It was also observed that G-quadruplex ligands induce more rapid effects on cell growth than initially expected based on telomerase inhibition. Apoptosis and short-term responses were observed with triazine derivatives (12459, 115405), telomestatin, and more recently with the pyridine dicarboxamide derivatives (307A, 360A) (Riou et al., 2002; Tauchi et al., 2003; Pennarun et al., 2005).
The observation that BRACO-19 causes chromosome end-to-end fusions associated with the appearance of p16-associated senescence led to the proposal that G-quadruplex ligands mostly act to disrupt the telomere structure (Incles et al., 2004). Such telomeric dysfunction was also observed in cell lines treated with RHPS4 or with 307A and in cell lines resistant to 12459, with typical images of telophase bridges (Gomez et al., 2003a; Leonetti et al., 2004; Pennarun et al., 2005). Further indirect evidence that G-quadruplex ligands target telomere replication arose from experiments in mutant cell lines resistant to these ligands: These cells present telomere capping alterations, overexpression of human telomerase reverse transcriptase (hTERT), and telomere shortening cross-resistance for different classes of ligands (Gomez et al., 2003a,b; Leonetti et al., 2004; Pennarun et al., 2005).
We have demonstrated that G-quadruplex ligands interfere with the conformation and the length of the telomeric G-overhang, an effect that is thought to be more relevant than the double-stranded telomere erosion as a marker for telomestatin cellular activity (Gomez et al., 2004). G-overhang degradation was found to be associated with the onset of senescence (Gomez et al., 2004) or with the onset of apoptosis (Douarre et al., 2005). Recent publications also indicate that G-quadruplex ligands may act by dissociation of telomere binding proteins POT1 and TRF2, uncapping telomeres to make them available for extension (Gomez et al., 2006a,b; Tahara et al., 2006).
To search for novel and more potent G-quadruplex ligands, we have screened a bank of derivatives from natural and synthetic origin using a temperature melting fluorescence assay. Two steroids derived from the natural products malouetine and funtumine were identified and characterized for telomeric G-quadruplex stabilization and telomere elongation inhibition in vitro. Funtumine substituted by a guanylhydrazone moiety (steroid FG) induced antiproliferative activities in HT1080 cells associated with telomere shortening, G-overhang degradation, and anaphase bridge formation suggesting that this steroid ligand is a high-affinity G-quadruplex ligand that binds to telomeres in human cells. These steroid ligands represent a new class of telomere-targeted agents that have potential as antitumor drugs.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) and Funtumia latifolia (Janot et al., 1958; Quevauviller and Blanpin, 1958), respectively. Funtumine guanylhydrazone was prepared from funtumine by reaction with aminoguanidine bicarbonate as described previously (Meyer et al. (1967). The structure and purity of the compounds were verified by liquid chromatography/mass spectrometry and NMR spectroscopy (data not shown). Compounds were solubilized at 10 mM in dimethyl sulfoxide. Additional dilutions were made in water. HT1080 human lung carcinoma, H460 lung carcinoma, and HeLa cervix carcinoma were from the American Type Culture Collection (Manassas, VA). BJhTERT human foreskin fibroblast immortalized with human telomerase reverse transcriptase (hTERT) has been described previously (Bodnar et al., 1998). The cells were grown in Dulbecco's modified Eagle's medium with GlutaMAX (Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum and antibiotics. The HT1080GFP-POT1 cell line was described previously (Gomez et al., 2006b).
Fluorescence Experiments. Initial screening experiments were performed on a LightCycler real-time PCR instrument (Roche, Basel, Switzerland) as described previously (Darby et al., 2002), using a fluorescent oligonucleotide F21D (5'-FAM-GGGTTAGGGTTAGGGTTAGGG-DabCyl-3'), alone or in the presence of 10 µM compound. Assays were performed in a buffer containing 0.5 µM F21D, 10 mM cacodylate, pH 8.0, 0.1 M LiCl, and 5 mM KCl. Excitation wavelength was 470 nm, and emission of fluorescein was recorded at 530 nm.
Quantitative experiments and dose-response results were obtained by real-time PCR (MX3000P; Stratagene, La Jolla, CA) using F21D or F21T (analogous to F21D but with the 3' quencher tetramethylrhodamine) fluorescent oligonucleotides (0.2 µM). Melting profiles were recorded in a 10 mM Li cacodylate buffer, pH 7.2, with 50 to 100 mM NaCl or 5 to 10 mM KCl. Ionic strength was kept constant by the addition of LiCl. Excitation wavelength was 496 nm, and emission was recorded at 516 nm. For competition experiments, various concentrations of the double-stranded ds26 competitor were added before the melting experiment (Kaiser et al., 2006).
PCR Stop Assay. The stabilization of G-quadruplex structures was investigated by a PCR-stop assay (Lemarteleur et al., 2004) using a test oligonucleotide and a complementary oligonucleotide that partially hybridizes to the last G-repeat of the test oligonucleotide. Sequences of the test oligonucleotides (21G) and the corresponding complementary sequence (anti21G) used here are presented in Fig. 3.
|
Cell Growth Assays. For long-term cell growth studies, cells were seeded at 1 x 104 cells/ml (5 ml) into a 25-cm2 tissue-culture flask in the presence or the absence of steroid derivatives, cultured for 4 days, then trypsinized and counted. At each passage, 1 x 104 cells/ml were replated into a new culture flask with fresh medium containing drug solution. Results were expressed as the cumulated population doublings as a function of the time of culture as described previously (Riou et al., 2002). MTT survival assays (4 days or 2 days) were performed in quadruplicate in 96-well plates, as recommended by the manufacturer (Sigma, St. Louis, MO).
Anaphase Bridge Analysis. To determine the presence of anaphase bridges, cells were seeded on glass coverslips in complete culture medium and treated with steroid ligands for 24, 48, and 96 h, then stained with DAPI (Sigma) and mounted. Images of anaphases were recorded with an Axiovert 200 M inverted microscope (Carl Zeiss, Oberkochen, Germany) coupled with a Coolsnap HQ camera controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). At least 50 metaphases were examined.
Detection of SA--Galactosidase Activity. Endogenous senescence-associated -galactosidase activity was assessed by a staining using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside as described previously (Dimri et al., 1995) on HT 1080 cultures treated with steroid FG (0.7 µM) or steroid B1 (10 µM) at days 4, 8, and 12.
Solution Hybridization Experiments. The nondenaturing hybridization assay to detect the 3' telomere G-overhang was performed as described previously (Gomez et al., 2004). Aliquots (2.5 µg) of undigested genomic DNA were hybridized at 50°C overnight with 0.5 pmol of [
-32P]ATP-labeled (5'-(CCCTAA)3CCC-3')oligonucleotide (21C) in sodium hybridization buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, and 1 mM EDTA) in a volume of 20 µl. For competition with Pu22myc (5'-GAGGGTGGGGAGGGTGGGGAAG-3'), the reactions were performed in the presence of 10 µM Pu22myc. Reactions were stopped by the addition of 6 µl of loading buffer (20% glycerol, 1 mM EDTA, and 0.2% bromphenol blue). Hybridized samples were size-fractionated on 0.8% agarose gels in 1x TBE buffer containing ethidium bromide (EtBr). Gels were dried on Whatman filter paper. Ethidium fluorescence and radioactivity were determined using a Typhoon 9210 PhosphorImager (GE Healthcare). The procedure allows detection of the amount of single-strand overhang available for hybridization. Results were expressed as the relative hybridization signal normalized to the fluorescent signal of EtBr and represented the mean of three independent experiments.
TRF Analysis. Aliquots of 5 µg of undigested genomic DNA were hybridized at 37°C overnight with 0.5 pmol [-32P]ATP-labeled (5'-(CCCTAA)3CCC-3') oligonucleotide in sodium hybridization buffer in the presence of RsaI and HinfI restriction enzymes in a volume of 20 µl. Reaction was stopped with 2 µl of proteinase K solution (1% SDS and 1 mg/ml proteinase K) and incubated for 30 min at 50°C. Hybridized samples were size-fractionated on 0.8% agarose gels in 1x TBE buffer. The gels were stained with EtBr, washed, and dried on filter paper (Whatman, Maidstone, UK). Ethidium fluorescence and radioactivity were determined and telomeric smears were revealed by using a Typhoon 9210 PhosphorImager (GE Healthcare). The mean length of the TRF corresponds to the peak of the integration curve from three separate experiments.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Gomez et al., 2004). Each compound was first evaluated at 10 µM in a buffer containing 100 mM LiCl and 5 mM KCl. Under these conditions, the F21D probe has an apparent melting temperature (T1/2) of 48°C. Ligands that specifically interact with a G-quadruplex increase the melting temperature of the F21D, as evidenced by plotting fluorescence emission versus temperature. Two compounds, malouetine dichloride and funtumine guanylhydrazone (Fig. 1), gave significant variations of 
T1/2 (>5°C) (Table 1). These two compounds are steroids from the pregnane series substituted at positions 3 and 17. Malouetine is a steroidal alkaloid isolated from the leaves of Malouetia bequaertiana E. Woodson (genera Apocynaceae) (Janot et al., 1960), known for its curarizing properties (Huu Laine and Pinto Scognamiglio, 1964; McKenzie, 2000) and its ability to bind double-stranded DNA (Gourevitch et al., 1981). Funtumine guanylhydrazone is a funtumine derivative substituted with a guanylhydrazone moiety. Funtumine, a steroid isolated from the leaves of Funtuma latifolia stapf, was inactive in the fluorescence melting assay (T1/2 < 2°C at 10 µM). An epimer of the funtumine guanylhydrazone derivative was reported to present cardiotonic properties (Meyer et al., 1967).
|
|
The effect of the two steroids on the stabilities of G-quadruplex structures formed by F21D and F21T (these two probes differ by the nature of the 3' quencher, dabcyl or tetramethylrhodamine, respectively) in different cation conditions and ligand concentrations were studied. As summarized in Table 1, results obtained with the two oligonucleotides were in qualitative agreement. The steroid FG far more efficiently stabilized quadruplexes than malouetine. In K+ conditions (i.e., cation conditions; see Table 1) steroid FG induced a 2- to 3-fold higher T1/2 than malouetine (Table 1). It is noteworthy that malouetine had nearly no effect on G-quadruplexes in Na+ conditions, whereas the steroid FG had only a slightly lower T1/2 in Na+ than in K+ (T = 2–4°C).
The steroid FG was also compared with 360A, a pyridine dicarboxamide derivative (Pennarun et al., 2005), for the F21D fluorescence melting assay in K+ conditions. The steroid FG concentration necessary to achieve the same melting curve as that with 2 µM 360A was 20 µM (result not shown). Thus, we estimated that this ligand is approximately 10-fold less potent than 360A.
To determine the selectivity of the interaction for the telomeric G-quadruplex relative to duplex DNA, the melting temperature of F21T (0.2 µM) in the presence of steroid FG (5 µM) was monitored in the presence of a 26-nucleotide duplex oligonucleotide (ds26) competitor (at 3, 10, and 30 µM) (Fig. 2). In the presence of 3 µM ds26 oligonucleotide (i.e., a 15-fold molar excess), the stabilization induced by steroid FG was not significantly lowered. Higher competitor concentrations led to a partial loss of stabilization (Fig. 2); at 30 µM ds26 (i.e., a 150-fold molar excess), T1/2 was only + 2°C. Thus, the selectivity is at least 15-fold for G-quadruplex DNA relative to duplex DNA.
|
G-quadruplex stabilization was also evaluated by the PCR stop assay using the 21G and anti-21G oligonucleotides (Lemarteleur et al., 2004). In this assay, 5' to 3' extension by Taq polymerase to produce a final double-stranded DNA product is inhibited when the target 21G oligonucleotide folds in a G-quadruplex structure. As shown in Fig. 3, the steroid FG induced a dose-dependent inhibition of PCR product formation. Slight inhibition was evident at 3 µM and was complete at 30 µM. Together, these results indicate that steroid FG is a potent and selective G-quadruplex ligand able to impair telomere repeat elongation and/or replication.
Steroid Ligands Induced Senescence in HT1080 Cells. To examine the effects of malouetine and steroid FG on HT1080 cells, we first determined the drug concentrations that inhibited cell viability after 4 days of culture (Fig. 4A). Results indicated that malouetine had very limited antiproliferative properties with an IC50 value higher than 30 µM, whereas steroid FG had a potent inhibitory effect with an IC50 equal to 1.8 (± 0.3) µM. Steroid FG also had potent antiproliferative effects on H460 lung carcinoma cells (IC50 = 2 µM) and to a lesser extent against HeLa cells (IC50 = 5.5 µM) and BJhTERT immortalized foreskin fibroblast cells (IC50 = 6 µM).
|
-galactosidase activity (SA--gal) at day 12 of treatment (Fig. 5B). Cells harvested earlier (i.e., after 4 or 8 days of steroid treatment), did not express SA--gal activity (result not shown), suggesting that the senescence occurs at the terminal phase of the culture. At lower ligand concentrations (3 µM malouetine or 0.3 µM steroid FG), HT1080 cells were able to grow continuously up to 36 days with only a slight decrease in doubling time compared with control untreated cells (Fig. 4B).
|
). To determine whether steroid ligands inhibited telomere replication, a TRF analysis was performed on DNA samples from steroid-treated HT1080 cells (Fig. 6). For steroid FG (0.7 µM), a TRF shortening was clearly detectable at day 8 of the treatment (Fig. 6A). Telomestatin (2 µM) induced a detectable TRF shortening after 4 days of treatment (Fig. 6A); this concentration provoked a plateau arrest at day 12 (result not shown). In contrast, malouetine (10 µM) treatment did not result in significant telomere shortening at days 4 or 8. The quantification of these experiments indicated a mean TRF length shortening of 300 and 600 base pairs for steroid FG and telomestatin, respectively (Fig. 6B). These results suggest that steroid FG induced a rapid double-stranded telomere shortening associated with its cell growth arrest properties.
|
Steroid FG Induces a Decrease in Length of the Telomeric G-Overhang. Recent studies have indicated that the telomeric G-overhang represents one of the direct targets of quadruplex ligands (Gomez et al., 2004; Douarre et al., 2005). We analyzed the effect of steroid FG on the telomeric G-overhangs from HT1080 cells. As shown before, hybridization of a telomeric C-rich probe (21C) under nondenaturing conditions allowed the measurement of the relative single-stranded G-overhang signal in undigested genomic DNA samples (Cimino-Reale et al., 2001; Gomez et al., 2004). Treatment of HT1080 cells with 0.7 µM steroid FG had no effect after 4 days but a strong decrease in the G-overhang signal (55 ± 8.5%) was observed after 8 days (Fig. 7, A and B). In contrast, treatment of HT1080 cells with malouetine (10 µM) only induced a modest decrease in the G-overhang signal (16 ± 11%) after 8 days of treatment (Fig. 7, A and B).
|
) indicate that the apparent decrease in G-overhang signal may result from the stabilization of the quadruplex, making it less prone to hybridization to its complementary C-rich probe. To exclude this possibility, we performed the following experiment: Steroid FG (3–100 µM) was added to purified DNA just before the hybridization reaction. This resulted in a detectable inhibition of the G-overhang signal, only at a concentration equal to 30 µM, but in a cation-dependent manner (Fig. 8B). The inhibition was almost completely reversed in presence of an oligonucleotide that adopts a G-quadruplex structure (Pu22myc, 10 µM) (Fig. 8, A and B) (Gomez et al., 2004). Because the in vitro inhibition of the G-overhang hybridization assay is only observed at a 50-fold higher concentration of steroid FG than that used to treat cells (0.7 µM), and the cellular effect was detected after a lag time of 8 days, we reasonably concluded that the ligand induced an effective degradation of the telomeric G-overhang in vivo rather than a modification of the G-overhang conformation that interferes with the hybridization reaction.
|
Steroid FG Induced Anaphase Bridge Formation in HT1080 Cells. Alterations of telomere capping have been reported during the treatment with G-quadruplex ligands (Izbicka et al., 1999a; Leonetti et al., 2004; Burger et al., 2005; Douarre et al., 2005; Pennarun et al., 2005). These alterations may be evidenced by the formation of anaphase bridges. We examined the anaphase bridge formation in steroid-treated HT1080 cells. Typical images of anaphase bridges were obtained in HT1080 cells treated for 24 h with 0.7 µM steroid FG, which represented 59 ± 6% of the anaphases examined (n = 50), compared with 0% for controls (Fig. 8C). It is noteworthy that anaphase bridge formation is an early event, in that it is observed within 24 h, before any evidence for antiproliferative or cytotoxic activity. Treatment for 4 days strongly reduced the amount of mitotic cells in the preparation and therefore the number of anaphase bridges (result not shown). Thus, anaphase bridge formation seems to precede the antiproliferative effects of the ligand. These data suggest that steroid FG is able to induce the degradation of the telomeric G-overhang and to increase the formation of anaphase bridges, indicating that this ligand induces an alteration of the telomere capping in HT1080 cells.
Expression of GFP-POT1 Induced Resistance to Steroid FG. Expression of a green fluorescent protein-POT1 fusion (GFP-POT1) in HT1080 cells increases telomere length and G-overhang signal (Colgin et al., 2003; Gomez et al., 2006b). This cell line model is resistant to the cellular effect of telomestatin (Gomez et al., 2006b). We have examined whether the overexpression of POT1 modulates the cytotoxic activity of steroid FG for short-term treatment (48 h). HT1080GFP-POT1 cells had a noticeable resistance to the effects of steroid FG, compared with parental HT1080 cells (Fig. 9). These results suggest that the cellular effect of the compound is due to a direct effect on telomeres.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Mergny et al., 2002; Neidle and Parkinson, 2002; Pendino et al., 2006). We show here that steroid diamine derivatives are also potent G-quadruplex ligands that interact with the human telomeric sequences and inhibit telomere elongation. It is noteworthy that these derivatives differ greatly from previously characterized G-quadruplex ligands because these compounds are nonplanar and nonaromatic. Structural studies will be required to determine their precise mode of interaction with telomeric G-quadruplexes.
Steroidal diamines exert a variety of effects on cells (Mahler and Baylor, 1967) and include substances from natural origin, such as the plant alkaloids irehdiamine A and malouetine (Janot et al., 1960; Goutarel et al., 1967). Their chemical similarity to hormonal steroids suggests that these compounds are able to interact with DNA, as directly shown for malouetine and dipyrandium (Gourevitch et al., 1981; Hui et al., 1989). Biophysical studies led to the conclusion that these derivatives partially insert between base pairs and induce a kink in AT-rich DNA structures (Hui et al., 1989). DNA interacting properties have been the basis for the discovery of several classes of G-quadruplex ligands, including porphyrin, acridine, and ethidium derivatives (Harrison et al., 1999; Izbicka et al., 1999b; Koeppel et al., 2001; Kerwin et al., 2002; Guittat et al., 2003), and our results seems to confirm this rule. The chemical modification of these DNA-interacting agents (i.e., trisubstituted acridines for BRACO-19), led to an important improvement in the selectivity for G-quadruplexes over duplex DNA (Burger et al., 2005).
Competition with a double-stranded oligonucleotide (ds26) in the G-quadruplex fluorescence melting assay indicated that malouetine had a poor selectivity but that the steroid FG selectively bound to G-quadruplex rather than duplex DNA. Although the selectivity was less than that obtained for telomestatin or pyridine-dicarboxamide derivatives (Rosu et al., 2003b; Lemarteleur et al., 2004; Pennarun et al., 2005), these results indicate that it is possible to improve the quadruplex/B DNA selectivity for steroid ligands (Goutarel et al., 1967).
Preliminary experiments using a Pu22myc oligonucleotide, corresponding to the G-quadruplex-forming sequence from the c-myc promoter, indicated that steroid FG does not discriminate between these two types of G-quadruplexes (B. Brassart, unpublished results), similar to many previously reported G-quadruplex ligands (Lemarteleur et al., 2004). However, because the stabilization was very dependent on the nature of the cation (Table 1), one might propose that these molecules have a strong preference for the potassium over the sodium form of the telomeric quadruplex. This observation makes the recent determination of the potassium form of the telomeric quadruplex (Ambrus et al., 2006; Xu et al., 2006) very important, as rational drug design approaches may now be initiated on this physiologically relevant quadruplex.
Biochemical assays indicated that the steroid ligands are less potent than the pyridine dicarboxamide derivative 360A or telomestatin (Lemarteleur et al., 2004; Pennarun et al., 2005). For example, malouetine induced the senescence of HT1080 cells at a 20-fold higher concentration than did telomestatin. However, steroid FG induced senescence on HT1080 cells at submicromolar concentrations. Steroid FG exhibited all the characteristics of a telomere interacting agent (double-stranded telomere erosion, G-overhang degradation, anaphase bridge induction), previously reported for other potent G-quadruplex ligands (Izbicka et al., 1999b; Riou et al., 2002; Gomez et al., 2004; Leonetti et al., 2004; Burger et al., 2005; Douarre et al., 2005; Pennarun et al., 2005; Tahara et al., 2006). We observed differences between in vitro and cellular effects (Figs. 7 and 8) suggesting that steroid FG might have better cellular penetration or intracellular distribution than telomestatin or 360A. It is noteworthy that bis-guanidinium cholesterol derivatives have been developed as transfection agents, because of their membrane solubility and the ability of the two guanidinium functions to bind the phosphate group of DNA (Vigneron et al., 1996).
Steroid FG also had short-term antiproliferative effects. Preliminary observations indicated that the steroid FG (3 µM) induced apoptosis and DNA damage in HT1080 cells after 24-h treatment (B. Brassart, unpublished results). This feature was previously reported for other G-quadruplex ligands with mild selectivity for quadruplex relative to duplex DNA, such as triazine derivatives (Riou et al., 2002; Douarre et al., 2005). The guanylhydrazone side chain of steroid FG may lead to polyamine biosynthesis inhibition (Davidson et al., 1998). Therefore, we cannot exclude that this ligand has another mechanism of action related to a target in addition to G-quadruplexes. It is noteworthy that the overexpression of POT1 in HT1080 cells induced a significant resistance to the short-term effects of steroid FG emphasizing that telomere targeting contributes, at least in part, to the cytotoxic effect of the compound. On the other hand, because of the presence of many G-quadruplex-forming sequences in other parts of the genome (Huppert and Balasubramanian, 2005; Todd et al., 2005), it is possible that the ligand impairs gene transcription or DNA replication triggering the apoptotic response. Further experiments are undertaken to answer this point.
In conclusion, we reported here a new class of steroid telomere-interacting agents that bind G-quadruplexes and induce telomere uncapping. The steroid FG may have potential for antitumor treatment. The ease with which this steroid can be obtained, together with known chemistry for accessing modifications to this molecule, will allow improvements to the selectivity and the potency of these derivatives.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: hTERT, human telomerase reverse transcriptase; PCR, polymerase chain reaction; POT1, protection of telomere 1; TBE, Tris-borate-EDTA; DAPI, 4,6-diamidino-2-phenylindole; TRF, terminal restriction fragment; steroid FG, a funtumine derivative substituted witha guanylhydrazone moiety; EtBr, ethidium bromide; TRF, telomere repeat factor.
1 Current affiliation: Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5089, Toulouse, France. 
2 Current affiliation: Laboratoire de Physiologie Cellulaire Végétale, Institut de Recherches en Technologie et Sciences pour le Vivant, Commissariat à l'Energie Atomique Grenoble, Grenoble, France.
Address correspondence to: Jean-François Riou, Laboratoire d'Onco-Pharmacologie, JE 2428, UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, F-51096 Reims, France. E-mail: jf.riou{at}univreims.fr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Blackburn EH (2001) Switching and signaling at the telomere. Cell 106: 661–673.[CrossRef][Medline]
Blackburn EH, Chan S, Chang J, Fulton TB, Krauskopf A, McEachern M, Prescott J, Roy J, Smith C, and Wang H (2000) Molecular manifestations and molecular determinants of telomere capping. Cold Spring Harb Symp Quant Biol 65: 253–263.[CrossRef][Medline]
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, and Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349–352.
Burger AM, Dai F, Schultes CM, Reszka AP, Moore MJ, Double JA, and Neidle S (2005) The G-quadruplex-interactive molecule BRACO-19 inhibits tumor growth, consistent with telomere targeting and interference with telomerase function. Cancer Res 65: 1489–1496.
Caprio V, Guyen B, Opoku-Boahen Y, Mann J, Gowan SM, Kelland LM, Read MA, and Neidle S (2000) A novel inhibitor of human telomerase derived from 10H-indolo[3,2- b]quinoline. Bioorg Med Chem Lett 10: 2063–2066.[CrossRef][Medline]
Cimino-Reale G, Pascale E, Battiloro E, Starace G, Verna R, and D'Ambrosio E (2001) The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay. Nucleic Acids Res 29: E35.[CrossRef][Medline]
Clark GR, Pytel PD, Squire CJ, and Neidle S (2003) Structure of the first parallel DNA quadruplex-drug complex. J Am Chem Soc 125: 4066–4067.[CrossRef][Medline]
Colgin LM, Baran K, Baumann P, Cech TR, and Reddel RR (2003) Human POT1 facilitates telomere elongation by telomerase. Curr Biol 13: 942–946.[CrossRef][Medline]
Cuesta J, Read MA, and Neidle S (2003) The design of G-quadruplex ligands as telomerase inhibitors. Mini Rev Med Chem 3: 11–21.[Medline]
Darby RA, Sollogoub M, McKeen C, Brown L, Risitano A, Brown N, Barton C, Brown T, and Fox KR (2002) High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a Lightcycler. Nucleic Acids Res 30: e39.
Davidson K, Petit T, Izbicka E, Koester S, and Von Hoff DD (1998) Mitoguazone induces apoptosis via a p53-independent mechanism. Anticancer Drugs 9: 635–640.[Medline]
Davies JT (2004) G-quartet 40 years later: from 5'-GMP to molecular biology and supramolecular chemistry. Angew Chem Int Edit 43: 668–698.[CrossRef]
De Cian A, Delemos E, Mergny JL, Teulade-Fichou MP, and Monchaud D (2007) Highly efficient g-quadruplex recognition by bisquinolinium compounds. JAm Chem Soc 129: 1856–1857.[CrossRef][Medline]
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92: 9363–9367.
Dixon IM, Lopez F, Tejera AM, Esteve JP, Blasco MA, Pratviel G, and Meunier B (2007) A G-quadruplex ligand with 10000-fold selectivity over duplex DNA. JAm Chem Soc 129: 1502–1503.[CrossRef][Medline]
Douarre C, Gomez D, Morjani H, Zahm JM, O'Donohue MF, Eddabra L, Mailliet P, Riou JF, and Trentesaux C (2005) Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence. Nucleic Acids Res 33: 2192–2203.
Duan W, Rangan A, Vankayalapati H, Kim MY, Zeng Q, Sun D, Han H, Fedoroff OY, Nishioka D, Rha SY, et al. (2001) Design and synthesis of fluoroquinophenoxazines that interact with human telomeric G-quadruplexes and their biological effects. Mol Cancer Ther 1: 103–120.
Fedoroff OY, Rangan A, Chemeris VV, and Hurley LH (2000) Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism. Biochemistry 39: 15083–15090.[CrossRef][Medline]
Gomez D, Aouali N, Londono-Vallejo A, Lacroix L, Megnin-Chanet F, Lemarteleur T, Douarre C, Shin-ya K, Mailliet P, Trentesaux C, et al. (2003a) Resistance to the short term antiproliferative activity of the G-quadruplex ligand 12459 is associated with telomerase overexpression and telomere capping alteration. J Biol Chem 278: 50554–50562.
Gomez D, Aouali N, Renaud A, Douarre C, Shin-Ya K, Tazi J, Martinez S, Trentesaux C, Morjani H, and Riou JF (2003b) Resistance to senescence induction and telomere shortening by a G-quadruplex ligand inhibitor of telomerase. Cancer Res 63: 6149–6153.
Gomez D, Mergny JL, and Riou JF (2002) Detection of telomerase inhibitors based on G-quadruplex ligands by a modified telomeric repeat amplification protocol assay. Cancer Res 62: 3365–3368.
Gomez D, O'Donohue MF, Wenner T, Douarre C, Macadre J, Koebel P, Giraud-Panis MJ, Kaplan H, Kolkes A, Shin-ya K, et al. (2006a) The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells. Cancer Res 66: 6908–6912.
Gomez D, Paterski R, Lemarteleur T, Shin-Ya K, Mergny JL, and Riou JF (2004) Interaction of telomestatin with the telomeric single-strand overhang. J Biol Chem 279: 41487–41494.
Gomez D, Wenner T, Brassart B, Douarre C, O'Donohue MF, El Khoury V, Shin-Ya K, Morjani H, Trentesaux C, and Riou JF (2006b) Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells. J Biol Chem 281: 38721–38729.
Gourevitch MI, Sempere R, and Parello J (1981) UV spectroscopic studies of the binding of malouetine to double-stranded DNA. Biochimie 63: 743–754.[Medline]
Goutarel R, Mahler HR, Green G, Khuong-Huu Q, Cave A, Conreur C, Jarreau FX, and Hannart J (1967) Steroidal alkaloids. LXXI. Synthesis of the four 3,20-diaminopreg-5-enes (irehdiamine-A and stereoisomers). Comparison of their interactions with the DNA from E. coli. Bull Soc Chim Fr 12: 4575–4582.[Medline]
Gowan SM, Heald R, Stevens MF, and Kelland LR (2001) Potent inhibition of telomerase by small-molecule pentacyclic acridines capable of interacting with G-quadruplexes. Mol Pharmacol 60: 981–988.
Griffith JD, Comeau L, Rosenfield S, Stansel RM, Bianchi A, Moss H, and de Lange T (1999) Mammalian telomeres end in a large duplex loop. Cell 97: 503–514.[CrossRef][Medline]
Guittat L, Alberti P, Gomez D, de Cian A, Pennarun G, Lemarteleur T, Belmokhtar C, Paterski R, Morjani H, Trentesaux C, et al. (2004) Targeting human telomerase for cancer therapeutics. Cytotechnology 45: 75–90.[CrossRef]
Guittat L, Alberti P, Rosu F, Van Miert S, Thetiot E, Pieters L, Gabelica V, De Pauw E, Ottaviani A, Riou JF, et al. (2003) Interactions of cryptolepine and neocryptolepine with unusual DNA structures. Biochimie 85: 535–547.[Medline]
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, and Weinberg RA (1999) Creation of human tumour cells with defined genetic elements. Nature 400: 464–468.[CrossRef][Medline]
Han H, Cliff CL, and Hurley LH (1999) Accelerated assembly of G-quadruplex structures by a small molecule. Biochemistry 38: 6981–6986.[CrossRef][Medline]
Han H, Langley DR, Rangan A, and Hurley LH (2001) Selective interactions of cationic porphyrins with G-quadruplex structures. J Am Chem Soc 123: 8902–8913.[CrossRef][Medline]
Harrison RJ, Cuesta J, Chessari G, Read MA, Basra SK, Reszka AP, Morrell J, Gowan SM, Incles CM, Tanious FA, et al. (2003) Trisubstituted acridine derivatives as potent and selective telomerase inhibitors. J Med Chem 46: 4463–4476.[CrossRef][Medline]
Harrison RJ, Gowan SM, Kelland LR, and Neidle S (1999) Human telomerase inhibition by substituted acridine derivatives. Bioorg Med Chem Lett 9: 2463–2468.[CrossRef][Medline]
Hui XW, Gresh N, and Pullman B (1989) Modelling basic features of specificity in the binding of a dicationic steroid diamine to double-stranded oligonucleotides. Nucleic Acids Res 17: 4177–4187.
Huppert JL and Balasubramanian S (2005) Prevalence of quadruplexes in the human genome. Nucleic Acids Res 33: 2908–2916.
Huu Laine FK, and Pinto Scognamiglio W (1964) Curarizing activity of 3-beta,20-alpha-bistrimethylammonium-5-alpha-pregnane dichloride (malouetine) and of its stereoisomers. Arch Int Pharmacodyn Ther 147: 209–219.[Medline]
Incles CM, Schultes CM, Kempski H, Koehler H, Kelland LR, and Neidle S (2004) A G-quadruplex telomere targeting agent produces p16-associated senescence and chromosomal fusions in human prostate cancer cells. Mol Cancer Ther 3: 1201–1206.
Izbicka E, Nishioka D, Marcell V, Raymond E, Davidson KK, Lawrence RA, Wheelhouse RT, Hurley LH, Wu RS, and Von Hoff DD (1999a) Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos. Anticancer Drug Des 14: 355–365.[Medline]
Izbicka E, Wheelhouse RT, Raymond E, Davidson KK, Lawrence RA, Sun D, Windle BE, Hurley LH, and Von Hoff DD (1999b) Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells. Cancer Res 59: 639–644.
Janot MM, Laine F, and Goutarel R (1960) Steroid alkaloids. V. Alkaloids of Malouetia bequaertiana E. Woodson (Apocynaceae): funtuphyllamine B and malouetine. Preliminary communication. Ann Pharm Fr 18: 673–677.[Medline]
Janot MM, Qui KH, and Goutarel R (1958) Two new sterol alkaloids: funtumine and funtumidine.. C R Hebd Seances Acad Sci 246: 3076–3078.[Medline]
Kaiser M, De Cian A, Sainlos M, Renner C, Mergny JL, and Teulade-Fichou MP (2006) Neomycin-capped aromatic platforms: quadruplex DNA recognition and telomerase inhibition. Org Biomol Chem 4: 1049–1057.[CrossRef][Medline]
Karlseder J, Smogorzewska A, and de Lange T (2002) Senescence induced by altered telomere state, not telomere loss. Science 295: 2446–2449.
Kerwin SM (2000) G-quadruplex DNA as a target for drug design. Curr Pharm Des 6: 441–478.[CrossRef][Medline]
Kerwin SM, Chen G, Kern JT, and Thomas PW (2002) Perylene diimide G-quadruplex DNA binding selectivity is mediated by ligand aggregation. Bioorg Med Chem Lett 12: 447–450.[CrossRef][Medline]
Kim JH, Lee GE, Kim SW, and Chung IK (2003a) Quinoxaline derivative: a potent telomerase inhibitor leading to cellular senescence of human cancer cells. Biochem J 373: 523–529.[CrossRef][Medline]
Kim MY, Gleason-Guzman M, Izbicka E, Nishioka D, and Hurley LH (2003b) The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures. Cancer Res 63: 3247–3256.
Kim MY, Vankayalapati H, Shin-Ya K, Wierzba K, and Hurley LH (2002) Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex. J Am Chem Soc 124: 2098–2099.[CrossRef][Medline]
Koeppel F, Riou JF, Laoui A, Mailliet P, Arimondo PB, Labit D, Petitgenet O, Helene C, and Mergny JL (2001) Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes. Nucleic Acids Res 29: 1087–1096.
Lavelle F, Riou JF, Laoui A, and Mailliet P (2000) Telomerase: a therapeutic target for the third millennium? Crit Rev Oncol Hematol 34: 111–126.[Medline]
Lemarteleur T, Gomez D, Paterski R, Mandine E, Mailliet P, and Riou JF (2004) Stabilization of the c-myc gene promoter quadruplex by specific ligands inhibitors of telomerase. Biochem Biophys Res Commun 323: 802–808.[CrossRef][Medline]
Leonetti C, Amodei S, D'Angelo C, Rizzo A, Benassi B, Antonelli A, Elli R, Stevens M, D'Incalci M, Zupi G, et al. (2004) Biological activity of the G-quadruplex ligand RHPS4 is associated with telomere capping alteration. Mol Pharmacol 66: 1138–1146.
Li GZ, Eller MS, Firoozabadi R, and Gilchrest BA (2003) Evidence that exposure of the telomere 3' overhang sequence induces senescence. Proc Natl Acad Sci U S A 100: 527–531.
Mahler HR and Baylor MB (1967) Effects of steroidal diamines on DNA duplication and mutagenesis. Proc Natl Acad Sci U S A 58: 256–263.
Makarov VL, Hirose Y, and Langmore JP (1997) Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88: 657–666.[CrossRef][Medline]
McEachern MJ, Krauskopf A, and Blackburn EH (2000) Telomeres and their control. Annu Rev Genet 34: 331–358.[CrossRef][Medline]
McKenzie AG (2000) Prelude to pancuronium and vecuronium. Anaesthesia 55: 551–556.[CrossRef][Medline]
Mergny JL, Lacroix L, Teulade-Fichou MP, Hounsou C, Guittat L, Hoarau M, Arimondo PB, Vigneron JP, Lehn JM, Riou JF, et al. (2001) Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay. Proc Natl Acad Sci U S A 98: 3062–3067.
Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP, and Gilson E (2002) Natural and pharmacological regulation of telomerase. Nucleic Acids Res 30: 839–865.
Meyer K-H, Schütz S, Stoepel K, and Kroneberg H-G (1966), inventors; Bayer Corporation, assignee. Novel steroid guanylhydrazones and their production. U.S. Patent 3,277,081. 1966 Oct 4.
Neidle S and Parkinson G (2002) Telomere maintenance as a target for anticancer drug discovery. Nat Rev Drug Discov 1: 383–393.[CrossRef][Medline]
Parkinson GN, Lee MP, and Neidle S (2002) Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417: 876–880.[CrossRef][Medline]
Pendino F, Tarkanyi I, Dudognon C, Hillion J, Lanotte M, Aradi J, and Segal-Bendirdjian E (2006) Telomeres and telomerase: pharmacological targets for new anticancer strategies? Curr Cancer Drug Targets 6: 147–180.[CrossRef][Medline]
Pennarun G, Granotier C, Gauthier LR, Gomez D, Hoffschir F, Mandine E, Riou JF, Mergny JL, Mailliet P, and Boussin FD (2005) Apoptosis related to telomere instability and cell cycle alterations in human glioma cells treated by new highly selective G-quadruplex ligands. Oncogene 24: 2917–2928.[CrossRef][Medline]
Perry PJ, Gowan SM, Reszka AP, Polucci P, Jenkins TC, Kelland LR, and Neidle S (1998) 1,4- and 2,6-disubstituted amidoanthracene-9,10-dione derivatives as inhibitors of human telomerase. J Med Chem 41: 3253–3260.[CrossRef][Medline]
Perry PJ, Read MA, Davies RT, Gowan SM, Reszka AP, Wood AA, Kelland LR, and Neidle S (1999) 2,7-Disubstituted amidofluorenone derivatives as inhibitors of human telomerase. J Med Chem 42: 2679–2684.[CrossRef][Medline]
Quevauviller A and Blanpin O (1958) Pharmacodynamics of funtumine, alkaloid from the leaves of Funtumia latifolia stapf (apocynacées). J Physiol (Paris) 50: 469–471.[Medline]
Read MA, Wood AA, Harrison JR, Gowan SM, Kelland LR, Dosanjh HS, and Neidle S (1999) Molecular modeling studies on G-quadruplex complexes of telomerase inhibitors: structure-activity relationships. J Med Chem 42: 4538–4546.[CrossRef][Medline]
Riou JF, Guittat L, Mailliet P, Laoui A, Renou E, Petitgenet O, Megnin-Chanet F, Helene C, and Mergny JL (2002) Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proc Natl Acad Sci U S A 99: 2672–2677.
Rosu F, De Pauw E, Guittat L, Alberti P, Lacroix L, Mailliet P, Riou JF, and Mergny JL (2003a) Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis. Biochemistry 42: 10361–10371.[CrossRef][Medline]
Rosu F, Gabelica V, Shin-ya K, and De Pauw E (2003b) Telomestatin-induced stabilization of the human telomeric DNA quadruplex monitored by electrospray mass spectrometry. Chem Commun (Camb) (21): 2702–2703.[Medline]
Shay JW and Wright WE (2002) Telomerase: a target for cancer therapeutics. Cancer Cell 2: 257–265.
Shin-ya K, Wierzba K, Matsuo K, Ohtani T, Yamada Y, Furihata K, Hayakawa Y, and Seto H (2001) Telomestatin, a novel telomerase inhibitor from Streptomyces anulatus. J Am Chem Soc 123: 1262–1263.[CrossRef][Medline]
Smogorzewska A and de Lange T (2004) Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73: 177–208.[CrossRef][Medline]
Tahara H, Shin-Ya K, Seimiya H, Yamada H, Tsuruo T, and Ide T (2006) G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3' telomeric overhang in cancer cells. Oncogene 25: 1955–1966.[CrossRef][Medline]
Tauchi T, Shin-Ya K, Sashida G, Sumi M, Nakajima A, Shimamoto T, Ohyashiki JH, and Ohyashiki K (2003) Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways. Oncogene 22: 5338–5347.[CrossRef][Medline]
Teulade-Fichou MP, Carrasco C, Guittat L, Bailly C, Alberti P, Mergny JL, David A, Lehn JM, and Wilson WD (2003) Selective recognition of G-qQuadruplex telomeric DNA by a bis(quinacridine) macrocycle. J Am Chem Soc 125: 4732–4740.[CrossRef][Medline]
Todd AK, Johnston M, and Neidle S (2005) Highly prevalent putative quadruplex sequence motifs in human DNA. Nucleic Acids Res 33: 2901–2907.
Vigneron JP, Oudrhiri N, Fauquet M, Vergely L, Bradley JC, Basseville M, Lehn P, and Lehn JM (1996) Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells. Proc Natl Acad Sci U S A 93: 9682–9686.
Wright WE, Tesmer VM, Huffman KE, Levene SD, and Shay JW (1997) Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev 11: 2801–2809.
Xu Y, Noguchi Y, and Sugiyama H (2006) The new models of the human telomere d[AGGG(TTAGGG)3] in K+ solution. Bioorg Med Chem 14: 5584–5591.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  D. J. Patel, A. T. Phan, and V. Kuryavyi Human telomere, oncogenic promoter and 5'-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics Nucleic Acids Res., December 3, 2007; 35(22): 7429 - 7455. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
