Interaction of G-rich GT oligonucleotides with nuclearassociated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines Bruna Scaggiante1, Barbara Dapas1, Gabriele Grassi2 and Giorgio Manzini1 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy Department of Clinical, Morphological, and Technological Sciences, Division of Internal Medicine, University of Trieste, Italy Keywords aptamers; CCRF-CEM; cell growth inhibition; eEF1A; G-rich GT oligonucleotides Correspondence B Scaggiante, Molecular Biology Section, Department of Biochemistry, Biophysics and Macromolecular Chemistry, via Giorgeri, 1, 34127-Trieste, Italy Fax: +39 040558 3691 Tel.: +39 040558 3678 E-mail: scaggiante@bbcm.units.it (Received 30 August 2005, revised 12 January 2006, accepted 18 January 2006) doi:10.1111/j.1742-4658.2006.05143.x G-rich GT oligonucleotides with a different content of G clusters have been evaluated for their ability to exert cytotoxicity and to bind to nuclear-associated proteins in T-lymphoblast CCRF-CEM cells Only the oligomers that did not form G-based structures or had a poor structure, under physiological conditions, were able to exert significant cellular growth inhibition effect The cytotoxicity of these oligomers was related to their binding to the nuclear-associated eEF1A protein, but not to the recognition of nucleolin or other proteins In particular, GT oligomers adopting a conformation compatible with G-quadruplex, did not exert cytotoxicity and did not bind to eEF1A The overall results suggest that the ability of oligomers to adopt a G-quadruplex-type secondary structure in a physiological buffer containing 150 mm NaCl is not a prerequisite for antiproliferative effect in haematopoietic cancer cells The cytotoxicity of G-rich GT oligomers was shown to be tightly related to their binding affinity for eEF1A protein Single-stranded DNA may act as aptamer in recognizing proteins with an affinity similar to or higher than that of antibodies [1] Novel strategic applications of aptameric single-stranded DNA encompass probes for protein localization [1], therapeutic oligomers [2–4] and microarrays of proteins [5] Among oligomers able to adopt structures that are recognized by specific proteins, there are those with a high G content Within eukaryotic cells, G-rich singlestranded structures appear to be involved in senescence and aging by affecting telomere structure [6] Chromosomes end with a G-rich single-stranded overhang, which is able to adopt a four-stranded G-quadruplex structure that is a poor substrate for telomerase and can be stabilized by ligands One of these, telomestatin, stabilizes G-quadruplexes, thus inhibiting telomerase activity [7] Moreover, a human protein named translin was recently shown to stimulate telomerase activity by specifically binding to the G-rich Tetrahymena and human telomeric repeats [8] Furthermore, the forma- tion of G-quadruplex structures is thought to contribute to nonantisense effects by their ability to bind to cellular proteins [9,10] In particular, some protein targets of these G-rich oligonucleotides have been identified as nucleolin and a helicase [10,11] Other proteins able to bind to G-quadruplex structures have been recently discovered For example, it has been demonstrated that the human ribosomal protein L7a interacts in vitro with a presumably G-rich RNA structure [12] G-quartet-forming oligodeoxynucleotides interacting with the SH2 domain of Stat3, a protein encoded by a proto-oncogene that is activated in many human cancer cells, represent a novel class of aptameric therapeutic agents for the treatment of metastasis in cancer [13] Stat3 mediates upregulation of bcl-x and mcl-1 gene expression and thus cell proliferation [14] GT oligomers have been demonstrated to exert a specific, dose-dependent growth inhibition effect on a variety of human cancer cell lines [15–17] The Abbreviations CRC, cytotoxicity-related complex; eEF1A, Elongation Factor A 1350 FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS B Scaggiante et al GT oligonucleotides, eEF1A and antiproliferative effect resultant cytotoxicity was tightly related to the aptameric behaviours of these GTs and in particular to their ability to specifically bind to nuclear proteins forming a major cytotoxicity-related complex (CRC) of apparent molecular mass 45 ± kDa [15–20] Recently, a component of this complex has been isolated from the nuclear enriched fraction of haematopoietic cancer cell lines and identified as the eukaryotic Elongation Factor A (eEF1A) [21] Factors involved in the translation of mRNA are known to contribute to development of cancer [22,23] It has been reported that the GT sequences with a G-rich content can exert antiproliferative effects and display aptameric properties by binding to nucleolin [10] or to SV40 large T antigen helicase [11] Moreover G-quartet-forming GTs have been shown to bind to Stat3 and to induce tumour cell apoptosis [13] Here, we wish to elucidate if GT oligomers with a G-rich content can exert their antiproliferative activity in human T-lymphoblast cancer cells and if they bind to eEF1A protein Results The GT sequences are listed in Table The 27-mer GT was the reference oligomer able to exert cytotoxicity and displaying the specific protein binding activities [15–21] Starting from the GT and GT-G4 sequences, the following oligomers were planned in order to contain different clusters of G: GT-G1 has one cluster of four guanines, GT-G2 has two clusters of four guanines, GT-G3 has one cluster of seven guanines and one of four guanines The human T-lymphoblast CCRF-CEM cell line was used to perform the analysis, being the reference cells extensively used in previous works on GT oligomers and their protein interactors [15–21] The electrophoretic mobility of these oligomers under native and denaturing conditions is illustrated in Fig To evidence different conformations the oligomers were labelled at their 5¢-end by [32P]dATP[cP] Figure 1A shows that under denaturing conditions, all Table Oligonucleotide sequences and names Sequence Name Length (-mer) 5¢-TGTTTGTTTGTTTGTTTGTTTGTTTGT-3¢ 5¢-TGGTGTGTGTGGGGTGGTTGGTG-3¢ 5¢-TGGGGTGTGTGGGGTGGTTGGTG-3¢ 5¢-TGGGGTGTGTGGGGGGGTTGGTG-3¢ 5¢-TGGTTGGGGTGGGGGGGGGGGTG-3¢ GT GT-G1 GT-G2 GT-G3 GT-G4 27 23 23 23 23 oligomers migrate according to their lengths It was previously demonstrated that GT does not fold into intra- or intermolecular structures and thus it migrates according to its length also in native conditions [18] Figure 1B shows the migrations in native conditions of the oligomers denatured and renatured overnight in a buffer with salt composition similar to that of the extracellular medium With respect to the unstructured GT, GT-G1 does not appear to form significant interor intramolecular structures, the electrophoretic mobility being in accord with its length GT-G2 shows a band migrating on the basis of its length, and a slightly slower nonresolved migrating band that might be due to a dynamic interconversion with a bimolecular structure GT-G3 can form an intermolecular structure of higher order demonstrated by the slowly migrating band, albeit a major band corresponding to its length was also present GT-G4 was shown to fold into an intramolecular structure (the faster migrating band), and to associate into an intermolecular one (the slower migrating band) Analogous results were obtained when the oligomers were renatured in a potassium phosphate buffer similar to the intracellular medium (data not shown) To test the effect of these oligomers on cellular growth we performed a cytotoxicity assay The oligomers were applied to human lymphoblast CCRFCEM cells in serum-containing medium and cell growth was evaluated after 72 h without changing the medium [15] As illustrated in Fig 2, GT-G1 and GT-G2 caused a reduction of cell growth at a level comparable to that of GT, showing almost complete inhibition at 15 lm This effect was cytotoxic, as previously demonstrated for GT [15], as no recovery of cell growth was observed by prolonging cell culture for up to a further days (data not shown) GT-G3 showed a moderate effect, giving not more than 50% of cell growth inhibition at the highest dose (15 lm), probably due to a cytostatic effect as shown by the absence of cellular debris by microscope observation On the contrary, no cell growth inhibition effect was observed for GT-G4 The ability of these sequences to bind to proteins forming the CRC of 45 ± kDa was checked by UV cross-linking assays in competition experiments As illustrated in Fig 3A, the nonlabelled GT-G1, GT-G2 and GT-G3 were able to displace the labelled GT from binding to the CRC (white arrow) in the order GT-G1 > GT-G2 > GT-G3 (lanes 3, 4, 5) GT-G3 (lane 5) was the least efficient in acting as competitor of GT, in agreement with the fact that it displayed a reduced cytotoxicity with respect to GT All competitors were able to displace GT from the minor complex FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1351 TG G TG G TG G TG G nt G T TG G TG G TG G TG G T G nt B A B Scaggiante et al GT oligonucleotides, eEF1A and antiproliferative effect 45 45 30 30 25 20 25 10 20 10 Fig Denaturing and native electrophoresis of G-rich GT oligomers (A) Denaturing electrophoresis Five micrograms of oligomers were denatured by heating at 95 °C for 10 in M urea and then cooled on ice The samples were then loaded onto a 20% polyacrylamide gel in 0.1 M sodium acetate ⁄ acetic acid buffer pH 5.0, containing M urea The gel was run in 0.1 M sodium acetate ⁄ acetic acid buffer pH 5.0, at 42 °C at 15 VỈcm)1 The gel was stained by 0.01% Stainsall dye in 50% formamide (v ⁄ v) The nucleotide length markers are noted on the left (nt) (B) Native electrophoresis The oligomers were 5¢-end labelled by using [32P]dATP[cP] polynucleotide kinase as described in Experimental procedures One microgram of unlabelled oligomers and about ng of corresponding labelled ones adjusted to a specific activity of 15 000 cpm, were added together in a total volume of 10 lL in 150 mM NaCl, 10 mM K2HPO4 ⁄ KH2PO4, mM EDTA pH 7.0 The samples were denatured by heating at 95 °C for and then slowly cooled overnight at room temperature After adding lL of 50% glycerol in TBE buffer, the samples were loaded onto 20% polyacrylamide gel in TBE buffer and run at 10 VỈcm)1, at °C The gel was fixed in 10% acetic acid, dried and then exposed to X-LS Kodak film The nucleotide length markers are noted on the left (nt) of 100 kDa (black arrow) Self-competition of GT was reported as reference (lane 2) On the contrary, GT-G4 did not displace GT from the CRC (lane 6) This evidence was in agreement with the finding that GT-G4 did not exert any growth inhibition effect It was able to displace the GT oligomer only from the minor 100-kDa complex probably formed with nucleolin [21] In fact, nucleolin was previously found to recognize the structured GT-G4 oligomer and to form with it the 100-kDa complex [21] Figure 3B shows the binding of labelled oligomers to the nuclear-enriched fraction of proteins The efficiency of labelling of the single oligomers being not homogenous, the ratios of the different binding signals within each lane instead of their absolute values have to be considered It can be seen that GT is present mainly in the CRC and in a minor complex of about 1352 100 kDa, but that GT-G1, GT-G2 and GT-G3 can form in addition to the CRC (white arrow) and the 100-kDa complex (black arrow), analogously to GT, a complex of about 70 kDa also Moreover, the G-rich oligomers produced a band of about 26 kDa, due to a nonspecific protein binding previously described, probably derived from a cytoplasmic contaminant [15] The ratios of oligomer bound in the CRC to that bound in the 70-kDa complex estimated for each lane by phosphoimager was shown to be 0.38, 0.37, 0.19, for GT-G1, GT-G2, GT-G3, respectively This may explain why GT-G3 exerted a reduced antiproliferative activity, i.e by its preferential binding to other proteins On the contrary, GT-G4 did not form the CRC, whereas it was found to produce the 70-kDa and the 100-kDa complexes, that therefore cannot be involved in the cell growth inhibition effect Thus, the lack of FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS GT oligonucleotides, eEF1A and antiproliferative effect 120 A Competitor – G T G TG G TG G TG G TG B Scaggiante et al kDa 119 % of cellular growth 100 GT 80 76 GT-G1 60 GT-G2 47 GT-G3 40 29 GT-G4 20 15 ODN concentration (µM) B Labelled oligomer TG G TG G TG G TG 10 T G G kDa 119 Fig Cytotoxicity of G-rich GT oligomers CCRF-CEM cells (5 · 103) in exponential growth phase were seeded in triplicate in 200 lL of serum-containing medium in a 96-well microtiter plate After overnight incubation, the oligomers were directly added to the cell medium at the indicated concentrations Cell growth was evaluated 72 h after oligonucleotide addition by incorporation of 0.5 mgỈmL)1 of MTT, as described in Experimental procedures The percentage cell growth was calculated by taking growth of an internal nontreated control as 100% The results are mean ± SD of 5–10 independent experiments formation of the CRC agrees with the absence of cell growth inhibition by GT-G4 By affinity chromatography, using a GT biotinylated 51-mer, the proteins interacting with GT were isolated from the pool of nuclear-enriched fraction and used to perform competition experiments by UV crosslinking assays Figure shows a western blot of the total nuclear extracts used for the affinity chromatography with antinucleolin and anti-eEF1A antibodies It is evident that at the level of eEF1A recognition no nucleolin fragments are present This excludes interference from possible proteolytic fragments of nucleolin to the GT oligomer binding at the level of the eEF1A band As illustrated in Fig 4B, the isolated proteins formed two complexes with GT (lane 1): the most abundant, compatible with the binding of eEF1A in the CRC (white arrow), the other of apparent molecular mass of about 100 kDa compatible with binding to nucleolin (black arrow) The competition experiments in UV cross-linking assays with the GT-recognizing proteins confirmed the results obtained with the total nuclear-enriched proteins: a decreasing ability from GT-G1 (lane 3) to GTG3 (lane 5) to displace GT from the CRC and a lack of competition by GT-G4 (lane 6) Competition by 76 47 29 Fig Binding of the G-rich GT oligomers to total nuclear proteins (A) Competition of binding to GT Three micrograms of total nuclear CCRF-CEM cell extract were incubated with ng of 5¢-end 32 P-labelled GT in buffer C in the presence of the nonspecific competitors (1 lg salmon sperm DNA, lg of CT oligomer) and in the absence or in the presence of the indicated specific competitors added at 1000-fold molar excess After 30 incubation at room temperature, the samples were exposed to UV light for 10 and then denatured by adding SDS ⁄ PAGE loading buffer and boiling the samples The samples were loaded onto an SDS ⁄ PAGE gel (10% acrylamide) and run at 15 VỈcm)1 The gel was dried and exposed to X-AR Kodak film The black arrow indicates the 100-kDa complex; the white arrow indicates the 45 ± 7-kDa complex (CRC) (B) UV crosslinking of G-rich GT oligomers Three micrograms of total nuclear extract of CCRF-CEM cells were incubated in buffer C in the presence of ng of the indicated 5¢-end 32P-labelled G-rich GT oligomers and with lg salmon sperm DNA and lg of CT oligomer as nonspecific competitors After 30 incubation at room temperature, the samples were exposed to UV light for 10 and then denatured by adding SDS ⁄ PAGE loading buffer and boiling the samples The samples were loaded onto an SDS ⁄ PAGE gel (10% acrylamide) and run at 15 VỈcm)1 ng of 5¢-end 32P-labelled GT sample were included as reference (lane 1) The gel was dried and exposed to X-LS Kodak film The black arrow indicates the 100-kDa complex; the white arrow indicates the CRC FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1353 GT oligonucleotides, eEF1A and antiproliferative effect αn u cl e αe olin EF 1A A B Scaggiante et al 116 kDa 85 47 36 kDa 116 G TG G TG Competitor B G T G TG G TG G TG G T G4 66 45 29 C 66 kDa WB:αeEF1A 45 kDa Fig (A) Western blotting of nuclear extract with anti-eEF1A and anti-nucleolin IgG Twenty micrograms of nuclear extract were separated by SDS ⁄ PAGE (12% acrylamide) and then blotted onto a 0.22-lm nitrocellulose membrane as described in Experimental procedures The blotted membrane was blocked with 3% nonfat dried milk in NaCl ⁄ Pi and incubated with eEF1A (lane 2) or nucleolin (lane 1) mAb (1 lgỈmL)1) in NaCl ⁄ Pi, overnight, at °C with constant rocking After washing, the membrane was incubated for 1.5 h with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody, then rinsed once with NaCl ⁄ Pi containing 0.05% Tween-20 and four times with deionized water The blot was developed as described in Experimental procedures (B) Binding of G-rich GT to affinity-purified proteins: (left) 3.5 lL of proteins purified by affinity chromatography as described in Experimental procedures were incubated in 10 lL buffer C containing ng of 5¢-end 32P-labelled GT and in the presence or in the absence of the indicated specific competitors added at 500-fold molar excess, or (right) in 10 lL buffer C containing ng of the indicated 5¢-end 32P-labelled oligomers without specific competitors After 30 incubation at room temperature, the samples were crosslinked by exposure to UV light for 10 min, denatured by adding SDS ⁄ PAGE loading buffer and boiling The samples were loaded onto 10% SDS ⁄ PAGE and run at 15 VỈcm)1 The gel was dried and exposed to X-AR Kodak film The black arrow indicates the 100-kDa complex; the white arrow indicates the CRC (C) Western blotting of the affinity-purified proteins with anti-eEF1A Thirty micrograms of affinity purified proteins (lane 1) or 7.5 lg total nuclear proteins (lane 2) were separated by SDS ⁄ PAGE (12% acrylamide) and then blotted onto a 0.22 lm nitrocellulose membrane as described in Experimental procedures The blotted membrane was blocked with 3% nonfat dried milk in NaCl ⁄ Pi and incubated with eEF1A mAb (1 lgỈmL)1) in NaCl ⁄ Pi overnight, at °C with constant rocking The blot was developed as described in (A) nonlabelled GT is shown as reference (lane 2) Moreover, full ability to compete in the binding to the higher molecular weight protein (i.e nucleolin; black arrow) 1354 was observed for all G-rich oligomers In panel B at right it can be observed that GT-G1 formed both complexes, whereas GT-G4 formed only the 100 kDa FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS B Scaggiante et al A Labelled oligomer - 5 µL 10 µL µL 5 µL 10 µL µ - L eEF1A protein GT GT-G4 G Competitor eEF1A protein T G TG G1 T G -G2 T G -G3 TG – B – complex The presence of eEF1A in the purified protein mix was confirmed by western blotting with the specific antibody (Fig 4C, lane 1) The eEF1A protein from total nuclear extract is shown as control in lane To test the binding abilities of the oligomers toward the isolated protein, eEF1A was excised from Coomassie-stained gel and recovered as previously demonstrated [21] Figure illustrates EMSA and UV cross-linking assays with purified eEF1A Figure 5A shows that in the absence of competition the noncytotoxic oligomer GT-G4 was found to bind very faintly to eEF1A, also with addition of increasing amounts of protein (lanes 2–4) In contrast, labelled GT was found to bind with stronger affinity to eEF1A in a manner directly proportional to protein quantities (lanes 5–7) The presence of a minor slower migrating band in lanes 5–7 might be due to a complex of higher molecularity The slightly faster mobility of the complex between GT-G4 and eEF1A (lanes 2–4) is probably accounted for by the difference in length and thus in migration of the free oligomer (lane vs lane 8) Moreover, it seems conceivable that GT-G4, forming the G-quartet structure, gave a more compact (i.e faster) complex than that generated by the nonstructured GT Figure 5B shows competition experiments performed with the isolated eEF1A in UV cross-linking assays GT-G1 (lane 3) and, to a lesser extent, GT-G2 (lane 4) were able to displace GT from eEF1A GT-G3 (lane 5) and GT-G4 (lane 6) resulted inefficient in producing competition On the left the western blot with the antieEF1A antibody of the protein recovered from the gel band is shown To completely elucidate the relationship between the structure of the GTs and their ability to inhibit cell growth by forming the CRC, we performed CD at 37 °C As a control we used two oligomers, GRO29A and GRO26A, whose structures were related to antiproliferative activity in tumour cells [10] As illustrated in Fig 6A, the weak CD bands of GT and GT-G1 indicate absence of appreciable secondary structure at 37 °C, under conditions similar to those of the extracellular medium The spectra of GT-G2 and GRO29A showed a small band at 263 nm, suggesting the formation of a limited structure GT-G3 was found to give a peak at 263 nm compatible with G-quartet structure However, no full structure in the G-quadruplex was detected as shown by the low intensity of the 263-nm band A clear structure formation was found for GRO26A and GT-G4: a positive peak at 263 nm and a negative one at 242 nm These CD spectra were compatible with parallel G-quadruplex Moreover, GRO26A showed a slight signal at 295 nm that might be related to a minor amount of antiparallel G-quad- GT oligonucleotides, eEF1A and antiproliferative effect + + + + + + – kDa 116 79 46 31 47 kDa WB:αeEF1A α Fig Binding of G-rich GTs to eEF1A protein The eEF1A protein was purified from a Coomassie blue-stained gel, as described in Experimental procedures (A) Band-shift assay From 2.5 to 10 lL of the isolated eEF1A protein were incubated with ng of 5¢-end 32 P-labelled GT-G4 (lanes 1–4) or ng of 5¢-end 32P-labelled GT (lanes 5–8) in 25 mM Tris ⁄ HCl pH 8.0, containing 0.05% SDS, 0.05 mgỈmL)1 BSA, 0.1 mM EDTA, 1.25% glycerol and 0.1 M NaCl, for 30 at room temperature The samples were then loaded onto 8% polyacrylamide in TBE buffer and run at 20 VỈcm)1 at °C The gel was then dried and exposed to X-AR Kodak film (B) UV cross-linking assay Ten microlitres of the isolated eEF1A protein were incubated in buffer C with ng of 5¢-end 32P-labelled GT in the presence or absence of the indicated specific nonlabelled competitors added at 10-fold molar excess After 30 incubation at room temperature, the samples were exposed to UV light for 10 min, denatured by adding SDS ⁄ PAGE loading buffer and boiling The samples were separated by SDS ⁄ PAGE (12% acrylamide) and run at 10 VỈcm)1 The gel was then dried and exposed to X-AR Kodak film On the left is shown western blotting of 50 lL of the recovered protein performed after SDS ⁄ PAGE (12% acrylamide) with an anti-eEF1A mAb as described in Experimental procedures ruplex The CD spectra of GRO29A and GRO26A agree with their electrophoretic mobilities under nondenaturing conditions: GRO29A, forming a poor FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1355 GT oligonucleotides, eEF1A and antiproliferative effect A B Scaggiante et al GT GT-G1 GT-G2 GT-G3 GT-G4 GRO26A GRO29A CD[mdeg] -3 220 B 240 260 280 300 Wavelength [nm] 320 GRO26A CD[mdeg] -2 220 240 260 280 300 Wavelength [nm] 320 20ºC 37ºC 50ºC 65ºC 80ºC 100ºC 20ºC, H2O 65ºC, H2O GT-G4 CD[mdeg] -4 220 240 260 280 300 Wavelength [nm] 320 structure, was found to run on the basis of its length, whereas GRO26A, demonstrating a full G-based structure, showed a fast and a slow migrating band similarly to GT-G4 (data not shown) Figure 6B illustrates the stability of GRO26A and GT-G4: these structures were not disrupted by increasing the temperature to 90 °C They showed CD spectra indicative of absence of structure only when they were resuspended in water and heated at 65 °C (dashed green spectrum) On the contrary, the structure of GT-G3 was not so stable and it was disrupted by increasing the temperature to 65 °C (data not shown) The effect of GRO29A and GRO26A on CCRF-CEM cell growth is shown in Fig 7A: in accordance with previous data, we found 1356 Fig Circular dichroism of oligomers (A) A 10 lM solution of the indicated oligomer was diluted in renaturation buffer (150 mM NaCl, 10 mM K2HPO4 ⁄ KH2PO4, mM EDTA pH 7.0) to a final concentration of 0.5 lM recording the spectra at 37°C as described in Experimental procedures (B) GRO26A and GT-G4 were diluted to 0.5 lM final concentration in water and the spectra were recorded at the indicated temperatures that GRO29A exerted a significant growth inhibition effect, similar to GT, whereas the G-quartet forming GRO26A did not alter cellular growth, similar to GTG4 Moreover, as illustrated in Fig 7B, GRO29A (lane 4) was able to compete in the binding to specific nuclear proteins (CRC) as did GT (lane 3) On the contrary, no competition was observed using GRO26A (lane 5) Discussion A series of guanosine-rich phosphodiester oligodeoxynucleotides strongly inhibits proliferation in a number of human tumour cell lines and the presence of FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS B Scaggiante et al GT oligonucleotides, eEF1A and antiproliferative effect 100 80 60 40 20 TG GT GR O GR 29A O2 6A G G RO 26 A G RO 29 G B A T % of cellular growth A Competitor -Proteins + + + + 116 kDa 70 kDa 46 kDa 32 kDa 23 kDa Fig Cytotoxic assay of GRO26A and GRO29A and their binding to nuclear proteins (A) CCRF-CEM cells (5 · 103) in exponential growth phase were seeded in triplicate in 200 lL of serum-containing medium in 96-well microtiter plates After overnight incubation, the oligomers were directly added to the cell medium at 10 lM concentration Cell growth was evaluated 72 h after oligonucleotide addition by incorporation of 0.5 mgỈmL)1 of MTT, as described in Experimental procedures As reference oligomers GT and GT-G4 were used in the same experiment (B) Two micrograms of total nuclear proteins (lanes 2–5) were incubated with ng of 5¢-end 32 P-labelled GT in buffer C in the presence of the nonspecific competitors (1 lg poly(dIdC) and lg of CT oligomer) and with the indicated specific competitors added at 500-fold molar excess (lanes 3–5) After 30 incubation at room temperature, the samples were cross-linked by UV exposure and then separated by SDS ⁄ PAGE (12% acrylamide) as described in Experimental procedures Reference lane shows the migration of the free oligomer; the open arrow shows the CRC G-quartets in the active oligonucleotides was found to determine cell growth inhibition activity [10,13,14] The G-rich oligonucleotides bind to specific cellular proteins in both nuclear and cytoplasmic extracts and to proteins derived from the plasma membrane, and their biological activity correlates with binding to these proteins Strong evidence showed that one of these proteins is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation in a variety of solid tumour cell lines [10] The biological activity of the G-rich oligomers was found to be associated with their ability to form stable G-quartetcontaining structures and with their binding to specific cellular proteins, most likely nucleolin [10] More recently, the antiproliferative activity of G-rich oligonucleotides has been directly related to their inhibition effect on DNA replication, resulting from negative modulation of a helicase activity [11] Independently, other authors found that G-quartet-forming oligomers bind to Stat3, a protein involved in tumour cell progression The oligomers inhibited Stat3 binding to DNA, thus blocking the transcription of Stat3-regulated genes and the progression of prostate and breast cancers in mice [13] Here we demonstrate that G-rich GT oligomers can exert cytotoxicity on haematopoietic T-lymphoblast CCRF-CEM cells only if the oligomers bind to nuclear proteins forming the CRC, derived from eEF1A recognition Similar results were confirmed in other cell lines of haematopoietic tumour origin, such as Jurkat, CEM-VLB, Raji, HL60, K562 (data not shown) The cytotoxicity and the formation of the CRC with nuclear proteins seem related to the presence of oligomers migrating according to their length, as demonstrated by electrophoresis for GT-G1, GT-G2 and GT-G3 GT-G4, demonstrating the formation of a full structure, did not form the CRC and did not exert any appreciable growth inhibition effect on the tumour cells The nontoxic GT-G4 has 78% G-content, two clusters of four and 11 consecutive Gs, and it appears to form structure in native electrophoresis, probably as a G-quadruplex Our CD spectrum clearly indicates a very stable G-quartet structure at physiological conditions A melting curve, recorded at 295 nm, compatible with the disruption of a G-quartet structure [10] was also found for GT-G4 (data not shown) A NMR study has shown that a DNA oligonucleotide containing different G clusters adopts an asymmetric bimolecular G-quadruplex structure in solution [24], and the topology of this structure is distinct from the folds of the closely related and well-characterized sequences d(G4T4G4) and d(G3T4G3) [25] Recently, the ability of the G-rich oligomers to exert an antiproliferative effect has been related to their binding to specific cellular proteins, rather than to G-quadruplex formation [26] The absence of cytotoxicity of GT-G4 appears not to be related to a reduced intracellular accumulation of this oligomer In fact, the incorporation of 32P-labelled oligomers into viable cells showed similar uptakes with the only exception of GT-G4, whose internalization rate was even higher (data not shown) FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1357 GT oligonucleotides, eEF1A and antiproliferative effect B Scaggiante et al GT-G4, showing a G-quartet-based structure, did not exert cytotoxic effects on haematopoietic cancer cell lines, whereas the G-rich GT oligomer GRO29A was successfully used by Bates to significantly inhibit the growth of a variety of human cancer cells derived from solid tumours [10] The same author used as a control GRO26A, that did not significantly alter cellular growth Accordingly with these authors, we found that GRO29A exerted on CCRF-CEM cells a growth inhibition effect similarly to GT, whereas GRO26A did not significantly alter cellular proliferation On the contrary, in our experimental conditions, which were similar to those of the extracellular medium, the CD spectra of GRO26A show that it formed a stable G-quadruplex structure as GT-G4 did, whereas GRO29A exhibited a CD indicative of a poor structure In experimental conditions similar to that of the intracellular medium (in 140 mm KCl containing buffer), GRO29A did not show a CD spectrum diagnostic of G-quartet-based structure (data not shown) In fact, the spectra clearly show only minor differences with that obtained in NaCl containing buffer, and this is indicative of a rather weak secondary structure The apparent discrepancy with literature results [10,26] can be explained by the fact that the formation of G-quartet based structures from a rather various repertoire often implies rather long kinetic processes, depending on molecularity, oligomer concentration, salt, temperature of annealing, and frequently different coexisting competing forms In particular GRO29A needs 56 h annealing at 60 °C in 140 mm KCl to assume a G-quartet containing structure [26] In agreement with our results, competition experiments demonstrated that GRO29A was able to displace the labelled oligomer from eEF1A, similarly to GT, whereas GRO26 was not Furthermore, in nondenaturing electrophoresis under our experimental conditions GRO29A migrated mostly on the basis of its length in accordance with other findings [26], whereas GRO26A demonstrated the formation of a full structure Thus it seems likely that GRO29A can exert a growth inhibition effect on human haematopoietic cancer cells because in physiological conditions it does not significantly form G-quartets and can bind to eEF1A The binding of GRO29A to eEF1A was not observed by Bates et al [10] in solid tumour, but this might be related to the absence in these cells of the eEF1A isoforms that we identified in the haematopoietic cell line [21], or to the buffer conditions used for the binding Thus the G-quartet structure is clearly not a prerequisite for the antiproliferative activity of G-rich oligomers in haematopoietic cancer cells The toxic GT-G1 has 60% G content and only one cluster of four guanines It migrates on the basis of its 1358 length in electrophoresis, it was as cytotoxic as GT and showed the formation of the CRC GT-G2, with 65% G-content, demonstrated a very faint structure and inhibited the cellular growth similarly to GT-G1 GT-G3, which, with 69% content, assumed ) in part ) intermolecular structures clearly related to the increase in the number of Gs in the cluster (from four in GT-G2 to seven in GT-G3), gave a reduced cellular growth inhibition; accordingly, it showed a reduced capacity to form CRC The lower ability of GT-G2 to compete for the binding of GT to eEF1A with respect to GT-G1 both in total nuclear extract and in affinitychromatography-purified proteins does not agree with cytotoxicity data, the two oligomers showing irrelevant differences in growth inhibition The overall results suggest that the kinetics of binding of GT-G2 to eEF1A might be slower with respect to that of GT-G1 explaining its reduced ability to displace GT from the CRC and this might be related to the mild grade of structure formation observed in its CD spectrum However, it cannot be completely excluded that GT-G1 and GT-G2 have a different in vivo intracellular localization, i.e GT-G2 being predominately nuclear in localization with respect to GT-G1, thus taking into account the different binding ability vs the same cytotoxicity Although a different intracellular localization could explain differences in antiproliferative effect, the protein binding ability suggests that the biological activity of the G-rich GT oligomers is related to their recognition of nuclear-associated eEF1A Furthermore, all of the oligomers were able to displace GT from nucleolin (the complex of highest molecular weight), both in assays with total nuclear extract and with affinity chromatography purified proteins, but not all were able to exert cell growth inhibition Thus it seems unlikely that nucleolin is related to the antiproliferative effect exerted by G-rich GTs Moreover, the oligomers that did not bind to eEF1A, such as GT-G4, did not exert growth inhibition It is interesting to note that GT-G1 recognizes also another nuclear protein, forming a complex of 70 kDa, just as GT-G2, GT-G3 and GT-G4 This complex is unlikely to involve already described proteins such as Stat3 [14] or a helicase [11], whose molecular masses are 80 and 124 kDa, respectively The formation of this complex clearly suggests that these G-rich oligomers target other proteins that GT does not engage, but this fact is not related to the cytotoxic effect Moreover, the binding of the G-rich oligomers GT-G1, GT-G2, GT-G3 and GT-G4 to proteins forming the 70-kDa complex might be due to the interaction with a proteolytic fragment of nucleolin observed by Bates [10] as well as by us (Fig 4A) This agrees FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS B Scaggiante et al with the observation that the nontoxic GT-G4 forms the two complexes of 100 kDa and of 70 kDa, both compatible with nucleolin recognition The possibility that a 48-kDa fragment of nucleolin [27] could be a major contaminant of eEF1A protein can be excluded by MALDI TOF analysis of the Coomassie blue band extract [21] and by the absence of a corresponding nucleolin signal in western-blotting of our nuclear extracts (Fig 4A) Thus these results indicate that in haematopoietic cancer cells G-rich GT oligomers exert a growth inhibition effect by binding to nuclear-associated eEF1A protein and this effect is inversely related to the ability of oligomers to adopt G-quartet structures in physiological conditions Experimental procedures Oligonucleotide sequences HPLC-purified phosphodiester oligomers were from MWGBiotech AG (Ebersberg, Germany) The oligomers were resuspended in physiological solution at 1000 lm stock solution and sterilized by centrifugation in 0.2 lm filter spin-X tubes Native and denaturing electrophoresis The oligomers (5 lg) were denatured by heating at 95 °C for 10 and supplemented with m urea The samples were loaded onto 20% polyacrylamide gel (acrylamide:bisacrylamide, 29 : w ⁄ w) in 0.1 m acetic acid, pH 5.0, 10 mm NaCl, 10 mm MgCl2 containing m urea The gel was run in 0.1 m acetic acid, pH 5.0, at 10 VỈcm)1 for 2.5 h at 42 °C In nondenaturing conditions, the oligomers (5 lg) were denatured by heating at 95 °C for 10 and renatured in 150 mm NaCl, 10 mm K2HPO4 ⁄ KH2PO4, mm EDTA, pH 7.0, by slowly cooling at room temperature overnight The samples were then electrophoresed through 20% polyacrylamide gel (acrylamide:bisacrylamide, 29 : w ⁄ w) in TBE buffer (0.09 m Tris ⁄ borate, pH 8.0, mm EDTA) at VỈcm)1 for h at room temperature The gels were stained by using 0.01% Stainsall dye (Sigma Chemical Co., St Louis, MO, USA) in 50% formamide (v ⁄ v) Alternatively, using 32P-labelled oligomers, the gels were fixed in 10% acetic acid, dried and then exposed to autoradiography on X-AR Omat Kodak film Cell cultures and cytotoxicity assay The human T-lymphoblastic leukaemic CCRF-CEM cell line was cultured in RPMI 1640 medium supplemented with 10% foetal serum (Euroclone, Celbio, Devon, UK), mm L-Gln, 100 mL)1 penicillin, 100 lgỈmL)1 streptomycin GT oligonucleotides, eEF1A and antiproliferative effect CCRF-CEM cells (5 · 103) in exponential growth phase were seeded in 200 lL foetal clone serum (Euroclone, Celbio, Devon, UK) containing medium, in 96-well microtiter plate in triplicate After overnight incubation, the oligomers were directly added to the cell medium at the indicated final concentrations Cell growth was evaluated days of culture after oligonucleotide administration by incorporation of 0.5 mgỈmL)1 MTT into viable cells [28] The percentage of cellular growth was estimated by considering 100% cell growth that of the internal-control nontreated cells Total nuclear extracts preparation Total nuclear extracts were obtained from approximately 20 · 106 CCRF-CEM cells by a small modification of Dignam’s method [15] The protein content was determined by the Bradford method [29] using BSA (Sigma Chemical Co.) as standard Affinity chromatography The 5¢-biotin labelled oligomer 5¢-T(GTTT)9GT-3¢ (MWGBiotech AG) was immobilized on streptavidin magnetic particles (Boehringer, Mannhein) in 10 mm Tris ⁄ HCl, mm EDTA, 100 mm NaCl, pH 7.5 (TEN 100) at a concentration of lg oligomerỈmg)1 beads After 30 incubation at room temperature, the beads were washed twice with 10 mm Tris ⁄ HCl, mm EDTA, m NaCl, pH 7.5 (TEN 1000) The beads were equilibrated in TEN 1000 For protein binding, the beads were preincubated with 0.5 mgỈmL)1 BSA in TEN 100 for 10 The beads were washed thrice with TEN 100 and then equilibrated in 20 mm Hepes, 1.5 mm MgCl2, 0.2 mm EDTA, 0.42 m NaCl, containing 10% glycerol and 0.05% NP40 (buffer C) The beads were incubated with mg proteins from the nuclear-enriched fraction supplemented with mm phenylmethanesulfonyl fluoride (PMSF), 0.01% NP40 and 0.05 mgỈmL)1 salmon sperm DNA, for h with gentle stirring The beads were washed twice with buffer C and twice with buffer C without NP40 The elution was made with three volumes of buffer C without NP40 adjusted to 1.5 m NaCl, incubating for 10 with gentle stirring The beads were then washed with TEN 100 and stored at °C with 0.01% NaN3 The eluted proteins were dialysed with a 3000-Da cutoff membrane (Centricon) in mm Hepes, 0.2 mm EDTA, 5% glycerol, mm dithiothreitol (DTT), mm PMSF and then lyophilized The protein was resuspended in 50 lL water containing 10% glycerol and mm DTT Purification of eEF1A from Coomassie blue-stained gel We have previously shown that the protein eEF1A can be isolated from a Coomassie blue stained gel [21] Briefly, the FEBS Journal 273 (2006) 1350–1361 ª 2006 The Authors Journal compilation ª 2006 FEBS 1359 GT oligonucleotides, eEF1A and antiproliferative effect B Scaggiante et al band corresponding to eEF1A was excised from the gel and recovered in 50 mm Tris ⁄ HCl pH 8.0, containing 0.1% SDS, 0.1 mgỈmL)1 BSA, 0.2 mm EDTA, 2.5% glycerol After two steps of freeze ⁄ thawing, followed by precipitation with cold acetone, the protein was rinsed with methanol, denatured with m urea and then renatured by overnight incubation in a fixed volume of 50 mm Tris ⁄ HCl pH 7.6, 100 mm KCl, mm DTT, 0.1 mm PMSF It was not possible to quantify the amount of recovered protein owing to the presence of a high molar excess of BSA remaining in the recovery buffer Therefore, a fixed aliquot of the protein was incubated with ng of the indicated probes After 30 incubation at room temperature, the samples were loaded onto a native 8% polyacrylamide gel in TBE and run at 10 VỈcm)1, at a temperature of °C 0.05% Tween-20 and four times with deionized water, the nitrocellulose blot was developed using enhanced chemiluminescence detection (Pierce, Rockford, IL) according to the manufacturer’s protocols, and then exposed to X-ray film Circular dichroism The oligomers were resuspended in 150 mm NaCl, 10 mm K2HPO4 ⁄ KH2PO4, mm EDTA, pH 7.0 at 10 lm and denatured at 100 °C and then renatured by slowly cooling overnight The oligomers were diluted in 150 mm NaCl, 10 mm K2HPO4 ⁄ KH2PO4, mm EDTA pH 7.0 buffer or in water at a final concentration of 0.5 lm and then analysed with a Jasco JT-710 CD spectrophotometer equipped with a thermostatic bath The spectra were recorded at different temperatures by a Spectra manager analyser software EMSA and UV cross-linking assay Samples containing lg total nuclear proteins or 2.5– 10 lL of proteins purified by affinity chromatography or by PAGE, were incubated with ng [c-32P]ATP-labelled probe and with lg of poly(dIdC) and lg of CT oligomer (5¢-TCTTTCTTTCTTTCTTTCTTTCTTTCT-3¢), as nonspecific competitors, in the absence or in the presence of 500-fold molar excess of the indicated nonlabelled specific competitors For EMSA, after 30 incubation at room temperature, the samples were loaded onto a 12% polyacrylamide gel (acrylamide:bisacrylamide, 29 : 1, w ⁄ w) in TBE and run at 10 VỈcm)1 for h at °C The gels were fixed in 10% acetic acid, then dried and exposed to KodaK XAR-OMAT film For the UV crosslinking, after 30 incubation at room temperature, samples were exposed to UV light at 302 nm for 10 using a transilluminator, denatured and loaded onto 12% SDS ⁄ PAGE gel The gels were dried and exposed to KodaK XAR-OMAT film Western blotting analysis Five micrograms total nuclear proteins and 30 lL affinity purified proteins, separated by 12% SDS ⁄ PAGE were electrophoretically transferred onto a 0.22-lm nitrocellulose membrane (Schleicher & Schuell, Keene, NH) in 50 mm Tris ⁄ HCl, 40 mm glycine, 0.4% SDS, 20% methanol buffer, using a transblot semidry apparatus system (Amersham Pharmacia Biotech, Uppsala, Sweden) The membrane was stained with Ponceau S (Sigma Chemical Co.) and destained with deionized water The blotted membrane was blocked with 3% nonfat dried milk in NaCl ⁄ Pi and incubated with eEF1A mAb (1 lgỈmL)1) (Upstate Biotechnology, Lake Placid, NY) or with nucleolin mAb (Santa Cruz Biotechnology Inc., CA) in NaCl ⁄ Pi, overnight, at °C with constant rocking Then, it was washed twice with deionized water and incubated for 1.5 h with a horseradish peroxidase-conjugated antimouse IgG secondary antibody (Promega, Madison, WI) After washing once with NaCl ⁄ Pi containing 1360 Acknowledgments This work was supported in part by FIRB number RBNE0155LB and in part by the program ‘Rientro cervelli’ art DM n.13 of Italian Ministry for University and Research, MIUR References Stanlis KKH & Mcintosh JR (2003) Single-strand DNA aptamers as probes for protein localization in cells J Histochem Cytochem 51, 708–808 Farokhzad OC, Jon S, Khademhosseini A, Tran TN, Lavan DA & Langer R (2004) Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells Cancer Res 64, 7668–7672 Zhang Z, Blank M & Schluesener HJ (2004) Nucleic acid aptamers in human viral disease Arch Immunol Ther Exp 52, 307–315 Lee JF, 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Length (-mer) 5¢-TGTTTGTTTGTTTGTTTGTTTGTTTGT-3¢ 5¢-TGGTGTGTGTGGGGTGGTTGGTG-3¢ 5¢-TGGGGTGTGTGGGGTGGTTGGTG-3¢ 5¢-TGGGGTGTGTGGGGGGGTTGGTG-3¢ 5¢-TGGTTGGGGTGGGGGGGGGGGTG-3¢ GT GT-G1 GT- G2 GT- G3 GT- G4... these results indicate that in haematopoietic cancer cells G-rich GT oligomers exert a growth inhibition effect by binding to nuclear-associated eEF1A protein and this effect is inversely related... differences in growth inhibition The overall results suggest that the kinetics of binding of GT- G2 to eEF1A might be slower with respect to that of GT- G1 explaining its reduced ability to displace GT