Eur J Biochem 270, 3251–3262 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03713.x Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers Barbara Dapas1, Gianluca Tell2, Andrea Scaloni3, Alex Pines2, Lino Ferrara3, Franco Quadrifoglio1 and Bruna Scaggiante1 Department of Biomedical Sciences and Technologies, University of Udine, Italy; 2Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy; 3Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples, Italy GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC) On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain Oligonucleotides, widely used as agents to specifically inhibit gene expression by antisense [1] or antigene [2] strategies, often display unexpected effects by interacting with cellular proteins In fact, they are able to bind to either membrane or intracellular proteins, probably by their polyanionic nature and/or by nonspecific or specific sequence-related mechanisms [3] In the last decade, oligonucleotides have progressively gained aptameric function, specifically recognizing proteins as natural or non-natural ligands [4] Constitutive proteins that bind to singlestranded DNA oligomers are widely recognized to be involved in important mechanisms associated with DNA replication, repair and recombination [5–7] Furthermore, many reports evidenced that modulation of gene expression [8,9], and stimulation or inhibition of cellular replication [10,11], are influenced by single-stranded DNA sequences specifically interacting with cellular proteins Oligonucleotides composed exclusively of G and T bases have previously been shown to exert a specific, selective and dose-dependent effect of cell growth inhibition on a variety of human cancer cell lines [12] The cytotoxic effect of these GT oligomers was shown to be highly related to their ability to form complexes with nuclear proteins, as measured by UV cross-linking assays [12–15] However, the nature of these nuclear proteins behaving as single-stranded DNAbinding proteins has not yet been identified [12–15] A protein isolated from fibroblasts with such an activity has been already described [16], but it was able to tightly bind either GA or GT oligomers On the contrary, the nuclear proteins binding to our GT oligomers did not specifically recognize GA sequences [12] More recently, it has been shown that GT oligonucleotides, capable of forming G-quartet structures, exerted a cytotoxic effect on human cancer cell lines By UV cross-linking assay, these oligomers have been reported to interact with nucleolin, forming a main complex of >100 kDa molecular mass [17] This complex was not formed when GT oligomers unable to form a G-quartet structure were used [17,18] The oligonucleotide under our investigation (a 27-mer; see the Materials and methods, below) did not present appreciable G-quartet structures, as deduced by gel electrophoresis and circular dichroism analysis [14]; on the contrary, it was able to form a cytotoxic-related complex (CRC), with an apparent molecular mass of 45 ± kDa, with nuclear proteins of different tumour cell lines [12–15] Thus, the characterization of these nuclear species seemed particularly interesting, Correspondence to B Scaggiante, Department of Biomedical Sciences and Technologies, University of Udine, p.le Kolbe 4, 33100 Udine, Italy Fax: + 39 432 494301; Tel.: + 39 432 494311; E-mail: bscaggiante@makek.dstb.uniud.it Abbreviations: CRC, cytotoxicity-related complex; CRS, control rabbit total serum; eEF1A, eukaryotic elongation factor alpha; Egr1, early growth response protein 1; IPG, immobilized pH gradient; PSD, postsource decay; TBP, TATA-binding protein (Received March 2003, revised 27 May 2003, accepted 10 June 2003) Keywords: aptameric oligonucleotides; eEF1A; proteomics; CCRF-CEM cells; cytotoxicity Ó FEBS 2003 3252 B Dapas et al (Eur J Biochem 270) either for using to elucidate new potential molecular targets in tumour biology or to highlight the mechanism of action of our cytotoxic GT oligonucleotides In this article, we report the identification of eukaryotic elongation factor alpha (eEF1A) as a nuclear component of the CRC in T-lymphoblastic CCRF-CEM cancer cells In these cells, we found a striking relationship between the growth-inhibition effect exerted by cytotoxic GT oligomers and their selective binding to nuclear eEF1A In fact, in normal human lymphocytes no appreciable binding of GT oligomer to nuclear eEF1A was shown and, accordingly, these cells were not sensitive to its cytotoxic action A possible role for nuclear eEF1A in tumour cell growth or maintenance is suggested by the selective identification of more basic isoforms of eEF1A in cancer cells, but not in normal lymphocytes Materials and methods Oligonucleotides Oligonucleotides were purchased from MWG Biotech (Ebersberg, Germany) as HPLC pure species and their purity was confirmed by electrophoresis on an 18% polyacrylamide/7 M urea gel For cell cultures, oligonucleotides were resuspended in water and sterilized by centrifugation on a spin-X tube provided with a 0.22-lM filter (Costar, Cambridge, MA, USA) The GT oligomer sequence was: 5¢-TGT TTG TTT GTT TGT TTG TTT GTT TGT-3¢; and the control CT sequence was: 5¢-TCT TTC TTT CTT TCT TTC TTT CTT TCT-3¢ The oligomers were 5¢ end-labelled by [c-32P]ATP with T4 polynucleotide kinase (MBI, Fermentas, MGMBH, St Leon-Rot, Germany) Cell culture and cytotoxic assay The T-lymphoblastic leukaemic cell line (CCRF-CEM) and normal human lymphocytes, obtained from peripheral blood by separation on Ficoll–Isopaque (Gibco BRL, Life Technologies, Milan, Italy), were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), mM )1 )1 L-glutamine, 100 mL penicillin and 100 lgỈmL streptomycin (Euroclone, Celbio, Devon, UK) CCRF-CEM cells in exponential growth phase, and lymphocytes, were seeded at 104 cells in 100 lL of complete medium containing 10% fetal clone serum (Euroclone, Celbio) in a 96-well microtiter plate The oligonucleotides were added directly to the medium h after seeding After 24 h of incubation, 100 lL of fresh medium was added The cellular growth was evaluated 72 h after addition of oligonucleotides by assessing the incorporation of 0.5 mgỈmL)1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co., St Louis, MO, USA) into viable cells [19] The percentage of viable cells in the treated samples was estimated, taking, as 100% cellular viability, that of the internal-control nontreated cells Preparation of total nuclear extracts Total nuclear extracts were obtained from · 107 cells by using a minor modification of the Dignam’s method, as previously described [12] The protein content was determined by the Bradford method [19] using BSA as standard EMSA, UV crosslinking and supershift assays In the EMSA, ng of [c-32P]ATP-labelled oligonucleotide was incubated with lg of total nuclear or cytoplasmic extracts supplemented with protease inhibitors (2 lgỈmL)1 apoprotinin, lgỈmL)1 pepstatin, mM dithiothreitol) (Sigma Chemical Co.) in 20 mM Hepes, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, pH 7.0, containing nonspecific competitors [1 lg of salmon-sperm DNA or lg of poly(dIdC)] (Pharmacia, Uppsala, Sweden) and the indicated amounts of unlabelled specific CT oligomer competitor When indicated, the protein excised from the Coomassie-stained gel was 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 dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride 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 buffer Therefore, a fixed aliquot of the protein was incubated with the indicated probes, as previously described After 25 of incubation at room temperature, the samples were loaded onto a native 7% polyacrylamide gel in 20 mM Tris/borate/0.5 mM EDTA buffer (TBE) and electrophoresed at 10 V cm)1, at a temperature of °C In the supershift gel-mobility assay, samples of total nuclear extracts were diluted : (v/v) in water and 0.5 lg of protein was incubated for 2.5 h at room temperature with the indicated amounts of specific rabbit polyclonal antieEF1A serum or with corresponding amounts of control total serum obtained from unimmunized rabbits Then, ng of specific [c-32P]-labelled GT oligonucleotide was added to 30 lL of 20 mM Tris/HCl buffer, pH 7.5, containing 75 mM KCl, mM dithiotreitol, lg BSA, 0.1% Tween 20, 0.025 mM Escherichia coli DNA, and 15% glycerol After 30 of incubation at room temperature, the samples were loaded onto a 7% native polyacrylamide gel in 20 mM TBE buffer and electrophoresed for 90 at 10 V cm)1, at 15 °C The gel was then dried and autoradiographed In the UV cross-linking assay, the samples were incubated at room temperature, as described above for the EMSA assay, and then irradiated at 302 nm for 10 using a transilluminator (Bio-Rad Laboratories) The samples were denatured by adding Laemmli sample buffer and boiled before electrophoresis through a 12% SDS polyacrylamide gel, according to the procedure of Laemmli [20] Electroblotting Nuclear proteins separated by SDS/PAGE were electrophoretically transferred onto a 0.22-lm nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) in 50 mM Tris, 40 mM glycine, 0.4% SDS, 20% methanol buffer using a transblot semidry apparatus system Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur J Biochem 270) 3253 (Amersham Pharmacia Biotech) The membrane was stained with Ponceau S (Sigma Chemical Co.) and destained with deionized water SouthWestern blotting analysis Fifty micrograms of total nuclear protein was separated by SDS/PAGE (29 : 1, acrylamide/bisacrylamide) (8% gel) Proteins were transferred to nitrocellulose and then denatured and slowly renatured by washing for 30 at room temperature consecutively with 6-, 4-, 3-, 2- and M guanidine hydrochloride solution in water The membrane was incubated overnight on a rocking shaker, at °C, in 50 mM Hepes, pH 7.2, containing 0.1 M KCl, mM MgCl2, mM dithiothreitol, mM EDTA and 10% glycerol Membranes derived from bidimensional PAGE blotting were processed without the denaturation/renaturation procedure Membranes were blocked by washing with the same buffer, containing 5% nonfat dried milk and mM dithiothreitol, for h Protein–DNA interaction was performed overnight, at °C, with 10 pmol of [c-32P]-labelled oligonucleotide Membranes were then washed between two and four times for 10 at room temperature, until the background radioactivity started to decline, and were then exposed to autoradiography Expression of recombinant eEF1A Full-length eEF1A cDNA was cloned into pET11a (kindly provided by Dr George M C Janssen, University of Leiden) at the Nde1/BamH1 site for bacterial expression Recombinant eEF1A protein was obtained from overexpression in E coli [21] Briefly, E coli BL21 cell culture (2 mL), transformed with pET11a–eEF1A, was grown overnight at 37 °C in LB (Luria–Bertani) medium supplemented with 50 lgỈmL)1 ampicillin Fresh and prewarmed (37 °C) LB medium was inoculated with the overnight culture to an absorbance (A) value of 0.05–0.1 The culture was grown until the A reached a value of 0.5–0.7, then isopropyl thio-bD-galactoside was added to a final concentration of mM Expression of eEF1A protein was induced by culturing the cells for additional h at 37 °C Cells were harvested by centrifugation (10 000 g, 10 min, °C), resuspended in 10 mL of lysis buffer (20 mM Tris/HCl, mM dithiothreitol, 250 mM NaCl, mM EDTA, 0.25% Tween-20, 0.3 lgỈlL)1 lysozyme) per gram of bacterial pellet and disrupted by sonication The lysate was centrifuged (10 000 g, 20 min, °C) and the recombinant eEF1A protein collected in the supernatant as a soluble protein Western blotting analysis The blotted membrane was blocked with 3% nonfat dried milk in PBS (NaCl/Pi) and incubated with eEF1A monoclonal antibody (mAb) (1 lg/mL) (Upstate Biotechnology, Lake Placid, NY, USA) in NaCl/Pi, overnight, at °C with constant rocking Then, it was washed twice with deionized water and incubated for 1.5 h with an anti-mouse IgGconjugated horseradish peroxidase secondary antibody (Promega, Madison, WI, USA) After washing once with NaCl/Pi containing 0.05% Tween-20 and four times with deionized water, the nitrocellulose blot was developed using enhanced chemiluminescence detection (Pierce, Rockford, IL, USA) according to the manufacturer’s protocols, and then exposed to X-ray film The same filter was stripped by a 10-min incubation in M guanidine hydrochloride, rinsed with 10 volumes of NaCl/Pi, blocked with 5% nonfat dried milk and then probed with b-actin antibody (VWR International Oncogene) for h, at room temperature, followed by incubation for h with a goat anti-mouse IgM-conjugated horseradish peroxidase secondary antibody (Sigma Chemical Co.) The blot was developed by using the chemiluminescence detection kit Band intensities were evaluated by scanning with a Gel Doc2000 phosphoimager densitometer equipped with a multianalyst PC software analysis system (Bio-Rad Laboratories) To test the nuclear enrichment, the presence of two nuclear transcription factors – the early growth response protein (Egr1) and the TATA-binding protein (TBP) – was confirmed by probing with specific rabbit antibodies (Santa Cruz) on the cytoplasmic and nuclear extracts The membrane was incubated with the antibodies for h at room temperature After three washes with NaCl/Pi containing 0.1% Tween-20, the membrane was incubated with anti-rabbit IgG–horseradish peroxidase conjugate (Sigma Chemical Co.) for 60 at room temperature The filter was then washed several times with NaCl/Pi containing 0.1% Tween-20, and the blot was developed using the enhanced chemiluminescence procedure (Amersham Pharmacia Biotech) MS analysis Bands from SDS/PAGE were excised from the gel, triturated and washed with water Proteins were in-gel reduced, S-alkylated and digested with trypsin, as previously described [22] Digest aliquots were removed and used directly or subjected to a desalting/concentration step on lZipTipC18 (Millipore Corp., Bedford, MA, USA) before analysis by MALDI-MS Peptide mixtures were loaded onto the MALDI target, using the dried droplet technique and a-cyano-4-hydroxycinnamic as matrix, and analysed by using a Voyager-DE PRO mass spectrometer (Applied Biosystems, Framingham, MA, USA) Internal-mass calibration was performed with peptides deriving from trypsin autoproteolysis The mass spectra were acquired in either reflectron or linear mode with delayed extraction Postsource decay (PSD) fragment ion spectra were acquired for intense signals after isolation of the appropriate precursor by using timed ion selection Fragment ions were refocused onto the detector by stepping the voltage applied to the reflectron in the following ratios: 1.000 (precursor ion segment), 0.960, 0.750, 0.563, 0.422, 0.316, 0.237, 0.178, 0.133, 0.100, 0.075, 0.056 and 0.042 (fragment segments) Individual segments were superimposed by using the DATA EXPLORER 4.0 software (Applied Biosystems) All precursor ion segments were acquired at low laser power (variable attenuator ¼ 1950), for less than 200 laser pulses, to avoid saturating the detector The laser power was increased to 200 units for all the remaining segments of the PSD acquisitions Typically, 300 laser pulses were acquired for each fragment-ion segment The PSD data were acquired with an Acquiris digitizer at a digitization rate of 500 MHz 3254 B Dapas et al (Eur J Biochem 270) Ó FEBS 2003 PROTEINPROSPECTOR and PROWL software packages were used to identify spots unambiguously from independent nonredundant sequence databases [23,24] Candidates from peptide-matching analysis were further evaluated by comparison with their calculated mass and pI using the experimental values obtained from bidimensional PAGE Bidimensional gel analysis Proteins of total nuclear extract (30 lg) were precipitated at )20 °C with four volumes of acetone, washed with cold methanol, and dried Pellets were dissolved in 120 lL of rehydration buffer (Amersham Pharmacia Biotech) containing M urea, 2% CHAPS, 0.5% immobilized pH gradient (IPG) buffer (pH 6–11), 65 mM dithiothreitol and 0.01% Bromophenol Blue, and used immediately in bidimensional PAGE experiments IEF was performed on 7-cm IPG strips (range: pH 6–11) by using the IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech) The second dimension was performed on a 12% SDS/PAGE system after equilibrating the strips for 10 in SDS Equilibration buffer containing 50 mM Tris/HCl (pH 8.8), M urea, 30% glycerol, 2% SDS, 2% dithiothreitol and 2.5% iodoacetamide Gels were then used for Western or SouthWestern blot analysis, as described above As internal normalizer, the presence of the nuclear protein, Ran-GTP, was detected by Western blot on the same filters used to analyse eEF1A protein, by using a specific mAb (BD Pharmingen, CA, USA) Results Identification of eEF1A as a nuclear protein specifically related to the cytotoxicity of GT oligomer in cancer cells In order to identify the nuclear proteins that specifically recognize cytotoxic GT oligomers, a 27-mer GT sequence was used [12] This oligomer forms a specific CRC with an apparent molecular weight of 45 ± kDa [12–15] The T-lymphoblastic CCRF-CEM cancer cell line was chosen for this investigation as it was previously used to demonstrate the specific cytotoxic action of the GT oligomers [12–15] Figure 1A shows that in SouthWestern blots, the labelled GT oligomer bound (in a specific manner) two main proteins, named P1 and P2, compared with the binding of a labelled nontoxic CT oligomer, used as a control The latter showed a weak interaction with P1 and P2 proteins, whereas it preferentially bound to a nuclear protein with a mass of 70 kDa (marked by an asterisk), recognized to the same extent also by the GT sequence Binding of the GT oligomer was not a result of DNA interaction with the more abundant components of the nuclear extract, as revealed by Ponceau staining of the immobilized proteins In fact, many other bands, equally or more intense than those recognized by the GT oligomer, were also detected (data not shown) To test for nuclear enrichment, nuclear and cytoplasmic extracts were blotted and assayed for the nuclear proteins Egr1 and TBP The results displayed in Fig 1B clearly indicate that the proteins Egr1 and TBP were detected only in the nuclear fraction This demonstrated that nuclear extracts are effectively enriched in nuclear proteins Moreover, Fig SouthWestern blot analysis of GT oligomer binding to nuclear proteins and immunoblot of the subcellular fractions (A) SouthWestern blot on the nuclear extract Fifty micrograms of total nuclear extract derived from CCRF-CEM cells was separated by SDS/PAGE (8% gel) and then transferred, by semidry blotting, onto a nitrocellulose filter The proteins were denatured and renatured as described in the Materials and methods One half of the filter was tested for protein– DNA interactions with a c32P-labelled GT probe (GT) and the other half with a c32P-labelled CT oligomer (CT) as a control, at °C (each probe counted 650 000 c.p.m.) After incubation overnight, the filters were rinsed and then exposed to Omat XAR Kodak film (B) Immunoblotting of subcellular fractions Twenty micrograms of cytoplasmic (lane 1) or nuclear extract (lane 2) fractions of CCRF-CEM cells was separated by SDS/PAGE (8% gel) After blotting onto nitrocellulose membrane, the filter was probed with the nuclear-specific antibodies anti-TBP or anti-Egr1, as described in the Materials and methods As a loading control, the presence of b-actin protein was also confirmed by using specific antibody, as described in the Materials and methods b-actin, used as a loading control, occurred at a higher level in the cytoplasmic fraction, similar to the cellular distribution of the protein P1 migrated with an apparent molecular mass similar to that previously reported for the CRC (45 ± kDa) [12] In contrast, P2 showed a higher apparent molecular mass P1 and P2 were excised from a Coomassie-stained gel, alkylated and digested MALDI-MS analysis of the P1 digest yielded a series of peptide-mass values that were used for nonredundant sequence database searching (Fig 2A) Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur J Biochem 270) 3255 Fig MALDI-MS analysis of P1 and P2 proteins (A) MALDI-MS analysis of component P1 following digestion with trypsin The mass values reported in the spectrum represent average values Numbers in parentheses indicate amino acid residues in the eEF1A sequence Possible methylation sites are shown and assigned based on the observed mass values, eEF1A sequence and previously published results [25] Peptides originating from trypsin autoproteolysis are indicated as open circles (B) Postsource decay (PSD)-MALDI fragment ion mass spectrum of the P2 tryptic peptide, GLSEDTTEETLK ESFDGSVR, with MH+ at m/z 2201.3 (average value) The mass values reported in the spectrum are indicated as monoisotopic values The comparison of this peptide-mass fingerprint with the theoretical ones, calculated by an in silico digestion of all human sequences occurring in the databases, identified P1 as eEF1A Furthermore, PSD experiments performed on selected peptide precursor ions (i.e m/z 1405.4 and 1780.9) generated internal sequence tags that unambiguously confirmed the nature of this protein (data not shown) Moreover, the spectrum reported in the figure showed the occurrence of a series of signals that were not interpreted simply on the basis of the eEF1A sequence, but according to the post-translational modifications already described for this protein [25] It demonstrated the occurrence of Ne-dimethyllysine (Lys55, Lys165) and Ne-trimethyllysine (Lys79, Lys318) in the eEF1A sample purified from T-lymphoblastic CCRF-CEM cancer cells No data on the modification status of Lys36 (methylation), Glu301 and Glu374 (glyceryl-phosphoryl-ethanolamine addition) were inferred MS analysis allowed a 50% coverage of the entire eEF1A sequence All signals occurring in the spectrum were assigned to this protein, thus ruling out the possibility that other polypeptide species comigrated in SDS/PAGE with eEF1A Moreover, the possibility that P1 was the oncogenic N-terminal truncated form of eEF1A protein, already known as PTI-1 [26,27], seemed unlikely on the basis of the signals occurring in the spectrum reported in Fig 2A In fact, at least eight signals matched perfectly with those expected for eEF1A (MH+ at m/z 1492.9, 2501.8, 2516.9, 2997.6, 3023.6, 3151.9, 3980.3 and 4108.5) and demonstrated the absence of seven of the eight amino acid substitutions described for PTI-1 (Ala65Met, Glu66Gln, Arg67Ser, Lys100Gln, Arg247Gly, Ala281Gly and Arg423Cys, respectively) Moreover, clear MH+ signals, corresponding to the N-terminal region of eEF1A, were present; this region is totally deleted in PTI-1 Similar considerations were taken into account to exclude the possibility that P1 corresponded to isoforms of eEF1A other than eEF1A1, already described Similarly, peptide-mass fingerprint analysis by MALDIMS identified P2 as nucleolin (data not shown) Different authors have already reported this protein as being able to specifically generate a 100-kDa complex with GT oligomers that form a G-quartet structure, thus exerting a cytotoxic effect on human cancer cell lines [10,17] PSD experiments performed on selected precursor ions (i.e m/z 2201.3 and 1649.7) allowed internal sequence tags to be obtained, definitively demonstrating the nature of this species (Fig 2B) The identity of P1 was also assayed by Western-blotting experiments with a mAb for eEF1A As illustrated in Fig 3A, the protein excised from the Coomassie-stained gel was recognized by the specific eEF1A antibody (Fig 3A, lane 3) As controls, recombinant eEF1A protein (Fig 3A, lane 1) and a sample obtained from total nuclear extracts (Fig 3A, lane 2) were tested The EMSA with the protein eluted from the P1 band excised from the Coomassie-stained gel of CCRF-CEM cell nuclear extracts showed that this protein selectively recognized the GT oligomer with respect to control CT sequence, similarly to results obtained with the total nuclear extracts [12–15] It is noteworthy that all the EMSA and UV crosslinking assays were performed using a buffer containing 25% glycerol to preserve the activity of eEF1A As illustrated in Fig 3B, the eEF1A recovered from the P1-excised band showed a stronger interaction when incubated with the labelled GT oligomer (Fig 3B, lane 1) than when incubated with the labelled control CT oligomer (Fig 3B, lane 7) Moreover, the presence of a fivefold molar excess of CT-unlabelled competitor (Fig 3B, lane 5), did not completely displace the GT oligonucleotide from the protein interaction On the contrary, only a fivefold molar excess of GT-unlabelled oligonucleotide competitor removed all the labelled CT control oligomer from the complex (Fig 3B, lane 8) To explore the possibility that eEF1A was the protein component present in the CRC [12], supershift assays were performed under native conditions The results shown in Fig demonstrate that a rabbit polyclonal antibody recognizing eEF1A elicited a specific supershift (marked by an arrow; Fig 4, lanes and 5) from the complex No supershift resulted from incubation of the nuclear protein extract with the same amounts of a total rabbit preimmune serum, which displayed only nonspecific competition (Fig 4, lanes and 7) The slight reduction in DNA-binding 3256 B Dapas et al (Eur J Biochem 270) Fig P1 Western blotting analysis and affinity measurements for GT oligomer (A) Western blotting analysis Protein samples were separated by SDS/PAGE (12% gel) and then transferred onto a nitrocellulose filter and incubated with mAb for eEF1A, as described in the Materials and methods Lane 1, bacterial recombinant eEF1A protein (R eEF1A); lane 2, eEF1A protein from total nuclear extracts (NE eEF1A); lane 3, P1 band excised from an SDS/PAGE gel (P1) (B) P1 affinity for GT oligomer P1 protein, excised from an SDS/PAGE gel loaded with 50 lg of total nuclear extract, was renatured as described in the Materials and methods Five microlitres of sample was then incubated with ng of [c-32P]-labelled GT probe (GT) in buffer (200 mM Tris/HCl, pH 7.5, containing 750 mM KCl, 10 mM dithiothreitol, 50 lgỈmL)1 BSA) in the absence (lane 1) or in the presence of 10 ng (lane 2), 20 ng (lane 3), 50 ng (lane 4), 100 ng (lane 5) or 200 ng (lane 6) of nonlabelled CT oligomer An identical aliquot was incubated with ng of c32P-labelled CT probe (CT) in the absence (lane 7) or in the presence of 10 ng (lane 8), 20 ng (lane 9), 50 ng (lane 10), 100 ng (lane 11) or 200 ng (lane 12) of nonlabelled GT oligomer The two probes were added to the sample at the same specific activity ( 10 000 c.p.m.) Labelled GT oligomer incubated without P1 (lane 13) and labelled CT oligomer incubated without P1 (lane 14) were used as controls After 20 of incubation at room temperature, the samples were loaded onto an 8% polyacrylamide gel in 0.5 · Tris/ borate/EDTA (TBE) buffer and electrophoresed at °C The dried gel was exposed to autoradiographic film The arrow indicates the specific complex activity shown in the presence of control rabbit total serum (CRS) could be caused by nonspecific sequestration of eEF1A by serum proteins More interestingly, as illustrated in Fig 5A, with respect to the protein of the nuclear extracts, the soluble eEF1A recovered from the cytoplamic fraction did not bind to a GT oligomer in SouthWestern blotting Although comparable Ó FEBS 2003 Fig Supershift assay experiments Proteins from CCRF-CEM cell nuclear extracts (0.5 lg) were incubated with or without the indicated amounts of specific polyclonal antibody (Ab eEF1A) or control rabbit total serum (CRS), for 2.5 h at room temperature Then, ng of c32Plabelled GT oligomer was added to the samples, as reported in the Materials and methods After a further 30 of incubation at room temperature, the samples were loaded onto a 7% polyacrylamide gel in 0.5 · Tris/borate/EDTA (TBE) buffer and electrophoresed at °C The gel was dried and exposed to Omat XAR Kodak film c32PLabelled GT oligomer was incubated with buffer (lane 1), with lg of polyclonal anti-eEF1A (lane 2), with lg of total CRS (lane 3), with nuclear proteins and lg of polyclonal anti-eEF1A (lane 4), with nuclear proteins and 0.9 lg of polyclonal anti-eEF1A (lane 5), with nuclear proteins and lg of total CRS (lane 6), with nuclear proteins and 0.9 lg of total CRS (lane 7) or with nuclear proteins only (lane 8) The arrow indicates the supershift quantities of the protein were loaded onto the gel, as evidenced by Western blotting, in the cytoplasmic extract, only the nucleolin band was evident Similarly to this and to the previous results [12], in the UV cross-linking assay the specific CRC displayed by the nuclear extract was not present in the cytoplasmic sample (Fig 5B) On the contrary, the cytoplasmic extract showed a band of about 28 kDa, previously demonstrated to bind to GT in a nonspecific manner [12] Characterization of eEF1A in normal and cancer cells In order to compare the binding properties of the nuclear eEF1A in normal cells compared with those in cancer CCRF-CEM cells, human lymphocytes were isolated from the peripheral blood of normal donors These cells were not sensitive to the cytotoxic effect of GT oligomers Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur J Biochem 270) 3257 Fig SouthWestern blot, Western blot and UV cross-linking analysis of cytoplasmic extracts (A) SouthWestern blot Twenty-five micrograms of total protein from cytoplasm or nuclear extracts, previously normalized by comparison on a Coomassie-stained gel, were separated by SDS/PAGE (8% gel) and transferred onto a nitrocellulose filter, as described in the Materials and methods The proteins were denatured, renatured and the filter hybridized with a c32P-labelled GT probe at °C After overnight incubation, the filter was rinsed and then exposed to Omat XAR Kodak film, as described in the Materials and methods The same samples were used for Western blotting analysis performed with the same amount of the cytoplasm and nuclear proteins used in the SouthWestern blot The specific protein was confirmed by using the monoclonal anti-eEF1A with the conditions described in the Materials and methods (B) UV crosslinking assay Two micrograms of total proteins derived from the cytoplasm or nuclear extracts were incubated in buffer containing 25% glycerol, with c32P-labelled GT probe in the presence of lg poly(dIdC) and lg of CT as competitors, as described in the Materials and methods After 25 of incubation at room temperature, the samples were cross-linked by UV exposure and then denatured and separated by SDS/PAGE (12% gel) The dried gel was then exposed to Omat XAR Kodak film NE, nuclear extract; CE, cytoplasmic extract (Fig 6A), and their nuclear proteins did not form the CRC with the GT sequence (marked by arrow), as shown by EMSA or UV cross-linking assays (Fig 6B,C) To investigate the binding properties of the lymphocyte Fig Effect of GT oligomer on cellular growth and on nuclear protein binding in human lymphocytes (A) Effect of GT oligomer on cellular growth or viability A total of 104 CCRF-CEM cells or peripheral normal human lymphocytes were seeded in 100 lL of complete medium on 96-well microtiter plates After h of incubation, 7.5 lM of GT oligomer or control CT sequence were added to the cells The percentage of viable cells was assayed after 72 h of incubation by determining the incorporation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, as described in Materials and methods (B) EMSA assay Two micrograms of total nuclear proteins derived from CCRF-CEM cells or from human lymphocytes were incubated with 1.5 lg of poly(dIdC), lg of CT and ng of c32P-labelled GT oligomer in a buffer containing 25% glycerol, as described in the Materials and methods After incubation at room temperature for 30 min, the samples were loaded onto a 7% polyacrylamide gel in Tris/ borate/EDTA (TBE) buffer and run at °C (C) UV cross-linking assay Two micrograms of total nuclear proteins derived from CCRFCEM cells or from human lymphocytes were incubated with 1.5 lg of poly(dIdC), lg of CT and ng of c32P-labelled GT oligomer, as described in the Materials and methods The samples were then exposed for 10 to a 302 nm UV light, added to SDS/PAGE loading buffer and separated by SDS/PAGE (12% gel) The arrows indicated the specific cytotoxicity-related complex eEF1A protein, SouthWestern blots were performed It was found that the CCRF-CEM nuclear extracts contained higher amounts of eEF1A than those of human lymphocytes In fact, Western blotting experiments demonstrated that the relative amount of eEF1A recovered from lymphocyte nuclear extracts was 2.7 ± 0.8-fold less than that obtained from cancer CCRF-CEM T lymphoblasts (mean of three independent experiments) For this reason, the SouthWestern assay was performed after 3258 B Dapas et al (Eur J Biochem 270) normalizing the quantities of loaded proteins on a Coomassie-stained gel by referring to the P1 band (known to correspond to eEF1A) As reported in Fig 7A, in the CCRF-CEM sample the GT oligomer recognized nucleolin (marked by a black arrow), eEF1A (marked by a white arrow) as well as the nonspecific 70 kDa protein (marked by an asterisk) On the contrary, in human lymphocytes the GT oligomer did not bind to eEF1A, but it significantly recognized nucleolin To rule out that protein-degradation artefacts might account for the lack of specific recognition, the same amounts of protein used in SouthWestern experiments were assayed by Western blotting As shown in Fig 7B, the amount of eEF1A in human lymphocytes was comparable to that in CCRFCEM cancer cells On the contrary, b-actin showed significant differences in the two samples, in agreement with the higher quantity of total proteins loaded in the lymphocyte sample, as mentioned above Fig Comparative SouthWestern blot for CCRF-CEM cells and normal lymphocytes (A) SouthWestern blot Twenty-five micrograms of total nuclear protein from CCRF-CEM cells and 50 lg of total nuclear protein from normal human lymphocytes were separated by SDS/PAGE (8% gel) and transferred onto a nitrocellulose filter, as described in the Materials and methods The proteins were denatured and renatured as described above, and the filter was hybridized with c32P-labelled GT probe at °C After overnight incubation with the probe, the filters were rinsed, as described in the Materials and methods, and then exposed to Omat XAR Kodak film (B) Westernblotting analysis Protein samples reported in (A) were analysed for eEF1A and b-actin content Ó FEBS 2003 Previous data demonstrated that the eEF1A protein is post-translationally modified [25,28,29] In order to detect whether this protein presented a different molecular nature in normal and cancer cells, we performed a comparative bidimensional PAGE analysis of nuclear extracts coupled to Western blotting analysis with an eEF1A mAb As an internal normalizer of loading amounts and focusing position, the nuclear protein, Ran-GTP, was used and identified by a specific mAb The data reported in Fig 8A clearly showed, in T-lymphoblastic CCRF-CEM cancer Fig Bidimensional PAGE analysis of nuclear elongation factor alpha (eEF1A) (A) Thirty micrograms of nuclear extracts from CCRF CEM cells and normal human lymphocytes, calculated from evaluation of the protein content in a Coomassie-stained gel, were analysed by bidimensional PAGE, as described in the Materials and methods The presence of eEF1A was tested by Western blot analysis by using the specific antibody anti-eEF1A As an internal normalizer of loading amount and focusing position, the presence of the constitutive nuclear transporter, Ran-GTP, was also tested by using a specific monoclonal antibody (B) Bidimensional PAGE analysis of other samples, confirming the reproducibility of data obtained Thirty micrograms of nuclear extracts from normal human lymphocytes were similarly analysed by bidimensional PAGE and compared with CCRF-CEM nuclear extracts The presence of eEF1A was confirmed by Western blot analysis using the specific antibody, anti-eEF1A Only the higher magnification of IEF of the eEF1A region is reported Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur J Biochem 270) 3259 cells, the presence of two different clusters of eEF1A isoforms (cluster and cluster 2) The apparent pI of cluster ( pH 9.0), was calculated theoretically by considering the pH gradient linear, as indicated by the manufacturer In nuclear extracts from lymphocytes, a similar species focused near the same pH Interestingly, cluster 2, which was absent in lymphocytes, focused at an apparent pH of 10.5 Thus, it accounted for a more basic pI and for species different from those present in lymphocytes However, taking apart the undoubtedly much more basic nature of the fastermigrating isoform of eEF1A in cancer cells, the estimation of its pI remains merely indicative A magnified image of this analysis, performed with a different nuclear extract, is reported in Fig 8B It is clear that human lymphocytes displayed only the main constitutive species that migrates in correspondence to cluster of CCRF-CEM cells (see Fig 8B) To elucidate the GT oligomer binding behaviour of the isoforms found in the cancer cell sample, a SouthWestern assay was performed after analysis by bidimensional PAGE The antibody recognition of the proteins performed on the same filter was prevented by the SouthWestern treatment Therefore, identification of the eEF1A–oligomer interaction was carried out by matching the SouthWestern results with those of the Western blotting of bidimensional PAGE performed on the same sample, Fig Comparative analysis of bidimensional PAGE analysis of SouthWestern and Western blots for CCRF-CEM cells Two samples of 50 lg of total nuclear protein from CCRF-CEM cells were separated, in parallel, by bidimensional PAGE and blotted onto nitrocellulose filters, as described in the Materials and methods (A) Western blotting The filter was assayed using anti-eEF1A mAb, as described in the Materials and methods The position of the eEF1A protein was confirmed by revealing the presence of the Ran–GTP protein (B) SouthWestern blotting The filter was assayed for SouthWestern blotting, as described in the experimental section using, as probe, c32Plabelled GT oligomer and then exposed to Omat XAR Kodak film under identical experimental conditions The perfect match between the Western blot signals of bidimensional PAGE (Fig 9A) and the autoradiographic signals found in the SouthWestern blot (Fig 8B), unequivocally demonstrated that the protein reacting in SouthWestern blots was eEF1A Furthermore, the SouthWestern blot, reported in Fig 9B, showed that the labelled GT oligomer mainly recognized the more basic form of eEF1A, whereas a very weak interaction was found for the isoform of eEF1A focusing at a pH of 9.0 The recognition seemed highly specific because, under these experimental conditions, no other interactions were detected on the filter Moreover, no significant interaction in bidimensional PAGE SouthWestern blots was found on human lymphocytes at the position corresponding to eEF1A (see Fig 10), once more indicating that normal eEF1A did not react with the GT oligomer Fig 10 Comparative analysis of bidimensional PAGE, SouthWestern and Western blotting for human lymphocytes Two samples of 50 lg of total nuclear protein of normal human lymphocytes, previously normalized with respect to CCRF-CEM cell protein by a Coomassiestained gel, were separated, in parallel, by bidimensional PAGE and blotted onto nitrocellulose filters, as described in the Materials and methods (A) Western blotting The filter was assayed for Western blot using anti-eEF1A mAb, as described in the Materials and methods The position of the eEF1A protein was focused by revealing the presence of the Ran–GTP protein (B) SouthWestern blotting The filter was assayed for SouthWestern blot as described in the Materials and methods using, as probe, c32P-labelled GT oligomer and then exposed to Omat XAR Kodak film 3260 B Dapas et al (Eur J Biochem 270) Discussion Eukaryotic EF1A is a protein belonging to the GTPbinding elongation factor family, which promotes the GTPdependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis and the capture of deacylated tRNA at the exit site and its delivery to synthase [30] It has been suggested that eEF1A might serve also as a downstream component of growth-signalling pathways, possibly through its capability to interact with actin, thus promoting cell transformation [31] An overexpression of eEF1A has been shown in many tumours [32], and eEF1A has been associated with a highly metastatic potential in cancer cells [33] The regulation of eEF1A expression by extracellular stimuli depends on human epidermal growth factor (EGF) receptor family members that are widely deregulated in human cancers [34] eEF1A is the second most abundant protein in the cell; whereas the b, c and d subunits of eEF1 are predominantly located in the cytoplasm, a considerable fraction of the a subunit can be found either in the cytoplasm or the nucleus [35,36] The involvement of eEF1A in the regulation of nuclear processes includes the accumulation of a nuclear complex with vigilin, for exporting tRNA [37], and with ZPR1, for inducing cell proliferation upon mitogen stimulation [38] Thus, the elucidation of a possible role for the nuclear fraction of eEF1A in modulating nuclear function and gene expression could gain new insights in tumorigenesis In this manuscript, we demonstrate that eEF1A, isolated from nuclear extracts of CCRF-CEM cancer cells, is specifically recognized by a cytotoxic GT sequence This protein was found to be the polypeptide component of the CRC, based on MALDI-MS analysis, Western blotting experiments and supershift assays In contrast, the GT oligomer did not bind to the eEF1A of normal human lymphocytes and these cells were not sensitive to the cytotoxic action of the GT It should be noted that nucleolin was also recognized by the GT oligomer [10,17] However, under native conditions, the more abundant CRC observed, migrated with an apparent mass not associated with the nucleolin–oligomer complex [12–15], probably because the GT sequence used does not form, in appreciable quantity, the G-quartet structure specifically recognized by this protein, as revealed by gel electrophoresis and circular dichroism studies [12,14] Moreover, nucleolin, and not eEF1A, was bound by the GT oligomer in lymphocyte sample on one-dimensional SouthWestern assay; however, lymphocyte viability was not affected by GT In cytoplasmic extracts, the nucleolin was found to bind to GT in a SouthWestern assay, but not in EMSA or UV crosslinking assays Furthermore, we analysed a G-rich GT sequence able to form the G-quartet structure and thus to bind to nucleolin We found that this oligomer did not elicit cytotoxicity on CCRF-CEM cells, although it was efficiently taken up by the cells More interestingly, in UV cross-linking competition experiments, this sequence did not displace the labelled GT from the CRC but from the less represented lower migrating complex (corresponding to nucleolin) (data not shown) On this basis, we can hypothesize a minor involvement of nucleolin in the mechanism of cytotoxicity elicited by the GT oligomers [12] It seemed Ó FEBS 2003 probable that the reactivity of the nucleolin was related much more to the experimental conditions of the immobilized protein on the SouthWestern assay, than to a native binding affinity for the GT Nevertheless, we cannot completely exclude that other, less-abundant proteins can contribute to this effect Binding assays by SouthWestern experiments demonstrated that nuclear eEF1A affinity for GT oligomers was significantly higher than that measured for the control CT sequence Moreover, in EMSA assays a significant quantity of GT oligomer remained bound to the protein, derived from the excised P1 band, in the presence of a 50-fold molar excess of CT oligomer competitor, whereas only a fivefold molar excess of GT oligomer was sufficient to release all control CT oligomer from the complex It is noteworthy that the P1 band excised from the Coomassie-stained gel of a normal lymphocyte sample failed to form complexes in EMSA with GT oligomer (data not shown) Furthermore, overloaded protein samples from normal lymphocytes did not show significant interaction between the eEF1A protein and the cytotoxic GT oligomer in SouthWestern assays Accordingly, GT oligomers did not elicit cytotoxic action on these cells, and did not form the CRC with the nuclear proteins when a fourfold increase in protein content was loaded onto the gel (data not shown) These results underline that eEF1A from CCRF-CEM cell nuclear extracts displays specificity in recognizing GT oligomers, and the selective cytotoxic action on CCRF-CEM cells suggests a possible role for eEF1A in maintaining the viability and proliferative activity of cancer cells One hypothesis may be that these oligomers exert their action by blocking the binding of eEF1A to its ligand in cancer cells, perhaps to zinc finger proteins involved in the modulation of cell proliferation, as proposed by Gangwani et al [38] Bidimensional PAGE analysis of eEF1A combined with a specific Western blotting assay showed the occurrence of two distinct clusters of spots in T-lymphoblastic CCRFCEM cells, whereas normal lymphocytes presented only one cluster In particular, the newly occurring components in cancer cells (cluster 2) focused at a more basic pH This result could hypothetically explain the higher affinity of the protein towards oligonucleotides simply on the basis of a charge increase at specific amino acids in its nucleotidebinding site, but not its binding selectivity for the GT sequences Different post-translational modifications have been reported to occur in the eEF1A polypeptide chain, such as phosphorylation, methylation and glyceryl-phosphorylethanolamine addition [25,27,29,39,40] but, to date, their functional significance has not been totally solved Differences in the level of phosphorylation of eEF1A have already been reported to be associated with variation in binding affinity towards viral genomic RNA, as well as to regulative interconversion between active and inactive forms [41] Our findings should not sustain the hypothesis that eEF1A propensity for recognition of GT oligomers in CCRF-CEM cancer cells might be related merely to an increase of the phosphorylation state On the contrary, the modification of eEF1A mobility on bidimensional PAGE by increasing its pI value should be associated with the presence of other post-translational modifications Methylation of eEF1A has been significantly associated with SV40 transformation in Ó FEBS 2003 eEF1A binding to aptameric cytotoxic GT oligomers (Eur J Biochem 270) 3261 mouse 3T3B cells [42] It was suggested that this modification should account for differences in growth properties for the different cell types Similarly, in Mucor racemous it was observed that, during morphogenesis, a sixfold elevation in eEF1A specific activity is accompanied by a dramatic increase in protein methylation at as many as nine lysine residues, without any change in the protein or mRNA levels [43] This modification, as well as other post-translational modifications, not deduced by simple mass fingerprint analysis on the resolved spots, cannot be excluded Furthermore, the eventual presence of specific amino acid substitutions in the eEF1A gene, producing the more basic isoform observed by bidimensional PAGE, has to be considered However, the possibility that this basic species corresponded to the eEF1A2 isoform already known, described in brain and skeletal muscle [44], or to PTI-1, an oncogenic truncated form of eEF1A [26], was ruled out on the basis of the peptide-fingerprint experiments reported above By using SouthWestern assays on bidimensional PAGE of CCRF-CEM cell nuclear extracts, we observed that the more basic isoform of eEF1A binds to the GT oligomer, whereas only a weak interaction was found for the eEF1A migrating like the normal constitutive molecule Accordingly, eEF1A from normal human lymphocytes did not recognize GT oligomer at all On this basis, it could be tempting to speculate that different isoforms of nuclear eEF1A, in particular a more basic molecule, should account for the different sensitivities to cytotoxic effect exerted by GT oligomers These differences might be related to a variable degree of post-translational processing, although the high shift in the apparent pI of the more basic molecules cannot alone be simply explained by modifications such as methylation of the lysine residues Even if theoretically possible, this phenomenon might account for a low probable high number of methylated lysine residues in a protein with a total low-density charge Thus, in these high basic proteins it might be that either methylation of the lysine or specific substitutions in the amino acid sequence, increasing the number of basic residues of lysine, arginine and histidine, could occur However it seemed probable that the eEF1A isoforms could act as a controlling event in maintaining the viability and/or promoting the growth of T-lymphoblastic tumour cells Supporting evidence for the specific role of this 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Identification of eEF1A as a nuclear protein specifically related to the cytotoxicity of GT oligomer in cancer cells In order to identify the nuclear proteins that specifically recognize cytotoxic GT oligomers, ... by cytotoxic GT oligomers and their selective binding to nuclear eEF1A In fact, in normal human lymphocytes no appreciable binding of GT oligomer to nuclear eEF1A was shown and, accordingly, these... protein belonging to the GTPbinding elongation factor family, which promotes the GTPdependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis and the capture of