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GToligomers, showingadose-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 1 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) | Eur. J. Biochem. 270 3251-3262 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03713.x Identification of different isoforms of eEFIA in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers Barbara Danas1 Gianluca Tell2. Andrea Scaloni3 Alex Pines2 Lino Ferrara3 Franco Quadrifoalio1 and Bruna Scaggiante1 1 Department of Biomedical Sciences and Technologies University of Udine Italy department 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 Irmam Tllymphoblatiic CCRF-CEM cells and by using MS and Southwestern blotting we identified eukaryotic elongation factor 1 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 Ịm l 3ỵytfô did not show nuclear proteins able to produce CRC in a Southwestern blot. Comparative bídimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of c specific cluster of spots focusing at more basic pH in nuclear extracts of cancer colls but absent in those of normal lymphocytes. Moreover a bidimansi illal PAGE Oouth-western blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer colls. TOeve eesulSs suggest tOe 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. Keywords aptameric oligonucleotides eEF1A proteomics CCRF-CEM colls .
