Tài liệu Báo cáo khoa học: Identification of different isoforms of eEF1A in the nuclear fraction of

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

1

Department of Biomedical Sciences and Technologies, University of Udine, Italy; 2

Department of Biochemistry, Biophysics

and Macromolecular Chemistry, University of Trieste, Italy; 3

Proteomics 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 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 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 South￾Western 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.

Keywords: aptameric oligonucleotides; eEF1A; proteomics;

CCRF-CEM cells; cytotoxicity.

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, oligo￾nucleotides have progressively gained aptameric function,

specifically recognizing proteins as natural or non-natural

ligands [4].Constitutive proteins that bind to single￾stranded 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 DNA￾binding 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 oligonu￾cleotide 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 ± 7 kDa, with nuclear proteins of

different tumour cell lines [12–15].Thus, the characteriza￾tion 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: [email protected]

Abbreviations: CRC, cytotoxicity-related complex; CRS, control

rabbit total serum; eEF1A, eukaryotic elongation factor 1 alpha;

Egr1, early growth response protein 1; IPG, immobilized pH gradient;

PSD, postsource decay; TBP, TATA-binding protein.

(Received 7 March 2003, revised 27 May 2003, accepted 10 June 2003)

Eur. J. Biochem. 270, 3251–3262 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03713.x