Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers.
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 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.
Dapas
Barbara
B
Department of Biomedical Sciences and Technologies, University of Udine, Italy.
Tell
Gianluca
G
Scaloni
Andrea
A
Pines
Alex
A
Ferrara
Lino
L
Quadrifoglio
Franco
F
Scaggiante
Bruna
B
D001665:Binding Sites; D026501:Blotting, Southwestern; D002455:Cell Division / Q000187:drug effects; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D006801:Humans; D015458:Leukemia, T-Cell / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D020648:Peptide Elongation Factor 1 / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013941:Thymine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D009687:Nuclear Proteins; D009841:Oligonucleotides; D020648:Peptide Elongation Factor 1; D020033:Protein Isoforms; D006147:Guanine; D013941:Thymine