Identification of different isoforms of eEF1A in the nuclear fraction of human T‐lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers

Abstract

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.

Increased Proteasome Activity, Ubiquitin-Conjugating Enzymes, and eEF1A Translation Factor Detected in Breast Cancer Tissue

“…4). The increased level of eEF1A in cancers has been described previously (20,21), and the results shown here suggest that a common cellular response to protein Figure 5. Composite data.…”

“…Immunoblotting studies revealed dramatic increase in eEF1A levels, suggesting that defects in protein synthesis quality control are associated with breast cancer. Because eEF1A expression is also increased in other cancers (20,21), it is likely that this result reflects a more general response to aberrant growth (22), unlike the specific expression of certain Ubconjugating enzymes in breast cancer. Significantly, none of our findings were reproduced in a well-defined cell culture system that is used as a model system for breast cancer (23,24).…”

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

“…61 Another eEF1A isoform has also been identified as the main nuclear protein in human T-lymphoblastic CCRF-CEM cells that specifically recognizes GT oligomers to form a cytotoxicity-related complex that shows a dose-dependent cytotoxic effect on a variety of human cancer cell lines but not on normal human lymphocytes. 62 This ability of both eEF1A1 and eEF1A2 to form complexes with so many different proteins, which might be explained by the partially unstructured conformation that has been detected in solution for eEF1A, 30 reveals a more complex role for these eEF1 subunits beyond protein biosynthesis. In fact, the multicomponent eEF1 system appears to behave as a sophisticated regulatory factor rather than as a mere "housekeeping" element of the cell, and its activity appears to be coordinated with cell replication.…”

EEF1A1 (eukaryotic translation elongation factor 1 alpha 1)

“…An isoform of eEF1A1 with a more basic isoelectric point was identified in human haematopoietic cancer cell lines but not in normal lymphocytes raising the possibility that posttranslation modifications of eEF1A1 could be involved in cancer development and progression of haematopoietic tumours (Dapas et al, 2003). The selective inhibition of eEF1A1 isoform by specific GT aptameric oligonucleotides, carried by the ethoxylated polyethylenimine, a weak basic polycation, has been demonstrated to exert specific cytotoxic effect and dose-dependent cell growth inhibition on lymphoblastic cancer cells (Scaggiante et al, 2005).…”