Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers
Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomers
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DOI：
10.1046/j.1432-1033.2003.03713.x
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发表时间：
2003-08-01
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影响因子：
--
通讯作者：
Scaggiante, B
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文献类型：
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作者：
Dapas, B;Tell, G;Scaggiante, B
GT寡聚体，显示出剂量依赖性的细胞毒性作用，对各种人类癌细胞系，但不对正常的人类淋巴细胞，识别和形成复合物与核蛋白。通过与人T淋巴母细胞CCRF-CEM细胞的工作，并通过使用MS和SouthWestern印迹，我们确定了真核延伸因子1 α（eEF 1A）作为主要的核蛋白，特异性地识别这些寡核苷酸。Western印迹和supershift测定证实了这种蛋白质的性质及其参与形成细胞毒性相关复合物（CRC）。相反，正常人淋巴细胞在Southwestern印迹中没有显示能够产生CRC的核蛋白。比较双向PAGE和蛋白质印迹分析eEF 1A揭示了一个特定的集群点的存在下，集中在更碱性的pH值，在癌细胞的核提取物，但在正常淋巴细胞中不存在。此外，双向PAGE SouthWestern印迹表明，细胞毒性GT寡聚体选择性地识别仅在癌细胞中表达的更基本的eEF 1A同种型。这些结果表明，参与的eEF 1A，与核富集部分，在肿瘤细胞的生长和维护，可能调制的多肽链的翻译后加工。
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.