Construction of the plasmid for expression of ETA-EGFP fusion protein under control of the cytomegalovirus promoter and its effects in HeLa cells.
Tumor-targeted vectors encoding toxic protein genes are promising tools for treating malignant tumors. We used the pEGFP-N1 vector to construct a novel plasmid (pCMV-ETA-EGFP) for eukaryotic expression of a truncated Pseudomonas aeruginosa exotoxin A (ETA) that is known to inhibit protein synthesis, and subsequently induce cell death, by inactivation of elongation factor-2. ETA was linked to the enhanced green fluorescent protein (EGFP) gene, and ETA-EGFP gene expression was driven by the cytomegalovirus (CMV) promoter. The time-lapse effects of pCMV-ETA-EGFP expression were examined in transiently transfected HeLa cells. HeLa cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and small er, Cyrilliccapital IE, CyrillicGFP-N1 showed lower fluorescence intensity than cells transfected with pEGFP-N1 alone. Analysis of the number of dead cells further confirmed the highly toxic effect of the ETA-EGFP fusion protein on cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and small er, Cyrilliccapital IE, CyrillicGFP-N1. ETA-EGFP fusion protein induced apoptotic cell death through the caspase-3 activation. By using the antibody against a marker nucleolar antigen A3 [Grigoryev, A.A., Bulycheva, T.I., Sheval, E.V., Kalinina, I.A., Zatsepina, O.V., 2008. Cytological indicators of the overall suppression of protein synthesis revealed by staining with new monoclonal antibody. Cell Tissue Biol. 2, 191-199], the distribution of which changes when HeLa cells are treated with known translation inhibitors, we obtained evidence to support the idea that protein synthesis is inhibited in transfected cells in situ. ETA-EGFP fusion protein was identified in lysates of transfected cells using anti-GFP-BL antibodies. Collectively, our results indicate that HeLa cells transfected with pCMV-ETA-EGFP synthesize the ETA-EGFP fusion protein that efficiently inhibits protein synthesis, leading to massive cell death by an apoptosis-mediated pathway with a participation of caspase-3. The constructed vector can be used in suicidal gene therapy of cancer and may also be useful for investigating the general effects of translational downregulation in human cancer cells. We also suggest a novel approach for detecting the activity of new vectors in transfected cells, which is based on the redistribution of nucleolar proteins in transfected cells.
Glinka
Elena M
EM
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. em_glinka@mail.ru
Andryushchenko
Alla S
AS
Sapozhnikov
Alexander M
AM
Zatsepina
Olga V
OV
D036002:ADP Ribose Transferases / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D005098:Exotoxins / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection; D037521:Virulence Factors / Q000235:genetics*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D049452:Green Fluorescent Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C485184:enhanced green fluorescent protein