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http://hdl.handle.net/10773/18869
Title: | Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA |
Author: | Bordeira-Carriço, Renata Ferreira, Daniel Mateus, Denisa D. Pinheiro, Hugo Pêgo, Ana Paula Santos, Manuel A. S. Oliveira, Carla |
Keywords: | hereditary diffuse gastric cancer (HDGC) nonsense suppression premature termination codon (PTC) suppressor-trna |
Issue Date: | 2014 |
Publisher: | Nature Publishing Group |
Abstract: | Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/E-cadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin- deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset. |
Peer review: | yes |
URI: | http://hdl.handle.net/10773/18869 |
DOI: | 10.1038/ejhg.2013.292 |
ISSN: | 1018-4813 |
Appears in Collections: | CESAM - Artigos DBio - Artigos |
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Bordeira-Carrico et al. - 2014 - Rescue of wild-type E-cadherin expression from non.pdf | 1.56 MB | Adobe PDF | ![]() |
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