Suppression of a nonsense mutation in mammalian cells<i>in vivo</i>by the aminoglycoside anthiotics G–418 and paromomycin

Burke, Julian F.; Mogg, Anne E.

doi:10.1093/nar/13.17.6265

Cited by 194 publications

(161 citation statements)

References 11 publications

Supporting

Mentioning

156

Contrasting

Unclassified

Order By: Relevance

“…However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5,6).…”

mentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

View full text Add to dashboard Cite

Cells were irradiated with 10 Gy and were incubated for 45 min at 37°C before fixation on coverslips. ATM pS1981 IRIFs were not observed in untreated cells; maximum intensity of staining was seen in the nuclei of cells exposed to 75 g͞ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci. A taxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder with onset in early childhood, resulting from mutations in the A-T mutated (ATM) gene (1). The ATM protein is a hierarchical serine-threonine kinase, phosphorylating many substrates involved in repair of doublestranded DNA breaks, control of cell cycle checkpoints, and responses to oxidative stress, as well as in radiosensitivity, cancer susceptibility, immune function, and neurological development (2). ATM protein levels are undetectable in the cells of most A-T patients by conventional testing. However a few patients, often with a milder phenotype, have some detectable ATM protein (3,4). This finding encouraged us to seek compounds, such as aminoglycosides, that have the potential to read through premature termination codons and restore ATM protein function (5, 6).To test these effects, we selected 13 lymphoblastoid cell lines (LCLs) with primary premature termination codon (PTC) mutations from a repository of Ͼ400 lines derived from A-T patients. Herein, we show representative data from 5 of the 13 LCLs. Using protein truncation testing (PTT) driven by plasmid templates containing PTC mutations in the ATM gene (7), we observed in vitro read-through effects of various magnitudes with three of four aminoglycosides tested. Full-length ATM protein was also documented ex vivo by immunoprecipitation. Correction of radioresistant DNA synthesis and radiosensitivity, as well as autophosphorylation of ATM, suggested that this readthrough produces functional ATM protein. Experimental ProceduresCell Lines. Lymphoblastoid cell lines were maintained in RPMI medium 1640 (Invitrogen) with 15% FBS (HyClone) and 1% penicillin͞streptomycin (Invitrogen) at 37°C and 5% CO 2 . In ex vivo experiments, aminoglycosides were added daily into culture media at the indicated doses and times. Because streptomycin is also an aminoglycoside that can perturb proofreading by binding to another ribosomal site, ex vivo experiments were performed with or without streptomycin; no differences were noted.Mutations. We selected only PTC mutations that resulted directly from the mutation, i.e., primary PTC mutations, and not those caused indirectly by upstream mutations or aberrant splicing, because the latter would have no potential therapeutic benefits. The LCLs carried the following mutations (www.benaroyaresearch.org͞bri investigators͞atm.htm): AT185LA, homozygous 3673C 3 T (a UAA G stop codon in PTT fragment 4); TAT51, homozygous 5623C 3 T (a UGA C stop codon in PTT fragment 5); AT187LA, homozygous 5908C 3 T (a UAA G stop codon in PTT fragment 6); AT153LA, homozygous 8977C 3 T (a UGA A stop codon in fragment 8); and AT...

show abstract

mentioning

confidence: 99%

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Lai

Chun

Nahas

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

146

102

View full text Add to dashboard Cite

show abstract

“…Aminoglycoside antibiotics are active in the codonanticodon recognition of aminoacyl tRNAs during translation. 3,29,30 Significantly, 10-20% of the mutations identified in DMD and DMC are nonsense mutations resulting in premature stop codons. 18,21 In the analyses conducted to date, the role of the stop codon nucleotide context on readthrough efficiency has been investigated by directed mutagenesis of target sequences inserted into reporter genes.…”

Section: Discussionmentioning

confidence: 99%

“…Several reasons indicated an investigation of gentamicin: its effect on readthrough is the best documented to date, and it is known to present a much lower toxicity than other suppressive molecules like G418. 3 Moreover, this antibiotic is currently used in human therapy against bacterial infections, and its pharmacokinetics properties and side effects are well characterized. 17 However, one cannot exclude that a long-term use of gentamicin may induce other types of side effects due to generalized readthrough of normal termination codons.…”

Section: Introductionmentioning

confidence: 99%

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

et al. 2004

View full text Add to dashboard Cite

The suppression levels induced by gentamicin on premature stop codons, caused by primary nonsense mutations found in muscular dystrophy patients, were assessed using a very sensitive dual reporter gene assay. Results show that: (i) the effect of gentamicin on readthrough is similar in cultured cells and in vivo in murine skeletal muscle; (ii) a wide variability of readthrough efficiency is obtained, depending on the mutation tested; (iii) due to the complexity of readthrough regulation, efficiency cannot be predicted by the nucleotide context of the stop codon; (iv) only a minority of premature stop codons found in patients show a significant level of readthrough, and would thus be amenable to this pharmacological treatment, given our present understanding of the problem. These results probably provide an explanation for the relative failure of clinical trials reported to date using gentamicin to treat diseases due to premature stop codons, and emphasize that preliminary assays in cell culture provide valuable information concerning the potential efficiency of pharmacological treatments.

show abstract

“…Aminoglycoside antibiotics are able to disrupt translational fidelity in both species. There are now data showing that the growth of mammalian cells in the presence of an aminoglycoside antibiotic can suppress nonsense mutations [148]. In vitro and in vivo studies have suggested that this strategy may be relevant for human diseases.…”

Section: Models Of Dravet Syndromementioning

confidence: 99%

Novel Animal Models of Pediatric Epilepsy

et al. 2012

View full text Add to dashboard Cite

When mimicking epileptic processes in a laboratory setting, it is important to understand the differences between experimental models of seizures and epilepsy. Because human epilepsy is defined by the appearance of multiple spontaneous recurrent seizures, the induction of a single acute seizure without recurrence does not constitute an adequate epilepsy model. Animal models of epilepsy might be useful for various tasks. They allow for the investigation of pathophysiological mechanisms of the disease, the evaluation, or the development of new antiepileptic treatments, and the study of the consequences of recurrent seizures and neurological and psychiatric comorbidities. Although clinical relevance is always an issue, the development of models of pediatric epilepsies is particularly challenging due to the existence of several key differences in the dynamics of human and rodent brain maturation. Another important consideration in modeling pediatric epilepsy is that "children are not little adults," and therefore a mere application of models of adult epilepsies to the immature specimens is irrelevant. Herein, we review the models of pediatric epilepsy. First, we illustrate the differences between models of pediatric epilepsy and models of the adulthood consequences of a precipitating insult in early life. Next, we focus on new animal models of specific forms of epilepsies that occur in the developing brain. We conclude by emphasizing the deficiencies in the existing animal models and the need for several new models.

show abstract

Suppression of a nonsense mutation in mammalian cellsin vivoby the aminoglycoside anthiotics G–418 and paromomycin

Cited by 194 publications

References 11 publications

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

Novel Animal Models of Pediatric Epilepsy

Contact Info

Product

Resources

About