Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable

Readthrough of Premature Termination Codons in the Adenomatous Polyposis Coli Gene Restores Its Biological Activity in Human Cancer Cells

The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough (aminoglycosides, negamycin), as a means of reactivating endogenous APC. By quantifying the readthrough of 11 nonsense mutations in APC, we were able to identify those giving the highest levels of readthrough after treatment. For these mutations, we demonstrated that aminoglycoside or negamycin treatment led to a recovery of the biological activity of APC in cancer cell lines, and showed that the level of APC activity was proportional to the level of induced readthrough. These findings show that treatment with readthrough inducers should be considered as a potential strategy for treating cancers caused by nonsense mutations APC gene. They also provide a rational basis for identifying mutations responsive to readthrough inducers

Translecture, antibiotiques et stratégie thérapeutique

Environ 10% des maladies humaines sont liées à l'apparition d'une mutation non-sens qui provoque une terminaison prématurée de la traduction, conduisant à la synthèse d'une protéine tronquée. Depuis une dizaine d'années, une stratégie thérapeutique se développe dans le but de re-synthétiser une protéine complète en utilisant des molécules favorisant l'incorporation d'un ARNt au niveau de ce codon stop prématuré (= translecture). Actuellement, les molécules les plus utilisées sont les antibiotiques de la famille des aminoglycosides (gentamicine, amikacine) qui se fixent au niveau du centre de décodage du ribosome. Le taux de translecture dépend du contexte nucléotidique de part et d'autre du codon stop. Afin de mieux comprendre l'influence de celui-ci sur le taux de translecture et la réponse au traitement, j'ai analysé 66 mutations non-sens. J'ai mis en évidence que le nucléotide en amont du codon stop est un déterminant majeur dans la réponse à la gentamicine. J'ai analysé l'effet des aminoglycosides sur la ré-expression des gènes suppresseurs de tumeur p53 et APC (Adenomatous Polyposis Coli) altérés par une mutation non-sens et fréquemment mutés dans les cancers humain. En sélectionnant les mutations non-sens sensibles au traitement, j'ai démontré que les protéines p53 et APC ré-exprimées retrouvaient une activité biologique. Pour p53, nous avons également mis en évidence que la protéine entière ré-exprimée après traitement était capable d'induire l'apoptose dans une lignée cancéreuse humaine. Ces travaux constituent la première preuve de principe que la stratégie thérapeutique utilisant des inducteurs de translecture pourrait être utilisée pour certains cancers.Ten percent of human diseases are linked to the occurrence of a nonsense mutation that leads to premature translation termination and triggers the synthesis of a truncated protein. For the past ten years, therapeutic strategies have emerged in the attempt to use molecules that facilitate tRNA incorporation opposite to this premature stop codon (= readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin) that bind the decoding center of the ribosome. Readthrough level is depending on the nucleotide context in the vicinity of the stop codon, such that only few nonsense mutations are sensitive to aminoglycoside treatment. To better understand the influence of the stop codon sequence context on readthrough level and response to treatment, I studied 66 nonsense mutations for their basal and gentamicin induced readthrough levels. This analysis revealed that the nucleotide upstream to the stop codon is a major determinant in gentamicin response. I also investigated the possibility to rescue two tumor suppressor genes, p53 and APC, altered by a nonsense mutation and frequently mutated in human cancers. By selecting nonsense mutations sensitive to treatment, I demonstrated that p53 and APC proteins could recover biological activities. For p53, I also determined that the full length protein, re-expressed after treatment, was able to induce apoptosis in a human cancer ceIl line. This work provides the proof of principle that the use of readthrough promoting molecules as a therapeutic approach couId be applied to re-express nonsense mutated tumor suppressor genes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Thérapie spécifique d’allèle

Les nouvelles stratégies thérapeutiques qui ont pour objectif de traiter les patients en fonction de la mutation responsable de la maladie plutôt qu’en fonction de leur pathologie sont en plein développement. Les mutations non-sens, qui conduisent à la synthèse d’une protéine tronquée, représentent 10 % des mutations impliquées dans les maladies génétiques humaines. L’utilisation de molécules favorisant l’entrée d’un ARNt au niveau de mutations non-sens lors de la traduction permet de synthétiser une protéine entière. Les antibiotiques de la famille des aminoglycosides (en particulier la gentamicine) sont les plus largement étudiés pour cette stratégie, et ont permis d’obtenir un bénéfice thérapeutique pour certains patients. Nous discutons dans cette revue ces résultats et ceux de plusieurs équipes qui tentent actuellement de découvrir des molécules moins toxiques et/ou pouvant agir sur les mutations réfractaires aux aminoglycosides, afin d’élargir la proportion de patients qui pourront bénéficier du traitement

Identification of APC nonsense mutations responsive to aminoglycoside treatment.

<p>Readthrough efficiencies for 11 nonsense mutations in the APC gene were assessed in NIH3T3 cells with and without gentamicin (800 µg/ml) treatment for 24 h. Two nonsense mutations (L360X and R1114X) displayed levels of gentamicin-induced readthrough of more than 0.5%. Means values are presented, together with the standard error of the mean (SEM) (n = 5).</p

APC biological activity is correlated with antibiotic-induced readthrough level in human colorectal cancer cells.

<p>(A) Readthrough efficiencies for APC L360X nonsense mutations were determined in DLD-1 cells in the presence of G418 (10, 25, 50, 100 and 200 µg/ml), negamycin (1 mg/ml), amikacin (2 mg/ml) or gentamicin (800 µg/ml). (B) Readthrough efficiencies for APC R1114X nonsense mutations were determined in NIH3T3 cells in the presence of G418 (50, 100 and 200 µg/ml) or negamycin (1 mg/ml). The readthrough efficiencies in DLD-1 cells were consistent with those in NIH3T3 cells (data not shown). (C) Aminoglycoside treatment restored APC activity. APC binding to beta-catenin (reppression of the pTOPGlow reporter) is restored by the treatment of DLD-1 cells transiently transfected with cDNA APC L360X with G418 (10, 25, 50, 100 and 200 µg/ml), negamycin (1 mg/ml), amikacin (2 mg/ml) or gentamicin (800 µg/ml). (D) APC binding to beta-catenin (repression of pTOPGlow reporter) is restored by the treatment of LoVo cells carrying APC R1114X with G418 (50, 100 and 200 µg/ml) or negamycin (1 mg/ml). As a control, LoVo cells were cotransfected with the pTOPGlow reporter plasmid and either the APC-targeting siRNA (siRNA APC) or a non-targeting siRNA (siRNA NT) and treated with G418 (200 µg/ml). (E) Effect of the siRNA targeting APC mRNA. We transiently transfected LoVo cells with an siRNA targeting (siRNA APC) or not targeting (siRNA NT) APC mRNA. Quantitative PCR was used to determine mRNA levels. Results are expressed relative to the amount of mRNA in the presence of siRNA NT. Mean values are presented, together with the SEM (n = 3).</p

Western blot.

<p>LoVo cells were left untreated or were treated with G418 (200 µg/ml) for 72 hours. Western blots were probed with the FE-9 antibody directed against the N-terminus of APC. The truncated forms corresponding to the mutated alleles present in LoVo cells are indicated by arrows. An extract from HeLa cells (APC WT) was used as a control; a band was detected at 311 kDa.</p

APC nonsense mutations.

<p>*Mutations are named by the position and the nature of the wild-type amino acid in APC protein sequence.</p>§<p>These nonsense mutation sequences were inserted into the dual reporter vector in order to determine readthrough level.</p>†<p>Frequencies were given relative to total nonsense mutations listed for APC gene.</p>‡<p>This nonsense mutation was reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024125#pone.0024125-Oh1" target="_blank">[33]</a>.</p