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

Abstract

Approximately 14% of genetic mutations in patients with ataxia-telangiectsia (A-T) are single-nucleotide changes that result in primary premature termination codons (PTCs), either UAA, UAG, or UGA. The purpose of this study was to explore a potential therapeutic approach for this subset of patients by using aminoglycosides to induce PTC read-through, thereby restoring levels of full-length ATM (A-T mutated) protein. In experiments using a modified in vitro cDNA coupled transcription/translation protein truncation test, 13 A-T cell lines carrying PTC mutations in different contexts exhibited read-through expression of ATM fragments, with three of four aminoglycosides tested. In ex vivo experiments with lymphoblastoid cell lines, we used radiosensitivity, radioresistant DNA synthesis, and irradiation-induced autophosphorylation of ATM Ser-1981 to show that the aminoglycoside-induced full-length ATM protein was functional and corrected, to various extents, the phenotype of A-T cells. These results encourage further testing of other compounds in this class, as well as follow up animal studies. Because some A-T patients with 5-20% of normal levels of ATM protein show slower neurological progression, A-T may prove to be a good model for aminoglycoside-induced read-through therapy.

Figures

Fig. 1.

Autoradiograms show in vitro effects of geneticin on read-through of various PTT fragments carrying different PTC mutations in the ATM gene. As the aminoglycoside concentrations increased, the densitometry readings (OD ratio = OD of band/OD of background) for full-length PTT product (filled diamonds) increased from 0 to 800 (A and C). (A) Experiment using plasmid with TAT51 mutation (UGA C) as template. (B) Experiment using plasmid with the corrected TAT51 mutation (CGA C, i.e., normal sequence). (C) Experiment using plasmid with AT153LA mutation (UGA A). (D) Experiment using plasmid with the corrected AT153LA mutation (CGA A). In all experiments, both the full-length and truncated PTT products (open squares) were inhibited by higher concentrations of geneticin.

Fig. 2.

Read-through effects for aminoglycosides in vitro (A–C) and ex vivo (D). Autoradiograms show in vitro read-through effects of gentamicin (A), paromomycin (B), and tobramycin (C) on the TAT51 (UGA C) mutation. Expression of a truncated 52-kDa PTT product (open squares) was inhibited by all three compounds at highest concentrations. The full-length 54-kDa read-through product (filled diamonds) was observed with gentamicin and paromomycin, but not with tobramycin. (D) Ex vivo geneticin-induced read-through of full-length ATM protein in A-T cells carrying the AT185LA (UAA G) mutation. LCLs were treated with varying concentrations of geneticin for 4 days. Nuclear lysates were immunoprecipitated with antibody to ATM and analyzed by Western blot. Control panel shows a nonspecific band from the same blot to evaluate loading of immunoprecipitates.

Fig. 3.

Geneticin-induced effects on normal cells. Lanes marked with 0, no irradiation; lanes marked with 10, 10 Gy of irradiation. First two lanes, no drug treatment; second two lanes, 25 μg/ml; third two lanes, 75 μg/ml; fourth two lanes, 125 μg/ml. Two upper blots were developed with antibodies to SMC1 phosphor-Serine S957 or S966. Two lower blots show the same blots developed with antibodies to SMC1 or β-actin.

Fig. 4.

Functional analyses of expressed ATM protein. (A) Radioresistant DNA synthesis. A-T cells carrying the AT160LA mutation (UGA A) (filled triangles) were treated with increasing concentrations of geneticin, 25 μg/ml (asterisks), 75 μg/ml (filled circles), and 125 μg/ml (open circles), and were exposed to increasing doses of irradiation. Note that [3H]thymidine uptake of treated cells approached that of normal cells (filled diamonds). (B) Radioresistant DNA synthesis. Absence of geneticin-induced effect on an A-T cell line (BRAT-16.3) that did not carry a PTC mutation is shown. Standard error bars represent typical triplicate results for each curve. (C) Colony survival assay. The graph depicts radiosensitivity of A-T cells after 1-Gy irradiation, after 4 days of treatment with varying concentrations of geneticin. Experiments a, b, and c were performed by using cells with the AT187LA mutation (UAA G) (open bars). Experiment d (filled bars) used cells with the AT160LA mutation (UGA A). Note the correction of radiosensitivity at concentration of 25 μg/ml. Asterisks denote “not tested” for that concentration. N, normal; Int, intermediate; RS, radiosensitive.

Fig. 5.

ATM pS1981 IRIFs of AT187LA cells after treatment with geneticin. 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 μg/ml. Nonirradiated cells, untreated or exposed to comparable concentrations of geneticin, showed only occasional foci.