Readthrough of stop codons by use of aminoglycosides in cells from xeroderma pigmentosum group C patients

Abstract

Readthrough of premature termination (stop) codons (PTC) is a new approach to treatment of genetic diseases. We recently reported that readthrough of PTC in cells from some xeroderma pigmentosum complementation group C (XP-C) patients could be achieved with the aminoglycosides geneticin or gentamicin. We found that the response depended on several factors including the PTC sequence, its location within the gene and the aminoglycoside used. Here, we extended these studies to investigate the effects of other aminoglycosides that are already on the market. We reasoned that topical treatment could deliver much higher concentrations of drug to the skin, the therapeutic target, and thus increase the therapeutic effect while reducing renal or ototoxicity in comparison with systemic treatment. Our prior clinical studies indicated that only a few percent of normal XPC expression was associated with mild clinical disease. We found minimal cell toxicity in the XP-C cells with several aminoglycosides. We found increased XPC mRNA expression in PTC-containing XP-C cells with G418, paromomycin, neomycin and kanamycin and increased XPC protein expression with G418. We conclude that in selected patients with XP, topical PTC therapy can be investigated as a method of personalized medicine to alleviate their cutaneous symptoms.

Background

Xeroderma pigmentosum (XP) is a rare, skin cancer prone, autosomal recessive disorder caused by defective nucleotide excision repair (NER) of ultraviolet radiation (UV) damaged DNA (1). Premature termination codons (PTC) have been reported in 15% of 159 XP complementation group C (XP-C) patients (2) (s10–15). Aminoglycosides bind to the small ribosomal subunit leading to conformational changes that reduce discrimination between tRNA substrates resulting in incorporation of an amino acid into the elongating peptide (s16). The affinity for the prokaryotic decoding centre is much higher than for the eukaryotic ribosome allowing for successful antibiotic usage. Some aminoglycoside antibiotics are capable of PTC readthrough and induction of functional eukaryotic protein (3). Clinical and cellular studies, primarily with systemic gentamicin, have been conducted in several human diseases including Duchenne muscular dystrophy and cystic fibrosis, with variable results (3) (s19, s23–35) (Table S1). Topical gentamicin (0.1%, 1 mg/ml) was reported to reduce symptoms of autosomal dominant Hailey–Hailey disease in a patient with PTC (4). In vitro gentamicin and paromomycin improved molecular disease markers in recessive dystrophic epidermolysis bullosa cells containing PTC (5). We demonstrated that aminoglycosides and small molecular weight non-aminoglycoside compounds can induce XPC protein expression and function in some XP-C cells with different PTC (2). Systemic administration of aminoglycosides is associated with risk of severe renal and ototoxicity (s17). Topically administered aminoglycosides could safely achieve about 100-fold greater concentrations in the skin than therapeutic antibacterial serum concentrations and might avoid much of the toxicity seen in systemic exposure (4) while directly increasing DNA repair protein expression in skin cells.

Questions addressed

We assessed cellular toxicity and efficiency of in vitro XPC read-through to determine which compounds should be studied for topical application in XP-C patients. We chose aminoglycosides that are already on the market and compared them to the known readthrough compound G418 (Geneticin) (2) which is not used clinically.

Experimental design

Normal primary human skin fibroblasts (AG13354), from the Human Genetic Mutant Cell Repository, Camden, NJ, USA, and XP-C fibroblasts (Table S2), were cultured as described (2). Cytotoxicity of aminoglycosides was assessed using a CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA). Total RNA isolated from cells 3 days after incubation with compounds was used to quantitate the XPC mRNA as described (2). Local UVC irradiation (254 nm, 100 J/m²), immunofluorescent labelling with an XPC antibody and confocal microscopy imaging were performed as described (2).

Results

We tested the effects of the aminoglycoside antibiotics on cell survival (Fig. 1). With XP-C cells containing different PTC, G418 decreased cell survival to 40–60% at 100 μM, whereas cell survival was >80% with 100 μM gentamicin, kanamycin, neomycin, paromomycin or tobramycin. Thus, these aminoglycoside compounds were less toxic than G418 to the XP-C cells in vitro.

Figure 1.

Survival of XP-C cells following treatment with different aminoglycosides. Treatment of PTC containing XP-C cells (TGA-A_1,2, TGA-T₁/TAG-A₂ and TAG-A₁) with (a) G418, (b) gentamicin, (c) paromomycin, (d) neomycin (e) tobramycin or (f) kanamycin for 72 h and assessment of cell survival with an MTT assay as described previously (2). In the XP-C cells with 100 μM treatment (vertical line), G418 showed greater toxicity compared to all other compounds.

To determine readthrough of the XPC gene, we measured the levels of XPC mRNA after aminoglycoside treatment (Fig. 2). As in earlier studies (2,6) (s12), the baseline XPC message levels were very low in the XP-C cells. This is a result of nonsense-mediated message decay (NMD), a protective pathway degrading PTC-bearing transcripts, thus preventing the expression of truncated proteins (7) (s18). Treatment with the NMD inhibitor cycloheximide increased XPC mRNA in all XP-C cells 2.5-fold to eight-fold (Fig. 2 bars 9 and 10), indicating that NMD-prone XPC mRNA could be restored. A high NMD efficiency may degrade a large amount of PTC-bearing transcripts before they are readthrough by aminoglycosides (s19). There was a low level of NMD in normal cells as reported (s20). As in earlier studies (2), the response to treatment with G418 varied with different XPC PTCs. With 100 μg/ml G418, there was a significant increase in XPC mRNA with TGA-A_1,2 and the heterozygous cell line TGA-T₁/TAG-A₂, but not with TAG-A₁ (Fig. 2 bar 2 for each). Treatment with paromomycin or neomycin led to a significant increase of XPC mRNA in 2 XP-C cell lines compared to non-treated cells or to cells treated with tobramycin (Fig. 2 bars 4, 5 and 7 compared to bar 8). Treatment with kanamycin (Fig. 2 bar 6) resulted in a significant increase in XPC mRNA in one cell line (TGA-T₁/TAG-A₂). Amino-glycosides show different binding affinities to the ribosome which may contribute to the varying PTC readthrough efficiencies.

Figure 2.

Increased XPC mRNA following treatment with aminoglycosides or cycloheximide in XP-C cells with PTC. XP-C cells containing different PTC (TGAA_1,2, TGA-T₁/TAG-A₂ and TAG-A₁) were incubated with aminoglycosides (bars 1 and 9 – no treatment; 2–100 μg/ml G418; 3–1000 μg/ml G418; 4–100 μg/ml paromomycin; 5–1000 μg/ml paromomycin; 6–1000 μg/ml kanamycin; 7–1000 μg/ml neomycin; 8–1000 μg/ml tobramycin) for 3 days or with 200 μg/ml cycloheximide (bar 10) for 5 h. XPC mRNA was measured using real-time QRT-PCR as described previously (2). Values are percentage femtogram, untreated is 100% (corresponding to 289 fg in normal, 57 fg in TGA-A1, 2, 67 fg in TGA-T1/TAG-A2 and 129 fg in TAG-A1). Data are mean ± SD of 1–3 experiments each in triplicate. *P < 0.05, **P ≤ 0.005.

We used an immunofluorescence assay using local UV irradiation and confocal microscopy (2) to determine whether the increased XPC mRNA after aminoglycoside treatment was translated into XPC protein. Normal cells but none of the XP-C cell lines showed foci of XPC protein 1 h after localized UVC exposure (Figure S1). All 3 XP-C cell lines tested had detectable XPC protein 72 h after G418 treatment. The XP-C cells TGA-A_1,2 and TGA-T₁/TAG-A₂ had detectable XPC protein after gentamicin treatment. However, XPC protein localization was not detected after incubation with neomycin, paromomycin or tobramycin.

There was a significant increase in post-UV cell survival following G418 treatment for TGA-A_1,2 cells (18 J/m²) and TAG-A₁ cells (36 J/m²) but not with paromomycin treatment (Figure S2).

Conclusion

We evaluated the toxicity of several aminoglycosides and their efficiency of in vitro XPC readthrough. Small increases in XPC mRNA after readthrough (Fig. 2) may be sufficient to lower the skin cancer risk in selected XP-C patients. In an XP-C family, 3–5% of normal XPC mRNA was associated with 29% of normal XPC protein, increased cell survival and mild disease while no detectable (<0.1%) XPC mRNA or XPC protein was associated with severe disease in another family (6,8). Only 0.1–1% PTC readthrough reduced severity of other diseases (9) (Table S1). Current skin cancer prevention therapy for XP-C patients relies on early diagnosis and rigorous sun protection (s21,22). In selected XP patients, topical PTC therapy can be investigated as a method of personalized medicine to alleviate their cutaneous symptoms.