Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons

Abstract

About 12% of human genetic disorders involve premature termination codons (PTCs). Aminoglycoside antibiotics have been proposed for restoring full-length proteins by readthrough of PTC. To assess the efficiency of readthrough, we selected homozygous and compound heterozygous skin fibroblasts from xeroderma pigmentosum (XP) patients with different PTCs in the XPC DNA repair gene. XP patients have a nucleotide excision repair defect and a 10,000-fold increased risk of UV-induced skin cancer. In six of eight PTC-containing XP-C cells, treatment with Geneticin and gentamicin resulted in (i) stabilized XPC-mRNA, which would have been degraded by nonsense-mediated decay; (ii) increased expression of XPC protein that localized to UV-damaged sites; (iii) recruitment of XPB and XPD proteins to UV DNA damage sites; and (iv) increased repair of 6-4 photoproducts and cyclobutane pyrimidine dimers. Expression of PTC in a transfected vector revealed that readthrough depends on the PTC sequence and its location within the gene. This sensitive DNA repair assay system demonstrates the complexity of response to PTC readthrough inducers. The efficiency of aminoglycoside-mediated readthrough depends on the type and copy number of PTC, the downstream 4+ nucleotide, and the location within the exon. Treatment with small-molecule nonaminoglycoside compounds (PTC124, BZ16, or RTC14) resulted in similarly increased XPC mRNA expression and photoproduct removal with less toxicity than with the aminoglycosides. Characterizing PTC structure and parameters governing effective PTC readthrough may provide a unique prophylactic therapy for skin cancer prevention in XP-C patients.

Keywords: UV radiation; readthrough compounds.

Fig. 1.

Increased XPC mRNA with Geneticin but not gentamicin. XP-C cells containing PTC were incubated with Geneticin or gentamicin for 3 d and mRNA was measured. Data are mean ± SD of three experiments each in triplicate. *P < 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Fig. 2.

Effect of Geneticin and gentamicin on XPC, XPB, and XPD proteins. (A) XP-C cells were incubated with geneticin for 4 d and immunoblot was performed. XPC protein was detectable via immunoblotting in Geneticin-treated TGA-T_1,2 and TGA-A_1,2 only (arrows). The ratio (%) of the intensity of the XPC band to b-actin band is indicated. In total, three different experiments were performed. Shown are three representative western blots. (B) Cells were incubated with Geneticin or gentamicin for 3 d, and an immunofluorescence assay 1 h after local UV irradiation was performed. Geneticin and gentamicin induce post-UV XPC protein localization in TGA-T_1,2 (yellow arrows) but not in TGA-G_1,2exon 6. For the gentamicin-treated normal cells (Lower Left), two representative areas of the same coverslip are shown. (C) Quantification of XPC protein detected via immunofluorescence at sites of UV damage 1 h after UV exposure. One hundred nuclei were scored. Bars indicate mean ± SD of the percent positive cells for XPC. *P < 0.05, **P < 0.005, ***P < 0.0005. (D) Cells were incubated with Geneticin for 3 d, and an immunofluorescence assay 1 h after local UV irradiation was performed. Geneticin induced post-UV XPB or XPD protein recruitment in TGA-T_1,2 (yellow arrows) but not in TGA-G_1,2exon 6. (E) Quantification of XPB and XPD proteins detected via immunfluorescence at sites of UV damage 1 h after UV exposure. Bars indicate mean ± SD of the percent positive cells for XPB and XPD, and 100 nuclei were scored. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 3.

Effect of Geneticin and gentamicin in removal of 6–4PPs and CPDs. XP-C cells were incubated with Geneticin or gentamicin for 3 d, and an immunofluorescence assay for detection of 6–4PPs and CPDs after local UV irradiation was performed. (A–C) Quantification of 6–4PP removal 0, 1, 3, 6, and 24 h after UV in (A) untreated, (B) Geneticin-treated, and (C) gentamicin-treated cells. (D–F) Quantification of CPD removal 0, 6, 24, and 48 h after UV irradiation in (D) untreated, (E) Geneticin-treated, and (F) gentamicin-treated cells. One hundred nuclei were scored. Bars indicate mean ± SD of the percent positive cells for 6–4PPs and CPDs. Legend: Normal; ATG > AGG; TGA-T_1,2; TGA-T₁; TGA-A_1,2; TGA-G_{1,2 Exon 6}; TGA-C₁/TAA-G₂; TAA-A₁; TGA-G_{1,2 Exon 9}; TGA-T₁/TAG-A₂.

Fig. 4.

Increased readthrough of TGA-G but not TAA-A luciferase expression vectors with Geneticin. XP-C and normal cells incubated with or without Geneticin for 2 d were transfected with wild-type or mutated luciferase expression vectors containing indicated PTCs. Relative luciferase activity at 48 h after transfection is expressed as percent activity of mutated plasmid in Geneticin-treated cells compared with untreated cells. Bars indicate mean ± SD of the relative luciferase activity of three different experiments each in triplicate. The expression levels of the wild-type plasmid varied between 300,000 and 700,000 relative light units. *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 5.

Increased readthrough of TGA-G_1,2 with nonaminoglycosides. XP-C cells with TGA-G_1,2 were incubated with nonaminoglycosides and aminoglycosides for 3 d. (A) MTT survival assay. The effective doses of PTC124, BZ16, and RTC14 used were less toxic in TGA-A_1,2 compared with Geneticin and gentamicin. (B) Measurement of XPC mRNA. Treatment with PTC124, BZ16, and RTC14 and Geneticin resulted in significantly increased XPC mRNA. **P < 0.005. (C) Immunofluorescence assay in normal and XP-C TGA-G_1,2 cells 1 h after local UV irradiation. Geneticin, gentamicin, PTC124, BZ16, and RTC14 induce post-UV XPC protein localization. (D) Quantification of XPC protein at sites of UV damage 1 h post UV (from C). Bars indicate mean ± SD of the percent positive cells for XPC. One hundred nuclei were scored for each bar. *P < 0.05, **P < 0.005, ***P < 0.0005. (E and F) Immunofluorescence assay for detection of 6–4PPs (E) and CPDs (F) 6 and 24 or 48 h after local UV irradiation. Bars indicate mean ± SD of the percent positive cells for 6-4PP and CPDs; 100 nuclei were scored.

Fig. 6.

Summary of assays and XP-C PTC readthrough results. Summary of XP-C cell lines tested (top row), the type of PTC mutation in the XPC gene, the assays used to assess various steps of the post-UV NER pathway (first column: IF, immunofluorescence; WB, Western blot), the response to Geneticin and gentamicin in each assay, and summary assay sensitivity of the eight PTC cell lines for Geneticin (last column). The efficiency of correction with Geneticin for all seven assays (bottom row) is indicated by ++++ positive in seven assays, +++ positive in five assays, ++ positive in four assays, + positive in one assay, and – none. , Geneticin response; , gentamicin response; x, no response to Geneticin; O, no response to gentamicin.