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. 2015 Sep 15;29(18):1891–1896. doi: 10.1101/gad.261867.115

Figure 3.

Figure 3.

A uORF in the 5′ UTR of ERCC5 promotes resistance to cisplatin in vitro. (Ai,Aii) Neuroblastoma cell lines (Ai) or B-cell lines (Aii) derived from healthy individuals for all three rs751402 genotypes were treated with cisplatin at the doses shown, and methionine incorporation was measured to assess the degree of inhibition of global protein synthesis rates. There were no significant differences in the degrees of inhibition of protein synthesis between the three B-cell lines or the three neuroblastoma-derived cell lines. (Solid bars) Control conditions; (hatched bars) cells treated with cisplatin. (Bi–Biii,Ci–Ciii) The three neuroblastoma cell lines (Bi–Biii) or the three B-cell lines (Ci–Ciii) of each ERCC5 5′ UTR rs751402 genotype were treated with the doses of cisplatin shown, and protein and RNA samples were obtained. (Bi,Ci) Protein samples were separated by SDS-PAGE and immunoblotted. The data show that, in all cases, eIF2α is phosphorylated by treatment of cells with cisplatin with no change in the total levels of eIF2α. In each case, with increasing doses of cisplatin in the cell lines that were homozygous for the “G” allele, there was a marked decrease in ERCC5. In contrast, when the cells lines that contain the ERCC5 5′ UTR “A” allele (either the homozygous or heterozygous cell lines) were treated with cisplatin, the expression of ERCC5 protein was maintained. (Bii,Cii) The level of ERCC5 mRNA was assessed by qPCR, and no significant increases were detected. (Biii,Ciii) A competitive ELISA was used to measure platinum adduct levels in cisplatin-treated neuroblastoma and B cells. Cells were treated with cisplatin at the doses shown. DNA was then extracted, and equal concentrations were used to perform a competitive ELISA against a standard of platinated DNA with the monoclonal antibody (CP9/19) that recognizes guanine intrastrand cross-links formed by cisplatin. Plotted is the mean absorbance normalized to competition with DMF DNA (100%) for the neuroblastoma cells (Biii) or B cells (Ciii). In both cell lines, the G/G genotype provided significantly more competition, demonstrating a higher adduct level. (*) P < 0.05; (**) P < 0.01.