Figure 7.
Inhibitors of hSMG-1 suppress NMD and cause accumulation of truncated p53 proteins in human cancer cell lines with PTC mutations. (A) Wortmannin or caffeine inhibits protein kinase activity of hSMG-1 in vitro. In vitro kinase assays were performed using immunoprecipitated His-hSMG-1 and a GST- hUPF1/SMG-2 fusion protein, hUPF1/SMG-2 S1096, as a substrate in the presence of an indicated concentration of wortmannin or caffeine. Autoradiograms of hUPF1/SMG-2 S1096 phosphorylation are indicated in the corresponding insets. (B) The BGG reporter mRNA with a PTC is specifically accumulated by inhibitors of hSMG-1. The MEF Tet-Off cells were transfected with a wild-type (upper) or mutant (lower) BGG reporter gene (Fig. 4C) and reseeded. Cells were treated for 2 h with caffeine (caff.), wortmannin (wort.), rapamycin (rap.), or cycloheximide (CHX) in the absence of doxycycline. Total RNAs were analyzed by Northern blotting using a BGG probe (top) or a GAPDH probe (bottom). (C) Schematic representation of p53 gene structure with PTC mutations in human lung cancer cell lines, Calu6 (196PTC) and N417 (298PTC). Boxes show exons. (D) Wortmannin induces the accumulation of transcripts and their corresponding proteins from the p53 gene with PTC. N417 and A549 cells were treated with or without (cont.) 10 μM wortmannin (wort.) or 100 μg/mL cycloheximide (CHX) for 4 h. Total RNA and cell lysates were analyzed by Northern blotting using a p53 probe and by Western blotting using an anti-p53 antibody, DO-1, respectively. The CBB image showing actin staining is also shown (bottom). (E) Wortmannin induces expression of p53 transcripts and p53 proteins of lower molecular weights in a dose-dependent manner. Calu6 or N417 cells were treated with an indicated concentration of wortmannin or CHX for 2 h. The total RNA and cell lysate were prepared and analyzed, as in E, by Northern blotting using a p53 probe and by Western blotting using DO-1, respectively.