Figure 6
Roc-A inhibits upregulation of p53 via inhibition of protein synthesis. (a) Inhibition of proteasome-mediated degradation does not influence Roc-A-mediated chemoprotection. T cells were treated with solvent (DMSO) or 100 nM Bortezomib to block proteasome-mediated protein degradation and treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for 4 h. Cell lysates were subjected to immunoblot analysis with antibodies against p53 and Actin. Data are representative of three independent experiments. (b) Roc-A does not influence ubiquitination of p53. T cells were treated with Bortezomib, Etoposide and Roc-A as in (a). After 4 h of treatment, cell lysates were used for immunoprecipitation against p53. Isotype-matched antibody-coupled beads and uncoupled beads were used as controls. Ubiquitinated p53 ((UB)n-p53) is indicated. Data are representative of two independent experiments. (c) Inhibition of protein translation by Roc-A or its derivatives correlates with their chemoprotective effects. Effects of Roc-A and its derivatives (-AB, -J, -AR, -Q, -I, -AF and -AA) on protein synthesis in T cells was determined by measuring the amounts of incorporation of [35S]-labeled methionine. Cell death was determined by DNA fragmentation of T cells treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM of different Roc-derivatives or solvent (DMSO) for 24 h. The percentage of chemoprotection was determined by calculating the percentage of protection against Etoposide-induced cell death. The data are shown by plotting the percentage of translation inhibition against the percentage of chemoprotection. Data are an average of three independent experiments. Error bars (S.E.M.) are shown. An allosteric sigmoidal regression curve was plotted against the experimental data. R2=0.96. (d) Roc-A inhibits p53 protein translation. T cells were treated according to (a), followed by metabolic pulse-labeling for indicated time periods and immunoprecipitation. Data are representative of three independent experiments. (e) Roc-A does not reduce the mRNA levels of p53. T cells were treated with solvent (DMSO) or 50 μM Etoposide in the presence of 75 nM Roc-A or solvent (DMSO) for indicated time periods. Total RNA was isolated and was subjected to quantitative real-time PCR for TP53. Data are presented as fold increase in mRNA levels determined by comparison of HPRT1 gene expression levels with TP53 and normalized to control treatment with solvent (DMSO). Data are an average of three independent experiments. Error bars (S.D.) are shown
