Translational Control Protein 80 Stimulates IRES-Mediated Translation of p53 mRNA in Response to DNA Damage

Figure 4

(a) MCF-7/shTCP80 cells express lower levels of TCP80 and RHA as compared to MCF-7 cells. MCF-7 and MCF-7/shTCP80 cells were grown to subconfluency. Cells were then lysed and equal amounts of protein were subjected to SDS-PAGE and western blotting. TCP80, RHA, and β-actin were detected by immunoblotting. (b) MCF-7/shTCP80 cells exhibit reduced induction of p53 and its downstream target PUMA following DNA damage. MCF-7 and MCF-7/shTCP80 cells were grown to subconfluency. Cells were then treated with 10 μM etoposide for 2 hours. After the treatment, cells were lysed and equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes. p53, PUMA, and β-actin proteins were detected with their respective antibodies. (c) Statistical analysis of the expression levels of p53 (p53/β-actin) between individual groups as seen in (b) was carried out using one-way ANOVA with a Newman–Keul post hoc test from 3 sets of experimental results. Significance was assumed at . (d) Statistical analysis of the expression levels of PUMA (PUMA/β-actin) between individual groups as shown in (b) was performed using one-way ANOVA with a Newman–Keul post hoc test from 3 sets of experimental results. Significance was assumed at . (e) A diagram showing proposed regulation of p53 IRES activity by TCP80 and RHA. During the basal conditions, the secondary structure of the p53 IRES is largely stabilized and has limited translational activity due to inadequate interaction between TCP80/RHA and the p53 IRES. Following DNA damage, increased binding of TCP80 to the p53 IRES and enhanced interaction between TCP80/RHA and the p53 IRES facilitate the unwinding of the secondary structure of the p53 IRES, allowing increased translation of the p53 mRNA in response to DNA damage.

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