Regulation of p53 translation in response to DNA damage by the p53 internal ribosome entry site and its trans-activating factors, TCP80 and RHA

The p53 protein is a tumor suppressor that plays a key role in the regulation of cell cycle arrest, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low in the cell. However, following a stressful event, such as DNA damage, p53 levels will increase dramatically and it will become activated. While it is known that increased p53 translation is necessary for its accumulation following DNA damage, the mechanisms underlying p53 translation are still poorly understood.;In our study, we sought to understand the mechanisms behind increased p53 translation following genotoxic stress. Cap-independent or internal ribosome entry site (IRES)-mediated translation is an important alternative mechanism of translation initiation for the synthesis of proteins needed under stressful conditions. Using a bicistronic reporter vector, we showed that the p53 5'-UTR sequence exhibits IRES activity in both cellular and in vitro systems. We also showed that p53 IRES activity increased following treatment with the DNA damaging agent etoposide, suggesting that IRES-mediated translation is an important mechanism for p53 accumulation following DNA damage.;Translation initiation at IRESs is often regulated by proteins known as IRES transacting factors (ITAFs). We have identified two proteins, TCP80 and RHA, that can act as positive p53 ITAFs by binding to the p53 IRES and increasing its activity. Overexpression of TCP80 and RHA led to increased expression and synthesis of p53 in p53-null cells transfected with a p53 expression vector containing the p53 5'-UTR. We also discovered two breast cancer cell lines, ZR75-1 and MDA-MB-175 that harbored wild-type p53, but had defective p53 induction and p53 IRES activity following DNA damage. We found that expression of TCP80 and RHA is low in ZR75-1 and MDA-MB-175 cells and that co-expression of both proteins was required to induce p53 IRES in these two cell lines, indicating that these two proteins cooperate to stimulate p53 IRES activity. Our results suggest an important role for TCP80 and RHA in the regulation of IRES-mediated p53 translation following genotoxic stress. Our findings also suggest that poor expression of TCP80 and RHA may be involved in the development of cancer.