Regulation of protein synthesis during doxorubicin-induced toxicity

In response to DNA damage, cells decrease global rates of protein synthesis to conserve energy and selectively translate mRNAs of proteins involved in the DNA damage response. Doxorubicin is a widely used chemotherapeutic that induces double strand DNA breaks. It might be expected that doxorubicin-induced DNA damage would rapidly inhibit global protein synthesis through the phosphorylation of eIF2α, as has been observed in response to UVB-induced DNA damage. However, in MCF10A cells, a delay of 9 hours was observed between DNA damage recognition and protein synthesis inhibition. Furthermore, eIF2α phosphorylation was not observed until 12 hours, and global protein synthesis inhibition was subsequently shown to be independent of eIF2α phosphorylation status. An alternative regulator of translation initiation is the mTORC1 target protein 4E-BP1. Doxorubicin-induced mTORC1 inhibition preceded eIF2α phosphorylation and correlated with the inhibition of global protein synthesis, suggesting that the DDR signalled through mTOR to regulate protein synthesis. Experiments using p53-/- MCF10A cells suggested that doxorubicin-induced mTORC1 inhibition was mediated by p53 activity, and p53-/- cells were shown to be more sensitive to doxorubicin-induced cell death. Interestingly, doxorubicin-and catalytic-inhibition of mTORC1 activity mediated the phosphorylation of eIF2α in a signalling mechanism that may be dependent on PP6, DNA-PKcs and GCN2 or PERK. Importantly, eIF2α phosphorylation was absent in response to doxorubicin in p53-/- cells, whereas catalytic inhibition of mTORC1 activity enhanced eIF2α phosphorylation. These data suggested a mechanism where p53-mediated mTORC1 inhibition signalled to enhance the phosphorylation of eIF2α.