A Live Cell DNA Damage Response Indicator System Based On p53 Regulation Mechanism

Abstract

p53 is one of the most essential tumor suppressor and its abundance is tightly regulated in cells: in normal state, the amount of p53 is kept low through ubiquitylation by MDM2 and subsequent degradation by proteasome. In response to DNA damage, p53 undergoes phosphorylation by ATM, which interferes with its association with MDM2, and, as the result, its abundance is greatly increased. We sought to develop a live cell imaging system to monitor the cellular status of DNA damage response based on this life cycle of p53. We first introduced a plasmid vector (pEGFP-C1) expressing GFP-p53 fusion protein into human cancer cells with normal p53, mutant p53 and p53 deletion and analyzed p53 abundance and its change after irradiation by Western blotting and flowcytometry. Furthermore, we are in progress to engineer cells to express p53-GFP fusion gene from chromosome by CRISPR/Cas9 genome editing technology.