Abstract

Background
The DNA-damage response (DDR) has evolved to maintain genome stability and optimise cell survival following DNA-damage. One of the key regulators of the mammalian DDR is p53, a protein which is itself tightly regulated by HDM2.

In response to double-strand DNA (dsDNA) breaks, several important mediators are recruited to the site of DNA-damage, including the MRN Complex (MRE11, Rad50 and NBS1) and ATM, although the order in which they become recruited remains to be fully elucidated. Subsequently, ATM and its downstream target Chk2 cause phosphorylation of HDM2 and p53, lifting the negative regulation of p53 by HDM2, resulting in p53 stabilisation and transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis.

Results
We investigated stabilisation and activation of p53 and other associated DDR proteins in response to treatment of HCT116 p53+/+ cells with Nutlin-3, an MDM2 antagonist currently undergoing clinical trials. Nutlin-3 treatment led to stabilisation of p53, as determined by western blotting. Unexpectedly, Nutlin-3 also caused phosphorylation of p53 at key serine residues (Ser15,20,37,392) that are known to undergo phosphorylation in response to DNA-damage. We next sought to establish whether these unexpected phosphorylation events were due to a Nutlin-3-mediated DDR. Indeed, western blotting showed Nutlin-3-induced activation of Chk2 and ATM, proteins required for DNA-damage-dependent phosphorylation and activation of p53. Moreover, we observed phosphorylation of Brca1 and H2AX, known to be activated specifically in response to dsDNA breaks, and additionally, using immunofluorescence confocal microscopy, we showed formation of H2AX foci, an event recognised as occurring early in the mammalian DDR.

Conclusion
These data provide the first evidence for an unexpected role of Nutlin-3 as a DNA damaging agent, or at the very least as an activator of the mammalian DDR. Future use of Nutlin-3 may need to consider the clinical implications of this novel role.