MDM2 Antagonist Nutlin-3 Stimulates Global DNA Hydroxymethylation by Enhancing p53-TET1 Signaling Axis

DNA hydroxymethylation is involved in many biologicalprocesses,including nuclear reprogramming, embryonic development, and tumorsuppression. In this study, we report that an anticancer agent, nutlin-3,selectively stimulates global DNA hydroxymethylation in TP53 wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine(5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNAhydroxymethylation in TP53-mutated cancer cells.Consistently, nutlin-3 as a MDM2 antagonist only activates wild-typebut not mutated TP53. Furthermore, nutlin-3 doesnot alter the expression of TET1 but slightly reduces the expressionof TET2 and TET3 proteins. These TET family proteins are responsiblefor converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1knockdown could significantly block the nutlin-3-induced DNA hydroxymethylationas well as TP53 and P21 activation.Immunoprecipitation analysis supports that p53 strongly interactswith TET1 proteins. These results suggest that nutlin-3 activates TP53 and promotes p53-TET1 interaction. As positivefeedback, the p53-TET1 interaction further enhances p53 activationand promotes apoptosis. Collectively, we demonstrate that nutlin-3stimulates DNA hydroxymethylation and apoptosis via a positive feedbackmechanism.