Wu Danni, Li Yao, Li Cuiping, Zhong Shangwei, Liu Baodong, Hang Haiying, Wang Hailin
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
China General Microbiological Culture Collection Center, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
ACS Chem Biol. 2023 Oct 20;18(10):2240-2248. doi: 10.1021/acschembio.3c00247. Epub 2023 Jul 18.
DNA hydroxymethylation is involved in many biological processes, including nuclear reprogramming, embryonic development, and tumor suppression. In this study, we report that an anticancer agent, nutlin-3, selectively stimulates global DNA hydroxymethylation in wild-type cancer cells as manifested by the elevation of 5-hydroxymethylcytosine (5hmC) in genomic DNA. In contrast, nutlin 3 fails to enhance DNA hydroxymethylation in -mutated cancer cells. Consistently, nutlin-3 as a MDM2 antagonist only activates wild-type but not mutated . Furthermore, nutlin-3 does not alter the expression of TET1 but slightly reduces the expression of TET2 and TET3 proteins. These TET family proteins are responsible for converting 5-methylcytosine (5mC) to 5hmC. Interestingly, TET1 knockdown could significantly block the nutlin-3-induced DNA hydroxymethylation as well as and activation. Immunoprecipitation analysis supports that p53 strongly interacts with TET1 proteins. These results suggest that nutlin-3 activates and promotes p53-TET1 interaction. As positive feedback, the p53-TET1 interaction further enhances p53 activation and promotes apoptosis. Collectively, we demonstrate that nutlin-3 stimulates DNA hydroxymethylation and apoptosis via a positive feedback mechanism.
DNA羟甲基化参与许多生物学过程，包括细胞核重编程、胚胎发育和肿瘤抑制。在本研究中，我们报道了一种抗癌药物nutlin-3，它能选择性地刺激野生型癌细胞中的整体DNA羟甲基化，这表现为基因组DNA中5-羟甲基胞嘧啶（5hmC）水平的升高。相比之下，nutlin-3无法增强突变癌细胞中的DNA羟甲基化。一致地，作为MDM2拮抗剂的nutlin-3仅激活野生型而非突变型。此外，nutlin-3不会改变TET1的表达，但会略微降低TET2和TET3蛋白的表达。这些TET家族蛋白负责将5-甲基胞嘧啶（5mC）转化为5hmC。有趣的是，敲低TET1可显著阻断nutlin-3诱导的DNA羟甲基化以及和激活。免疫沉淀分析支持p53与TET1蛋白强烈相互作用。这些结果表明，nutlin-3激活并促进p53-TET1相互作用。作为正反馈，p53-TET1相互作用进一步增强p53激活并促进细胞凋亡。总体而言，我们证明nutlin-3通过正反馈机制刺激DNA羟甲基化和细胞凋亡。
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