Human MDM2 Knockout Cell Line-HeLa

MDM2 (murine double minute 2) is a key inhibitor of p53-mediated tumor suppressor functions, including cellular senescence. Werner syndrome (WS) is caused by mutations in the WRN gene and is an autosomal recessive disorder. Loss of WRN function induces senescence in human cancer cells. Here, the researchers show that MDM2 acts as an E3 ligase for the WRN protein. MDM2 interacts with WRN both in vivo and in vitro. MDM2 induces WRN ubiquitination and significantly downregulates the levels of WRN protein in human cells. During the DNA damage response, WRN is translocated to the nucleoplasm to promote its DNA repair function. However, it is degraded by the MDM2-mediated ubiquitination pathway. Furthermore, the senescence phenotype induced by DNA damaging agents, such as etoposide, is at least partially mediated by MDM2-dependent WRN degradation, as ectopic expression of WRN can significantly attenuate this degradation. These results demonstrate that MDM2 plays a critical role in regulating WRN function through ubiquitin-dependent degradation and reveal an unexpected role for MDM2 in promoting cellular senescence in a p53-independent manner.

Here, the researchers performed cycloheximide (CHX) chase experiments to examine whether MDM2 could regulate the half-life of WRN protein by transfecting HCT116 cells with a combination of plasmids producing MYC-WRN and MDM2. When MDM2 was co-expressed, the half-life of WRN decreased dramatically (Figure 1a). In addition, the half-life of WRN was increased in the MDM2 knockout cell line compared to the wild-type cell line (Figure 1b).

Figure 1. a When MDM2 was coexpressed, the half-life of WRN decreased dramatically. B WRN half-life was increased in MDM2 knockout cell lines compared with that in wild-type cell lines. (Liu, Boya, et al. 2019)