Abstract
The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.
Highlights
In addition to its role as a ubiquitin protein ligase for p53, HDM2 has been implicated in facilitating p53 nuclear export, an activity that appears necessary for its ability to degrade the protein [7, 8]
We utilized a panel of NB cell lines to examine the relationship between p53 stability and HDM2 function, and we further investigated whether the p53 protein present in Nand S-type cells is susceptible to HDM2-mediated degradation
We employed only wild type (WT) p53-containing cell lines, and we further confirmed that the p53 in LAN5 is WT by sequencing exons 2–11
Summary
We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2 In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway. Neuroblastoma is complex, in that tumors are composed of a mixture of neuroblastic (N-type) and Schwannian (S-type) cell morphologies that differ in tumorigenicity [22] and in p53 subcellular localization [23] Taken together, these observations suggest that subversion of p53 function by mechanisms independent of mutation potentially play a role in the progression of NB. We suggest that direct inactivation of HDM2 ubiquitin ligase function may contribute significantly to the stabilization of p53
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