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Summary In vitro studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. To study the physiological significance of the RP-Mdm2 interaction, we generated mice carrying a cancer-associated cysteine-to-phenylalanine substitution in the zinc finger of Mdm2 that disrupted its binding to RPL5 and RPL11. Mice harboring this mutation, although retain normal p53 response to DNA damage, lack p53 response to perturbations in ribosome biogenesis. Loss of RP-Mdm2 interaction significantly accelerates Eμ-Myc induced lymphomagenesis. Furthermore, ribosomal perturbation induced p53 response does not require tumor suppressor p19Arf. Collectively, our findings establish RP-Mdm2 interaction as a genuine p53 stress-signaling pathway activated by aberrant ribosomal biogenesis and essential for safeguarding against oncogenic c-Myc-induced tumorigenesis.

The U.S. Environmental Protection Agency (EPA) is faced with the challenge of efficiently and credibly evaluating chemical safety often with limited or no available toxicity data. The expanding number of chemicals found in commerce and the environment, coupled with time and resource requirements for traditional toxicity testing and exposure characterization, continue to underscore the need for new approaches. In 2005, EPA charted a new course to address this challenge by embracing computational toxicology (CompTox) and investing in the technologies and capabilities to push the field forward. The return on this investment has been demonstrated through results and applications across a range of human and environmental health problems, as well as initial application to regulatory decision-making within programs such as the EPA’s Endocrine Disruptor Screening Program. The CompTox initiative at EPA is more than a decade old. This manuscript presents a blueprint to guide the strategic and operational direction over the next 5 years. The primary goal is to obtain broader acceptance of the CompTox approaches for application to higher tier regulatory decisions, such as chemical assessments. To achieve this goal, the blueprint expands and refines the use of high-throughput and computational modeling approaches to transform the components in chemical risk assessment, while systematically addressing key challenges that have hindered progress. In addition, the blueprint outlines additional investments in cross-cutting efforts to characterize uncertainty and variability, develop software and information technology tools, provide outreach and training, and establish scientific confidence for application to different public health and environmental regulatory decisions.

The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.The mammalian p53 transcription factor mediates a major tumor suppression pathway that is negatively controlled by the proto-oncoprotein MDM2 (HDM2 in humans; henceforth denoted MDM2) and is altered in most, if not all, human cancers. The gene for mouse Mdm2 (murine double minute 2) was originally identified in a spontaneously transformed mouse BALB/c cell line (13). The Mdm2 protein was found to be responsible for transformation of NIH 3T3 and Rat2 cells when overexpressed (13), and this transforming function is believed to stem from its ability to bind with and inhibit the transactivation activity of p53 (39). Subsequently, the HDM2 gene, the human homologue of Mdm2, was found to be amplified in over one-third of those human sarcomas that still retain wild-type p53 (41), suggesting that overexpression of MDM2 could be a common mechanism by which cells inactivate p53. Mice with targeted deletion of the Mdm2 gene die during early embryonic development, and this lethality can be rescued by concomitant deletion of p53, indicating that a major in vivo function of MDM2 is to keep p53 activity in check (27,33).It is believed that MDM2 controls p53 through two mechanisms: inhibition of the transcriptional activity of p53 (39) and promotion of p53 ubiquitination and degradation (18,29). Mdm2 binds to and masks the N-terminal transactivation domain of p53 by directly interfering with the interaction between p53 and the basal transcriptional machinery (42, 54). Mdm2 belongs to a large family of RING finger ubiquitin ligases (25). Studies have demonstrated that Mdm2 is a ubiquitin ligase (19) and that the ubiquitin ligase activity of Mdm2 is responsible for degradation of p53 both in vitro (14,20) and in transfected cells (14). MDM2-mediated p53 degradation also depends on its ability to promote p53 nuclear export (46). Mutation of a nuclear export signal (NES) in MDM2 abolishes ...