p53-Regulation in Liver Regeneration

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DESCRIPTION (provided by applicant): Renewed organogenesis of the fully differentiated liver is stimulated in response to surgical removal (partial hepatectomy, PHx) or destruction of liver tissue by chemicals or disease. An induced network of signal transduction pathways triggers re-entry of hepatocytes (followed by non-parenchymal cells) into cell cycle, proliferation and compensatory growth, which terminates at a precisely regulated endpoint. We hypothesize that critical functions of the p53 tumor suppressor protein, in promoting cell cycle arrest and apoptosis, must be blocked or attenuated in response to partial hepatectomy. Our goal is to define the mechanisms of this regulatory process. The wealth of information, regarding regulation and functions of p53, stems primarily from studies of tumor-derived cells, cells under long term, continuous culture, and/or cells expressing dysfunctional, mutant or exogenous p53. Although evidence continues to mount that p53 acts in normal cells during development, aging and senescence, major gaps in our knowledge exist regarding mechanisms of endogenous p53-regulation. Tumor suppressor p53 is expressed at low levels, which are tightly controlled by multiple pathways. This offers a considerable challenge to biochemical purification and mechanistic analyses of endogenous p53, especially in normal cells. We have developed a new mouse model to address these gaps in our knowledge. The mouse model, created by knock-in methodology, expresses endogenous p53 fused in-frame with a C-terminal epitope-tag (TAP-p53). TAP or Tandem Affinity Purification is a proven means of purifying low-abundance, large protein complexes under relatively physiological conditions. TAP-p53 purification and subsequent peptide analyses by mass spectrometry will be used to define p53-protein interactions (the p53 "proteome") and to determine post-translational modifications of p53. Epitope-tagging of p53 further facilitates studies of p53-chromatin interactions by chromatin immunoprecipitation (ChIP) and genome-wide analysis of p53-targets (ChIP-chip). We will use these and other approaches to establish the ground state of p53 function and regulation in normal hepatic cells and to determine how these functions may be altered during liver regeneration. The proposed research will address how p53 functions in normal, differentiated cells, an area generally overlooked in investigations of tumor suppressor activities. This work will further molecular understanding of tissue regeneration with a long-term goal of understanding how the surveillance status of the p53-network may be temporally controlled to facilitate cellular renewal and tissue regeneration. PROJECT NARRATIVE: These studies are focused on understanding how a fully differentiated tissue circumvents regulation and surveillance by tumor suppressor p53 to regenerate itself in response to partial hepatectomy. We believe that p53, which normally stops growth and proliferation, is temporarily blocked from functioning during regeneration and then restored to normal capacities when regeneration is finished. How p53 functions in normal cells and during regeneration is essentially unknown.