SummaryHepatocytes and cholangiocytes self renew following liver injury. Following severe injury hepatocytes are increasingly senescent, whether Hepatic Progenitor Cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where Mdm2 is inducibly deleted in over 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease.

One Sentence Summary:Inhibiting injury-induced senescence mediated by TGFβ signaling in regenerative epithelium improves liver regeneration. Accessible Summary:The liver is a paradigm of organ regeneration, however regeneration may fail in a previously normal liver following acute severe injury such as acetaminophen poisoning. We show that, a process with prevents proliferation termed senescence, which is classically associated with aging and carcinogenesis, stops the liver's regenerative cells. This senescence can be spread from cell to cell by the signaling molecule TGFβ. When TGFβ signaling is inhibited during acetaminophen poisoning in mice, senescence is impeded, regeneration accelerates, and survival is improved. Therefore targeting senescence induced by acute tissue injury is an attractive therapeutic approach to improve regeneration. Abstract:Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy.However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction 4 of paracrine senescence was observed within twenty four hours, and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended upon macrophage-derived TGFβ1 ligand. In acetaminophen poisoning, inhibition of TGFβ receptor 1 (TGFβR1) improved mouse survival. TGFβR1 inhibition reduced senescence and enhanced liver regeneration even when delivered after the current therapeutic window for acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.

Germ-line heterozygosity of the BRCA2 gene in women predisposes to breast and ovarian cancers. Successful therapies targeted specifically at these neoplasms have thus far remained elusive. Recent studies in mice have shown that inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) targets cells lacking Brca2 and xenografts derived from BRCA2-deficient ES cells or Chinese hamster ovary cells. We set out to develop a more relevant preclinical model that will inform and accelerate translation into the clinic. As such, we conditionally deleted Brca2 and p53 within murine mammary epithelium and treated the resulting tumors in situ with a highly potent PARP-1 inhibitor (AZD2281) alone or in combination with carboplatin. Daily exposure to AZD2281 for 28 days caused significant regression or growth inhibition in 46 of 52 tumors. This response was shown to be specific to tumors lacking both Brca2and p53. AZD2281/carboplatin combination therapy for 28 days showed no advantage over carboplatin monotherapy. However, if PARP inhibitor treatment was continued, this significantly increased the time to tumor relapse and death in these mice. This preclinical study is the first to show in vivo hypersensitivity of spontaneously arising Brca2-deficient mammary tumors to PARP-1 inhibition monotherapy or combination therapy. As such, our data add substantial weight to the argument for the use of PARP inhibitors as therapeutic agents against human breast cancers in which BRCA2 is mutated. Moreover, the specificity that we have shown further suggests that PARP inhibitors will be generally effective against tumors caused by dysregulation of components of the homologous recombination pathway.

The MDM2 oncoprotein is a negative regulatory partner of the p53 tumour suppressor. MDM2 mediates ubiquitination of p53 and targets the protein to the cytoplasm for 26S proteosome-dependent degradation. In this paper, we show that MDM2 is modified in cultured cells by multisite phosphorylation. Deletion analysis of MDM2 indicated that the sites of modification fall into two clusters which map respectively within the N-terminal region encompassing the p53 binding domain and nuclear export sequence, and the central acidic domain that mediates p14 ARF binding, p53 ubiquitination and cytoplasmic shuttling. The data are consistent with potential regulation of MDM2 function by multisite phosphorylation. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.