Identification of functional p53-binding motifs in the mouse wig-1 promoter

AbstractWe previously identified wig-1 as a p53-induced mouse gene that encodes a nuclear zinc finger protein with unknown function. To investigate whether wig-1 is a direct target of p53-dependent transactivation, a DNA fragment corresponding to the promoter region was cloned and sequenced. Three regions containing consensus p53-binding sites were identified. Two p53-binding motifs formed DNA–protein complexes with p53 and were able to drive p53-dependent transcription in a luciferase reporter assay. Our results demonstrate that wig-1 is a direct target of p53-mediated transcriptional transactivation

Novel Allosteric Mechanism of Dual p53/MDM2 and p53/MDM4 Inhibition by a Small Molecule

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising treatment strategy. However, several high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which promotes inhibition of both p53/MDM2 (murine double minute 2) and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA (reactivation of p53 and induction of tumor cell apoptosis) and protoporphyrin IX (PpIX). Ion mobility-mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions

The p53-induced gene wig-1 : Regulation of expression and role in embryonic development

The p53 tumor suppressor is a transcription factor that responds to various forms of cellular stress. Activated p53 induces growth arrest or apoptosis through transcriptional regulation of a wide range of target genes. More than 50% of human tumors carry mutations in the p53 gene, most of which reside within the DNAbinding core domain of p53. Most mutant p53 proteins are unable to bind to p53 motifs in DNA and activate transcription of target genes, and are therefore unable to initiate cell cycle arrest or apoptosis in response to cellular stress. The aims of this thesis were to characterize the regulation and function of the p53-induced gene wig-1 (wild type p53-induced gene-1). Wig-1 encodes a protein containing three Cys2His2 zinc fingers and a potential bipartite nuclear localization signal, NLS. Wig-1 is upregulated in response to activation of wild type p53 and this lead us to investigate whether wig-1 is a direct transcriptional target of p53. The promoter region of wig- I was cloned and partially characterized. Three putative p53-binding motifs were found in the mouse wig-1 promoter region. Two of the sites were found to form DNA/p 53 -complexes in electrophoretic mobility shift assay (EMSA) and were able to drive p53-dependent transcription in a cellular reporter assay. This demonstrates that wig-1 is a bona fide p53-target gene. When analysing the zinc finger structure of wig- I we observed that it shared some structural features with two other proteins, JAZ and dsRBP-Zfa, that have been found to bind dsRNA. Using EMSA we demonstrated that a recombinant GST-wig-1 fusion protein preferentially binds dsRNA over ssRNA or dsDNA. The first zinc finger in wig- I was found to be essential for the binding to dsRNA. Wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA-binding plays a role in the p53dependent stress response. To investigate whether the p53 paralogs, p73 and p63, could also regulate wig-1, we generated cell lines that express temperature sensitive mutant p73 or p63 that obtain wild type properties at the permissive temperature. Using these cells we could see that both p73 and p63 upregulate wig-1 on mRNA and protein level after temperature shift. Moreover, ectopically expressed wig- I stabilized p53 protein levels and inhibition of endogenous wig- I by siRNA reduced p53 protein in wild type p53-expressing MCF7 and HCT116 cells. In contrast, overexpression of wig-1 reduced p73 protein half-life and inhibition of endogenous wig-1 resulted in an increase in p73á and p73â protein levels. Our results demonstrate that wig-1 is transcriptionally induced by all members of the p53 family, and that wig-1 in turn acts as a positive regulator of p53 but antagonizes p73. Thus, wig- I can be viewed as a rheostat that regulates the relative activity of the p53 and p73 pathways. In order to study the role of wig-1 in embryonic development and its possible role in tumorigenesis, we generated mice carrying inactivated wig-] -alleles. We found that complete lack of wig-1 is associated with embryonic lethality. The wig-1 null mouse embryos died before E 10.5 during gestation and showed severely deficient development of the brain. These results demonstrate that wig- I is essential for normal embryonic development

Zukunft (die Zeitdimension des Handelns)

Le but de ce mémoire est d'apporter une contribution à l analyse de la structure phénoménologique et de la théorie de la constitution de l action humaine. Dans la théorie traditionnelle de l action, le temps est considéré comme l une des dimensions fondamentales et créatrices de l action. On essaiera de comprendre l action en soi et de l expliquer. Les thèses présentées dans ce travail se basent sur le postulat suivant : la structure et la fonction de l action ne peuvent être suffisamment comprises, si l action n'est pas saisie et conçue à partir du rapport des conditions existant entre le temps et l action. Ces thèses se limitent ainsi : l avenir qui constitue la dimension temporelle centrale pour l action et celui qui agit. Ce travail se divise en trois parties : l existence de l avenir, les critères de l avenir et l action et l avenir.PARIS1-BU Pierre Mendès-France (751132102) / SudocSudocFranceF

The Tumor Microenvironment of Medulloblastoma: An Intricate Multicellular Network with Therapeutic Potential

Medulloblastoma (MB) is a heterogeneous disease in which survival is highly affected by the underlying subgroup-specific characteristics. Although the current treatment modalities have increased the overall survival rates of MB up to 70–80%, MB remains a major cause of cancer-related mortality among children. This indicates that novel therapeutic approaches against MB are needed. New promising treatment options comprise the targeting of cells and components of the tumor microenvironment (TME). The TME of MB consists of an intricate multicellular network of tumor cells, progenitor cells, astrocytes, neurons, supporting stromal cells, microglia, immune cells, extracellular matrix components, and vasculature systems. In this review, we will discuss all the different components of the MB TME and their role in MB initiation, progression, metastasis, and relapse. Additionally, we briefly introduce the effect that age plays on the TME of brain malignancies and discuss the MB subgroup-specific differences in TME components and how all of these variations could affect the progression of MB. Finally, we highlight the TME-directed treatments, in which we will focus on therapies that are being evaluated in clinical trials

Generation of induced pluripotent stem cell lines from two Neuroblastoma patients carrying a germline ALK R1275Q mutation

Neuroblastoma (NB) is an embryonic tumor of the peripheral nervous system and one of the most common solid cancers in infants. Mutations in the Anaplastic lymphoma tyrosine kinase (ALK) gene are common in NB. To study the contribution of ALK mutations in NB initiation and progression, we reprogrammed fibroblasts from two related NB patients carrying germline mutations in ALK (R1275Q) using non-integrating Sendai virus. The iPS cells are grown in a feeder- and xeno-free conditions, have normal karyotype, retain the ALK R1275Q mutation, have been characterized by expression of pluripotency markers and differentiation to all three germ layers