title: Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway.
abstract: BACKGROUND
  The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins.
  
  
  METHODS
  Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins.
  
  
  RESULTS
  More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2.
  
  
  CONCLUSIONS
  Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. ping.zhang@ludwig.ox.ac.uk.|Faculty of Biosciences, University of Heidelberg, 69120, Heidelberg, Germany. ping.zhang@ludwig.ox.ac.uk.|Present address: Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Oxford, OX3 7DQ, UK. ping.zhang@ludwig.ox.ac.uk.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. annesophie.kratz@yahoo.de.|Present address: Cell Cycle Control and Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120, Heidelberg, Germany. annesophie.kratz@yahoo.de.
authors.forename: Anne Sophie
authors.initials: AS
authors.lastname: Kratz
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. mohammed.salama@kit.edu.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Salama
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. seham.elabd@alex-mri.edu.eg.
authors.forename: Seham
authors.initials: S
authors.lastname: Elabd
authors.affiliation: Department of Anti-Aging Medicine, University of Tokyo, Tokyo, 113-8865, Japan. thorsten@cbcmp.icou.osaka-u.ac.jp.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Heinrich
authors.affiliation: Department of Developmental Biology and Physiology, University of Heidelberg, 69120, Heidelberg, Germany. jochen.wittbrodt@cos.uni-heidelberg.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Wittbrodt
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. christine.blattner@kit.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Blattner
authors.affiliation: Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76021, Karlsruhe, Germany. gary.davidson@kit.edu.
authors.forename: Gary
authors.initials: G
authors.lastname: Davidson
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D019143:Evolution, Molecular; D005786:Gene Expression Regulation / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D009990:Oryzias / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2