title: Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts.
abstract: BACKGROUND
  In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.
  
  
  METHODS
  We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.
  
  
  RESULTS
  Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.
  
  
  CONCLUSIONS
  Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, P,O, Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Kristiaan
authors.initials: K
authors.lastname: Lenos
authors.forename: Job
authors.initials: J
authors.lastname: de Lange
authors.forename: Amina F A S
authors.initials: AF
authors.lastname: Teunisse
authors.forename: Kirsten
authors.initials: K
authors.lastname: Lodder
authors.forename: Matty
authors.initials: M
authors.lastname: Verlaan-de Vries
authors.forename: Eliza
authors.initials: E
authors.lastname: Wiercinska
authors.forename: Marja J M
authors.initials: MJ
authors.lastname: van der Burg
authors.forename: Karoly
authors.initials: K
authors.lastname: Szuhai
authors.forename: Aart G
authors.initials: AG
authors.lastname: Jochemsen
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D018797:Cell Cycle Proteins; D049109:Cell Proliferation; D048430:Cell Shape; D002471:Cell Transformation, Neoplastic / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology*; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D009687:Nuclear Proteins / Q000378:metabolism*; D009857:Oncogenes; D010879:Piperazines / Q000378:metabolism; D061251:Primary Cell Culture; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012160:Retina / Q000196:embryology / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D007093:Imidazoles; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D010879:Piperazines; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3