title: An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence.
abstract: BACKGROUND
  Cellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism.
  
  
  RESULTS
  To identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells.
  
  
  CONCLUSIONS
  The senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action.
authors: Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.
authors: Emilie
authors: E
authors: Rovillain
authors: Louise
authors: L
authors: Mansfield
authors: Christopher J
authors: CJ
authors: Lord
authors: Alan
authors: A
authors: Ashworth
authors: Parmjit S
authors: PS
authors: Jat
mesh_terms: D016922:Cellular Senescence / Q000235:genetics* / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D056426:Genetic Loci / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D009363:Neoplasm Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
chemical_list: C493956:CDKN1A protein, human; C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53