title: Hypoxia inducible prolyl hydroxylase PHD3 maintains carcinoma cell growth by decreasing the stability of p27.
abstract: BACKGROUND
  Hypoxia can halt cell cycle progression of several cell types at the G1/S interface. The arrest needs to be overcome by cancer cells. We have previously shown that the hypoxia-inducible cellular oxygen sensor PHD3/EGLN3 enhances hypoxic cell cycle entry at the G1/S boundary.
  
  
  METHODS
  We used PHD3 knockdown by siRNA and shRNA in HeLa and 786-0 renal cancer cells. Flow cytometry with cell synchronization was used to study cell growth at different cell cycle phases. Total and phosphospecific antibodies together with cycloheximide chase were used to study p27/CDKN1B expression and fractionations for subcellular protein localization.
  
  
  RESULTS
  Here we show that PHD3 enhances cell cycle by decreasing the expression of the CDK inhibitor p27/CDKN1B. PHD3 reduction led to increased p27 expression under hypoxia or VHL mutation. p27 was both required and sufficient for the PHD3 knockdown induced cell cycle block. PHD3 knockdown did not affect p27 transcription and the effect was HIF-independent. In contrast, PHD3 depletion increased the p27 half-life from G0 to S-phase. PHD3 depletion led to an increase in p27 phosphorylation at serine 10 without affecting threonine phosphorylation. Intact serine 10 was required for normal hypoxic and PHD3-mediated degradation of p27.
  
  
  CONCLUSIONS
  The data demonstrates that PHD3 can drive cell cycle entry at the G1/S transition through decreasing the half-life of p27 that occurs by attenuating p27S10 phosphorylation.
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6B, 20520, Turku, Finland. hhogel@btk.fi.|Department of Medical Biochemistry, Faculty of Medicine, University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland. hhogel@btk.fi.
authors.forename: Heidi
authors.initials: H
authors.lastname: Högel
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6B, 20520, Turku, Finland. pmiikkul@btk.fi.|Department of Medical Biochemistry, Faculty of Medicine, University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland. pmiikkul@btk.fi.
authors.forename: Petra
authors.initials: P
authors.lastname: Miikkulainen
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6B, 20520, Turku, Finland. lucia.bino@gmail.com.|Present address: Institute of Biophysics, The Academy of Sciences of the Czech Republic, Brno, Czech Republic. lucia.bino@gmail.com.
authors.forename: Lucia
authors.initials: L
authors.lastname: Bino
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6B, 20520, Turku, Finland. panjaa@utu.fi.|Department of Medical Biochemistry, Faculty of Medicine, University of Turku, Kiinamyllynkatu 10, 20520, Turku, Finland. panjaa@utu.fi.|Department of Oncology and Radiotherapy, Turku University Hospital, Hämeentie 11, 20520, Turku, Finland. panjaa@utu.fi.
authors.forename: Panu M
authors.initials: PM
authors.lastname: Jaakkola
mesh_terms: D002277:Carcinoma / Q000235:genetics* / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics*; D015687:Cell Hypoxia / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis* / Q000235:genetics; D059585:G1 Phase Cell Cycle Checkpoints / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000037:antagonists & inhibitors / Q000235:genetics*; D010766:Phosphorylation; D034741:RNA, Small Interfering
chemical_list: C436291:CDKN1B protein, human; D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases