Prolyl Hydroxylase PHD3 Enhances the Hypoxic Survival and G1 to S Transition of Carcinoma Cells Heidi Ho¨ gel1., Krista Rantanen1., Terhi Jokilehto1, Reidar Grenman2,3, Panu M. Jaakkola1,4* 1 Turku Centre for Biotechnology, Turku University and A˚ bo Akademi University, Turku, Finland, 2 Department of Medical Biochemistry, Turku University, Turku, Finland, 3 Department of Otorhinolaryngology, Head and Neck Surgery, Turku University Hospital, Turku, Finland, 4 Department of Oncology and Radiotherapy, Turku University Hospital, Turku, Finland Abstract Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells. Citation: H o¨gel H, Rantanen K, Jokilehto T, Grenman R, Jaakkola PM (2011) Prolyl Hydroxylase PHD3 Enhances the Hypoxic Survival and G1 to S Transition of Carcinoma Cells. PLoS ONE 6(11): e27112. doi:10.1371/journal.pone.0027112 Editor: Fabio Martelli, Istituto Dermopatico dell’Immacolata-IRCCS, Italy Received April 29, 2011; Accepted October 10, 2011; Published November 8, 2011 Copyright: 2011 H o¨gel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by The Academy of Finland (grant 200462), Emil Aaltonen Foundation, Turku University Hospital (EVO13031) and The Turku University Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: panjaa@utu.fi . These authors contributed equally to this work. Introduction Oxygen level in tissues is reduced in many pathological conditions including cancer progression [1]. Solid tumours develop areas of poor oxygenation as they outgrow their blood supply. Mammalian cells have developed an array of mechanisms in order to adapt to and survive under varying oxygen tensions. The responses to hypoxia include upregulation of angiogenic factors and the switch from oxidative to glycolytic metabolism [2]. In cancer cells low oxygen tension induces genetic instability [3,4] and is a strong driving force in the clonal selection that supports more aggressive disease. When cells are exposed to acute hypoxia cell proliferation is inhibited by several mechanisms such as activation of quiescence, apoptosis, necrosis and differentiation. In hypoxia also cell cycle is arrested occuring at the G1/S interface [5,6]. The key molecular event needed for the G1 to S transition is the hyperpho- sphorylation of the retinoblastoma protein (Rb) that is performed by cyclin-dependent kinase (CDK) –cyclin complexes. Hyperpho- sphorylation of Rb at late G1 releases E2F transcription factor and allows S-phase to proceed. Under hypoxic conditions Rb is hypophosphorylated thus inhibiting the transcription of S-phase genes and cell cycle progression [7,8,9]. Several possible mechanisms as to how hypoxia may regulate cell cycle progression have been suggested. Hypoxia has been reported to upregulate p21(Cip1/Waf1/CDKN1A) and p27(Kip1/CDKN1B) which regulate the inactivation of cyclin E – CDK2 –complex. While there is some controversy on the role of p27, the hypoxic cell cycle arrest does not seem to require p21 [10,11]. Hypoxia may also activate cell cycle arrest by inhibiting c-Myc transcriptional activity [12]. Also p16(INK4a) that attenuates cell cycle progres- sion has been shown to be hypoxia-inducible [13]. Noticeably, a subpopulation of cancer cells need to escape the hypoxic cell cycle arrest in order to maintain growth. Many of the responses to hypoxia are mediated by hypoxia- inducible transcription factor (HIF) that is rapidly degraded in normoxia but stabilized under hypoxia [14]. In normoxia the alpha subunit (HIF-a) is hydroxylated by a family of prolyl hydroxylase domain containing enzymes (PHDs) [15,16]. Hy- droxylation of HIF-a allows its recognition by the von Hippel- Lindau protein and leads to subsequent proteosomal degradation of HIF-a [17,18,19,20]. PHDs are enzymes that require molecular oxygen as substrate. Therefore under hypoxic conditions the activity of PHDs is lowered thus allowing HIF-a to escape degradation. The stabilized HIF-a is translocated to nucleus where it dimerizes with HIF-1beta forming an active transcription factor PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27112