title: Normoxic accumulation of HIF1α is associated with glutaminolysis.
abstract: OBJECTIVE
  The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood.
  
  
  METHODS
  We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR.
  
  
  RESULTS
  Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine.
  
  
  CONCLUSIONS
  Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions.
  
  
  CONCLUSIONS
  Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany. matthias.kappler@medizin.uni-halle.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kappler
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Pabst
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
authors.forename: Swetlana
authors.initials: S
authors.lastname: Rot
authors.affiliation: Department of Urology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany.
authors.forename: Helge
authors.initials: H
authors.lastname: Taubert
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
authors.forename: Henri
authors.initials: H
authors.lastname: Wichmann
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schubert
authors.affiliation: Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Halle(S), Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Bache
authors.affiliation: Institute of Computer Science, Martin Luther University Halle-Wittenberg, Halle(S), Germany.
authors.forename: Claus
authors.initials: C
authors.lastname: Weinholdt
authors.affiliation: Institute of Legal Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany.
authors.forename: Uta-Dorothee
authors.initials: UD
authors.lastname: Immel
authors.affiliation: Institute of Computer Science, Martin Luther University Halle-Wittenberg, Halle(S), Germany.
authors.forename: Ivo
authors.initials: I
authors.lastname: Grosse
authors.affiliation: Department of Radiotherapy, Martin Luther University Halle-Wittenberg, Halle(S), Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Vordermark
authors.affiliation: Department of Oral and Maxillofacial Plastic Surgery, Martin Luther University Halle-Wittenberg, Ernst-Grube-Str. 40, 06097, Halle(S), Germany.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Eckert
mesh_terms: D000641:Ammonia / Q000378:metabolism; D015153:Blotting, Western; D000071231:Carbonic Anhydrase IX / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D005973:Glutamine; D000641:Ammonia; D000071231:Carbonic Anhydrase IX
keywords: Ammonia; Ammonium; Glutamine; Glutaminolysis; HIF1α; Metabolism; Normoxia; Warburg effect