title: Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors.
abstract: OBJECTIVE
  Carbonic anhydrase (CA) IX expression is increased in response to hypoxia. Recently, sulfonamide based carbonic anhydrase inhibitors (CAI) showing specificity for CA IX have been designed. Aim was to investigate the CAI binding properties under normoxia, hypoxia and reoxygenation.
  
  
  METHODS
  Cells with varying CA IX expression were incubated with fluorescein labeled CAI (1mM) during normoxia, hypoxia (0.2%) and reoxygenation. CA IX expression levels were assessed using Western blotting. CAI binding was determined by immunostaining and flow cytometry.
  
  
  RESULTS
  CAI binding in hypoxic cells was significantly higher compared with normoxic cells and correlated with upregulated CA IX levels. Binding occurred within 15min of hypoxia, but was gradually lost upon reoxygenation. Interestingly, although CA IX levels remained high upon reoxygenation, CAI binding was dramatically reduced and no longer correlated with CA IX expression. Similarly, RCC4 cells, constitutively expressing CA IX, do not bind CAI under normoxic conditions.
  
  
  CONCLUSIONS
  Our results confirm and extend previous results showing that CAI binding occurs only under hypoxia. The inability of CAI to bind CA IX in RCC4 cells and following reoxygenation in other cells demonstrates that formation of the active site not only depends on HIF-1alpha-dependent gene activity, but also on the absence of oxygen per se.
authors.affiliation: Maastricht Radiation Oncology (MaastRO) Lab, Grow Research Institute, University of Maastricht, The Netherlands.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Dubois
authors.forename: Kim
authors.initials: K
authors.lastname: Douma
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Roland K
authors.initials: RK
authors.lastname: Chiu
authors.forename: Marc A M J
authors.initials: MA
authors.lastname: van Zandvoort
authors.forename: Silvia
authors.initials: S
authors.lastname: Pastoreková
authors.forename: Andrea
authors.initials: A
authors.lastname: Scozzafava
authors.forename: Bradly G
authors.initials: BG
authors.lastname: Wouters
authors.forename: Philippe
authors.initials: P
authors.lastname: Lambin
mesh_terms: D000951:Antigens, Neoplasm / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D005260:Female; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D010100:Oxygen / Q000378:metabolism*; D013449:Sulfonamides / Q000493:pharmacokinetics
chemical_list: D000951:Antigens, Neoplasm; D002257:Carbonic Anhydrase Inhibitors; D013449:Sulfonamides; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D010100:Oxygen