title: Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells.
abstract: OBJECTIVE
  Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells.
  
  
  METHODS
  HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy.
  
  
  RESULTS
  Hypoxia-induced CA IX protein expression was seen after >6 h at < or =5% O(2), and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy.
  
  
  CONCLUSIONS
  CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1alpha. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro and can be applied to sort chronically hypoxic cells from tumors.
authors.affiliation: Department of Radiation Oncology, University of Würzburg, Josef-Schneider-Str, 97080 Würzburg, Germany. vordermark_d@klinik.uni-wuerburg.de
authors.forename: Dirk
authors.initials: D
authors.lastname: Vordermark
authors.forename: Anja
authors.initials: A
authors.lastname: Kaffer
authors.forename: Susanne
authors.initials: S
authors.lastname: Riedl
authors.forename: Astrid
authors.initials: A
authors.lastname: Katzer
authors.forename: Michael
authors.initials: M
authors.lastname: Flentje
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor / Q000528:radiation effects; D005434:Flow Cytometry; D006801:Humans; D011836:Radiation Tolerance; D013997:Time Factors
chemical_list: D000951:Antigens, Neoplasm; D015415:Biomarkers; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases