Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors

Background and purposeCarbonic 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.Material and methodsCells with varying CA IX expression were incubated with fluorescein labeled CAI (1 mM) 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.ResultsCAI binding in hypoxic cells was significantly higher compared with normoxic cells and correlated with upregulated CA IX levels. Binding occurred within 15 min 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.ConclusionsOur 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-1α-dependent gene activity, but also on the absence of oxygen per se.