Induction by hypoxia combined with low glucose or low bicarbonate and high posttranslational stability upon reoxygenation contribute to carbonic anhydrase IX expression in cancer cells

Hypoxia is an important factor of tumor microenvironment that significantly influences behaviour of tumor cells via activation of genes whose products are involved in adaptation to hypoxic stress, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT-1). Carbonic anhydrase IX (CA IX) is one of the most strongly hypoxia-inducible proteins with potential value as an intrinsic marker of hypoxia. However, intratumoral distribution of CA IX only partially overlaps with distribution of VEGF and GLUT-1 indicating that regulation of CA IX differs from the regulation of other hypoxic markers. Therefore, we analysed CA IX expression in response to hypoxia combined with other stresses, and determined the stability of CA IX protein upon reoxygenation using HeLa cells as a model. We found that both hypoxia-induced transcription and CA IX protein level are further increased by reduced glucose or bicarbonate concentrations. Post-translational stability of CA IX was assessed by monitoring the quantity of biotinylated protein extracted at different time points from the cells labelled immediately after shift to reoxygenation. CA IX protein half-life in reoxygenated cells was 38 h and was independent of the duration of the foregoing hypoxia. This finding has potential implications for interpretation of clinical data as it suggests that CA IX expression may detect not only actually hypoxic tumor regions, but also the regions affected by hypoxia and adverse microenvironmental stresses before biopsy or tumor removal.