Abstract
Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using (18)O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO(2) species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.
Highlights
Catalytically active members of this family, carbonic anhydrase IX (CAIX) and carbonic anhydrase XII, are tumorrelated [3]
Using the 18O exchange method, which directly measures CA activity, we demonstrate that exofacial CA activity increases in intact MDA-MB-231 cells in response to hypoxia
This conclusion is based on the significant changes to the rates of depletion of 18O from CO2 in whole cell suspensions of MDA-MB-231 cells, an experimental system thoroughly characterized in red blood cells [22, 28, 30, 31]
Summary
Cell Culture—The MDA-MB-231 breast cancer cell line was provided by Dr Kevin Brown (University of Florida). Cells were washed three times with cold PBS (2.7 mM KCl, 10 mM phosphate salts, 120 mM NaCl (pH 7.4)) and exposed to hypotonic buffer (1 ml/plate of a solution containing 2.7 mM KCl, 10 mM phosphate salts (pH 7.4)) in the presence of protease inhibitors (Roche) for 15 min at 4 °C. The protonation of the zincbound, 18O-labeled hydroxide results in the release of H218O, which is essentially infinitely diluted by H216O in the solvent (Equation 2) This method is used to obtain catalytic rates R1 from the 18O exchange catalyzed by carbonic anhydrase [26, 28]. The membrane inlet was immersed in the medium in the reaction vessel and used to detect the atom fraction of 18O in extracellular 13CO2 This activity was measured after addition of cells (exposed to normoxic or hypoxic conditions) in medium at specific pH values and with specific CA inhibitors. The cells were isolated and assayed under anoxic conditions
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