Abstract 2056: Evidence against hypoxic-dependent activation of carbonic anhydrase IX in MDA-MB-231 breast cancer cells

Abstract

Abstract Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme the expression of which is often considered a marker for hypoxia, an indicator of poor prognosis, and associated with acidification of the tumor microenvironment. Many studies have shown that CAIX expression is induced by hypoxia exposing its catalytic domain to the interstitial milieu. Several recent studies have suggested that hypoxic conditions may also permit activation of CAIX, perhaps by causing a conformational change which exposes the catalytic pocket. Our goal was to assess the effect of anoxic conditions on CAIX activity in MDA-MB-231 cells, previously exposed to hypoxia which increases CAIX expression in the absence of other membrane-bound carbonic anhydrase (CA) family members. We have taken advantage of membrane inlet mass spectrometry (MIMS) to directly analyze CA activity in intact cells by measuring the 18O exchange between CO2 and H2O. This method distinguishes between intracellular and extracellular CA activity. MDA-MB-231 cells were exposed to 1% oxygen for 16 hours after which they were isolated under normoxic or anoxic conditions. CA activity was then measured, again under normoxic or anoxic conditions. These data show biphasic depletion of 18O from CO2 under both normoxic and anoxic assay conditions. The first phase (which occurs over the first 20-40 seconds) represents a rapid diffusion of CO2 into cells where it is exposed to intracellular CAII. A catalytic cycle depletes 18O followed by efflux of CO2 from the cell. There was no difference in this phase between cells prepared and assayed under normoxic or anoxic conditions. The second phase (from 200-500 sec) is dominated by the hydration-dehydration reaction of CO2/HCO3− catalyzed by exofacial CA activity (CAIX). In cells exposed to anoxia, the slope of the second phase was greater than that observed with cells exposed to normoxic conditions indicating elevated CAIX activity. While this provided evidence that oxygen limitation might influence CAIX activity, the Ki values for two impermeant CA inhibitors, Cpd 5C and a polymeric sulfonamide, did not differ between anoxic and normoxic cells. We conclude from these data that the catalytic site of CAIX is exposed and functional under both anoxic or normoxic conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2011-2056