Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors
Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors
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DOI：
10.1016/j.radonc.2007.04.018
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发表时间：
2007-06-01
影响因子：
5.7
通讯作者：
Lambin, Philippe
中科院分区：
文献类型：
--
作者：
Dubois, Ludwig;Douma, Kim;Lambin, Philippe
背景与目的：碳酸酐酶（CA） IX在缺氧条件下表达增加。近年来，基于磺胺的碳酸酐酶抑制剂（CAI）已被设计出对caix具有特异性。目的是研究CAI在常氧、缺氧和再氧作用下的结合特性。材料和方法：用荧光素标记的CAI （1 mM）培养不同CA IX表达的细胞，分别在常氧、缺氧（0.2%）和复氧条件下培养。Western blotting检测CA IX表达水平。免疫染色和流式细胞术检测CAI结合情况。结果：缺氧细胞中CAI结合明显高于常氧细胞，且与caix水平上调相关。结合在缺氧15分钟内发生，但在再氧后逐渐消失。有趣的是，虽然CA IX水平在再氧化后仍然很高，但CAI结合显著降低，不再与CA IX表达相关。同样，组成性表达caix的RCC4细胞在常温条件下不结合CAI。结论：我们的结果证实并扩展了先前的结果，即CAI仅在缺氧情况下发生结合。CAI无法在RCC4细胞中结合CA IX，并在其他细胞中再氧化，这表明活性位点的形成不仅取决于HIF-1 α依赖性基因的活性，还取决于缺氧本身。2007爱思唯尔爱尔兰有限公司版权所有。
Background and purpose: Carbonic 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 methods: Cells 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.Results: CAI 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.Conclusions: Our 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-1 alpha-dependent gene activity, but also on the absence of oxygen per se. (C) 2007 Elsevier Ireland Ltd. All rights reserved.