Carbonic Anhydrase IX/CA9 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Carbonic anhydrase alpha, isozyme IX, belongs to a family of zinc-containing metalloproteins which hydrate carbon dioxide to generate bicarbonate ions and protons (1). This main catalytic function allows carbonic anhydrase IX to participate in cellular pH regulation. The large family of carbonic anhydrase metalloproteins includes three major classes which have been identified based on sequence and structure analysis. The alpha class is a monomer found in mammals. The beta class may occur as a dimer, tetramer, hexamer or octamer and is found in plants, algae, and bacteria. Lastly, the gamma class is a trimer found in bacteria and represents the most ancient carbonic anhydrase. These three classes of carbonic anhydrase enzymes lack sequence or structural similarities, but all share a conserved active site zinc atom (1).

Carbonic anhydrase IX (theoretical molecular weight 50kDa) belongs to the monomeric alpha class and is a single pass-transmembrane protein with two extracellular domains which serve catalytic and cell adhesion functions (2, 3). By cooperating with sodium bicarbonate cotransporters (NBC), lactate and proton exporting monocarboxylic acid transporters (MCT), and a sodium/hydrogen exchanger (NHE), carbonic anhydrase IX is involved in pH regulation across the cell membrane. This functional property protects cancer cells from intracellular acidification and partly explains the role of carbonic anhydrase IX in cancer cell survival and proliferation. In contrast, the pH regulating activity of carbonic anhydrase IX induces extracellular acidification, which has been implicated in epithelial to mesenchymal transition (EMT) and promoting cancer invasion. Carbonic anhydrase IX is frequently overexpressed in cancer cells (e.g., colorectal-, breast-, lung-carcinoma and brain tumors), an effect promoted by hypoxia within the tumor microenvironment (4). An exception are tumors carrying pVHL inactivating mutations, such as clear cell renal cell carcinoma (ccRCC), where HIF-alpha is stabilized due to dysfunctional proteasomal targeting and can induce HRE (Hypoxia Response Element) containing genes even under physiological normoxia (5). Carbonic anhydrase IX may be detected by immunostaining in tumors, which is found in association with necrotic tissue and metastatic cells. Because the expression of carbonic anhydrase IX correlates with both tumor grade and stage, analysis of its expression in tumors serves as a prognostic factor (4, 6).

References

1. Tripp, B. C., Smith, K., & Ferry, J. G. (2001). Carbonic Anhydrase: New Insights for an Ancient Enzyme. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.R100045200

2. Nishimori, I., & Onishi, S. (2001). Carbonic anhydrase isozymes in the human pancreas. Digestive and Liver Disease. https://doi.org/10.1016/s1590-8658(01)80138-9

3. Zavadova, Z., & Zavada, J. (2005). Carbonic anhydrase IX (CA IX) mediates tumor cell interactions with microenvironment. Oncology Reports. https://doi.org/10.3892/or.13.5.977

4. Pastorekova, S., & Gillies, R. J. (2019). The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond. Cancer and Metastasis Reviews. https://doi.org/10.1007/s10555-019-09799-0

5. Haase, V. (2009). The VHL Tumor Suppressor: Master Regulator of HIF. Current Pharmaceutical Design. https://doi.org/10.2174/138161209789649394

6. Young, J. R., Coy, H., Kim, H. J., Douek, M., Sisk, A., Pantuck, A. J., & Raman, S. S. (2018). Association of the gross appearance of intratumoral vascularity at MDCT with the carbonic anhydrase IX score in clear cell renal cell carcinoma. American Journal of Roentgenology. https://doi.org/10.2214/AJR.18.19725

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-08619

• Carbonic Anhydrase IX/CA9 Antibodies
• Carbonic Anhydrase IX/CA9 ELISA Kits
• Carbonic Anhydrase IX/CA9 Lysate
• Carbonic Anhydrase IX/CA9 Proteins
• Carbonic Anhydrase IX/CA9 ELISAs
• Carbonic Anhydrase IX/CA9 Proteins and Enzymes