Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1α stabilization: A role for phosphatidylinositol 3'-kinase

• Published in: Cancer research (Chicago, Ill.) Vol. 62; no. 15; pp. 4469 - 4477
• Main Authors: KALUZ, Stefan, KALUZOVA, Milota, CHRASTINA, Adrian, OLIVE, Peggy L, PASTOREKOVA, Silvia, PASTOREK, Jaromir, LERMAN, Michael I, STANBRIDGE, Eric J
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01.08.2002
• Subjects: Antigens, Neoplasm; Biological and medical sciences; Carbonic Anhydrase IX; Carbonic Anhydrases - biosynthesis; Carbonic Anhydrases - genetics; Cell Count; Enzyme Activation; Fibrosarcoma - enzymology; Fibrosarcoma - genetics; Gene Expression Regulation, Enzymologic - physiology; Gene Expression Regulation, Neoplastic - physiology; Glucose - deficiency; HeLa Cells; Host-tumor relations. Immunology. Biological markers; Humans; Hydrogen-Ion Concentration; Hypoxia-Inducible Factor 1, alpha Subunit; Medical sciences; Neoplasm Proteins - antagonists & inhibitors; Neoplasm Proteins - biosynthesis; Neoplasm Proteins - genetics; Oxygen - metabolism; Oxygen - physiology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphatidylinositol 3-Kinases - metabolism; Phosphatidylinositol 3-Kinases - physiology; Promoter Regions, Genetic; Transcription Factors - metabolism; Transcription Factors - physiology; Tumors; Human; Tumor associated antigen; Enzyme; Isozyme; Transcription promoter; Transferases; Biological marker; Lyases; Gene expression; Oxygen content; Response element; In vitro; 1-Phosphatidylinositol 3-kinase; Cell density; Signal transduction; Animal; Established cell line; Hypoxia; Carbonate dehydratase; Transcription factor HIF1; Mechanism of action; Oxygenation; Carbon-oxygen lyases; Hydro-lyases
• ISSN: 0008-5472; 1538-7445

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.