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Lowered Oxygen Tension Induces Expression of the Hypoxia Marker MN/Carbonic Anhydrase IX in the Absence of Hypoxia-inducible Factor 1 Stabilization

A Role for Phosphatidylinositol 3'-Kinase¹

Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic [S. K., M. K., A. C., S. P., J. P.]; British Columbia Cancer Research Centre and British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 1L3 Canada [P. L. O.]; Laboratory of Immunobiology, National Cancer Institute, NIH, Bethesda, Maryland 20889 [M. I. L.]; and Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92717 [E. J. S.]

Transcription of the gene coding for the tumor-associated antigen MN/carbonicanhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1).Previous studies identified CAIX expression in areas adjacentto 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 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 O₂ 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 CoCl₂ 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-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. These interdependent pathways function at a lowered O₂ concentration that is, however, above that necessary for HIF-1 stabilization.