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Stabilization
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.
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