Vascular endothelial growth factor gene expression in the human breast cancer cell line MX-1 is controlled by O 2 availability in vitro and in vivo

Abstract

The vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. Mediated by the hypoxia-inducible transcription factor HIF-1alpha/beta, a reduction in O2 tension (pO2) leads to increased VEGF gene expression in nonmalignant tissues. In tumor cells VEGF mRNA levels are often constitutively elevated. We examined pO2-dependent VEGF mRNA expression and VEGF protein formation in the human breast cancer cell line MX-1 in vitro and in vivo. For in vitro study MX-1 cultures were grown on dishes with a gas-permeable bottom to expose the cells to defined O2 concentrations (from 95% to 0%) for 4 h. Northern blot analysis showed significant VEGF mRNA in MX-1 cultures under normoxic conditions which was further increased by hypoxia. The amount of secreted VEGF was also elevated in hypoxic cultures. Western blot analysis revealed a correlation between the severity of hypoxia and HIF-1alpha protein amounts in the nucleus. Furthermore, DNA-binding activity of HIF-1 could be demonstrated by gel-shift assays. For in vivo study immunodeficient nude mice bearing MX-1 tumor transplants were exposed to inspiratory hypoxia (10% O2). Northern blot and immunohistochemical analyses of MX-1 tumor transplants showed that VEGF mRNA and VEGF protein levels were increased in mice 17 h after the induction of inspiratory hypoxia. Thus, pO2-dependence of VEGF gene expression can be maintained in cancer cells, even in vivo, which may be relevant in regard to therapeutic attempts to inhibit tumor angiogenesis by increasing tumor oxygenation.