Effects of oxygen and calcium on the expression of wild-type and mutated hypoxia-inducible factor-1alpha eukaryotic vectors in HEK293 cells / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To study the effects of oxygen and calcium on the expression of eukaryotic vectors harboring wild-type or mutated hypoxia-inducible factor-1alpha (HIF-11alpha) in HEK293 cells.</p><p><b>METHODS</b>HEK293 cells were transiently transfected with pcDNA3.1+/HIF-11alpha, pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha-564Ala-803Ala via lipofectin. Western blotting were used to detect HIF-11alpha protein after normoxic or hypoxic exposure of the transfected HEK293 cells in the presence or absence of Ca(2+). The levels of vascular endothelial growth factor (VEGF) mRNA in the transfected cells in normoxic condition was detected using RT-PCR.</p><p><b>RESULTS</b>The levels of HIF-11alpha protein and VEGF mRNA increased in HEK293 cells transfected with the vectors harboring mutated HIF-11alpha, but not in the cells transfected with wild-type HIF-11alpha vectors in normoxia. Hypoxia increased the levels of HIF-11alpha protein in the cells transfected with wild-type HIF-11alpha vectors, which was inhibited by the application of Ca(2+). Ca(2+) showed no inhibitory effect on HIF-11alpha levels in HEK293 cells transfected with the vectors containing mutated HIF-11alpha.</p><p><b>CONCLUSION</b>The protein products of pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha- 564Ala-803Ala in HEK293 cells enhance the cell tolerance to oxygen and protease.</p>