[Effects of oxygen and calcium on the expression of wild-type and mutated hypoxia-inducible factor-1alpha eukaryotic vectors in HEK293 cells].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Construction of eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 and their expressions in human microvascular endothelial cells].

OBJECTIVE: To construct eukaryotic expression vectors of two mutants of hypoxia-inducible factor-1 (HIF-1alpha) and study their expressions in human microvascular endothelial cells (HMVECs). METHODS: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Pro564 (ccc) in HIF-1alpha into gcc (Ala) in pcDNA3.1(+)-HIF-1alphato obtain single-site-mutated vector pcDNA3.1(+)-HIF-1alpha-564Ala, which was then subjected to a second site-directed mutagenesis to convert the codons for Asn803 into that of Ala (gct) to acquire double-site-mutated pcDNA3.1(+)-HIF-1alpha-564Ala-803Ala. After lipofectin-mediated transient transformation of HMVECs with the 3 recombinant plasmids including the two plasmids containing the mutations and the one without mutation, respectively, the expression levels of HIF-1alpha mRNA and protein were determined using RT-PCR, immunofluorescent staining and Western blotting. RESULTS: DNA sequence analysis demonstrated success of the two-step mutagenesis and the two plasmids of pcDNA3.1+-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha-564Ala- 803Ala were obtained, both of which could produce HIF-1alpha protein resistant to oxidation degradation in HMVECs as compared with the non-mutated one. CONCLUSION: The recombinant plasmids pcDNA3.1(+)-HIF-1alpha-564Ala and pcDNA3.1(+)-HIF-1alpha- 564Ala-803Ala have been successfully constructed with efficient expressions in HMVECs.