[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.


CONCLUSIONS
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.
Fu
Rui-Bin
RB
Department of Cardiology, Guangzhou General Hospital of Guangzhou Command, Guangzhou 510010, China.
Wu
Ping-Sheng
PS
Qiu
Jian
J
Xiang
Ding-Cheng
DC
Ma
Jun
J
Hong
Chang-Jiang
CJ
He
Jian-Xin
JX
D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D010100:Oxygen; D002118:Calcium