HIF-1-dependent VEGF reporter gene assay by a stable transformant of CHO cells.
For the establishment of a screening system to detect inhibitors of vascular endotherial growth factor (VEGF) expression, a stable transformant of Chinese hamster ovary cells was isolated and cloned by transfection of a hypoxia-inducible factor 1 (HIF-1)-dependent VEGF promoter reporter gene. The expression of the reporter gene in the clone cells, as measured by luciferase activity, was stable. Hypoxic responses were best observed at an initial cell density of 2 x 10(4)/well. The maximal increase of luciferase activity was 30 fold. In the highest cell density of 8 x 10(4)/well (2.1 x 10(5)/cm(2)), basal activity was increased 13-15 fold compared to that at the lower cell densities, and did not respond to hypoxia. Addition of CoCl(2), which is known to mimic hypoxia, increased luciferase activity more than 10 times in normoxia. Nitric oxide donors, which are known to suppress the activation of HIF-1, inhibited expression of the VEGF promoter reporter gene under hypoxia. Histone deacetylase inhibitors, trichostatin A and sodium n-butyrate which are known to stimulate transcription of many genes enhanced its transcription in hypoxia. These results indicate that the stable transformant is a useful tool for screening of HIF-1 modifiers.
Yamazaki
Yohko
Y
Institute of Microbial Chemistry, Tokyo, Japan. yamako@bikaken.or.jp
Egawa
Kiyoshi
K
Nose
Kiyoshi
K
Kunimoto
Setsuko
S
Takeuchi
Tomio
T
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology*; D051793:Hypoxia-Inducible Factor 1; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D009687:Nuclear Proteins; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A