Overexpression of Ha-ras enhances the transcription of human arachidonate 12-lipoxygenase promoter in A431 cells.
The effect of transient transfection with expression vectors of Ha-ras on the promoter activity of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. Overexpression of Ha-ras increased the promoter activity in a dose- and time-dependent manner, which correlated closely with the cellular expression of Ras protein. Promoters of different gene lengths for human 12-lipoxygenase were used to prepare the luciferase fusion vectors. Following transfection by Ha-ras for 68 h, an approx. 40-fold increase in luciferase reporter activity was observed in plasmids with the 5'-flanking region ranging from -951 to -224 bp upstream from translation starting site. There was no obvious stimulation in cells transfected with a vector-bearing promoter with a length of -100 bp. These results indicate that the promoter region ranging from -224 to -100 bp was important for the Ha-ras response. With the aid of additional 5'-deletion and site-directed mutagenesis, three Sp1 binding sequences residing at -169 to -161 bp, -158 to -150 bp and -123 to -114 bp were found to be critical for the Ha-ras response of activating the transcription of human 12-lipoxygenase gene.
Chen
B K
BK
Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC.
Liu
Y W
YW
Yamamoto
S
S
Chang
W C
WC
D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics*; D001483:Base Sequence; D015870:Gene Expression; D017930:Genes, Reporter; D011905:Genes, ras; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D016329:Sp1 Transcription Factor; D001092:Arachidonate 12-Lipoxygenase