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.