INDUCTION OF 12-LIPOXYGENASE EXPRESSION BY PHORBOL 12-MYRISTATE 13-ACETATE IN HUMAN EPIDERMOID CARCINOMA A431 CELLS

Phorbol 12-myristate 13-acetate (PMA) increased the expression of 12-l ipoxygenase activity and mRNA in a time-dependent manner in human epid ermoid carcinoma A431 cells. The increase of 12-lipoxygenase was accom panied by the increase in protein level in microsomes prepared from A4 31 cells. The PMA-induced expression of 12-lipoxygenase activity and m RNA was inhibited by the treatment of cells with a protein kinase C in hibitor GF 109203X. Promoters of different DNA lengths for human 12-li poxygenase gene were used to prepare the luciferase fusion vectors. Th ese plasmid constructs were transiently transfected into A431 cells. F ollowing treatment of PMA for 18 h, a 4- to 5-fold increase in lucifer ase reporter activity was observed in plasmids with the 5'-flanking re gion length of -951 bp and that of -224 bp upstream from translation s tarting site. A time-dependent induction of luciferase activity by PMA was found to parallel the PMA-induced enzyme activity and mRNA expres sion. Transient transfection with a series of 5'-deletion constructs s howed that the 5'-flanking region spanning from -224 to -100 bp from t ranslation starting site played an important role for PMA response. Ge l mobility shift assay and site-directed mutagenesis indicated that tw o Spl binding sequences residing at -158 to -150 bp and -123 to -114 b p were responsible for the PMA response in activating the transcriptio n of human 12-lipoxygenase gene. (C) 1998 Elsevier Science B.V.