title: Stimulation of synthesis de novo of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase in human promyelocytic leukaemia (HL-60) cells by phorbol ester.
abstract: Human promyelocytic leukaemia (HL-60) cells were employed to study the induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme in controlling prostaglandin inactivation. Phorbol 12-myristate 13-acetate (PMA) stimulated 15-PGDH activity in a time- and concentration-dependent manner. Dimethyl sulphoxide (DMSO) also stimulated the enzyme activity, although a much delayed stimulation was observed. Western blot studies indicated that PMA increased significantly a 28 kDa immunoreactive protein characteristic of 15-PGDH. L-[35S]Methionine labelling of the PMA-treated cells showed a similar enhancement over the control cells. These studies indicate that PMA induced synthesis of 15-PGDH. Stimulation of 15-PGDH activity by PMA or DMSO appears to be mediated by protein kinase C activation, since an inactive analogue of PMA failed to induce the effect, and both staurosporine and H-7 blocked the stimulation. Stimulation by PMA was optimal at 10 nM and less effective at higher concentrations. Western blot studies indicated that a similar, if not greater, amount of enzyme protein was induced at high concentrations of PMA, suggesting that enzyme inactivation might be occurring. Possible enzyme inactivation by protein kinase C activation was further examined by incubating DMSO-treated cells with a high concentration of PMA (50 nM). Time-dependent inactivation of 15-PGDH within the first 1 h was observed and this inactivation was partially blocked by staurosporine and H-7. Pulse-chase experiments indicated that 15-PGDH had a rapid turnover rate (t 1/2 = 47 min), and PMA shortened the half-life of the enzyme (t 1/2 = 33 min), suggesting that PMA might have an additional effect on 15-PGDH degradation. The rapid turnover of 15-PGDH indicates that the enzyme activity depends on continued enzyme synthesis, and this could be susceptible to hormone and drug control mechanisms.
authors.affiliation: Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
authors.forename: C Q
authors.initials: CQ
authors.lastname: Xun
authors.forename: Z G
authors.initials: ZG
authors.lastname: Tian
authors.forename: H H
authors.initials: HH
authors.lastname: Tai
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D015153:Blotting, Western; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000096:biosynthesis*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000470:Alkaloids; D007546:Isoquinolines; D010879:Piperazines; D009243:NAD; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase; D011493:Protein Kinase C; D019311:Staurosporine; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide