epidermal-growth-factor and 4-bromophenacyl-bromide

We demonstrated the effect of epidermal growth factor (EGF) on the production of PGE2 in human squamous carcinoma A431 cells. The production of PGE2 was increased by stimulating the cells with EGF for 2 h and reached a maximum for 10 h. EGF was also found to augment the release of arachidonic acid (AA) following the increase in phospholipase A2 (PLA2) activity (1.7-fold). The induced PLA2 activity was diminished by 4-bromophenacyl bromide, but not by dithiothreitol, indicating that the EGF-induced release of AA was due to the increase in the activity of cytosolic PLA2 (cPLA2). On the other hand, cyclooxygenase (COX) activity was increased (1.6-fold) within 2 h after the EGF-treatment and the induced activity was inhibited by cycloheximide. In addition, Northern blot analysis showed that the level of COX-2 mRNA was increased by the EGF-treatment, whereas no COX-2 mRNA was detected in the untreated cells, indicating that the EGF-induced COX activity was resulted from the increase in the production of COX-2. These results suggest that EGF augments the production of PGE2 by increasing not only the activity of cPLA2 but also the production of COX-2 in A431 cells.

Topics: Acetophenones; Arachidonic Acid; Blotting, Northern; Carcinoma, Squamous Cell; Cloning, Molecular; Cycloheximide; Cyclooxygenase 2; Dinoprostone; Dithiothreitol; DNA, Complementary; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Membrane Proteins; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Cells, Cultured

The effect of phospholipase A2 (PLA2) on intestinal epithelial cell migration was investigated using an in vitro wounding model of confluent monolayers of IEC-6. PLA2 (0.001-2 U/ml) enhanced IEC-6 cell migration in a dose dependent manner. Addition of 4-bromophenacyl bromide (BPB) (a PLA2 inhibitor) to PLA2 completely blocked the migration-promoting effect. However, addition of piroxicam (a cyclooxygenase inhibitor) or nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no influence on the effect. Lysophosphatidylcholine (lysoPC) (0.01-5,000 ng/ml), one of the products of phosphatidylcholine by PLA2, dose-dependently enhanced IEC-6 cell migration as well. A combination of PsLA2 (1 U/ml) and lysoPC (1,000 ng/ml) had no additive effect or migration. Moreover, the migration-promoting effect of PLA2 that was blocked by BPB was recovered by lysoPC. After pretreatment of IEC-6 cells with replication-inhibiting doses of mitomycin C, the enhanced migration induced by PLA2 or lysoPC was still observed. These observations suggest that PLA2 may, independently of proliferation, enhance intestinal epithelial cell migration mainly via lysoPC.

Topics: Acetophenones; Animals; Cell Division; Cell Movement; Cells, Cultured; Dinoprostone; Enteritis; Epidermal Growth Factor; Intestine, Small; Lysophosphatidylcholines; Lysophospholipids; Masoprocol; Mitomycin; Phospholipases A; Phospholipases A2; Piroxicam; Rats

Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.

Topics: Acetophenones; Animals; Arachidonate 5-Lipoxygenase; Calcium Channels; Enzyme Activation; Epidermal Growth Factor; Humans; Lipoxygenase Inhibitors; Mice; Models, Biological; Neoplastic Stem Cells; Phospholipases A; Phospholipases A2; Proto-Oncogene Mas; Signal Transduction; SRS-A; Tumor Cells, Cultured

Topics: Acetophenones; Animals; Azo Compounds; Bacitracin; Cells, Cultured; Chlorpromazine; Endocytosis; Epidermal Growth Factor; Methylamines; Peptides; Phenylglyoxal; Phospholipases; Phospholipases A; Quinacrine; Rats