Abstract
Specific metabolites of arachidonic and linoleic acid have been proposed as serving a regulatory function in growth factor signal transduction in fibroblasts. In studies with Syrian hamster embryo (SHE) fibroblasts, we found lipoxygenase inhibitors to be potent blockers of epidermal growth factor (EGF)-dependent mitogenesis. Analytical chemical characterization of arachidonic and linoleic acid metabolism in SHE cells demonstrated that the major lipoxygenase product was 13-hydroxyoctadecadienoic acid (HODE). EGF stimulation of quiescent SHE cells resulted in an enhancement of HODE biosynthesis. The primary arachidonate products were prostaglandin E2 and F2 alpha formed via the cyclooxygenase pathway. Inhibition of cyclooxygenase activity did not alter the EGF-mitogenic response in SHE cells. Addition of lipoxygenase-derived linoleate metabolites (10(-10)-10(-6) M) produced a 2-4-fold potentiation of EGF-stimulated [3H]thymidine incorporation in SHE cells. Interestingly, the linoleate products did not enhance the EGF mitogenic effect in variant SHE cells that had lost tumor suppressor gene function. These results were confirmed by autoradiographic studies of DNA synthesis and suggest that loss of tumor suppressor phenotype correlates with a lack of responsiveness to linoleate products in signal transduction. In studies on the mechanism of EGF regulation of linoleic acid metabolism, inhibitors of EGF receptor tyrosine kinase activity were observed to block EGF-stimulated HODE biosynthesis. In addition, both cyclohexamide and actinomycin D attenuated the ability of EGF to increase linoleic acid metabolism in SHE cells. EGF induction of the linoleate pathway appears to be linked to activation of the EGF receptor and may be modulated at transcriptional or translational levels.
Highlights
Specific metabolites of arachidonic andlinoleic acid biochemical processes in transducingthe mitogenic signal has have been proposed as serving a regulatory function yet to be clearly delineated
EGF treatment of SHE cells did not increase the biosynthesis of PGE2 and seen in Fig. 8,addition of methyl-2,5-dihydroxycinnamate PGF2, the primary arachidonate metabolites
Studies with produced a dose-dependent decrease in EGF-mediated DNA inhibitors of lipoxygenase and prostaglandin H synthase acsynthesis in SHE cells. These findings are similar to those tivity revealed that low doses of NDGA were very effectivein reported for this compound in other cell systems [18].blocking EGF-dependent DNA synthesis
Summary
Materials-The sources of isotopes, reagents, and chemicals were as follows. [n~ethyl-~HlThymidin(e70 Ci/mmol) was from ICN Radiochemicals (Irvine, CA). [l-'4C]Linoleic acid (40-60 mCi/mmol) and [5,6,8,9,11,12,14,15-3H]arachidoanciicd (60-100 Ci/mmol) were from Du Pont-New England Nuclear. Cells were made quiescent by incubation for 16 hrin serum-free DMEM After this point, the media was decanted and thecells were again washed twice with 15 ml of Hanks' balanced metabolism, the mobile phase was composed of70% methanol, 30% water, 0.01% acetic acid, with a flow rate of 1.0 ml/min. The cells were treated with 10 ml of serum-free with phosphate-buffered saline and fixed with cold methanol and DMEM containing EGF(10 ng/ml) with either [3H]arachidonicacid 10%trichloroacetic acid. The column was washed with 10 ml of water and the sample 10 PM) was added in 5 p1 of ethanol and incubated with the enzyme eluted with 5 ml of methanol and subsequently evaporated to dryness solution for 5 min prior to theaddition of the linoleic acid substrate. The formation of compounds containinga diene chromophore was monitored by UV detection at 235 nm
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