12(S)-HYDROXYEICOSATETRAENOIC ACID INCREASES THE ACTIN MICROFILAMENT CONTENT IN B16A MELANOMA-CELLS - A PROTEIN KINASE-DEPENDENT PROCESS

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabo lite of arachidonic acid, has been shown to be involved in a wide vari ety of cellular activities (i.e., adhesion, spreading, motility, invas ion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay ) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods in dependently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) result ed in an increase in the F-actin content in the cytoskeletal preparati ons. Since the integrity of cytoskeletal networks (i.e., actin filamen ts) can be dynamically regulated through protein phosphorylation, we i nvestigated the potential role of several protein kinases in the 12(S) -HETE-induced actin polymerization. By flow cytometric analysis, 12(S) -HETE was found to increase the actin filament contents. This effect c ould be inhibited by protein kinase C (PKC) inhibitors (calphostin C a nd staurosporine) as well as by protein tyrosine kinase (PTK) inhibito r (genistein) but not by protein kinase A inhibitor (H8), suggesting t hat the 12(S)-HETE effect involves PKC and PTK. This conclusion is con sistent with the observations that phorbol 12-myristate-13-acetate (PM A) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphor ylation studies indicate that 12(S)-HETE treatment led to enhanced pho sphorylation of myosin light chain, which may contribute to the increa sed stress fiber formation following 12(S)-HETE stimulation. (C) 1998 Wiley-Liss, Inc.