omega-6 polyunsaturated fatty acid-stimulated cellular internalization of phosphorothioate oligodeoxynucleotides - Evidence for protein kinase C-zetadependency

The rate of cellular internalization of phosphorothioate oligodeoxynucleoti des is determined predominantly by adsorptive plus fluid-phase endocytosis. Internalization of a 5'-fluoresceinated phosphorothioate 15mer homopolymer of thymidine (FSdT15) in K562 cells in medium containing lipid-depleted al bumin was reduced consistently versus nondepleted albumin. Treatment of K56 2 and several other cell lines with omega-6 polyunsaturated fatty acids (om ega-6 PUFAs; e.g. arachidonic and linoleic acids) but not saturated fatty a cids dramatically increased FSdT15 internalization in a concentration-depen dent manner and over a wide albumin concentration range. The rate of efflux of FSdT15 from K562 cells was not affected by the omega-6 PUFA, implying t hat an increase of cellular fluorescence was due to an increase in the in-r ate. These data were consistent with the observation that the binding of FS dT15 to the cell surface was also increased in the presence of omega-6 PUFA s. omega-6 PUFAs are stimulators of protein kinase C (PKC) activity. Inhibi tion of PKC activity in K562 cells by Go6976, an inhibitor of the classical PKC isoforms, did not block the linoleic acid-induced stimulation of FSdT1 5 internalization. On the other hand, treatment of cells with Ro318220, whi ch has considerably less isoform specificity, almost totally blocked the ef fect of linoleic acid on FSdT15 internalization, implying the involvement o f a nonclassical PKC isoform in the process. Finally, since the only PKC is oform expressed in K562 cells that also is activated by omega-PUFAs is PKC- xi, we obtained NIH 3T3 cells expressing a doxycycline-repressible dominant negative PKC-xi mutant. Expression of the mutant blocked the stimulation o f FSdT15 internalization by linoleic acid. Stimulated internalization also was blocked by wortmannin and LY 294002, which are relatively specific inhi bitors of phosphatidylinositol 3-kinase (PI 3-K). Taken together, our data suggest that w-6 PUFA stimulation of fluoresceinated phosphorothioate oligo mers may be PKC-xi dependent, and perhaps PI-3K dependent as well. (C) 1999 Elsevier Science Inc.