Omega-6 polyunsaturated fatty acid-stimulated cellular internalization of phosphorothioate oligodeoxynucleotides: evidence for protein kinase C-zeta dependency.
The rate of cellular internalization of phosphorothioate oligodeoxynucleotides 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 albumin was reduced consistently versus nondepleted albumin. Treatment of K562 and several other cell lines with omega-6 polyunsaturated fatty acids (omega-6 PUFAs; e.g. arachidonic and linoleic acids) but not saturated fatty acids dramatically increased FSdT15 internalization in a concentration-dependent 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 that an increase of cellular fluorescence was due to an increase in the in-rate. These data were consistent with the observation that the binding of FSdT15 to the cell surface was also increased in the presence of omega-6 PUFAs. Omega-6 PUFAs are stimulators of protein kinase C (PKC) activity. Inhibition of PKC activity in K562 cells by Go6976, an inhibitor of the classical PKC isoforms, did not block the linoleic acid-induced stimulation of FSdT15 internalization. On the other hand, treatment of cells with Ro318220, which has considerably less isoform specificity, almost totally blocked the effect of linoleic acid on FSdT15 internalization, implying the involvement of a nonclassical PKC isoform in the process. Finally, since the only PKC isoform expressed in K562 cells that also is activated by omega-PUFAs is PKC-zeta, we obtained NIH 3T3 cells expressing a doxycycline-repressible dominant negative PKC-zeta mutant. Expression of the mutant blocked the stimulation of FSdT15 internalization by linoleic acid. Stimulated internalization also was blocked by wortmannin and LY 294002, which are relatively specific inhibitors of phosphatidylinositol 3-kinase (PI 3-K). Taken together, our data suggest that omega-6 PUFA stimulation of fluoresceinated phosphorothioate oligomers may be PKC-zeta dependent, and perhaps PI-3K dependent as well.
Khaled
Z
Z
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Ho
Y Y
YY
Benimetskaya
L
L
Deckelbaum
R J
RJ
Stein
C A
CA
D002518:Ceramides / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects*; D043371:Fatty Acids, Omega-6; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D012709:Serum Albumin / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000378:metabolism*; D000099221:Protein Kinase C zeta
D002518:Ceramides; D043371:Fatty Acids, Omega-6; D005231:Fatty Acids, Unsaturated; D009838:Oligodeoxyribonucleotides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C; D012709:Serum Albumin; D013755:Tetradecanoylphorbol Acetate; D013873:Thionucleotides; D000099221:Protein Kinase C zeta