Presence Of Antisense Oligodeoxynucleotides Research Articles

Lysophosphatidylcholine Stimulates the Release of Arachidonic Acid in Human Endothelial Cells

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Detection of Ribonuclease H-Generated mRNA Fragments in Human Leukemia Cells Following Reversible Membrane Permeabilization in the Presence of Antisense Oligodeoxynucleotides

The involvement of ribonuclease H (RNase H) in antisense phenomena in intact cells has, to date, only been adequately demonstrated for microinjected Xenopus systems. The significance of RNase H for the antisense effects of oligodeoxynucleotides observed in human and other mammalian cell cultures has remained obscure, in part because of inadequate analytic methods. In this report we show that the "reverse ligation-mediated PCR" (RL-PCR) procedure permits amplification of RNA fragments produced by oligodeoxynucleotide-directed RNase H activity. We have used this procedure to demonstrate RNase H-dependent antisense effects in irreversibly permeabilized (dead) cells and reversibly permeabilized (live) cells.

The role of primer recognition proteins in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides.

Primer recognition proteins (PRP) are cofactors for DNA polymerase alpha and may have a role in lagging-strand DNA replication. PRP is composed of two subunits, which we have previously identified as the protein-tyrosine kinase substrate annexin II and phosphoglycerate kinase (PGK). In this study, we have examined the physiological involvement of these proteins in DNA synthesis and cell proliferation. When exponentially growing human HeLa cells are exposed to antisense phosphorothioate oligodeoxynucleotides to annexin II, ongoing DNA synthesis is reduced. The extent of reduction with antisense oligodeoxynucleotide to PGK was much less than with the antisense annexin II oligodeoxynucleotide. Reductions in the labeling and mitotic indices of HeLa cell cultures are seen after exposure to antisense oligodeoxynucleotides. Flow cytometric analyses indicate that progression from S phase to G2 phase of the cycle is retarded by exposure of cells to the antisense oligodeoxynucleotides. Corresponding sense oligodeoxynucleotides have no inhibitory effects on these parameters. The new synthesis of annexin II and PGK is specifically reduced in the presence of antisense oligodeoxynucleotides, indicating that the complex of newly synthesized annexin II and PGK may participate in PRP function. These experiments indicate that annexin II and PGK may have a physiological role in DNA synthesis and cell cycle progression, and represent the first physiological role for annexin II monomer in cells.

Antisense Inhibition of β-Glucuronidase Expression in Preimplantation Mouse Embryos: A Comparison of Transgenes and Oligodeoxynucleotides

Using as a model the inhibition of beta-glucuronidase expression in preimplantation embryos, we have compared injections of transgenes directing the synthesis of antisense RNA and antisense oligodeoxynucleotides to our previous results with cytoplasmic injections of antisense RNAs. Pronuclear injection of an antisense DNA construct containing 1.4 kb of coding region of beta-glucuronidase fused to the mouse metallothionein I promoter results in transient inhibition of gene expression in preimplantation mouse embryos. Pronuclear injection of a smaller antisense DNA construct, overlapping the start codon, failed to inhibit gene expression. Injection of two 20-mer antisense oligodeoxynucleotides, one complementary to sequences including the initiation codon and the second complementary to exon 7 sequences of the beta-glucuronidase gene, failed to inhibit gene expression. In addition, cultures of embryos in the presence of antisense oligodeoxynucleotides had no effect on gene activity. Using radiolabeled oligomers added to the culture medium, we found poor uptake of oligodeoxynucleotides by embryos.

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.

Antisense oligodeoxynucleotides provide insight into mechanism of translation initiation of two Sendai virus mRNAs.

Translation of Sendai virus nucleocapsid protein (NP) and phosphoprotein (P/C) mRNAs in rabbit reticulocyte lysates in the presence of antisense oligodeoxynucleotides (15-20-mers) showed that oligonucleotides having complementarity within the 5' noncoding region of the mRNAs blocked translation, oligonucleotides having complementarity within 20 nucleotides upstream from the initiator codon blocked translation only partially, and oligonucleotides complementary to the coding region of mRNA had no effect on translation. The results suggest the possibility that the 80 S initiation complex may form about 20 bases upstream from the initiator codon. Alternatively, the 40 S preinitiation complex may recognize an initiator codon at least 20 nucleotides upstream from the codon, activating a helix-destabilizing process.