Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

The extracellular domain of CD4 receptor possesses a protein kinase activity.

The CD4 receptor of T-helper cells is an essential participant in immune response formation and HIV infection. We report here that the extracellular domains of CD4 receptor can catalyze the phosphotransferase (kinase) reaction. Incubation of rsCD4 in solution with [gamma-32P]ATP results in the Ca2+-dependent autophosphorylation of the protein presumably at a His residue because the reaction is prevented by the diethylpyrocarbonate treatment. The rsCD4 phosphorylates milk casein or human plasma proteins as a Ser/Thr protein kinase.

Polydeoxyguanine motifs in a 12-mer phosphorothioate oligodeoxynucleotide augment binding to the v3 loop of HIV-1 gp120 and potency of HIV-1 inhibition independency of G-tetrad formation.

Phosphorothioate oligodeoxynucleotides belong to a class of polyanions that bind to the third variable domain (v3) of HIV-1 gp120 and inhibit infectivity of a wide variety of HIV-1 isolates. This potent v3 binding of phosphorothioate oligodeoxynucleotides, which is relatively independent of the nucleotide sequence of the oligodeoxynucleotides, decreases with chain length (below 18-mers) and is low for 8-mers. However, recent studies have observed a nucleotide sequence-dependent augmentation of phosphorothioate oligodeoxynucleotide binding to v3 for 8-mers that contain the S-dG4 motif (e.g., SdT2G4T2) and have suggested that formation of quadruple helical tetraplexes (G-tetrads) is associated with the acquisition of v3 binding ability by small phosphorothioate oligodeoxynucleotides. In the current study, a series of SdG4-containing oligodeoxynucleotides were synthesized with varying tandem length (including the 8-mer SdT2G4T2, the 12-mer SdG4T4G4, and the 28-mer SdG4(T4G4)3) and compared with phosphorothioate oligodeoxynucleotides (with similar lengths or related sequences) for (1) their inhibition of the binding of mAb 9284, which binds to the N-terminal portion of the v3 loop, (2) the values of Kc when these compounds are used as competitors of the rgp120-binding of an alkylating phosphodiester oligodeoxynucleotide probe, and (3) inhibition of HIV-1 infectivity in a cell-cell transmission model. The presence of S-dG4 motifs and the number of tandem motifs augmented v3 binding and anti-HIV-1 infectivity for small (8-mer or 12-mer oligodeoxynucleotides) but did not significantly augment the potency of 28-mers. Whereas tetraplex formation of SdT2G4T2 may contribute to its v3 binding, the 12-mer SdG4T4G4 does not migrate as the tetraplex on nonreducing gels, suggesting that S-dG4 motifs may augment anti-HIV activity by multiple mechanisms.

Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix.

We studied the interactions of phosphorothioate oligodeoxynucleotides and heparin-binding growth factors. By means of a gel mobility shift assay, we demonstrated that phosphodiester and phosphorothioate homopolymers bound to basic fibroblast growth factor (bFGF). Binding of a probe phosphodiester oligodeoxynucleotide could also be shown for other proteins of the FGF family, including acidic fibroblast growth factor (aFGF), Kaposi's growth factor (FGF-4) as well as for the bFGF-related vascular endothelial growth factor, VEGF. No binding to epidermal growth factor (EGF) was observed. In addition, using a radioreceptor assay, we have shown that phosphorothioate homopolymers of cytidine and thymidine blocked binding of not only 125I-bFGF, but also of 125I-PDGF to NIH 3T3 cells, whereas phosphodiester oligodeoxynucleotides were ineffective. The extent of blockade of binding was dependent on the chain length of the phosphorothioate oligodeoxynucleotide. Furthermore, we have examined the effects of 18-mer phosphorothioate oligodeoxynucleotides of different sequences on 125I-bFGF binding to low and high affinity sites on both NIH 3T3 fibroblasts and DU-145 prostate cancer cells. Despite the fact that we have observed inhibition of bFGF binding by the 18-mer phosphorothioate oligodeoxynucleotides for both the high and low affinity classes of bFGF receptor, the inhibition was sequence-selective only for the high affinity receptors. We have also demonstrated that phosphorothioate homopolymers of cytidine and thymidine release bFGF bound to low affinity receptors in extracellular matrix (ECM). Finally, the most potent phosphorothioate oligodeoxynucleotides used in these experiments (e.g. SdC28) were inhibitors of bFGF-induced DNA synthesis in NIH 3T3 cells.

Transport of oligonucleotides across natural and model membranes.

Oligo- and polynucleotides can not diffuse through lipid membrane, however they are taken up by eukaryotic cells by endocytosis mediated by the nucleic acid specific receptors. The compounds find some way to escape from endosomes and reach nucleic acids in both cell nucleus and cytoplasm. Oligonucleotides bind to a few cell surface proteins which take part in the virus-cell interaction and in the development of immune response. Interaction of nucleic acids with cell surface proteins may play a role in development of some pathologies. The biological role of this interaction is unclear. Efficient delivery of oligonucleotides into eukaryotic cells can be achieved in some conditions by natural mechanisms and by using artificial carriers--membrane vehicles and cationic polymer micelles.

Serum immunoglobulins interact with oligonucleotides.

The interaction of reactive derivatives of oligonucleotides bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamin residue at the 5'-terminal phosphate with serum blood proteins has been investigated. It was found that the compounds react with serum albumin and immunoglobulins M and G, the reactivity increasing in the order: albumin < IgG < IgM. The reactions with immunoglobulins were inhibited in the presence of different oligonucleotides, DNA and heparin, suggesting the oligonucleotide binding to some cationic region of the proteins. Myoglobin inhibited the interaction of oligonucleotide derivatives with myoglobin-specific monoclonal antibodies which indicates that the derivatives interact with the proteins within or near the antigen binding site.

Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: a comparative study.

Phosphorodithioate (PS2) oligodeoxynucleotides (oligos) represent a relatively new class of backbone-modified oligo that have potential use as antisense agents. PS2 oligos are isoelectronic with phosphodiester (PO) and phosphoromonothioate (PS) oligos, and are nuclease resistant. However, unlike their PS congeners, PS2 oligos do not contain chiral centers. Little is known about the manner in which PS2 oligos interact with biological systems. In this study, we compare the cellular pharmacology of PS and PS2 oligos in HL60 cells. Cell surface binding, internalization, and compartmentalization are examined. Furthermore, the ability of PS and PS2 oligos to bind to rsCD4 and bFGF and to inhibit the activity of protein kinase C (PKC) is examined. Although the behavior of PS2 oligos closely parallels that of PS oligos, PS2 oligos appear to interact with some biological systems in a slightly different manner than PS oligos. These results indicate that PS2 oligos may have therapeutic potential other than as antisense agents.

Iontophoretic delivery of oligonucleotide derivatives into mouse tumor.

Oligodeoxyribonucleotides (22-mers) were delivered through the skin of C3H mice in the region of a mammary gland tumor by means of iontophoresis. It was shown that the oligonucleotides enter the tumor, cross it, and reach all mouse organs. Electrophoretic analysis of the oligonucleotide extracted from tumor showed that the compounds were delivered in the tissue in the intact state.

Oligodeoxynucleotides interact with recombinant CD4 at multiple sites.

Phosphodiester oligodeoxynucleotides bearing the 5'-alkylating moiety 4-(N-2-chloroethyl-N-methyl)aminobenzylamine specifically modify recombinant soluble CD4 (rsCD4) in solution. This reaction is saturable with respect to the alkylating oligonucleotide reagent. The existence of at least two binding sites, with different affinities, on the rsCD4 molecule, were demonstrated. The values of apparent Kd for the sites are approximately 0.1 and 1 microM. The existence of two sites was confirmed by electrophoretic analysis of the modified protein, in which two distinct gel bands were seen. The modification is inhibited by excess non-alkylating oligonucleotide, as well as by phosphorothioate oligonucleotides. Quantitative estimates of the competition constants (Kc), for the binding of these competitors of the binding of the alkylating oligonucleotide reagent with rsCD4, have been made. By use of this method, several anionic dyes as well as potential anti-HIV therapeutic agents were also demonstrated to interact with rsCD4. Phosphorothioate oligonucleotides also inhibit binding of rsCD4 with the monoclonal antibody L71.1.1 This monoclonal antibody recognizes the CDR3-like loop (D1 domain) of the rsCD4 molecule. Thus, oligonucleotide binding sites exist on two remote regions (i.e. both the CDR2- and CDR3-like loops) of the D1 domain of CD4.

Penetration of oligonucleotides into mouse organism through mucosa and skin.

Benzylamide 5'-32P-oligonucleotide derivatives were shown to penetrate into mice organism when administered by various routes; intranasally, per os, intravaginally and per rectum. In all cases, the compounds are rapidly accumulated in blood and guts. Analysis of the radioactive material from blood and pancreas revealed intact oligonucleotides. Although concentrations of oligonucleotides in tissues differ considerably by the various methods of administration, the efficiency of delivery is sufficient to consider all the routes as being of therapeutic value. Dose effect on the efficiency of oligonucleotide penetration into mice suggests the transport to be a saturable process. Application of an oligonucleotide lotion on mice ear helices results in reproducible accumulation of radioactivity in the animal tissues. Effectiveness of oligonucleotide delivery into mouse through skin can be improved by using electrophoretic procedure.

Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells.

We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.

Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

Phosphorothioate oligodeoxynucleotides bind to the third variable loop domain (v3) of human immunodeficiency virus type 1 gp120.

Although having variability in primary sequence, the v3 loop of gp120 in pathogenic strains of human immunodeficiency virus type-1 (HIV-1) is positively charged and known to interact with sulfated polysaccharides. Because the interaction of sulfated polysaccharides with the v3 loop inhibits HIV infection in vitro, we investigated the interaction of the v3 loop with phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos). In a solid-phase ELISA assay, a PS 28-mer homopolymer of cytidine, SdC28, blocked the binding of the v3 loop-specific monoclonal antibody (mAb) 9284 to rgp120 more potently than did dextran sulfate. In addition, like dextran sulfate, SdC28 appeared to bind specifically to the v3 loop, because neither compound inhibited the binding of other anti-gp120 mAbs. In contrast to PS oligos, PO oligos did not inhibit mAb 9284 binding. The length dependence of the interaction of PS oligos with the v3 loop was studied by using a series of PS oligos. A discrete loss of inhibiting activity occurred as a function of decreasing PS oligo length, which was most marked between PS oligos of 18-mer and 12-mer in length. We further probed the chemical nature of the interaction of oligos with gp120 by measuring the gp120 binding affinities of PS and PO oligos of various lengths. We employed a 5'-32P-labeled alkylating oligo, ClRNH32P-OdT15, and determined that the Km of gp120 binding is 4 microM. We also determined values of competition constant (Kc) for PS competitors of ClRNH32P-OdT15 binding. The binding constant (= 1/Kc) for PS oligos showed a discrete increase in gp120 binding for PS oligos > 12- to 18-mer in length, with no further increment beyond an 18-mer. Given the important role of the v3 loop in HIV-1 pathogenicity, these data suggest that therapeutic trials of PS oligos should be considered.

Phosphorothioate oligodeoxynucleotides--anti-sense inhibitors of gene expression?

Phosphorothioate (PS) oligodeoxynucleotides are relatively nuclease resistant, water soluble analogs of phosphodiester (PO) oligodeoxynucleotides. These molecules are chiral but still hybridize well to their RNA targets. While considered for use as in vivo anti-sense inhibitors of gene expression, their biology, especially in the anti-viral area, is dominated by non-sequence specific effects. This review discusses both the sequence and non-sequence specific biologic effects of PS oligomers, and attempts to more clearly indicate their ultimate therapeutic potential.