Multiple Mechanisms May Contribute to the Cellular Anti-Adhesive Effects of Phosphorothioate Oligodeoxynucleotides

Abstract

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.

Novel E2F decoy oligodeoxynucleotides inhibit in vitro vascular smooth muscle cell proliferation and in vivo neointimal hyperplasia

“…4,5 Although the stability of ODN against nucleases was enhanced by these chemical modifications, other problems were encountered due to the introduction of foreign materials into the DNA sequence. [9][10][11][12] Dumbbell-type ODN is reported to increase nuclease resistance and uptake into cells, compared to the chemically modified linear ODN. 28,29 Accordingly, we designed a novel circular dumbbell ODN containing two binding sites for E2F in a single decoy molecule without an open end, thus allowing the multiple targeting of a target promoter site or more than one promoter site.…”

“…However, these modified ODNs exhibited problems, such as insensitivity to RNase H, mutational potential by hydrolyzed modified nucleotides, lack of sequencespecific binding effects of ODN-based gene therapy and immune activation. [9][10][11][12] On the other hand, circular dumbbell ODNs constructed by the enzymatic ligation of the 3 0 and the 5 0 ends of ODNs exhibit increased stability to exonucleases, easy uptake and a nontoxic unmodified backbone, which resembles natural DNA.…”

Molecular strategy using cis-element ‘decoy’ of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model

“…Higher doses over prolonged periods of time may cause kidney damage, as shown by proteinuria and leukocytes in urine in animals. 22 Liver enzymes may also be increased in animals treated with moderate to high doses. Several phosphorothioate ODN have been shown to cause acute hypotensive events in monkeys, 23,24 probably due to complement activation.…”

relA over-expression reduces tumorigenicity and activates apoptosis in human cancer cells

“…On the contrary, in our model we evaluated the effect of NF-kB over-expression in the absence of stimuli inducing apoptosis. Moreover, even though a few studies have demonstrated reduced cell growth, adhesion and in vivo tumour growth by antisense inhibition of the p65 subunit (Kitajima et al, 1992;Higgins et al, 1993;Khaled et al, 1996), some authors disputed the specificity of these effects (Andela et al, 2000), and other authors found the lack of an effect of NF-kB suppression on in vivo tumour growth (Khaled et al, 1996).…”