MULTIPLE MECHANISMS MAY CONTRIBUTE TO THE CELLULAR ANTIADHESIVE EFFECTS OF PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A ) subunit of the NF-kappa B nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhes ion. 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, Pho sphorothioate 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 subun it of laminin with a probe 12mer phosphodiester oligodeoxynucleotide c ould be demonstrated. This interaction was described by a single-site binding equation (K-d = 14 mu M). Human Rel A antisense and sense olig odeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleo tide binding occurred at or near the heparin-binding site. Competition for 5' P-32-SdT18 (an 18mer phosphorothioate homopolymer of thymidine ) binding to fibronectin with the discrete heparin analogs, as well as with SdC(28), was also observed. Phosphorothioate oligodeoxynucleotid es (Rel A antisense >> Rel A sense) inhibited the binding of laminin t o 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 non-specific ally block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate-treated Jurk at cells. Blockade of specific binding occurred in the oligodeoxynucle otide treated cells in the presence or absence of oligomer in the medi a.