An Oligonucleotide Which Blocks EGFr Tyrosine Phosphorylation Modulates the Rapid Morphological Change Induced by EGF Stimulation of A431 Cells

Abstract

We have previously shown that an antisense phosphorothioate oligonucleotide, erbB1AS15 (PS), targeted against the initiation codon of the epidermal growth factor receptor (EGFr) mRNA, inhibited receptor tyrosine kinase activity via a non-antisense mechanism in A431 cells. The aims of this study were to evaluate the effect of this oligonucleotide on A431 cells in the presence of exogenous stimulation with high concentrations of the EGF ligand and to further elucidate the mechanism of action of erbB1AS15 (PS). The morphological effects of EGF stimulation were monitored in A431 cells in culture. The results show that the rapid rounding induced by stimulation with 500 ng mL−1 EGF in A431 cells was modulated by the erbB1AS15 (PS) oligonucleotide. The presence of the oligonucleotide reduced the initial rapid morphological change and increased the rate at which the cells returned to normal following removal of EGF. Control oligonucleotides had no effect, suggesting that the morphological effects of erbB1AS15 (PS) were sequence dependent. The possibility of a site of action at the level of the ligand was discounted by band shift analysis of radiolabelled oligonucleotide in the presence of excess EGF. These results therefore indicate that the oligonucleotide is acting at a point in the EGF-mediated signal transduction pathway that ultimately leads to these cytoskeletal changes, probably as a result of modulation of downstream phosphorylation. These data, along with the longer-term morphological change in the A431 cells caused by erbB1AS15 (PS) in the absence of EGF stimulation, demonstrate a potent non-antisense mediated activity of a phosphorothioate oligonucleotide.