G-rich Oligonucleotide Inhibits the Binding of a Nuclear Protein to the Ki-ras Promoter and Strongly Reduces Cell Growth in Human Carcinoma Pancreatic Cells

Abstract

Oligonucleotides are able to recognize both nucleic acids and proteins with a high degree of specificity and are therefore investigated as a new and innovative class of therapeutic anticancer drugs. In the present study, we have constructed from Panc-1 cells a stable transfectant (AG transfectant) generating constitutively a short transcript (T-22AG), which is potentially capable of forming a triplex with a critical polypurine/polypyrimidine (pur/pyr) motif located in the Ki-ras promoter. Because of the presence of a G-rich element in its sequence, transcript T-22AG was also capable, under physiological conditions, of adopting a tetraplex conformation. We found that the levels of Ki-ras mRNA and p21^RAS protein in the AG transfectant were, respectively, 52 ± 8 and 40 ± 4% of those observed in the control cell lines: untransfected Panc-1 cells and stably transfected Panc-1 cells producing a control transcript (T-22SCR). The downregulation of Ki-ras resulted in a strong reduction of colony formation (42 ± 7% of the control) and cell proliferation (34 ± 5% of the control) capacity. As in vitro experiments showed that the G-rich element of T-22AG (22AG) formed with the Ki-raspur/pyr motif a triplex of low thermodynamic stability, it is unlikely that the strong bioactivity exhibited by transcript T-22AG is mediated by a triplex-based mechanism, although we cannot totally exclude that in vivo polyamine levels may increase the triplex stability. We found that 22AG adopted a tetraplex conformation and competitively inhibited the binding of a nuclear factor to the Ki-raspur/pyr sequence. This effect was specific and virtually entirely abrogated when 22AG was denatured by heating. Our data showed that transcript T-22AG acted as a molecular aptamer, binding specifically to a nuclear factor essential for Ki-ras expression. The biological implications of this study are discussed.