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.
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-ras pur/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-ras pur/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.
Department of Biomedical Science and Technology, School of Medicine, University of Udine, Piazzale Kolbe, 4, 33100 Udine, Italy.
Susanna
S
Cogoi
Franco
F
Quadrifoglio
Luigi E
LE
Xodo
D017209:Apoptosis / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D015536:Down-Regulation / Q000235:genetics; D054856:G-Quadruplexes; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006147:Guanine / Q000096:biosynthesis / Q000737:chemistry*; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D011685:Purine Nucleotides / Q000378:metabolism; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D006131:Growth Inhibitors; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009843:Oligoribonucleotides; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; C051802:triplex DNA; D006147:Guanine; D004247:DNA; D015689:Oncogene Protein p21(ras)