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

被引:51
作者
Cogoi, S [1 ]
Quadrifoglio, F [1 ]
Xodo, LE [1 ]
机构
[1] Univ Udine, Sch Med, Dept Biomed Sci & Technol, I-33100 Udine, Italy
关键词
D O I
10.1021/bi035754f
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:2512 / 2523
页数:12
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