Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei.
Decoy oligonucleotides have been used for functional sequestering of transcription factors. Efficient introduction into cells is a prerequisite for the oligonucleotides to exert their blocking function. Lipofection is the most widely used technique for that purpose because of its convenience and relatively high efficiency. However, the transduction efficiency of lipofection largely depends on cell types and experimental conditions and the introduced nucleotides are not specifically directed to nuclei where they exert their major function. In the present study, we designed a new system for transporting oligonucleotides into cell nuclei. The vehicle is composed of glutathione-S-transferase, 7 arginine residues, the DNA-binding domain of GAL4 and a nuclear localization signal, which are linked with flexible glycine stretches. The p53-responsive element linked to the GAL4 upstream activating sequence was efficiently transferred by the vehicle protein into nuclei of primary cultures of neuronal cells, embryonic stem cells and various human normal cells. Transcriptional activation of p21(WAF1/CIP1) and Bax by p53 on exposure to cisplatin was completely blocked by introducing the p53 decoy oligonucleotide. Thus, the system developed in the present study can be a convenient and powerful tool for specifically disrupting the function of DNA-binding proteins in culture.
Sakaguchi
Masakiyo
M
Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shikatachou, Okayama 700-8558, Japan.
Nukui
Takamasa
T
Sonegawa
Hiroyuki
H
Murata
Hitoshi
H
Futami
Junichiro
J
Yamada
Hidenori
H
Huh
Nam-ho
NH
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D019913:Nuclear Localization Signals; D009841:Oligonucleotides / Q000378:metabolism*; D015202:Protein Engineering; D051381:Rats; D020218:Response Elements; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors*
D002352:Carrier Proteins; D019913:Nuclear Localization Signals; D009841:Oligonucleotides; D016159:Tumor Suppressor Protein p53