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Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei.

Sakaguchi M, Nukui T, Sonegawa H, Murata H, Futami J, Yamada H, Huh NH - Nucleic Acids Res. (2005)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shikatachou, Okayama 700-8558, Japan.

ABSTRACT
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.

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Stability of the decoy oligonucleotide in cells. (A) Radiolabeled decoy oligonucleotide was applied with GST-7GR-Ga-NLS to HCT116 or NHK cells and amounts of the intact oligonucleotide (95 bp in length) recovered from the cells was monitored by electrophoresis and autoradiography. (B) Northern blot analysis for p21WAF1/CIP1 mRNA after the introduction of the oligonucleotides. NHK cells were subjected to application of the wild-type or mutated decoy oligonucleotide and GST-7GR-Ga-NLS at the time point 0, exposed to cisplatin (10 μg/ml) from 1 h later and harvested at indicated time points. GAPDH was used as a control for applied amounts of RNA.
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fig6: Stability of the decoy oligonucleotide in cells. (A) Radiolabeled decoy oligonucleotide was applied with GST-7GR-Ga-NLS to HCT116 or NHK cells and amounts of the intact oligonucleotide (95 bp in length) recovered from the cells was monitored by electrophoresis and autoradiography. (B) Northern blot analysis for p21WAF1/CIP1 mRNA after the introduction of the oligonucleotides. NHK cells were subjected to application of the wild-type or mutated decoy oligonucleotide and GST-7GR-Ga-NLS at the time point 0, exposed to cisplatin (10 μg/ml) from 1 h later and harvested at indicated time points. GAPDH was used as a control for applied amounts of RNA.

Mentions: Finally, we examined stability of the incorporated oligonucleotide and duration of the functional sequestering of p53 protein. Labeled decoy oligonucleotide was introduced into HCT116 cells or NHK cells, and the radioactivity recovered from the cells was monitored by autoradiography after electrophoresis. As shown in Figure 6A, the intracellular oligonucleotide levels remained essentially unchanged until 12 h, gradually declined afterwards reaching a level of about half around 24 h, and decreased to an undetectable level around 48 h after the application in both cell types. Duration of the biological effects of the introduced oligonucleotide was studied in NHK cells because HCT116 cells underwent apoptotic cell death by treatment with cisplatin. NHK cells were treated with cisplatin 1 h after the introduction of the wild-type or mutated decoy oligonucleotide into the cells, and then p21WAF1/CIP1 mRNA level was monitored (Figure 6B). In the cells exposed to the mutated p53 decoy oligonucleotide, p21WAF1/CIP1 mRNA was induced and remained at similar levels during the observation period of 72 h. Introduction of the wild-type p53 decoy oligonucleotide completely suppressed the induction of p21WAF1/CIP1 mRNA up to 24 h. A low level of p21WAF1/CIP1 mRNA was detected at 36 h and the amount had increased to a level comparable with that of untreated cells at 48 h. This kinetics accorded well with the amount the decoy oligonucleotide in the cells (Figure 6A).


Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei.

Sakaguchi M, Nukui T, Sonegawa H, Murata H, Futami J, Yamada H, Huh NH - Nucleic Acids Res. (2005)

Stability of the decoy oligonucleotide in cells. (A) Radiolabeled decoy oligonucleotide was applied with GST-7GR-Ga-NLS to HCT116 or NHK cells and amounts of the intact oligonucleotide (95 bp in length) recovered from the cells was monitored by electrophoresis and autoradiography. (B) Northern blot analysis for p21WAF1/CIP1 mRNA after the introduction of the oligonucleotides. NHK cells were subjected to application of the wild-type or mutated decoy oligonucleotide and GST-7GR-Ga-NLS at the time point 0, exposed to cisplatin (10 μg/ml) from 1 h later and harvested at indicated time points. GAPDH was used as a control for applied amounts of RNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1140756&req=5

fig6: Stability of the decoy oligonucleotide in cells. (A) Radiolabeled decoy oligonucleotide was applied with GST-7GR-Ga-NLS to HCT116 or NHK cells and amounts of the intact oligonucleotide (95 bp in length) recovered from the cells was monitored by electrophoresis and autoradiography. (B) Northern blot analysis for p21WAF1/CIP1 mRNA after the introduction of the oligonucleotides. NHK cells were subjected to application of the wild-type or mutated decoy oligonucleotide and GST-7GR-Ga-NLS at the time point 0, exposed to cisplatin (10 μg/ml) from 1 h later and harvested at indicated time points. GAPDH was used as a control for applied amounts of RNA.
Mentions: Finally, we examined stability of the incorporated oligonucleotide and duration of the functional sequestering of p53 protein. Labeled decoy oligonucleotide was introduced into HCT116 cells or NHK cells, and the radioactivity recovered from the cells was monitored by autoradiography after electrophoresis. As shown in Figure 6A, the intracellular oligonucleotide levels remained essentially unchanged until 12 h, gradually declined afterwards reaching a level of about half around 24 h, and decreased to an undetectable level around 48 h after the application in both cell types. Duration of the biological effects of the introduced oligonucleotide was studied in NHK cells because HCT116 cells underwent apoptotic cell death by treatment with cisplatin. NHK cells were treated with cisplatin 1 h after the introduction of the wild-type or mutated decoy oligonucleotide into the cells, and then p21WAF1/CIP1 mRNA level was monitored (Figure 6B). In the cells exposed to the mutated p53 decoy oligonucleotide, p21WAF1/CIP1 mRNA was induced and remained at similar levels during the observation period of 72 h. Introduction of the wild-type p53 decoy oligonucleotide completely suppressed the induction of p21WAF1/CIP1 mRNA up to 24 h. A low level of p21WAF1/CIP1 mRNA was detected at 36 h and the amount had increased to a level comparable with that of untreated cells at 48 h. This kinetics accorded well with the amount the decoy oligonucleotide in the cells (Figure 6A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shikatachou, Okayama 700-8558, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus