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Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

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Inhibition of p53 expression in MCF-7 cells using different concentration of GAP oligomer no. 7b.The cells were transfected with GAP oligomer no. 7b with its final concentration of 0.5–0.1 µM and then 10 hours from transfection the cells were lysed and endogenous p53 was determined by western blot. The data was provided from at least two independent experiments.
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pone-0078863-g008: Inhibition of p53 expression in MCF-7 cells using different concentration of GAP oligomer no. 7b.The cells were transfected with GAP oligomer no. 7b with its final concentration of 0.5–0.1 µM and then 10 hours from transfection the cells were lysed and endogenous p53 was determined by western blot. The data was provided from at least two independent experiments.

Mentions: Finally, GAP oligomer no. 7b was used in MCF-7 cells in various concentrations. It turned out that a decrease in p53 expression of approximately 40% can be reached over a short time, 10 hours post transfection, in the presence of this oligomer used at 100–150 nM concentration while with 500 nM oligomer inhibition of almost 70% was observed (Fig. 8).


Modulation of p53 expression using antisense oligonucleotides complementary to the 5'-terminal region of p53 mRNA in vitro and in the living cells.

Gorska A, Swiatkowska A, Dutkiewicz M, Ciesiolka J - PLoS ONE (2013)

Inhibition of p53 expression in MCF-7 cells using different concentration of GAP oligomer no. 7b.The cells were transfected with GAP oligomer no. 7b with its final concentration of 0.5–0.1 µM and then 10 hours from transfection the cells were lysed and endogenous p53 was determined by western blot. The data was provided from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824000&req=5

pone-0078863-g008: Inhibition of p53 expression in MCF-7 cells using different concentration of GAP oligomer no. 7b.The cells were transfected with GAP oligomer no. 7b with its final concentration of 0.5–0.1 µM and then 10 hours from transfection the cells were lysed and endogenous p53 was determined by western blot. The data was provided from at least two independent experiments.
Mentions: Finally, GAP oligomer no. 7b was used in MCF-7 cells in various concentrations. It turned out that a decrease in p53 expression of approximately 40% can be reached over a short time, 10 hours post transfection, in the presence of this oligomer used at 100–150 nM concentration while with 500 nM oligomer inhibition of almost 70% was observed (Fig. 8).

Bottom Line: Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells.A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins.One of these oligomers might be used in the future as a support treatment in anticancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

ABSTRACT
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5'-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2'-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5'-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.

Show MeSH
Related in: MedlinePlus