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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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In vitro translation with 2′-OMe antisense oligomers no. 1 and no. 7b.The autoradiogram shows the protein products of the translation reaction in RRL with the 5′-capped ΔNp53utr-Luc RNA in the presence of 2′-OMe antisense oligomers no. 1, no. 7b and the control oligomer (+), respectively. The control oligomer anneals to Firefly luciferase sequence. Lane (−) indicates control reaction with no antisense oligonucleotide addition.
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pone-0078863-g005: In vitro translation with 2′-OMe antisense oligomers no. 1 and no. 7b.The autoradiogram shows the protein products of the translation reaction in RRL with the 5′-capped ΔNp53utr-Luc RNA in the presence of 2′-OMe antisense oligomers no. 1, no. 7b and the control oligomer (+), respectively. The control oligomer anneals to Firefly luciferase sequence. Lane (−) indicates control reaction with no antisense oligonucleotide addition.

Mentions: Further, in order to test the impact of RNase H activity on protein synthesis 2′-OMe oligomers no. 1 and no. 7b were used in a translation assay in RRL. In the presence of 2′-OMe oligomer no. 1 the efficiency of protein synthesis from both initiation codons was reduced (Fig. 5), however, not as strongly as in the case of unmodified oligomer (see Fig. 3). Since 2′-OMe oligomers form heteroduplexes which are unable to recruit RNase H the 2′-OMe oligomer no. 1 might impair translation only via the steric-blocker action. Additionally, breakage of RNA in the G161-G168 region upon binding of oligomer no. 1 (see Fig. 4) might also influence the translation efficiency.


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)

In vitro translation with 2′-OMe antisense oligomers no. 1 and no. 7b.The autoradiogram shows the protein products of the translation reaction in RRL with the 5′-capped ΔNp53utr-Luc RNA in the presence of 2′-OMe antisense oligomers no. 1, no. 7b and the control oligomer (+), respectively. The control oligomer anneals to Firefly luciferase sequence. Lane (−) indicates control reaction with no antisense oligonucleotide addition.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078863-g005: In vitro translation with 2′-OMe antisense oligomers no. 1 and no. 7b.The autoradiogram shows the protein products of the translation reaction in RRL with the 5′-capped ΔNp53utr-Luc RNA in the presence of 2′-OMe antisense oligomers no. 1, no. 7b and the control oligomer (+), respectively. The control oligomer anneals to Firefly luciferase sequence. Lane (−) indicates control reaction with no antisense oligonucleotide addition.
Mentions: Further, in order to test the impact of RNase H activity on protein synthesis 2′-OMe oligomers no. 1 and no. 7b were used in a translation assay in RRL. In the presence of 2′-OMe oligomer no. 1 the efficiency of protein synthesis from both initiation codons was reduced (Fig. 5), however, not as strongly as in the case of unmodified oligomer (see Fig. 3). Since 2′-OMe oligomers form heteroduplexes which are unable to recruit RNase H the 2′-OMe oligomer no. 1 might impair translation only via the steric-blocker action. Additionally, breakage of RNA in the G161-G168 region upon binding of oligomer no. 1 (see Fig. 4) might also influence the translation efficiency.

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