Limits...
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.

Show MeSH

Related in: MedlinePlus

Disturbance of the p53 translation initiation process in MCF-7 cells in the presence of 2′-OMe antisense oligomers.(A) Cells were transfected with 2′-OMe oligomers: no. 1, no. 7b and control, respectively. Cells were harvested and lysed after specified time points and the level of endogenous p53 was determined by western blot using monoclonal antibody p53– Pab 1801. The GAPDH level was used to normalize the data. The p53 level in the presence of 2′-OMe oligomer no 1 or no. 7b was compared to the value of p53 protein level with control oligomer which was defined as 100%. The bar graph shows average and standard deviations for at least three independent experiments. (B) RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cell after the transfection of 2′-OMe oligomers at specified time points, respectively. The results from at least two independent experiments show no changes in the p53 mRNA level in the presence of 2′-OMe oligomers.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3824000&req=5

pone-0078863-g006: Disturbance of the p53 translation initiation process in MCF-7 cells in the presence of 2′-OMe antisense oligomers.(A) Cells were transfected with 2′-OMe oligomers: no. 1, no. 7b and control, respectively. Cells were harvested and lysed after specified time points and the level of endogenous p53 was determined by western blot using monoclonal antibody p53– Pab 1801. The GAPDH level was used to normalize the data. The p53 level in the presence of 2′-OMe oligomer no 1 or no. 7b was compared to the value of p53 protein level with control oligomer which was defined as 100%. The bar graph shows average and standard deviations for at least three independent experiments. (B) RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cell after the transfection of 2′-OMe oligomers at specified time points, respectively. The results from at least two independent experiments show no changes in the p53 mRNA level in the presence of 2′-OMe oligomers.

Mentions: Both 2′-OMe oligomers no. 1 and no. 7b impaired p53 translation over a short time (Fig. 6A). Six hours after transfection the p53 expression was lower by approximately 30% compared with the level of p53 protein in the control sample in which 2′-OMe oligonucleotide complementary to Firefly luciferase sequence was used (Fig. 6A). Despite the lack of efficient inhibition of protein synthesis from AUG1 with 2′-OMe oligomer no. 7b in RRL (see Fig. 5), its effect in vivo was significant. Ten hours post transfection a further decrease in p53 expression, up to 40%, was observed. Twenty four hours after cell transfection with 1 µM 2′- OMe oligomer no. 7b more than 50% decrease of the p53 protein level was still observed (data not shown). The RT-PCR revealed no changes in the amount of p53 mRNA in the presence of 2′-OMe oligomers no. 1 or no. 7b (Fig. 6B) which confirmed that no RNase H-dependent mechanism was involved in down-regulation of the p53 protein level.


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)

Disturbance of the p53 translation initiation process in MCF-7 cells in the presence of 2′-OMe antisense oligomers.(A) Cells were transfected with 2′-OMe oligomers: no. 1, no. 7b and control, respectively. Cells were harvested and lysed after specified time points and the level of endogenous p53 was determined by western blot using monoclonal antibody p53– Pab 1801. The GAPDH level was used to normalize the data. The p53 level in the presence of 2′-OMe oligomer no 1 or no. 7b was compared to the value of p53 protein level with control oligomer which was defined as 100%. The bar graph shows average and standard deviations for at least three independent experiments. (B) RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cell after the transfection of 2′-OMe oligomers at specified time points, respectively. The results from at least two independent experiments show no changes in the p53 mRNA level in the presence of 2′-OMe oligomers.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078863-g006: Disturbance of the p53 translation initiation process in MCF-7 cells in the presence of 2′-OMe antisense oligomers.(A) Cells were transfected with 2′-OMe oligomers: no. 1, no. 7b and control, respectively. Cells were harvested and lysed after specified time points and the level of endogenous p53 was determined by western blot using monoclonal antibody p53– Pab 1801. The GAPDH level was used to normalize the data. The p53 level in the presence of 2′-OMe oligomer no 1 or no. 7b was compared to the value of p53 protein level with control oligomer which was defined as 100%. The bar graph shows average and standard deviations for at least three independent experiments. (B) RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cell after the transfection of 2′-OMe oligomers at specified time points, respectively. The results from at least two independent experiments show no changes in the p53 mRNA level in the presence of 2′-OMe oligomers.
Mentions: Both 2′-OMe oligomers no. 1 and no. 7b impaired p53 translation over a short time (Fig. 6A). Six hours after transfection the p53 expression was lower by approximately 30% compared with the level of p53 protein in the control sample in which 2′-OMe oligonucleotide complementary to Firefly luciferase sequence was used (Fig. 6A). Despite the lack of efficient inhibition of protein synthesis from AUG1 with 2′-OMe oligomer no. 7b in RRL (see Fig. 5), its effect in vivo was significant. Ten hours post transfection a further decrease in p53 expression, up to 40%, was observed. Twenty four hours after cell transfection with 1 µM 2′- OMe oligomer no. 7b more than 50% decrease of the p53 protein level was still observed (data not shown). The RT-PCR revealed no changes in the amount of p53 mRNA in the presence of 2′-OMe oligomers no. 1 or no. 7b (Fig. 6B) which confirmed that no RNase H-dependent mechanism was involved in down-regulation of the p53 protein level.

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