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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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Down-regulation of p53 protein level in MCF-7 cells in the presence of GAP antisense oligomers.(A) Cells were transfected with GAP 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. The bar graph shows average and standard deviations for at least three independent experiments, while normalization was performed as described in the legend to Figure 6. (*) p-values were calculated using Student’s t-test (B) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after GAP oligomer transfection at specified time points, respectively. The data were obtained from at least two independent experiments.
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pone-0078863-g007: Down-regulation of p53 protein level in MCF-7 cells in the presence of GAP antisense oligomers.(A) Cells were transfected with GAP 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. The bar graph shows average and standard deviations for at least three independent experiments, while normalization was performed as described in the legend to Figure 6. (*) p-values were calculated using Student’s t-test (B) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after GAP oligomer transfection at specified time points, respectively. The data were obtained from at least two independent experiments.

Mentions: Subsequently, GAP oligomers no. 1 and no. 7b which could induce RNase H activity were used in MCF-7 cells to test their effects on the p53 expression in the living cells. A strong reduction of the level of the p53 protein was observed for both gapmers (Fig. 7A). Inhibition of approximately 50% was achieved in the presence of GAP oligomer no. 1 and of 80% in the presence of GAP oligomer no. 7b. Moreover, both oligomers reduced the mRNA level compared with the control reaction (Fig. 7B) which was in line with the conclusion that RNase H activity was mainly responsible for changes in the amount of p53 protein.


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)

Down-regulation of p53 protein level in MCF-7 cells in the presence of GAP antisense oligomers.(A) Cells were transfected with GAP 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. The bar graph shows average and standard deviations for at least three independent experiments, while normalization was performed as described in the legend to Figure 6. (*) p-values were calculated using Student’s t-test (B) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after GAP oligomer transfection at specified time points, respectively. The data were obtained from at least two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078863-g007: Down-regulation of p53 protein level in MCF-7 cells in the presence of GAP antisense oligomers.(A) Cells were transfected with GAP 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. The bar graph shows average and standard deviations for at least three independent experiments, while normalization was performed as described in the legend to Figure 6. (*) p-values were calculated using Student’s t-test (B) The RT-PCR analysis of p53 mRNA and β-actin mRNA (as a control) extracted from the cells after GAP oligomer transfection at specified time points, respectively. The data were obtained from at least two independent experiments.
Mentions: Subsequently, GAP oligomers no. 1 and no. 7b which could induce RNase H activity were used in MCF-7 cells to test their effects on the p53 expression in the living cells. A strong reduction of the level of the p53 protein was observed for both gapmers (Fig. 7A). Inhibition of approximately 50% was achieved in the presence of GAP oligomer no. 1 and of 80% in the presence of GAP oligomer no. 7b. Moreover, both oligomers reduced the mRNA level compared with the control reaction (Fig. 7B) which was in line with the conclusion that RNase H activity was mainly responsible for changes in the amount of p53 protein.

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