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Decreasing expression of the G1-phase inhibitors, p21Cip1 and p16INK4a, promotes division of corneal endothelial cells from older donors.

Joyce NC, Harris DL - Mol. Vis. (2010)

Bottom Line: Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment.The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers.Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA. nancy.joyce@schepens.harvard.edu

ABSTRACT

Purpose: The current studies were conducted to determine whether the cyclin-dependent kinase inhibitors, p21Cip1 (p21 cyclin-dependent kinase-interacting protein 1) and p16INK4a (p16 cyclin-dependent kinase inhibitor 1A), help mediate G(1)-phase inhibition in human corneal endothelial cells (HCEC) by testing the effect of siRNA (small interfering RNA)-mediated down-regulation of the expression of these inhibitors on cell cycle entry and proliferation in HCEC cultured from older donors.

Methods: HCEC were obtained from National Disease Research Interchange, Philadelphia, PA, and cultured according to published methods. Cells were electroporated in the presence of either a non-silencing siRNA control or p21+p16 siRNA. The efficiency of siRNA transfer was observed by fluorescence microscopy of Cy3-labeled control siRNA. Viability was determined by direct counting of cells before and after electroporation. The ability of p21+p16 siRNA to decrease the protein expression of p21Cip1 and p16INK4a was determined by semi-quantitative analysis of western blots. The effect of siRNA treatment on cell cycle progression and proliferation was determined 1, 5, and 11 days after electroporation by counting Ki67-positive cells and total DAPI-stained nuclei.

Results: siRNA was efficiently transferred to HCEC by the electroporation method. The average cell loss was 41.25% at 24 h following electroporation. Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment. This treatment significantly increased the average number of Ki67-positive cells over controls and increased the total number of cells in a time-dependent manner.

Conclusions: Both p21Cip1 and p16INK4a are involved in negative regulation of the cell cycle in HCEC and, thereby, provide an effective barrier to cell division. The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers. Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.

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Effect of treatment with 3 different p21 siRNAs on p21Cip1 and p16INK4a protein levels. The graph of western blot results in (A) shows the effect of p21 siRNAs s415, s416, and s417 on the protein level of p21Cip1. The graph in (B) shows the effect of the same 3 p21 siRNAs on the protein level of p16INK4a. In the graphs, each bar shows the average relative expression of p21Cip1 or p16INK4a (+/− SEM) in HCEC cultured from 3 older donors. Beta-actin was used to normalize all results. Conditions included no electroporation or siRNA treatment (-E/-siRNA), electroporation with Silencer Cy3-labeled Negative Control #1 siRNA (+E/Cy3control), and electroporation with each of three siRNAs (+E/p21 s415, s416, or s417).
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f2: Effect of treatment with 3 different p21 siRNAs on p21Cip1 and p16INK4a protein levels. The graph of western blot results in (A) shows the effect of p21 siRNAs s415, s416, and s417 on the protein level of p21Cip1. The graph in (B) shows the effect of the same 3 p21 siRNAs on the protein level of p16INK4a. In the graphs, each bar shows the average relative expression of p21Cip1 or p16INK4a (+/− SEM) in HCEC cultured from 3 older donors. Beta-actin was used to normalize all results. Conditions included no electroporation or siRNA treatment (-E/-siRNA), electroporation with Silencer Cy3-labeled Negative Control #1 siRNA (+E/Cy3control), and electroporation with each of three siRNAs (+E/p21 s415, s416, or s417).

Mentions: Preliminary western blot studies were conducted to determine the effect of three different p21 siRNAs (Ambion s415, s416, or s417) on the protein levels of both p21Cip1 and p16INK4a in cultured HCEC. As shown by the graphs in Figure 2, at 72 h after electroporation, all three p21 siRNAs significantly reduced the level of p21Cip1 compared with non-electroporated controls (p≤0.05) and with controls electroporated with Silencer Cy3-labeled Negative Control #1 siRNA (p≤0.02). None of the p21 siRNAs induced a significant change in the expression of p16INK4a (p=0.93 for all 3 siRNAs tested). These results demonstrate the specificity of the p21 siRNA treatment and provide evidence that lowering the protein level of p21Cip1 does not result in a compensatory increase in the level of p16INK4a. Three p16 siRNAs (Ambion s216, s217, s218) were similarly tested and all three efficiently reduced p16INK4a protein levels, but did not affect the expression of p21Cip1 (data not shown).


Decreasing expression of the G1-phase inhibitors, p21Cip1 and p16INK4a, promotes division of corneal endothelial cells from older donors.

Joyce NC, Harris DL - Mol. Vis. (2010)

Effect of treatment with 3 different p21 siRNAs on p21Cip1 and p16INK4a protein levels. The graph of western blot results in (A) shows the effect of p21 siRNAs s415, s416, and s417 on the protein level of p21Cip1. The graph in (B) shows the effect of the same 3 p21 siRNAs on the protein level of p16INK4a. In the graphs, each bar shows the average relative expression of p21Cip1 or p16INK4a (+/− SEM) in HCEC cultured from 3 older donors. Beta-actin was used to normalize all results. Conditions included no electroporation or siRNA treatment (-E/-siRNA), electroporation with Silencer Cy3-labeled Negative Control #1 siRNA (+E/Cy3control), and electroporation with each of three siRNAs (+E/p21 s415, s416, or s417).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875254&req=5

f2: Effect of treatment with 3 different p21 siRNAs on p21Cip1 and p16INK4a protein levels. The graph of western blot results in (A) shows the effect of p21 siRNAs s415, s416, and s417 on the protein level of p21Cip1. The graph in (B) shows the effect of the same 3 p21 siRNAs on the protein level of p16INK4a. In the graphs, each bar shows the average relative expression of p21Cip1 or p16INK4a (+/− SEM) in HCEC cultured from 3 older donors. Beta-actin was used to normalize all results. Conditions included no electroporation or siRNA treatment (-E/-siRNA), electroporation with Silencer Cy3-labeled Negative Control #1 siRNA (+E/Cy3control), and electroporation with each of three siRNAs (+E/p21 s415, s416, or s417).
Mentions: Preliminary western blot studies were conducted to determine the effect of three different p21 siRNAs (Ambion s415, s416, or s417) on the protein levels of both p21Cip1 and p16INK4a in cultured HCEC. As shown by the graphs in Figure 2, at 72 h after electroporation, all three p21 siRNAs significantly reduced the level of p21Cip1 compared with non-electroporated controls (p≤0.05) and with controls electroporated with Silencer Cy3-labeled Negative Control #1 siRNA (p≤0.02). None of the p21 siRNAs induced a significant change in the expression of p16INK4a (p=0.93 for all 3 siRNAs tested). These results demonstrate the specificity of the p21 siRNA treatment and provide evidence that lowering the protein level of p21Cip1 does not result in a compensatory increase in the level of p16INK4a. Three p16 siRNAs (Ambion s216, s217, s218) were similarly tested and all three efficiently reduced p16INK4a protein levels, but did not affect the expression of p21Cip1 (data not shown).

Bottom Line: Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment.The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers.Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA. nancy.joyce@schepens.harvard.edu

ABSTRACT

Purpose: The current studies were conducted to determine whether the cyclin-dependent kinase inhibitors, p21Cip1 (p21 cyclin-dependent kinase-interacting protein 1) and p16INK4a (p16 cyclin-dependent kinase inhibitor 1A), help mediate G(1)-phase inhibition in human corneal endothelial cells (HCEC) by testing the effect of siRNA (small interfering RNA)-mediated down-regulation of the expression of these inhibitors on cell cycle entry and proliferation in HCEC cultured from older donors.

Methods: HCEC were obtained from National Disease Research Interchange, Philadelphia, PA, and cultured according to published methods. Cells were electroporated in the presence of either a non-silencing siRNA control or p21+p16 siRNA. The efficiency of siRNA transfer was observed by fluorescence microscopy of Cy3-labeled control siRNA. Viability was determined by direct counting of cells before and after electroporation. The ability of p21+p16 siRNA to decrease the protein expression of p21Cip1 and p16INK4a was determined by semi-quantitative analysis of western blots. The effect of siRNA treatment on cell cycle progression and proliferation was determined 1, 5, and 11 days after electroporation by counting Ki67-positive cells and total DAPI-stained nuclei.

Results: siRNA was efficiently transferred to HCEC by the electroporation method. The average cell loss was 41.25% at 24 h following electroporation. Protein levels of both p21Cip1 and p16INK4a were significantly decreased as the result of p21+p16 siRNA treatment. This treatment significantly increased the average number of Ki67-positive cells over controls and increased the total number of cells in a time-dependent manner.

Conclusions: Both p21Cip1 and p16INK4a are involved in negative regulation of the cell cycle in HCEC and, thereby, provide an effective barrier to cell division. The siRNA-induced reduction in expression of these proteins increased the number of cells entering the cell cycle, as well as total cell numbers. Thus, reduction of the levels of p21Cip1 and p16INK4a could be useful in the development of treatments to induce transient cell division to increase corneal endothelial cell density.

Show MeSH
Related in: MedlinePlus