Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of p21+p16 siRNA on p21Cip1 and p16INK4a protein expression 2 and 7 days following treatment. HCEC from a 68-year-old donor were electroporated with either control siRNA (+E/control siRNA) or p21+p16 siRNA (+E/p21+p16 siRNA). Western blots were used to analyze p21Cip1 (A) and p16INK4a (B) protein levels at the 2 time-points. Results are expressed relative to beta-actin. Note that both p21Cip1 and p16INK4a were decreased to similar levels at both time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875254&req=5

f4: Effect of p21+p16 siRNA on p21Cip1 and p16INK4a protein expression 2 and 7 days following treatment. HCEC from a 68-year-old donor were electroporated with either control siRNA (+E/control siRNA) or p21+p16 siRNA (+E/p21+p16 siRNA). Western blots were used to analyze p21Cip1 (A) and p16INK4a (B) protein levels at the 2 time-points. Results are expressed relative to beta-actin. Note that both p21Cip1 and p16INK4a were decreased to similar levels at both time points.

Mentions: A second series of studies tested the effect of combined treatment of HCEC with Ambion s416 p21 siRNA plus Ambion s217 p16 siRNA (p21+p16 siRNA). Cells were treated with both siRNAs, because previous work from this laboratory had indicated that both p21Cip1 and p16INK4a are expressed at significantly higher levels in HCEC from older donors, suggesting that, together, these two CKIs are responsible for mediating the reduced proliferative response of HCEC with increasing donor age [25]. HCEC cultured from three older donors were treated with p21+p16 siRNA and the effect on the protein expression of both CKIs was determined by western blot analysis. Results demonstrated that treatment of HCEC with p21+p16 siRNA consistently decreased the protein levels of both CKIs. Representative results from a 71-year-old donor are shown in Figure 3. The graphs in Figure 4 show representative western blot analyses of the relative protein levels of p21Cip1 and p16INK4a in HCEC from a 68-year-old donor 2 and 7 days after electroporation with control or p21+p16 siRNA. Results show similarly decreased levels of both p21Cip1 and p16INK4a at both time points, indicating that siRNA treatment remained effective in decreasing the expression of both CKIs for at least one week.


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 p21+p16 siRNA on p21Cip1 and p16INK4a protein expression 2 and 7 days following treatment. HCEC from a 68-year-old donor were electroporated with either control siRNA (+E/control siRNA) or p21+p16 siRNA (+E/p21+p16 siRNA). Western blots were used to analyze p21Cip1 (A) and p16INK4a (B) protein levels at the 2 time-points. Results are expressed relative to beta-actin. Note that both p21Cip1 and p16INK4a were decreased to similar levels at both time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effect of p21+p16 siRNA on p21Cip1 and p16INK4a protein expression 2 and 7 days following treatment. HCEC from a 68-year-old donor were electroporated with either control siRNA (+E/control siRNA) or p21+p16 siRNA (+E/p21+p16 siRNA). Western blots were used to analyze p21Cip1 (A) and p16INK4a (B) protein levels at the 2 time-points. Results are expressed relative to beta-actin. Note that both p21Cip1 and p16INK4a were decreased to similar levels at both time points.
Mentions: A second series of studies tested the effect of combined treatment of HCEC with Ambion s416 p21 siRNA plus Ambion s217 p16 siRNA (p21+p16 siRNA). Cells were treated with both siRNAs, because previous work from this laboratory had indicated that both p21Cip1 and p16INK4a are expressed at significantly higher levels in HCEC from older donors, suggesting that, together, these two CKIs are responsible for mediating the reduced proliferative response of HCEC with increasing donor age [25]. HCEC cultured from three older donors were treated with p21+p16 siRNA and the effect on the protein expression of both CKIs was determined by western blot analysis. Results demonstrated that treatment of HCEC with p21+p16 siRNA consistently decreased the protein levels of both CKIs. Representative results from a 71-year-old donor are shown in Figure 3. The graphs in Figure 4 show representative western blot analyses of the relative protein levels of p21Cip1 and p16INK4a in HCEC from a 68-year-old donor 2 and 7 days after electroporation with control or p21+p16 siRNA. Results show similarly decreased levels of both p21Cip1 and p16INK4a at both time points, indicating that siRNA treatment remained effective in decreasing the expression of both CKIs for at least one week.

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