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S.I. Ho, D.M. Robertson, B.S. Hansen, A.M. Allam, P. Parmar, W.M. Petroll, H.D. Cavanagh; The Role of p63 as a Regulator of Proliferation in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3035.
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© ARVO (1962-2015); The Authors (2016-present)
In the human corneal epithelium, it has been suggested that specific isoforms of the putative stem cell marker p63 may regulate homeostatic renewal through control of cell proliferation. We recently reported that reduced expression of p63 in human corneal epithelial cells had no effect on cell cycle control. The purpose of this additional study was to further characterize the role of p63 in proliferation and expression of p63 as a function of the cell cycle.
To assess nuclear changes with the cell cycle, hTCEpi cells were grown on collagen–coated glass coverslips and double–labeled with a mouse monoclonal antibody to Ki–67 and a rabbit polyclonal antibody recognizing an epitope specific for ΔNp63 isoforms. To assess functional regulation, hTCEpi cells were transfected with siRNAs directed against p63 and assessed at 24, 48, and 72 hours. Mock transfection, Lamin A/C siRNA, and non–targeting controls were used. For BrdU labeling, cells were incubated in BrdU for 2 hours at the respective time points, fixed in 4% paraformaldehyde, and double–labeled with a sheep polyclonal BrdU antibody and propidium iodide (PI). Ki–67/p63 images were obtained with a Leica SP2 laser scanning confocal microscope; BrdU/PI images were obtained using a Leitz Diaplan epifluorescent microscope. All images were analyzed using MetaMorph Software. Western blotting and Real time PCR were used to confirm knockdown using ImageQuant and ABI Prism SDS 2.1, respectively.
Quantitative analysis of ΔNp63 protein expression levels throughout the cell cycle demonstrated no significant change regardless of cell cycle phase (p<0.490). Western blotting and real time PCR demonstrated efficient knockdown of p63 at 48 and 72 hours by siRNA. BrdU labeling following siRNA knockdown demonstrated no measurable effect on proliferation.
In support of our previously reported findings, these additional data show that ΔNp63 does not vary within the cell cycle and that induced reduction of ΔNp63 levels has no effect on proliferation. Taken together, these findings further suggest that ΔNp63 is not a direct regulator of corneal epithelial proliferation.
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