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S.R. Fisher, D.M. Robertson, H.D. Cavanagh; P63 Expression in hTERT–Immortalized Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2123.
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Purpose: p63 has been reported as a marker for corneal epithelial stem cells. The purpose of this experiment was to evaluate p63 expression in hTERT–immortalized human corneal epithelial cells (hTCEpi) throughout the cell cycle in primary culture conditions and during subsequent air–lifted epithelial differentiation and in normal cornea. Methods: To assess nuclear changes with the cell cycle, hTCEpi cells were plated and grown for 3 days on collagen–coated coverslips in serum–free media containing 0.15 mM calcium. Cells were fixed in 4% paraformaldehyde and double–labeled with a mouse monoclonal antibody to Ki–67 and a rabbit polyclonal antibody recognizing an epitope specific for the ΔNp63α isoform. To assess changes in p63 levels during subsequent differentiation, cells were plated on collagen coated tissue culture inserts in 1.15mM calcium and grown in sequential submersed and air– lifted culture. Cells were double–labeled with a p63 antibody and phalloidin and evaluated at 3 time points; 3 day low calcium, 7 days submerged, and after an additional 7 days air–lifted. Whole mount fresh (organ donor) human corneal tissue was used to establish patterns of p63 expression in vivo. All images were obtained with a Leica SP2 laser scanning confocal microscope. Results: Double–labeling with Ki–67 and p63 demonstrated the expected characteristic cell cycle changes for Ki–67 in nuclear staining; however, there was no change in nuclear p63 expression at any stage of the cell cycle defined by concomitant Ki–67 staining. Both confluent cell cultures and 7 day submerged conditions demonstrated robust p63 nuclear staining. p63 nuclear staining was also observed to persist in 7 day air–lifted constructs and was detected in fresh normal whole–mount human corneal controls. Conclusions: Taken together, these data show: (1) that all hTCEpi immortalized cells express p63 in cultured, submersed, and air–lifted conditions. There appears to be a tendency to lose p63 expression as multi–layering occurs in both air–lifted and normal human controls; and, (2) nuclear p63 expression does not appear to vary during the cell cycle as shown by Ki–67 indicating that it is not a cyclin.
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