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X.Q. Guo, A.E. Hutcheon, J.D. Zieske; P15 Is Sufficient to Inhibit Proliferation of Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3836.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In previous investigations, we demonstrated that the cell cycle inhibitor p15 was localized in migrating corneal epithelial cells. We also efficiently transduced functionally active proteins into corneal epithelial cells using TAT-fusion protein technology. Using this technology, we examined the effect of p15 on human corneal epithelial cell proliferation and migration. Methods: Human p15, obtained by RT-PCR, was cloned into TAT-HA vector (a gift from Steven Dowdy, UCSD). TAT-HA-p15 fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct. Various dilutions of TAT-HA-p15 were applied to cultured primary human epithelial cells and incubated for 18 hours to test potency. Optimal exposure time of TAT-HA-p15 was determined by incubating cells for 0, 2, 4, 6, 8 and 18 hours. TAT-HA-ß-gal served as a control. At the appropriate time, the cells were washed 3 times with PBS and labeled with BrdU. Indirect immunofluorescence was used to localize BrdU and HA. To assay cell migration, 5.6 X 10-4 primary human corneal epithelial cells were put into a cylinder in a 6-well plate. After 3 hours, cylinders were removed, the wells rinsed, and medium with 200nM of TAT-HA-p15 was added. After 2 days, the cells were fixed and stained and the area of cell coverage was calculated. TAT-HA-ß-gal served as a control. Results: TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells. After overnight exposure to different concentrations of TAT-HA-p15, the number of proliferating cells decreased in a concentration-dependent manner. The percentage of BrdU-labeled cells were 45.7%, 21.4%, 3.3% and 1.7% at 0, 60, 150 and 300nM of TAT-HA-p15 respectively and 42.9%, 38.9%, 39.0%, and 40.2% at 0, 60, 150 and 300nM of TAT-HA-ß-gal respectively. After 0, 2, 4, 6, 8 or 18 hours exposure to 150nM of TAT-HA-p15, the percentage of BrdU-labeled cells was 41.4%, 36.5%, 29.9%, 18.6%, 7.0% and 3.3% respectively. The migration assay showed that TAT-HA-p15 stimulated cell migration. The increase in area of cell coverage for control cells (no additions), TAT-HA-ß-gal and TAT-HA-p15 were 53.44%, 51.11% and 96.44%. Conclusion: p15 can be efficiently transduced into primary human corneal epithelial cells in culture using TAT-fusion protein technology. p15 appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration.
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