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TAT-Mediated Protein Transduction into Human Corneal Epithelial Cells: p15^INK4b Inhibits Cell Proliferation and Stimulates Cell Migration

From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.

PURPOSE. The cell cycle inhibitor p15^INK4b has been localized in migrating corneal epithelial cells. In this study, TAT-fusion protein technology was used to transduce p15^INK4b into human corneal epithelial cells to examine the effect on cell proliferation and migration.

METHODS. Human p15^INK4b, obtained by RT-PCR, was cloned into a TAT-HA vector, and the fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct. Various dilutions of TAT-HA-p15 were applied to primary human corneal epithelial cells to test potency. In addition, the effect of exposure time was examined. Cells were labeled with bromodeoxyuridine to detect proliferation, and indirect immunofluorescence was performed. Ki67 expression was also examined. To assay cell migration, human corneal epithelial cells were plated inside a cylinder and exposed to TAT-HA-p15. The cylinder was removed, the cells were allowed to spread for 2 days, and the area of cell coverage was calculated. TAT-HA-ß-galactosidase served as the control in all experiments. Finally, the extent of retinoblastoma protein phosphorylation was assayed by Western blot in cells cultured with and without TAT-HA-p15.

RESULTS. TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells. TAT-HA-p15 decreased proliferation in a concentration- and time-dependent manner. The migration assay showed that TAT-HA-p15 stimulated cell migration 1.8-fold. TAT-HA-ß-galactosidase had no effect on proliferation or migration. Finally, TAT-HA-p15 decreased the level of phosphorylated retinoblastoma protein by 4.9-fold.

CONCLUSIONS. Active p15^INK4b can be efficiently transduced into primary human corneal epithelial cells using TAT-fusion protein technology. p15^INK4b appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration.

This article has been cited by other articles:

				H. Sakai, B.-C. Park, X. Shen, and B. Y. J. T. Yue Transduction of TAT Fusion Proteins into the Human and Bovine Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4427 - 4434. [Abstract] [Full Text] [PDF]