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-beta-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-beta-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.