Protein kinase C alpha and gamma in N/N 1003A rabbit lens epithelial cell differentiation

Purpose: To determine if Protein Kinase C (PKC) plays a role in the initiat ion of lens epithelial cell differentiation into a lens fiber cell. Methods: PKC alpha or PKC gamma was overexpressed in N/N 1003A lens epithel ial cells for up to 7 days. Phase contrast microscopy was used to observe m orphological changes associated with PKC alpha or PKC gamma overexpression. Cell cycle changes in cells overexpressing PKC alpha or PKC gamma were mea sured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKC alpha or PKC gamma were measured using Western blots and RT-PCR. Results: Significant differences in cell cycling were observed between untr ansfected cells and those overexpressing PKC alpha or PKC gamma. Overexpres sion of PKC alpha and PKC gamma caused the cells to lose their epithelial-l ike appearance and elongate. alphaB-crystallin expression was detected in a ll the samples while alphaA-crystallin was detected only in cells after 7 d ays of PKC alpha or PKC gamma overexpression. Conclusions: The observations that alphaA-crystallin is only found in N/N 1 003A cells overexpressing PKC alpha or PKC gamma for 7 days along with the finding that a block in the G(0)/G(1) phase of the cell cycle and the conse quent morphological changes are observed, indicate that PKC alpha and PKC g amma may have a role in the initiation of differentiation in lens epithelia l cells.