Control of SV-40 transformed RCE cell proliferation by growth-factor-induced cell cycle progression

To determine in SV40-immortalized rabbit corneal epithelial cells (RCE), whether there is conservation of parent tissue serum growth-factor-stimulated cytokine receptor activation and downstream intracellular signaling events mediating control of cell cycle progression and differentiation.

Immunostaining and Western blot analysis were used to measure cytokeratin K3 and K12 expression with AE5 and AK12 antibodies. Karyotype analysis was performed based on comparison of the RCE chromosomal complement with its parent tissue. EGF receptor activation was evaluated based on immunochemistry and Western blot analyses of EGF receptor dimerization and phosphorylation. Functional status of EGF receptor was determined through measurements of EGF-induced stimulation of ERK-2 activity, which is a component of the mitogen-activated protein kinase cascade (MAPK). This was done by immunocomplex and kinase assay using anti-ERK antibodies and a specific substrate. EGF-induced increases in proliferation and cell cycle progression were determined based on measurements of [(3)H]-thymidine incorporation, G(2)-specific cyclin B1 expression and cell cycle mapping.

From days 7 to 14, K12 expression increased based on marked rises in the levels of a 55 kD band. At day 14, a 64 kD band also appeared indicative of K3 expression. Karyotype analysis showed that there were no chromosomal losses due to SV-40 transformation. Upon exposure to EGF (5 ng/ml) for 1 min, EGF receptors were activated and formed clusters indicating that autophosphorylation and multimerization of the EGF receptor were occurred. In the presence of serum growth factors or EGF, ERK-2 kinase activity was markedly increased with a bell-shaped time-dependent activation pattern. Cell cycle progression was analyzed in G(1)/S boundary synchronized RCE cells. After releasing the cells into modified Supplemented Hormonal Epithelium Medium containing 10% serum and DMEM/F-12 medium, 80% of the cells had entered the S phase within 2 h. In addition, time dependent changes in [(3)H]-hymidine incorporation over 8 h confirmed RCE passage through the G(1)/S checkpoint. There were more RCE cells entered the G(2)/M phase of cell cycle in the 6-8 h interval after their release. Another indication of cell cycle progression into the G(2)/M phase was that at 8-10 h cyclin B(1) expression reached its maximal level.

RCE in passage number 12-20 are a physiologically relevant model for studies on growth factor receptor mediated control of cell cycle progression and differentiation in its parent tissue as each of these phenomena were conserved: 1) EGF-induced EGF receptor activation; 2) EGF-activated ERK signaling; 3) expression of cornea-specific differentiation markers; 4) karyotype profile; and 5) cell cycle control and progression.