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K.V. Gonzalez, S.C. Fonseca, L.F. Melecio, J.R. Rodriguez–Medina; Changes in Cyclin Dependent Kinase 1 (Cdk–1/Cdc–2) in Galactosemic Bovine Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4115.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanism by which diabetic patients develop cataracts is unknown. It has been previously shown that one of the most significant changes in diabetic and galactosemic lens epithelial cells (LECs) are changes in the rate of mitosis when compared to controls, but the analysis of the cell cycle progression of bovine LECs using different techniques has produced contradictory results. The purpose of this project is to study the expression of cdk–1/cdc–2 in galactosemic LECs. Cdk–1/cdc–2 plays an important role in the cell cycle transition through the G2/M phase and its activity increases at the onset of the M phase. Cdk–1/Cdc–2 also has an important role in the activation of the cell cycle check point. The expression pattern of the cyclins was also studied.
Bovine LECs were maintained as primary cultures as described elsewhere (Curr. Eye Res. 14:269 (1995). Galactosemic LECs were kept under the same conditions, but the culture media contained 40mM galactose for a total of 7 days. Whole LECs protein homogenates were separated using SDS–PAGE on 12% gels and transferred to nitrocellulose membranes. Western blot analysis were performed using antibodies against cdk–1/cdc–2 (Upstate), cyclin A, B1, E, D1 (Rockland) and beta–actin (abcam) (internal control) were used to determine their levels of expression. Super Signal West Pico Chemiluminescent Substrate (Pierce) and horseradish peroxidase secondary antibody were used to visualize the bands.
The expression of Cdk–1/Cdc–2 increased by 7% in galactosemic LECs when compared to normal LECs. The corresponding cyclins associated with this kinase (cyclin A and B1) were also expressed in higher levels (by 32% and 15% respectively) in galactosemic LECs when compared to normal LECs.
The results showed that galactosemic LECs expressed cdk–1/cdc–2 at higher levels when compared to normal LECs, and cyclin B, one of the activators of this kinase, was also expressed in higher concentration in galactosemic LECs. Others have showed (Cell (1993)75:817) that during LECs differentiation, peak cdk–1/cdc–2–cyclin B activity was correlated with changes in chromatin structure and nuclear envelope breakdown in the terminally differentiating primary lens fiber cells. In other studies using diabetic rats (Exp. Eye Res. (2002) 74:245), p21 expression increased. P21 is a regulator of cdk–1/cdc–2. The relevance of this findings and the involvement of a cell cycle checkpoint in the process of cataractogenesis has yet to be determined.
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