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EA Blakely, MP McNamara, PY Chang, KA Bjornstad, CJ Rosen; Role Of Cdk-inhibitors In The Differentiation And Radiation Response Of Cultured Human Lens Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3578.
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Purpose:Lens cells continue to differentiate throughout the lifetime of an organism. The cyclin-dependent kinase inhibitors (CDKI) p21CIP1 (CDKN1a), p27 KIP1 and p57KIP2 negatively regulate entry into S-phase of the cell cycle. p21 can be transiently induced by either p53-dependent or p53-independent regulation, DNA damage-induced growth arrest, and in the terminal differentiation of post-mitotic cells. We have investigated changes in the expression of the CDKIs during normal lens cell differentiation and after exposure to Xrays or protons. Methods:Low passage number human lens epithelial (HLE) cells were grown on extracellular matrix (ECM) derived from bovine corneal endothelial cells (Blakely et al., IOVS 41:3898, 2000) in Dulbecco's Modified Eagle Medium supplemented with fetal calf serum and 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cultures of different stages of lens cell differentiation were prepared to provide cells in exponential growth and growth-arrested confluence showing markers of differentiation. Total RNA and protein were isolated in a time course before and after exposure to graded doses of 150 kVp Xrays or 55 MeV protons. RT/PCR, and Western analyses were completed for the expression of the CDKIs. Separate cultures were fixed and prepared for immunofluorescence to study p21 expression and localization within the cell. Parallel, irradiated cultures were treated with neutralizing antibody to basic fibroblast growth factor (FGF-2) to examine the effects of elimination of the rapid and cyclical upregulation expression of FGF-2 after particle radiation exposures (Chang et al., Radiat. Res. 154:477, 2000). Results:Our evidence indicates statistically significant radiation-induced changes in the expression of the CDKIs leading to a disruption of normal lens cell differentiation compared to the unirradiated and FGF-2-neutralizing-antibody-treated controls. Conclusion:Our data suggest that upstream modulation of FGF-2 expression after radiation exposure affects subsequent CDKI regulation in cultured HLE cells.
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