Gene Expression of CDK-Inhibitors During Lens Differentiation and the Radiation Response of Cultured Human Lens Cells

Abstract

Abstract: : Purpose:We have previously reported changes in protein expression of the cyclin-dependent kinase inhibitors (CDKI), p21CIP1 (CDKN1a), p27KIP1 and p57KIP2 during normal lens cell differentiation and after exposure to Xrays or protons. We now report quantitative gene expression of p21 and p57 during normal lens cell differentiation using our in vitro model system and in a time course after radiation exposure. To validate our model system, we examined p21 protein expression in normal human adult lens tissue sections. Methods:Low passage number human lens epithelial (HLE) cells were grown on extracellular matrix (Blakely et al., IOVS 41:3898, 2000). Multiple cultures of HLE cells were prepared to provide different stages of lens cell differentiation. Total RNA from triplicate experiments was isolated in a time course before and after exposure to graded doses of 150 kVp Xrays or 55 MeV protons. Quantitative RT/PCR was completed for the expression of the CDKIs. p21 immunofluorescence was performed in the anterior and bow sections of normal human lens. Results:Our evidence indicates 4-fold changes in p21 gene expression when lens epithelial cells begin to differentiate into fiber cells in vitro. In lens tissues, we noted dramatic punctate p21 positive signals, specifically localized at the bow region of the lens. These signals were absent in the anterior and antibody control sections. We observed dose-dependent statistically significant radiation-induced changes in the expression of p21 in HLE cells in vitro. Xrays induce a 16-fold increase in p21 expression in epithelial cells 3 hrs. after 4 Gy. Equidoses of proton radiation induced a 4-fold increase in p21 at the same time point. Parallel studies using the same doses of radiation with differentiating fiber cells show that Xrays induce a 9-fold increase in p21 expression at 4 hrs., whereas a 5.5-fold increase was observed after protons. Protons induce a nearly 5-fold change in p57 in epithelial cells within an hr. after radiation. Conclusions:Our results suggest that CDKI expression in our in vitro differentiating lens model system parallels the pattern of differentiation seen in normal adult lens tissues. Radiation triggers a premature upregulation of CDKIs that signals a defective lens cell differentiation.

Keywords: gene/expression • radiation damage: light/UV • proliferation