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G. Chandrasekher, D. Sailaja; Influence of Akt overexpression and induction of apoptosis on lens epithelial cell differentiation. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1154.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Transformation of lens epithelial cells into fibers has been suggested to involve an apoptosis–like process that does not lead to cell death. Our previous studies showed that IGF–1–mediated activation of anti–apoptotic factor PI–3K/Akt decreases in differentiating lens epithelial cells, and inhibition of PI–3K/Akt signaling promotes differentiation (IOVS, 2003, 44, 4411). The purpose of this study was to investigate the connection between Akt signaling and apoptosis during epithelial cell differentiation. Methods: Lens epithelial cell primary cultures from 10–day’s embryonic chicken eggs were grown in DMEM/10% FCS. Transient transfection of epithelial cells with a eukaryotic plasmid containing Akt cDNA was performed using Fugene 6 reagent. Epithelial cells were treated with staurosporine (0–25 nM) for different times. Lentoid body development and growth were monitored in live cultures. Nuclear condensation in cells was assessed by Hoechst fluorescence staining. δ–crystallin synthesis was determined by metabolic labeling of cells with [35S]methionine. Immunoblotting was performed to identify the proteins. Results: Overexpression of constitutively active Akt (cAkt) produced significant changes in the normal events associated with differentiation of chicken lens epithelial cells in culture. Development of differentiating bodies, the lentoids, was decreased by more than 50% compared to untransfected cultures. The growth of the lentoids and synthesis of the differentiation marker protein, δ–crystallin were also inhibited in these cells. In contrast, 2–3 days of treatment of epithelial cultures with staurosporine resulted in dramatic changes in cell morphology: elongation of the cells and shrinkage of nuclei were prominent. Marked increase in the number of lentoid bodies and extensive spreading of these structures was observed thereafter. δ–crystallin synthesis was elevated by about 50%. A decrease in cyclin D expression and the degradation of apoptosis marker protein, poly(ADP–ribose) polymerase–1, due to caspase–3 activation accompanied staurosporine–induced differentiation. Overexpression of cAkt in lens epithelial cultures had no effect on the activation status of Erk–2 MAP kinase. Akt overexpression conferred protection in epithelial cells against apoptosis. Conclusions: Our studies demonstrate that down–regulation of PI–3K/Akt anti–apoptotic signaling and up–regulation of apoptosis–associated cellular events are prerequisites for lens epithelial cell differentiation.
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