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NC Joyce, DL Harris, J McAlister, RR Ali, DF P Larkin; E2F2 Overexpression Induces Cell Cycle Progression in Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):853.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Corneal endothelial cells in vivo do not normally proliferate although they retain proliferative capacity. Excessive loss of endothelial cells can result in loss of visual acuity. These studies test whether overexpression of the transcription factor, E2F2, can induce cell cycle progression in non-proliferating rabbit corneal endothelium using an ex vivo model. Methods: Rabbit corneas were incubated in 10% FBS to stabilize endothelial cells and then transfected for 3 hrs with a pIRES2-E2F2-EGFP or pIRES2-EGFP control plasmid using LipofectAmine-PLUS as transfection reagent. Corneas were washed and incubated for 24 or 48 hrs in medium containing 0.1% FBS (which does not promote proliferation). Viability was tested using a Live/Dead assay. E2F2 overexpression was visualized by confocal microscopy. RT-PCR detected expression of E2F2, Ki67 (a proliferation marker), and cyclin B1 (whose synthesis increases in G2-phase of the cell cycle). Results: E2F2 was co-expressed with EGFP with a transfection efficiency of 10-12%. No significant loss of viability was noted. RT-PCR revealed overexpression of E2F2, Ki67, and cyclin B1 mRNA in corneas transfected with full-length E2F2 cDNA, but not in those transfected with the control plasmid. Conclusion: Non-proliferating rabbit corneal endothelial cells can be induced to overexpress E2F2 using lipid-based transfection. High expression levels of Ki67 and cyclin B1 in E2F2-transfected endothelium indicate that E2F2 overexpression can promote cell cycle progression in non-proliferating cells.
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