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M. Kikuchi, D.L. Harris, N.C. Joyce; P27kip1 Antisense-Induced Proliferative Activity of Rat Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4723.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether antisense down-regulation of p27kip1 will overcome G1-phase arrest and promote cell cycle progression in rat corneal endothelial cells (CEC). Method: Confluent cultures of rat CEC were incubated for 24 or 48 hrs in the presence of p27kip1 antisense oligonucleotides using the technique of nonliposomal lipid transfection. Control cultures were incubated under one of the following conditions: 1) no oligonucleotides or lipid-containing buffer, 2) lipid-containing buffer alone, or 3) lipid-containing buffer plus mismatch p27kip1 oligonucleotides. Following incubation, cultures were fixed and immunostained for p27kip1 to test for down-regulation or Ki67 to detect actively cycling cells. Viability was tested by a Live/Dead assay after 0, 24, 48, and 72 hrs. Staining was visualized using by fluorescence microscopy. Results: Viability was not significantly affected by lipid-based oligonucleotide transfection for up to 48 hrs, after which a decline was noted. Nuclear staining for p27kip1 was greatly reduced in CEC incubated with antisense oligonucleotide by 48 hrs. No change in p27kip1 levels was observed in controls at any time-point tested. A time-dependent increase in the relative number of Ki67-positive cells was noted in CEC incubated with antisense oligonucleotide. In contrast, no-to-few Ki67-positive cells were observed in CEC incubated with mismatch oligonucleotide or buffer controls. Conclusion: The observation of Ki67-positive cells in antisense treated endothelium provides evidence that lowering of p27kip1 protein levels in confluent cells permits induction of proliferation.
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