Induction of replication in human corneal endothelial cells by E2F2 transcription factor cDNA transfer.
OBJECTIVE
Corneal endothelial cells in humans do not replicate to any meaningful extent. Diminishing density of the cell monolayer with age and in the disease states is a major cause of loss of corneal transparency. This study was conducted to test the hypothesis that overexpression of the transcription factor E2F2 results in replication in nonproliferating human corneal endothelial cells.


METHODS
Whole human corneas were incubated for 2 hours in a solution of recombinant E1(-)/E3(-) adenovirus incorporating cDNA encoding E2F2 and green fluorescent protein (GFP) under control of a bidirectional promoter and subsequently maintained in ex vivo culture. Control specimens were incubated with an identical virus bearing the GFP sequence only, or virus-free medium. Efficiency of gene transfer and localization was examined by fluorescence microscopy. En face confocal microscopy of the corneal endothelial surface was used to image recombinant E2F2 expression. 5-bromodeoxyuridine (BrdU) incorporation was used to examine progression to the S phase. Changes in density of the corneal endothelium were quantified by specular microscopy and counting of trypan-blue-stained cells. Apoptosis was tested with a TUNEL assay.


RESULTS
Recombinant proteins were expressed predominantly in the endothelium and in a high proportion of endothelial cells in the first week after exposure to virus, diminishing thereafter. Compared with the control, transduction with E2F2 resulted in progression from the G(1) to the S phase in a significant number of cells and in increased cell density. Apoptosis was not found to any significant extent.


CONCLUSIONS
Overexpression of the transcription factor E2F2 in nonmitotic human corneal endothelial cells results in short-term expression, cell-cycle progression, and increased monolayer cell density.
McAlister
James C
JC
Department of Molecular Genetics, Institute of Ophthalmology, University College London, UK.
Joyce
Nancy C
NC
Harris
Deshea L
DL
Ali
Robin R
RR
Larkin
Daniel F P
DF
D000260:Adenoviruses, Human / Q000235:genetics; D017209:Apoptosis; D002452:Cell Count; D002455:Cell Division / Q000502:physiology*; D002478:Cells, Cultured; D004261:DNA Replication / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics*; D050703:E2F2 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D018613:Microscopy, Confocal; D014162:Transfection
D018076:DNA, Complementary; D050703:E2F2 Transcription Factor; C493761:E2F2 protein, human; D049452:Green Fluorescent Proteins