Effect of overexpressing the transcription factor E2F2 on cell cycle progression in rabbit corneal endothelial cells.
OBJECTIVE
To test the hypothesis that overexpression of the transcription factor E2F2 promotes cell cycle progression in nonproliferating corneal endothelial cells.


METHODS
Ex vivo rabbit corneas were transfected with a lipid transfection reagent and either a control plasmid containing full-length cDNA for enhanced green fluorescent protein (pIRES2-EGFP) or a plasmid containing full-length cDNA for both E2F2 and EGFP (pIRES2-E2F2/EGFP). Transfection control experiments consisted of corneas incubated in buffer without transfection reagent or plasmid or incubated in reagent without plasmid. After transfection, corneas were incubated for various periods in 0.1% FBS (a concentration that maintains cell health, but does not promote proliferation). Immunocytochemical (ICC) localization tested for overexpression of E2F2 in transfected corneal endothelial cells and permitted calculation of transfection efficiency. Endothelial cell viability was tested in transfected ex vivo corneas and confluent cultures by using a cell-viability assay. Apoptosis was detected in confluent cultures by TUNEL assay. RT-PCR tested for mRNA expression of Ki67 (a marker of actively cycling cells) and cyclin B1 (a marker for the G2-phase of the cell cycle). Semiquantitative densitometric analysis compared the relative amounts of PCR reaction products.


RESULTS
ICC demonstrated the colocalization of E2F2 and EGFP in corneal endothelium with a transfection efficiency of 10% to 12%, using the pIRES2-based plasmid and transfection reagent. The cell-viability assay revealed very few dead cells in ex vivo corneal endothelium that overexpressed E2F2. Cell viability and TUNEL assays of confluent cultures revealed that approximately 27% of cells died in all cultures incubated with transfection reagent, but death appeared not to be due to apoptosis. No additional cell death was noted by either assay in cells that overexpressed E2F2. RT-PCR of endothelial samples obtained 48 hours after transfection showed the presence of higher levels of reaction product for Ki67 (a 5.1-fold increase) and cyclin B1 (a 2.3-fold increase) in cells that overexpressed E2F2 than in control samples.


CONCLUSIONS
Overexpression of the transcription factor E2F2 in nonproliferating rabbit corneal endothelial cells induces cell cycle progression without inducing significant apoptosis.
Joyce
Nancy C
NC
Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA. njoyce@vision.eri.harvard.edu
Harris
Deshea L
DL
Mc Alister
James C
JC
Ali
Robin R
RR
Larkin
Daniel F P
DF
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002470:Cell Survival; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D050703:E2F2 Transcription Factor; D004728:Endothelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000502:physiology*; D049452:Green Fluorescent Proteins; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D019394:Ki-67 Antigen / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D050703:E2F2 Transcription Factor; D019394:Ki-67 Antigen; D008164:Luminescent Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins