Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells.
Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)-based differential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3'-end of the coding sequence of the human RAS-GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA-PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation- and proliferation-dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy.
Department of Vitreoretinal Surgery, University Eye Hospital Cologne, Germany. norbert.kociok@uni-koeln.de
N
N
Kociok
P
P
Esser
K
K
Unfried
F
F
Parker
U
U
Schraermeyer
S
S
Grisanti
B
B
Toqué
K
K
Heimann
D000368:Aged; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D020690:GTPase-Activating Proteins; D011506:Proteins; D012333:RNA, Messenger; D020703:ras GTPase-Activating Proteins