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 express ion in proliferating RPE cells by polymerase chain reaction (PCR)-based dif ferential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with PO. Sequence alignment dis played its identity with the 3'-end of the coding sequence of the human RAS -GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of th e induced expression of G3BP was performed by gene-specific reverse transcr iption-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cel ls and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cu ltivated retinal pigment epithelial cells in an artificial lesion assay. Th e expression of G3BP mRNA increased with the number of passages. G3BP prote in expression increased in cells repopulating the artificial lesion. DEmRNA -PCR in RPE cells with subsequent sequence analysis led to the characteriza tion of dedifferentiation- and proliferation-dependent expression of a prev iously undetected gene product in cultured RPE cells with a possible role i n modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy. J. Cell. Biochem. 74:194-201 , 1999. (C) 1999 Wiley-Liss, Inc.