97th DOG Annual Meeting 1999
DIFFERENTIAL EXPRESSION mRNA ANALYSIS: EXPRESSION OF THE SERIN/THREONINE PROTEIN KINASE RAF-1 IS UPREGULATED IN PROLIFERATING RPE CELLS
A. Hueber, N. Kociok, R. Krott, T. Luther, P. Esser
Purpose: Proliferating RPE cells are thought to contribute significantly to membrane formation in PVR. As an in-vitro model we used proliferating RPE cells when routinely passaged during cell culturing. To find new genes that change their expression in proliferating RPE cells we conducted a differential mRNA expression analysis (DEmRNA-PCR) of cultured human RPE cells of different passages.
Methods: Total RNA was prepared from cultured human RPE cells of passage 0 and 3 followed by DEmRNA-PCR. A band with enhanced expression in P3 was excised, reamplified and directly sequenced. Semiquantitative RT-PCR with gene specific primers was used to estimate the upregulation of the gene expression in RPE cells from P0 to P3.
Results: DEmRNA-PCR revealed an enhanced expression of a specific RNA from P0 to P3. A sequence alignment search showed its 99% identity with the 3'-end of the coding sequence of human RAF-1. Upregulation of RAF-1 was estimated by semiquantitative RT-PCR. The expression of RAF-1 was confirmed immunhistochemically on the protein level.
Conclusions: Differential mRNA expression analysis of cultured RPE cells is a useful tool for the identification of previously unknown proteins that may play an important role in diseases involving proliferating cells like PVR. Further research on the role of the serin/threonine protein kinase RAF-1 in PVR may lead to new intervention strategies of inhibition of cell proliferation.
Supported by DFG (Es 82/5-1, He 840/6-1), Köln Fortune and the Retinovit Foundation.
Department of Ophthalmology, University of Cologne,
50924 Köln, Germany