EXPRESSION AND UP-REGULATION OF MICROTUBULE-ASSOCIATED PROTEIN 1B IN CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS

Purpose. During routine cell culture and under pathologic conditions, human retinal pigment epithelial (RPE) cells lose epithelial character istics and change their morphology. In this study, changes in gene exp ression in RPE cells of different generations were evaluated by polyme rase-chain-reaction-based differential display mRNA analysis (DD-RT-PC R). Methods. Total RNA was prepared from freshly isolated and cultured human RPE cells of passages P0 and P3 and was subjected to DD-RT-PCR. One band with enhanced expression was excised, reamplified, and parti ally sequenced, using a modified dideoxy chain termination approach. E xpression of the corresponding protein was ascertained by immunocytoch emical analysis. Results. Differential display RT-PCR showed enhanced expression of a specific RNA in P3 cells compared with that in PO cell s. Sequence alignment revealed 98% identity with the 3' end of the cod ing sequence of human microtubule-associated protein 1B (MAP1B). Confi rmation of induced expression of MAP1B mRNA was obtained by PCR with s pecific primers and by immunocytochemical analysis in cultured RPE cel ls and in surgically removed epiretinal membranes from patients with p roliferative vitreoretinopathy. No expression of MAP1B mRNA or protein was detected in freshly isolated RPE cells. Conclusions. Differential display RT-PCR in RPE cells with subsequent sequence analysis allows characterization of the maturation- and differentiation-dependent expr ession of previously undetected genes and gene products in cultured RP E cells.