2127 FAS/APO-1 mediated apoptosis in retinal pigment epithelial cells, modulation by cytokines

Pm-mme. The function of RPE is well known in PVR. Pharmacologic agents have been extensively shldied both experimentally and clinically. Few reports have derailled the interactions of a”timitctic drugs on the microtubule network. The aim of this study is to visualize by indirect immunofluwescence the modification of the microtubule network of cultured pig RPE cells by colchicina and taxol. Yetbuds. Pigs were killed at the slaughl;m-house&b&r eyes were enucleated. RPE cells were isolated and cultured as discribed by K. Gabrielian (cuwsnt eye research 1992,11,6,481487). RPE cells were plated onto glass coverslips at a density of 2000000 cells/ml.cuItured 1 day and treated with the drugs during 4 hours at 37°C at different concentrations. Immunofluorescence reaction was‘ dewlopped as discribed byP.Garcia (Cancer chemotherapy and Pharmacology 1994,34,335-343) using antitubulin (Sigma,diluted 1:40) and fluoresceinated ant-mouse antibodies (Sigma,diluted l::!O). The cytoskelettons were visualized employing a Zeiss photomicroscope equipped with epiilumination,a 63 x lens and appropriate filters for fluoresceine. I:&. The cytoplasmic microtubules of RPE cells were disrupted in s concentration and time-dependant manner by colchicine. Between I id 100 nmolar several degrees of d~olymtization of the microtubule network were observed (maximal at 50 nmolar). Tax01 (l-100 “molar) was found to induce several de8.mes of microtubule “b;ndling” after 4 hours of incubation. Comcltiou. The results show that antimitotic drugs inhibit the micmtubule network formation by depolymerization (colcbicine) or stabilize it (Taxol) with no action on the Actin network. These actions inhibit celI division,wbich is one of the mechanism!; implicated in PVR.