The Cytoskelefon of the Cynomolgus Monkey Trobeculor Cell //. Influence of Cytoskeleron-Acrive Drugs

The effects of cytochalasin B (10~6 M and 1 0 s M), taxol (10" 5 M), nocodozole (10~5 M), and colchicine (10~5 M) on the cytoskeleton of cynomolgus monkey trabecular cells were examined with Nomarski observations, fluorescent labeling as well as extraction, S-l labeling, and critical-point drying. Changes in actin, microtubules, and intermediate filaments, the three major cytoskeletal systems, were correlated with changes in the overall shape and organization of the monkey trabecular cell. Incubation with cytochalasin B caused a marked alteration on actin filament structure, as well as cell shape and cytoskeletal organization. Effects on microtubule structure were noted with taxol, nocodozole, or colchicine; however, no marked changes in overall cell shape or other cytoskeletal structures were observed. These studies demonstrate the importance of actin filaments in regulating the shape and cytoskeletal organization of cynomolgus monkey trabecular cells. Invest Ophthalmol Vis Sci 27: [1312][1313][1314][1315][1316][1317] 1986 Substantial evidence has accumulated during the past several years that the cytoskeleton is an essential component of many cell processes. 1 " 3 In a prior study, 4 transmission electron microscopy was employed to obtain stereo pairs of whole cynomolgus monkey trabecular cells that had been extracted, S-1 labeled, and critical-point dried in order to simultaneously identify actin, microtubules, and intermediate filaments, the three major cytoskeletal systems, and visualize their three-dimensional nature. A double fluorescence technique for actin and microtubules was also used to provide a broad view of cytoskeletal relationships within the cell. In the current investigation, we evaluated the effects of cytoskeleton-active drugs on these systems. Furthermore, we correlated changes in these cytoskeletal systems with changes in the overall shape and organization of the monkey trabecular cell. Materials and Methods Monkey trabecular cells were grown to confluence (7-10 days of culture) on 1 cm 2 glass coverslips (with or without attached grids), using previously described methods. 5 The cells were then incubated for 1 hr at 37°C with either: Results No difference in cell shape Light Microscopic Observations Incubation with the antiactin drug, cytochalasin B, caused marked alterations in cell shape and cytoskeletal organization. At 10~5 M cytochalasin B the cell body rounded u

Growth and differentiation of human lens epithelial cells in vitro on matrix,

PURPOSE. To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS. HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS. HLE cells had a male, human diploid (2N ϭ 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of ␣A-and ␤B2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (␣A-, ␣B-, and ␤B2-crystallin) with time in culture. By Western blot analysis, expression of p57 KIP2 , a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and ␥Ϫcrystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS. HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity. (Invest Ophthalmol Vis Sci. 2000;41:3898 -3907