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J.R. Reddan, A.L. Hitt, G. Duncan, L.W. Schovan, D.C. Dziedzic; Telomerase Overcomes Senescence in Cultured Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2626.
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Purpose: The establishment of continuous cultures of HLECs would facilitate the study of the lens epithelium and associated diseases of the lens. Cultured human lens epithelial cells (FHL124) complete 56 population doublings (pd) before senescence. The cells express Pax6, FoxE3, αA and αB crystallins, and are highly sensitive to TGFß2, one of the major driving forces in PCO. Microarray analysis shows 99.4% homology with native human epithelium (Wormstone, Tamiya, Eldred, Lazaridis, Chantry, Reddan, Anderson and Duncan, in press, EER). Here we determine if the catalytic unit of telomerase (hTERT) permits HLECs to overcome in vitro senescence. In addition, we determined the effect of a telomerase inhibitor, BIBR1632SE (Boehringer Ingelheim Pharma KG), on the growth of cells expressing the telomerase gene. Methods:A DNA expression construct encoding hTERT driven by the MPSV LTR promoter was introduced into FHL124 cells resulting in a line designated hTRT124. hTERT was also introduced into FHL124 cells using the tripartite FELIX vector system (Nolan and Curran, Humana Press, 2001). We modified the transfer vector, pPanther, to carry a bicistronic cassette of hTERT and EGFP driven by a CMV promoter. Three plasmids were cotransfected into 293T producer cells to assemble a replication deficient virus used to carry hTERT into FHL124 cells resulting in a line called 124VT. Results:The hTRT124 and 124VT cells are at 170 pd and continue to grow. 124VT cells showed EGFP, indicating expression of hTERT. Telomerase activity (TRAP assay) was low in FHL124 cells but elevated in hTRT124 and 124VT cells. When hTRT124 cells at pd 148 were grown in 10 µM BIBR1632SE, a marked inhibition of growth was noted. Discussion:The results show that telomerase can extend the lifespan of cultured human lens epithelial cells. This approach may permit routine establishment of HLEC lines from young, old and cataractous lenses. These cells should be useful to study the effects of cataractogenic agents and aging, as well as to investigate therapeutic interventions that might be useful in preventing secondary cataracts. Supported by NEI grant EY13123; The Humane Research Trust, Bramhall, UK.
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