Cataract is the world's leading cause of blindness, the most frequently performed surgery on the elderly in the U.S., and is the largest item in Medicare expenses. Our long-term goal is to identify changes in the human lens epithelium that precede cataract formation and to identify therapeutic agents that prevent cataract formation. The epithelium is responsible for the formation of the lens, has a major transport function, provides cells that produce lens fibers and is the primary locus of enzymes and other compounds that protect the lens from oxidative insult. The major factor limiting experimentation on human lens epithelial cells (LECs) is that there are insufficient numbers of cells to initiate detailed biochemical and physiological experiments. Well characterized human LEC lines would be particularly useful for studying the control of crystallin gene expression. We will determine the conditions required to generate cell lines from normal and cataractous human lenses and will obtain detailed baseline information on growth and biochemical properties of human LECs. We will compare the biochemical and metabolic properties of lines established from normal and cataractous lenses with these parameters in immortalized human LECs transfected with AD12-SV40. Metabolic and transport properties in cultured human lECs will be compared with those in intact human lenses. We will determine the specific growth factor(s) that trigger mitosis in the epithelium of human lenses in organ culture thus permitting comparison with tissue culture studies.
Aims are to 1) Determine the culture conditions required for the long-term growth of human LECs from normal lenses and frog tags obtained from human cataractous lenses. 2) Characterize the differentiated state of primary and long-term cultures of human LECs using biochemical markers (such as alphaA-crystallin, alphaB-crystallin, beta crystallin and gamma-crystallin) and ultrastructural markers (such as nuclear dissolution, loss of cytoplasmic organelles, and formation of gap junctions). 3) Determine the levels of selected biochemical parameters in newly established lens epithelial liens and in transfected human LECs. 4) Determine the conditions required to initiate DNA synthesis and mitosis in human lenses in organ culture.
