APOPTOSIS--LENS DIFFERENTIATION AND CATARACT DEVELOPMENT

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It has been well documented that cataractogenesis can be induced by a large number of agents. However, the common cellular mechanism by which these different agents cause cataract has remained unknown. Our recent studies of the lens epithelial cell viability in human normal and cataractous lenses and in rat lenses with or without insult by oxidative stress, calcimycin and UVB consistently suggest that induced lens epithelial cell apoptosis is such a common cellular mechanism. Therefore, whether inhibition of the stress-induced lens epithelial apoptosis in the mature lens can retard or prevent cataractogenesis under various stress environments becomes an important question related to human health and obtaining the answer to this question will certainly shed some light to the potential gene therapy for human cataract. Since apoptosis has been reported to occur in normal development of the rat lens and moreover, the differentiation process of lens fiber cells (both primary and secondary fibers) in the developing and mature lenses share certain common pathways with the apoptotic process, any attempt to delay or prevent cataractogenesis by simply inhibiting apoptosis in the developing lens may interfere with normal development of the lens. Therefore it is proposed here to conduct comparative studies of normal physiological apoptosis in the developing lens, pycnosis during both primary and secondary fiber cell differentiation in the developing and mature lenses and stress-induced pathological apoptosis in the mature lenses of rat, mouse and chicken with respect to two aspects: gene expression and signal transduction. To analyze gene expression, reverse-transcription-linked polymerase chain reaction and in situ hybridization will be used to determine the presence or absence of the mRNAs for the selected apoptosis-regulatory genes (Rb, p53, c-myc, c-jun, c-fos, p34cdc2, bcl-2, bcl-x, bax, bad, bag-1, mch-1, ice and ich-1). The presence of the mRNA expression for a particular gene will be further confirmed by immunocytochemistry analysis which detects the expression of the protein. To conduct signal transduction investigations, the effects of both RNA and protein synthesis inhibitors, various kinase and phosphatase inhibitors, and a kinase activator on the above mentioned three processes will be examined by in vitro culture followed by studies of their responses. These studies will elucidate the similarities and differences of the three processes and help to determine whether there is a specific experimental strategy to inhibit only stress- induced epithelial cell apoptosis in the mature lens while not interfering with normal lens growth and differentiation. Then, studies to determine whether inhibition of the stress-induced apoptosis could delay or prevent cataractogenesis in the mature lenses will be conducted with the in vitro lens organ culture systems. At the same time, the antiapoptotic abilities of a group of 6 genes: p35, crmA, bcl-xL, bag-1, mcl-1 and ich-IS known to inhibit apoptosis in non-lens systems will be tested in the in vitro cell lines, N/N1003A and alphaTN4-1. Following the confirmation of their ability to suppress stress-induced apoptosis, the positive genes will be used to develop over-expression transgenic models. The phenotype (e.g., lens transparency and antiapoptotic ability) of these transgenic mice will be examined with comparison to the normal mice. The results from these studies will not only provide valuable information for future design of gene therapy for human cataract prevention but also increase our understanding of the developmental biology of the normal lens.