Cataracts arise in uveal melanoma patients as a complication following their successful treatment with helium-ion radiotherapy. The objective of the proposed research is to determine the helium-ion-induced alterations in chromosomes and in protein expression that are important to cataractogenesis, and to develop strategies to diminish the incidence or severity of these changes. The current paradigm for cataractogenesis is focussed on genomic damage of lens epithelial cells leading to altered crystallin proteins. This proposal will examine two alternative mechanisms of cataractogenesis involving radiation-induction of basic Fibroblast Growth Factor (bFGF) in human lens epithelial (HLE) cells functioning either to alter the normal program of crystallin expression and thereby disrupting normal fiber formation, or the radiation-induced bFGF acting to hinder cell loss processes which leads to the formation of aberrant lens fiber formation. To this end experiments have been designed with three specific aims that will: Firstly, characterize acute and residual responses to helium-ion irradiation of cultured HLE cells grown on extracellular matrix. Quantitative dose-response measurements between helium-ion induced survival, repair capacity and yields of micronuclei, apoptotic nuclei, and persistent chromosomal rearrangements will be determined. Secondly, since radiation is known to induce bFGF in endothelial cells leading to inhibition of apoptosis, experiments are proposed that will determine whether helium-ion radiation changes levels of bFGF mRNA or protein in HLE cells, and whether there is a correlation with apoptotic events. Finally, an investigation of the possible modification of intracellular lens proteins by helium-ion radiation is proposed. The program will elucidate relationships between helium-ion- induced damage to the chromatin of HLE cells in vitro, and biological consequences to the cells surviving the resulting damage. In the revised application significant changes include: 1) an increased source of HLE cells, 2) new preliminary data from mRNA and protein analyses (SDS-PAGE) of exponential and confluent cell cultures, 3) new preliminary data with immunological evidence confirming feasibility to characterize lens cell differentiation, 4) some revised experimental protocols, and 5) and an updated review of literature published in this rapidly increasing area. This knowledge will allow correlative comparisons with available experimental work in vivo, and may provide a basis for potential changes to the treatment protocol that could reduce the risk of cataract. The biological mechanisms contributing to cataractogenesis may also be elucidated.
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