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Q Chen, D Liang, L Fromm, PA Overbeek; A mutant SV40 Large T Antigen (E107K) that Does Not Bind Rb Dramatically Inhibits Lens Fiber Cell Differentiation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2331.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Full-length SV40 large T antigen binds to the retinoblastoma protein and causes cellular transformation when expressed in lens fiber cells. In order to determine whether transformation involves inhibition of other factors in addition to Rb, we expressed a mutant version of SV40 large T antigen (E107K) that does not bind Rb in lenses of transgenic mice. Methods: E107K was linked to the αA-crystallin promoter in the CPV2 vector. Transgenic mice were characterized by histology, immunohistochemistry, in situ hybridization and western blotting assays. A cell culture system was used for crystallin reporter assays as well as immunoprecipitations to assay for interactions among E107K, full-length T antigen, CBP/p300 and c-maf. Results: At embryonic day 15.5 (E15.5), the E107K transgene was expressed in lens fiber cells and also, unexpectedly, in lens epithelial cells. Lens histology revealed small hollow lenses in the transgenic eyes due to the inhibition of fiber cell elongation. The fiber cells did not incorporate BrdU, supporting the prediction that E107K does not bind pRb. TUNEL assays showed no evidence for apoptosis in the fiber cells. In situ hybridization and immunohistochemistry assays showed that the expression of fiber cell differentiation markers (ß-, γ-crystallin, CP49 and MIP26) was significantly reduced. Western blotting showed that there were almost no ß- and γ-crystallin proteins in E107K transgenic lenses, while c-maf and CBP/p300 proteins were still synthesized. Cell culture studies revealed that full-length T antigen modestly inhibits c-maf plus CBP/p300 coactivation, while E107K almost completely blocks coactivation of the γF crystallin promoter. E107K does not block CBP/p300 nuclear localization. In addition, immunoprecipitations indicated that E107K binds to CBP/p300 much like full-length T antigen. However, E107K can completely block the binding of c-maf to CBP/p300. Conclusion: A point mutant of SV40 large T antigen that no longer binds to pRb has lost its transforming ability, but is nonetheless a potent repressor of fiber cell differentiation. E107K interferes with CBP/p300 coactivation, crystallins and other differentiation markers. Our data further demonstrate that CBP/p300 coactivation is critical during lens development.
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