RESUMEN
PURPOSE: Apoptosis, a type of programmed cell death, is observed in various types of cataract and in cultured lens epithelium subjected to oxidative damage. We have recently described oxidative DNA base damage in epithelium in age-related cataract and cultured cells, and we here aimed to examine such epithelium for markers for proliferation, initiation of apoptosis and morphological patterns of cell damage. METHODS: Samples (n = 75) were analysed by light microscopy/electron microscopy (LM/EM); immunohistochemistry (IHC) for PCNA and Ki67 (DNA synthesis/proliferation); TUNEL assay (DNA fragmentation/apoptosis); and protein/gene expression of Caspase-3 (apoptotic effector molecule) and BAX/Bcl2 (pro-/anti-apoptotic marker) in fresh/cultured epithelium by IHC and qRT-PCR. RESULTS: In fresh samples, the majority of cells were Ki67-/PCNA+. BAX/BCL-2-ratio was approximately 1, and Caspase-3 levels were low. TUNEL stained scattered nuclei/nuclear fragments (9/6302 cells). Main morphological signs of cell damage included rupture of cell membranes and hydration of cytoplasm and nuclei. Cultivation increased levels of BAX and Bcl2 by IHC and qRT-PCR (approximately 10-fold upregulation). Caspase-3 levels remained low by IHC with similar expression in fresh and cultured samples by qRT-PCR. CONCLUSION: Genomic stress and DNA repair may explain the contrasting expression of Ki67/PCNA in fresh epithelium. Despite low levels of Caspase-3 and similar expression of BAX/Bcl-2, a low incidence of apoptosis may be detected in epithelium in age-related corticonuclear cataract. Epithelium may be transferred to culture without an increase in expression of Caspase-3, one of the central mediators of apoptosis.
Asunto(s)
Apoptosis , Catarata/patología , Células Epiteliales/patología , Cápsula del Cristalino/patología , Biomarcadores/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Catarata/metabolismo , Proliferación Celular , Células Cultivadas , Fragmentación del ADN , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Antígeno Ki-67/metabolismo , Cápsula del Cristalino/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía de Contraste de Fase , Reacción en Cadena de la Polimerasa , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoAsunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 6/genética , Discapacidades del Desarrollo/genética , Translocación Genética , Adulto , Niño , Preescolar , Aberraciones Cromosómicas , Bandeo Cromosómico , Cromosomas Artificiales Bacterianos , Femenino , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Lactante , MasculinoRESUMEN
PURPOSE: DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. METHODS: Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. RESULTS: DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. CONCLUSION: The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.