Apoptosis after butyrate-induced differentiation in retinoblastoma cell line Y-79

Purpose: To study the fate of Y-79 human retinoblastoma cells after inducti on of differentiation. Methods: Y-79 cells were cultured in a synthetic medium and were induced to neuronal differentiation by butyrate treatment. Neurofilaments, p53, and D NA-synthesizing nuclei labeled with 5-bromodeoxyuridine were immunostained, and apoptotic cells were labeled by in situ DNA nick end labeling (TUNEL). We combined these immunostaining and labeling methods to determine whether the cells expressed these markers at the same time. DNA fragmentation and p53 levels were also determined by electrophoresis. Results: Y-79 cells proliferated in the synthetic medium. After butyrate tr eatment, they extended protrusions and increased neurofilament immunoreacti vity. The differentiated features were striking on day 7. Thereafter, diffe rentiated cells decreased and apoptotic cells increased. DNA synthesis was detected in the cells expressing immunoreactivity for neurofilaments or p53 . At day 7, most of the cells with p53-positive nuclei were alive and neuro filament-positive. However at day 20, the p53-positive cells were apoptotic and neu rofilament-positive apoptotic cells accumulated. Conclusious: We conclude that the Y-79 cells express p53 and undergo apopto sis after neuronal differentiation. There could be a p53-dependent apoptoti c pathway in butyrate-induced differentiated Y-79 cells due to the inabilit y to regulate cell cycling. (C) 2000 Japanese Ophthalmological Society.