Apoptosis after butyrate-induced differentiation in retinoblastoma cell line Y-79.
OBJECTIVE
To study the fate of Y-79 human retinoblastoma cells after induction of differentiation.


METHODS
Y-79 cells were cultured in a synthetic medium and were induced to neuronal differentiation by butyrate treatment. Neurofilaments, p53, and DNA-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 treatment, they extended protrusions and increased neurofilament immunoreactivity. The differentiated features were striking on day 7. Thereafter, differentiated 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 neurofilament-positive. However at day 10, the p53-positive cells were apoptotic and neurofilament-positive apoptotic cells accumulated.


CONCLUSIONS
We conclude that the Y-79 cells express p53 and undergo apoptosis after neuronal differentiation. There could be a p53-dependent apoptotic pathway in butyrate-induced differentiated Y-79 cells due to the inability to regulate cell cycling.
Karasawa
Y
Y
Department of Ophthalmology, National Defense Medical College, Tokorozawa, Saitama, Japan.
Murakami
A
A
Okisaka
S
S
D017209:Apoptosis; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D016900:Neurofilament Proteins / Q000378:metabolism; D019572:Retinal Neoplasms / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000378:metabolism / Q000473:pathology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D002087:Butyrates; D004273:DNA, Neoplasm; D016900:Neurofilament Proteins; D016159:Tumor Suppressor Protein p53