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Journal of Neuroscience, Vol 13, 3669-3680, Copyright © 1993 by Society for Neuroscience
A system for characterizing cellular and molecular events in programmed neuronal cell death
RN Pittman, S Wang, AJ DiBenedetto and JC Mills
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
A model system has been established in which PC12 cells are converted to
neuronal-like cells that undergo transcription-dependent cell death
following removal of NGF. Nineteen sublines of PC12 cells were tested to
establish parameters for making cells dependent on NGF for survival. In
most sublines, a relatively small percentage of cells become dependent on
NGF for survival, and following removal of NGF, most of the cells begin
proliferating in serum-containing medium. In several sublines, however, a
significant percentage of cells die following removal of NGF. One of these
sublines, PC6-3, can be grown under conditions in which 90% of the cells
undergo transcription-dependent cell death following removal of NGF in
either serum-free or serum- containing medium. Fourteen hours after
removing NGF, 50% of the cells are committed to die, while initial
morphological signs of cell death as determined by time-lapse
videomicroscopy occur 2-6 hr later and include loss of neurites followed by
a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death
can be blocked by the RNA synthesis inhibitor actinomycin D, the protein
synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or
dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the
endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF
activates an endonuclease that causes nucleosomal laddering of the DNA;
however, endonuclease activity does not appear to be required for cell
death. In agreement with previous studies (Batistatou and Greene, 1991;
Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo
transcription-independent cell death when shifted into serum-free medium in
the absence of growth factors, all cell lines tested except for one die
when cultured in RPMI medium lacking growth factors. DNA fragmentation is a
prominent feature of transcription-independent cell death, and death can be
blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl.
The PC12 model system described here should be useful for identifying cell
death genes and for characterizing cellular and molecular events in
programmed neuronal cell death.
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