A system for characterizing cellular and molecular events in programmed neuronal cell death

Abstract

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.

Persistent TrkA Activity Is Necessary to Maintain Transcription in Neuronally Differentiated PC12 Cells

“…Indeed, rapid regeneration of sheared neurites from differentiated PC12 cells is regulated by translation of short-lived mRNAs, indicating that continual transcription is needed to maintain this injury response (38). Differentiated PC12 cells also require continual exposure to NGF to maintain their survival (47). Despite the need for continual exposure to NGF, most kinetic analyses of NGF signal transduction have shown that activities of its TrkA receptor and downstream protein kinases rapidly peak and then fall to seemingly undetectable levels within hours after ligand stimulation (11,23,34,48).…”

Bcl-xS and Bax induce different apoptotic pathways in PC12 cells

“…In an attempt to gain a better understanding of the molecular mechanism underlying the proapoptotic action of Bcl-x S in cancer cells and to compare it to that of Bax, we examined the e ects of Bcl-x S and Bax in the rat pheochromocytoma line PC12, a well characterized cellular model system commonly used for the study of neuronal apoptosis (see, e.g., Haviv et al, 1998;Pittman et al, 1993;Stefanis et al, 1996). Our results suggest that Bcl-x S and Bax di er in their apoptotic e ects in PC12 cells.…”

p21WAF1 induces permanent growth arrest and enhances differentiation, but does not alter apoptosis in PC12 cells

“…In serum containing medium these cells proliferate; however, when treated with nerve growth factor (NGF), cells stop proliferating and di erentiate into neural cells similar to sympathetic neurons (Greene and Tischler, 1976). Additionally, there are numerous apoptotic paradigms that have been established for PC12 cells, including those involving removal of trophic support (Greene, 1978;Batistatou and Greene, 1991;Pittman et al, 1993). Given that some studies have implicated aberrant or inappropriate cell cycle in the death of trophic factor deprived PC12 cells and sympathetic neurons (Batistatao and Greene, 1993;Rubin et al, 1993;Farinelli and Greene, 1996;Park et al, 1996), this system provides a means for testing the hypothesis that p21 expression would block death resulting from trophic factor removal.…”