The PC12 clone is a noradrenergic cell line derived from a rat pheochromocytoma. In culture medium containing horse serum, PC12 cells undergo mitosis; when nerve growth factor (NGF) is included in the medium, the cells cease multiplication and extend neurites. It is shown here: (a) that PC12 cells are not viable in serum-free medium. When serum is withdrawn, 90% of the cells die within 4-6 days and 99% by 2-3 wk. Experiments with camptothecin demonstrate that the effects of NGF on survival and neurite outgrowth may be uncoupled and suggest that the survival effects are transcriptionally independent. The present results also suggest that PC12 cells have a requirement for NGF (similar to that of normal sympathetic neurons) and that serum may substitute for this requirement. In addition, the present system of maintaining a highly differentiated cell line in a chemically defined medium suggests certain experimental opportunities.KEY WORDS nerve growth factor differentiation 9 pheochromocytoma 9 PC12 cell line 9 serum-free medium neuritesNerve growth factor protein (NGF) is known to have a number of effects on responsive sympathetic and sensory ganglionic neurons (for reviews, see references 18, 30, and 34). These effects include stimulation of neurite outgrowth (18), increase in cell size (18) and induction of certain enzymes involved in neurotransmitter synthesis (26). In addition, NGF also appears to play a role J. CELL BIOLOGY 9 The Rockefeller University Press 9

Past studies revealed that NGF and fibroblast growth factor (FGF) prevent the death of PC 12 pheochromocytoma cells that otherwise occurs in serum-free medium. Additional agents were tested here for their abilities to promote long-term survival of naive and NGF-pretreated (primed) PC 12 cells in serum-free conditions. Forskolin and permeant cAMP analogs effectively prevented serum-free cell death, as did micromolar levels of insulin and 10–100-nM levels of insulin-like growth factors I and II. In contrast to NGF and FGF, none of these agents caused neuronal differentiation of naive cells or neurite regeneration by primed cells. Each of the agents also prevented rapid cell death in a balanced salt solution, thus apparently ruling out a mechanism dependent on regulation of nutrient uptake. Epidermal growth factor and elevated K+ appeared to slow the rate of cell death, but did not promote long-term survival; phorbol ester, dexamethasone, or vanadate did not prevent cell death. Each of the survival-promoting agents was effective even when macromolecular synthesis was blocked. Because the synthesis inhibitors themselves did not significantly prevent cell death, such findings indicate that survival was promoted by mechanisms that do not require synthesis of RNA or protein. In addition, various lines of experimental evidence (using the kinase inhibitor K-252a or PC 12 cell variants deficient either in protein kinase A activity or in responsiveness to NGF) further suggested that the effective agents maintain survival by independent initial pathways. Regulation of protein kinase activity appears to be a common feature of each pathway and may therefore play a key convergent role in mediating prevention of cell death.

Acidic (aFGF) and basic (bFGF) fibroblast growth factors are well-characterized peptide hormones that have potent angiogenic activity and that are mitogenic for a variety of cell types. The present findings demonstrate that FGFs can reproduce the entire spectrum of rat pheochromocytoma PC12 cell responses previously shown to be elicited by NGF. These include responses that are rapid (cell flattening, enhanced phosphorylation of tyrosine hydroxylase) or delayed (neurite outgrowth, induction of phosphorylated MAP 1.2, regulation of NILE and Thy-1 glycoproteins, cessation of mitosis, elevation of AChE activity), as well as responses that have been shown to be either transcription-independent (neurite regeneration, promotion of survival) or transcription-dependent (priming, regulation of NILE and Thy-1 glycoproteins, elevation of AChE activity). The only responses for which the FGFs and NGF consistently showed quantitative differences were in the rates for neurite initiation and elongation in serum-containing medium. Thus, while all 3 factors promoted the formation of stable neurites, the network of outgrowth elicited by NGF at any given time of treatment was always of greater density. Togari et al. (1985) have previously reported that bFGF can initiate transient neurite formation in PC12 cell cultures. The present observations describe a variety of additional actions of bFGF on a neuronal cell line, and demonstrate that aFGF is capable of mimicking many, if not all, of these actions. These observations thus extend the range of actions that aFGF and bFGF may potentially exert on nerve cells, either during their development, repair, or maintenance. In addition, this work suggests that the PC12 cell line may serve as a useful model system with which to study the mechanism of action of FGFs on neurons. Since all 3 factors appear capable of eliciting the same wide spectrum of responses, molecular events specifically associated with FGFs and NGF in PC12 cells may prove illuminating of the causal steps involved in neuronal differentiation.

We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.

Abstract. Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b)Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event.(D Extracellular Ca 2÷ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.