LA Greene

A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.

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 neuritis. It is shown here: (a) that PC12 cells are not viable in serum-free medium. When serum is withdrawn, 90 percent of the cells die within 4-6 days and 99 percent by 2-3 wk. (b) If NGF is added at the time of serum withdrawal, the cells undergo one doubling and remain viable for at least 1 mo. (c) Addition of NGF to cultures after more than 2 days in serum-free conditions results in maintenance of surviving cells, but not in an increase in cell number. (d) NGD also induces neurite outgrowth from PC12 cells in serum-free medium. (e) NGF-treated cells exhibit much less cell-cell and neurite-neurite aggregation in the absence than in the presence of serum. (f) The apparent minimum level of 2.5S NGF required for PC12 survival and morphological differentiation in serum-free medium is about 10 ng/ml (approximately 0.4 nM). (g) Withdrawal of NGF in serum-free conditions results in degeneration of neurites and loss of cell viability. (h) Experiments with campotothecin 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.

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.

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.

Nerve growth factor (NGF) is a neurotrophic agent for sympathetic and embryonic sensory neurons both in vivo and in vitro. We report here that the membrane-permeant cAMP analogs, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, can replace NGF in promoting long-term survival and neurite outgrowth in cultures of rat neonatal sympathetic and embryonic sensory neurons. N6-substituted analogs, including the more commonly used N6,O2'-dibutyryl-cAMP, are less efficacious. Additivity and switching experiments indicate that the cAMP analogs affect the same neuronal population as that maintained by NGF. However, unlike NGF, the cAMP analogs do not evoke somatic hypertrophy. Moreover, studies with sympathetic neurons reveal that the neurotrophic actions of the cAMP analogs, but not of NGF, are blocked by the axial diastereoisomer of adenosine 3',5'-phosphorothioate, a competitive cAMP antagonist. Thus, the mechanism by which cAMP analogs promote neuronal survival and differentiation appears to involve activation of cAMP-dependent protein kinases, whereas, in contrast, the same effects of NGF neither require nor are mediated by such a pathway. Furthermore, the different efficacies observed with N6- and C8-substituted cAMP analogs suggest that this neurotrophic pathway may involve differential activation of the regulatory subunits of cAMP-dependent protein kinases. The presence of this parallel, cAMP-responsive, neurotrophic pathway in at least two types of NGF-responsive neurons may be developmentally important and has the potential to be exploited for the treatment of injuries or diseases affecting these and possibly other nerve cells.