Nerve growth factor prevents the death and stimulates the neuronal differentiation of clonal PC12 pheochromocytoma cells in serum-free medium

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Abstract
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. 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 When serum is withdrawn, 90% of the cells die within 4-6 days and 99% by 2-3 wk. If NGF is added at the time of serum withdrawal, the cells undergo 1 doubling and remain viable for at least 1 mo. Addition of NGF to cultures after more than 2 days in serum-free conditions results in maintenance of surviving cells, but not an increase in cell number. NGF also induces neurite outgrowth from PC12 cells in serum-free medium. NGF-treated cells exhibit less cell-cell and neurite-neurite aggregation in the absence than in the presence of serum. The apparent minimum level of 2.5S NGF required for PC12 survival and morphological differentiation in serum-free medium is about 10 ng/ml (.apprx. 0.4 nM). Withdrawal of NGF in serum-free conditions results in degeneration of neurites and loss of cell viability. 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 serum may substitute for this requirement. The present system of maintaining a highly differentiated cell line in a chemically defined medium suggests certain experimental opportunities.