Priming of PC12 cells for semiquantitative microinjection studies involving Ras

Nerve growth factor and activated Ras can induce differentiation of rat pheochromocytoma cells (PC12 cells) [Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73, 2424-2428] from a chromaffin cell-like morphology into one that resembles sympathetic neurones. We developed a special treatment of PC12 cells which apparently synchronizes these cells such that they are more useful for semi-quantitative microinjection studies for signal transduction pathways. This treatment leads to a faster and more reproducible differentiation which faithfully reproduces the involvement of Ras in the process and allows a comparison of the biological activity of different Ras mutants. It shows that G12V and Q61L oncogenic mutants are not equally potent in inducing differentiation. Partial loss-of-function mutations T35S, E37G and Y40C are inactive and even a triple combination of these does not restore full biological activity.