Priming of PC12 cells for semiquantitative microinjection studies involving Ras

Nerve growth factor and activated Ras can induce differentiation of rat phe ochromocytoma cells (PC12 cells) [Greene and Tischler (1976) Proc, Natl. Ac ad. Sci, USA 73, 2424-2428] from a chromaffin cell-like morphology into one that resembles sympathetic neurones. We developed a special treatment of P C12 cells which apparently synchronises these cells such that they are more useful for semi-quantitative microinjection studies for signal transductio n pathways. This treatment leads to a faster and more reproducible differen tiation which faithfully reproduces the involvement of Ras in the process a nd allows a comparison of the biological activity of different Res mutants, It shows that G12V and Q61L oncogenic mutants are not equally potent in in ducing differentiation. Partial loss-of-function mutations T35S, E37G and Y 40C are inactive and even a triple combination of these does not restore fu ll biological activity. (C) 2000 Federation of European Biochemical Societi es.