Induction of apoptosis by rho in NIH 3T3 cells requires two complementary signals. Ceramides function as a progression factor for apoptosis.

We have previously reported that rho genes, members of the ras superfamily, are tumorigenic when overexpressed in NIH 3T3 cells. As other known oncogenes, they also induce apoptosis after serum deprivation but not in the presence of growth factors. In the present study, we provide evidence that overexpression of the Aplysia Rho protein in NIH 3T3 cells induces the generation of phosphatidylcholine (PC)-derived second messengers as a result of activation of a PC-specific phospholipase D (PC-PLD) as previously reported for ras-transformed cells. In contrast, removal of serum in the Rho transfectants, but not in normal NIH 3T3 cells or cells transformed by the ras oncogene, induced the production of ceramides as a result of activation of an sphingomyelinase (SMase). Furthermore, the rho-expressing cells underwent apoptosis in the presence of serum when exogenous ceramides were added, and this process was accelerated if cells were treated with exogenous SMase. Thus, Rho proteins act as an initiation signal that is necessary but not sufficient for the induction of apoptosis in NIH 3T3 cells. We propose here that induction of apoptosis in NIH 3T3 cells requires two complementary signals: an initiation signal generated even in the presence of serum which 'primes' the cells, making them sensitive to a progression signal, triggered by serum removal, which we have identified as generation of ceramides.