The role of oncogenic Ras in mediating apoptosis induced by the inhibition of protein kinase C

Ras serves as a molecular switch that controls a variety of cellular processes, including proliferation, differentiation, and senescence. In recent years, Ras has also been established as a modulator of apoptosis. The mechanisms by which Ras protects against apoptosis are becoming clear, but less is known about how Ras mediates apoptosis under certain conditions. We have previously shown that cells stably expressing oncogenic Ras selectively undergo apoptosis after PKC inhibition, unlike normal parental cells. However, due to the possibility of secondary changes in these stably-transformed cell lines, the causal role of Ras in mediating apoptosis was unclear. We now report that pretreatment of Ras-transformed mouse fibroblasts with a farnesyltransferase inhibitor prevented apoptosis after PKC inhibition, while transient expression of oncogenic Ras in normal mouse fibroblasts induced apoptosis in response to PKC inhibition. These results indicate that oncogenic Ras activity is both necessary and sufficient to mediate the apoptotic response to PKC inhibition. Although inhibition of PKC blocked cell cycle progression during PDGF stimulation of serum-starved mouse fibroblasts, no induction of apoptosis was observed, implying that physiologic Ras activation is not sufficient to mediate apoptosis. However, a variety of human tumor lines containing oncogenic Ras mutations showed sensitivity to PKC inhibition-induced apoptosis in vitro, and a PKC inhibitor reduced the in vivo growth of a pancreatic tumor expressing oncogenic Ras, suggesting that a clinical application may be feasible. The mechanism by which oncogenic Ras mediates the apoptotic response to PKC inhibition was unknown, but Ras has been reported to increase generation of reactive oxygen species, a known mediator of apoptosis. We now show that Ras-transformed cells express higher basal levels of reactive oxygen species compared to normal cells, and that PKC inhibition increases the generation of ROS. Treatment with the antioxidant, NAC, or incubation under hypoxic conditions, protected Ras-transformed cells from apoptosis, indicating that reactive oxygen species are necessary as downstream effectors of Ras-mediated apoptosis. Finally, oncogenic Ras is known to aberrantly drive cell cycle progression. Induction of cell cycle arrest inhibited Ras-mediated apoptosis, indicating that cell cycle progression may also be important for Ras-mediated apoptosis.