ACTIVATION OF THE RAF-1 MAP KINASE CASCADE IS NOT SUFFICIENT FOR RAS TRANSFORMATION OF RIE-1 EPITHELIAL-CELLS/

The potent transforming activity of membrane-targeted Raf-1 (Raf-CAAX) suggests that Ras transformation is triggered primarily by a Ras-medi ated translocation of Raf-1 to the plasma membrane. However, whereas c onstitutively activated mutants of Pas [H-Ras(61L) acid K-Ras4B(12V)] and Raf-1 (Delta Raf-22W and Raf-CAAX) caused indistinguishable morpho logic and growth (in soft agar and nude mice) transformation of NIH 3T 3 fibroblasts, only mutant Ras caused morphologic transformation of RI E-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE- 1 cells formed colonies in soft agar and developed rapid and progressi ve tumors in nude mice, We also observed that activated Ras, but not R af-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and Delta Raf-22W-express ing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (M AP) kinase activities, only Ras-transformed cells produced secreted fa ctors that promoted PIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not Delta Raf-22W-expressing, cells caused a rapid and stable mor phologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growt h mechanism may distinguish the transforming actions of Ras and Raf. I n summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorig enic transformation of RIE-1 epithelial cells. Thus, Raf-independent s ignaling events are essential for oncogenic Ras transformation of epit helial cells, but not fibroblasts.