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-mediated translocation of Raf-1 to the plasma membrane. However, whereas constitutively activated mutants of Ras [H-Ras(61L) and K-Ras4B(12V)] and Raf-1 (DeltaRaf-22W and Raf-CAAX) caused indistinguishable morphologic and growth (in soft agar and nude mice) transformation of NIH 3T3 fibroblasts, only mutant Ras caused morphologic transformation of RIE-1 rat intestinal cells. Furthermore, only mutant Ras-expressing RIE-1 cells formed colonies in soft agar and developed rapid and progressive tumors in nude mice. We also observed that activated Ras, but not Raf-1, caused transformation of IEC-6 rat intestinal and MCF-10A human mammary epithelial cells. Although both Ras- and DeltaRaf-22W-expressing RIE-1 cells showed elevated Raf-1 and mitogen-activated protein (MAP) kinase activities, only Ras-transformed cells produced secreted factors that promoted RIE-1 transformation. Incubation of untransformed RIE-1 cells in the presence of conditioned medium from Ras-expressing, but not DeltaRaf-22W-expressing, cells caused a rapid and stable morphologic transformation that was indistinguishable from the morphology of Ras-transformed RIE-1 cells. Thus, induction of an autocrine growth mechanism may distinguish the transforming actions of Ras and Raf. In summary, our observations demonstrate that oncogenic Ras activation of the Raf/MAP kinase pathway alone is not sufficient for full tumorigenic transformation of RIE-1 epithelial cells. Thus, Raf-independent signaling events are essential for oncogenic Ras transformation of epithelial cells, but not fibroblasts.
Oldham
S M
SM
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
Clark
G J
GJ
Gangarosa
L M
LM
Coffey
R J
RJ
Der
C J
CJ
D016475:3T3 Cells; D000818:Animals; D001943:Breast Neoplasms; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D002455:Cell Division; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D017077:Culture Media, Conditioned; D005260:Female; D011905:Genes, ras; D006801:Humans; D007422:Intestines; D051379:Mice; D008819:Mice, Nude; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)