Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.
The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.
Kerkhoff
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Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Germany.
Rapp
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D016475:3T3 Cells; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004965:Estrogen Antagonists / Q000494:pharmacology; D016147:Genes, Tumor Suppressor; D048031:JNK Mitogen-Activated Protein Kinases; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf; D011960:Receptors, Estrogen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; D050760:Cyclin-Dependent Kinase Inhibitor p27; C016601:afimoxifene; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases