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The serine / threonine kinase, c-Raf, plays an essential role in the control of proliferation and survival in many human malignant tumors. The aim of this work was to detect mechanisms by which c-Raf controls the apoptotic pathways. By using specific antisense oligo-nucleotides (AS-ODN) and “small interfering RNA“ (siRNA) for gene knockdown experiments, the c-Raf mRNA levels were reduced. These effects were reproduced by using a c-Raf Inhibitor BAY 43-9006. As suspected, the fraction of apoptotic cells increased and interestingly, simultaneously, the mRNA levels of different epidermal growth factor receptor (EGF-Receptor) ligands decreased. To confirm that stimulation of the EGF-receptor is critical for cell survival, the different cell lines were treated with an EGF-receptor blocker. As expected the fraction of apoptotic cells increased again. These data suggest an autocrine loop of the EGF-receptor ligand controlled by c-Raf. To identify other signalling pathways which are involved in c-Raf mediated protection of apoptosis, cDNA array experiments were performed. Surprisingly, it could be shown that the tumor necrosis factor receptor 1 (TNF-R1) is also transcriptionally controlled by c-Raf, suggesting crosstalk between the classical Ras-Raf-MEK-Erk pathway and TNF-receptor signalling in tumour cells to escape apoptosis.
This work supports the significance of c-Raf in the control of apoptosis and suggests two mechanisms as an explanation: an auotocrine EGF-receptor ligand loop and regulation of the TNFreceptor.