Depletion of Raf-1 protooncogene by geldanamycin causes apoptosis in humanluteinized granulosa cells

Objective: To investigate the hypothesis that epidermal growth factor (EGF) signaling in luteinized granulosa cells works through Raf-1 and mitogen-ac tivated protein (MAP) kinases and that depletion of Raf-1 by geldanamycin w ill inhibit the signaling pathway and cause apoptosis. Design: Laboratory study. Setting: University of Minnesota. Patient(s): Human luteinized granulosa cells from IVF patients. Intervention(s): The cells were treated with vehicle (DMSO), 0.5 mu M of ge ldanamycin, 10 ng/mL of EGF, and geldanamycin + EGF. Main Outcome Measure(s): Radiochemical MAP kinase assay, Western blotting, confocal microscopy, and flow cytometry. Result(s): Geldanamycin treatment depleted Raf-1 and lowered MAP kinase act ivity in luteinized granulosa cells. EGF treatment increased MAP kinase pho sphorylation and translocation of the phosphorylated MAP kinase to the nucl eus. Geldanamycin blocked this effect. Cleavage of caspase-3, the execution er protein in apoptosis, into an active 17 kD fragment was observed by West ern blotting in geldanamycin-treated cells. Finally, by flow cytometry we o bserved significantly increased percentages of subdiploid apoptotic nuclei in geldanamycin-treated cells. Conclusion(s): In human luteinized granulosa cells, EGF works through Raf-1 , and MAP kinase and depletion of Raf-1 by geldanamycin resulted in decreas ed MAP kinase activity, increased activated caspase-3, and, ultimately, apo ptosis. (C)2000 by American Society for Reproductive Medicine.