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

Abstract

Objective: To investigate the hypothesis that epidermal growth factor (EGF) signaling in luteinized granulosa cells works through Raf-1 and mitogen-activated protein (MAP) kinases and that depletion of Raf-1 by geldanamycin will 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 μM of geldanamycin, 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 activity in luteinized granulosa cells. EGF treatment increased MAP kinase phosphorylation and translocation of the phosphorylated MAP kinase to the nucleus. Geldanamycin blocked this effect. Cleavage of caspase-3, the executioner protein in apoptosis, into an active 17 kD fragment was observed by Western blotting in geldanamycin-treated cells. Finally, by flow cytometry we observed 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 decreased MAP kinase activity, increased activated caspase-3, and, ultimately, apoptosis.