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


METHODS
Laboratory study.


METHODS
University of Minnesota.


METHODS
Human luteinized granulosa cells from IVF patients.


METHODS
The cells were treated with vehicle (DMSO), 0.5 microM of geldanamycin, 10 ng/mL of EGF, and geldanamycin + EGF.


METHODS
Radiochemical MAP kinase assay, Western blotting, confocal microscopy, and flow cytometry.


RESULTS
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.


CONCLUSIONS
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.
Department of Obstetrics, Gynecology and Women's Health, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
S M
SM
Khan
R H
RH
Oliver
L M
LM
Dauffenbach
J
J
Yeh
D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D047029:Lactams, Macrocyclic; D008183:Luteal Phase; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D011809:Quinones; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C001277:geldanamycin