Difficulty demonstrating estradiol-mediated Erk1/2 phosphorylation in MCF-7 cells.
While some studies report that estradiol (E2) activates extracellular-signal regulated kinase (Erk1/2) in MCF-7 breast cancer cells, others report E2 does not activate this signaling pathway. This study attempted to resolve the conflicting reports by investigating experimental variables that could impact Erk1/2 activation using a high through-put assay that quantitatively assessed Erk1/2 phosphorylation. Variables tested included: cell staging and dosing regimes with and without charcoal-stripped serum, different MCF-7 cell sublines and culture densities and several E2 formulations and solvents. Levels of phosphorylated Erk1/2 were normalized to cellular protein rather than to total Erk1/2 protein because an antibody purported to recognize total Erk1/2 preferentially reacted with non-phosphorylated Erk1/2, potentially exaggerating the apparent level of Erk1/2 activation. Dosing MCF-7 cells with E2 containing small amounts of stripped serum induced Erk1/2 phosphorylation; however, this induction was largely attributed to serum factors. E2 administered in serum-free medium did not significantly alter Erk1/2 phosphorylation under any condition tested; immunocytochemistry corroborated this conclusion. While phosphatase inhibitors generally increased Erk1/2 phosphorylation, they did not impact E2-altered Erk1/2 phosphorylation. It remains important to resolve the basis of conflicting reports regarding E2-induced Erk1/2 activation due to the potential importance of this pathway on breast cancer and other processes.
Brower
Stacey L
SL
Department of Biochemistry and Molecular Pharmacology, Mary Babb Randolph Cancer Center, West Virginia University Health Sciences Center, Morgantown, WV 26506-9142, USA.
Roberts
Jenny R
JR
Antonini
James M
JM
Miller
Michael R
MR
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology; D002417:Cattle; D045744:Cell Line, Tumor; D002606:Charcoal; D004789:Enzyme Activation; D004958:Estradiol / Q000502:physiology*; D005260:Female; D006801:Humans; D015151:Immunoblotting; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D044967:Serum
D002606:Charcoal; D004958:Estradiol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3