Date of Graduation
Estradiol (E2), epidermal growth factor (EGF) and acetaminophen (APAP) each exert mitogenic activity in estrogen receptor-positive (ER+) MCF-7 human breast cancer cells. Antiestrogens inhibit the mitogenic activity of each of these compounds, suggesting that the ER plays a role. EGF is an established activator of the Erk/MAPK signal transduction pathway, and E2 reportedly activates this pathway as well. In addition, both E2 and EGF have been reported to induce ER phosphorylation, a modification of the receptor that is important in ER signaling. Therefore, the first goal of this research project was to determine the extent to which all three mitogens affect Erk/MAPK activation. In agreement with other reports, EGF induced robust Erk/MAPK activation; however, E2 did not stimulate Erk/MAPK. APAP had no effect on Erk1/2 activation. The next goal was to determine the extent to which phosphorylation of endogenous ERÎ± is altered by these three mitogens in MCF-7 cells. In accordance with established findings, E2 induced significant ERÎ± phosphorylation, as assessed by an upshift of the total ERÎ± population and detection of phosphorylation of ERÎ± at serine 118 in Western blotting. However, neither EGF nor APAP evoked phosphorylation of endogenous ERÎ±. Although E2 did not induce Erk1/2 activation, the Erk/MAPK pathway has been implicated in E2-induced ERÎ± phosphorylation. The third objective of this project was to identify signaling pathways that play a role in E2-induced ERÎ± phosphorylation through the use of a variety of kinase inhibitors as well as RNA-mediated interference (RNAi). The results of these studies suggest that the basal activity of Erk1/2 plays a role is E2-mediated ERÎ± phosphorylation. Finally, in an attempt to rectify conflicting reports regarding the effect of E2 on Erk/MAPK activation, more in-depth studies were undertaken to assess various factors that may impact experimental results. Although some experimental variables that may impact Erk/MAPK activation were identified, these studies could not completely resolve why some reports describe E2-induced Erk1/2 activation and others do not.
Brower, Stacey Lynn, "The mitogens estradiol, epidermal growth factor and acetaminophen differentially alter estrogen receptor phosphorylation and Erk/MAPK activation in MCF-7 cells." (2004). Graduate Theses, Dissertations, and Problem Reports. 8537.