Electronic Thesis and Dissertation Repository


Master of Science




Dr. Gregory M Kelly


Mouse teratocarcinoma F9 cells differentiate into primitive endoderm (PrE) when treated with retinoic acid (RA) or H2O2 and these changes are accompanied by an upregulation of Wnt6 and activation of the canonical WNT/β-catenin pathway. Data from our lab shows PrE differentiation is accompanied by an increase in reactive oxygen species (ROS), which induces a conformational change in Nucleoredoxin preventing its ability to bind and inhibit Dishevelled. This in turn positively impacts on the WNT/β- catenin signaling pathway leading to differentiation. The source of endogenous ROS seen following RA treatment was investigated and members of the NADPH oxidase (NOX) family were identified as candidates as Nox1-4 and Duox2 genes are up-regulated by RA. This study shows Nox1 and Nox4 are up-regulated when Gata6 is overexpressed in F9 cells. Furthermore, the pan-NOX inhibitor VAS2870 and NOX1-specific inhibitor ML171 significantly reduced the ability of RA to induce PrE differentiation. Additionally, a knockdown of Nox1 and/or Nox4 attenuate RA-mediated differentiation. Overexpression of Nox1 or Nox4 in F9 cells increases the levels of ROS, however, this is not sufficient for differentiation. Thus, the data suggest that the ROS produced during the differentiation of F9 cells into PrE is the result of an increase in NOX1 and NOX4 activity; however, overexpressing Nox1 or Nox4 alone is not sufficient to induce cells to form PrE.