Diffuse large B cell lymphoma (DLBCL) is a heterogeneous malignancy distinguished by aggressive clinical symptoms and abnormal B cell receptor... Show moreDiffuse large B cell lymphoma (DLBCL) is a heterogeneous malignancy distinguished by aggressive clinical symptoms and abnormal B cell receptor signaling. Current treatments are shown to have refractory or relapsed outcome post initial treatment signifying the need for novel chemotherapeutic targets. ERM family proteins have been shown to modify B cell immune response to antigen. Previous studies in our lab reveal that ERM proteins are phosphorylated in both DLBCL tumors and cell lines. Alteration of ERM function with Ezrin dominant negative mutant (EZ-DN) decreased the growth of DLBCL cells. This reduction of cell growth is thought to be mediated by interfering with the NF-kB and PI3K signaling. To understand the molecular mechanism of EZ-DN in a time dependent manner without impairing the cell growth immediately, Tet-on 3G based inducible expression system was utilized. Molecular cloning of the EZ-DN into the inducible system was performed, sequencing confirmed the insert of EZ-DN into the response vector (pTRE3G-BI-ZsGreen1) at desired locations. We have further evaluated the effect of doxycycline on the expression of fluorescent protein (Zsgreen1) and EZ-DN, also optimized its concentration for maximal expression of the proteins in DLBCL cell lines. Higher dose of doxycycline (above 5 μg/ml) induced reduction in cell viability. The expression of EZ-DN and Zsgreen1 is considered to be much stronger in the double stable cell lines containing the co-transfected regulator vector (pEF1α- TET3G) and response vector (pTRE3G-BI-ZsGreen1) with a selection marker. In conclusion, our data demonstrated the successful creation of inducible system that expresses EZ-DN, which can be employed to validate Ezrin as a therapeutic target in DLBCL. M.S. in Biology, May 2015 Show less
