All ETDs from UAB

Advisory Committee Chair

Yuhua Song

Advisory Committee Members

John D Montz

Donald Muccio

Tong Zhou

Document Type

Thesis

Date of Award

2013

Degree Name by School

Master of Biomedical Engineering (MBE) School of Engineering

Abstract

Breast cancer is one of the most commonly diagnosed cancers. Calmodulin (CaM) is involved in regulating breast cancer growth, proliferation, and transformation to malignancy. CaM has been shown to play a role in regulating Fas and DR-5 mediated apoptosis. The Fas receptor apoptotic pathway has been well characterized in cholangiocarcinoma; however, targeting Fas for anti-cancer therapy has shown hepatotoxicity in vivo. Thus, in vitro studies with breast cancer will focus on the DR-5 mediated apoptotic pathway. Targeting DR-5 to regulate breast cancer apoptosis is a promising strategy for breast cancer treatment. Understanding the mechanism of Fas and DR-5 interaction with CaM is imperative to unveil CaM-Fas/DR-5 binding as a potential target for novel strategies to modulate Fas or DR-5 apoptosis. Here the interactions of CaM with Fas death domain (DD) were characterized using a combined isothermal titration calorimetry (ITC) and circular dichroism spectroscopy (CD) approach. Furthermore, an interaction of CaM and DR-5 was characterized and the role of CaM-DR-5 binding in DR-5-mediated DISC formation was determined in MCF-7 and MDA-MB-231 breast cancer cells. ITC showed an endothermic and an entropy-driven CaM-Fas DD WT binding interaction at 37oC. The binding of CaM and Fas DD was significantly decreased by the Fas DD V254N mutation. Far UV CD showed that the V254N mutation resulted in secondary structure changes in the Fas DD. The structural changes of Fas DD V254N could affect CaM and Fas DD interaction. These results provided the structural and thermodynamic evidence to explicate the role of the V254N mutation of Fas in CAM-Fas interaction. For the characterization of CaM and DR-5 interaction, co-immunoprecipitation experiments using a DR-5 antibody demonstrated the interactions of CaM with DR-5. Further, upon DR-5 activation by TRA-8, CaM is recruited into the DR-5 mediated (DISC) and CaM antagonist trifluopherazine (TFP) attenuated DR-5 mediated DISC formation in MCF-7 and MDA-MB-231 breast cancer lines. The results showed the important role of CaM-DR-5 binding in DR-5 mediated DISC formation in breast cancer cells.

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