All ETDs from UAB

Advisory Committee Chair

Donald J Buchsbaum

Advisory Committee Members

Stephen Barnes

Peter J Detloff

Gabriel A Elgavish

George Y Gillespie

Document Type

Dissertation

Date of Award

2011

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Our laboratory reported that a combination of ionizing radiation (IR) or temozolomide (Tmz), a DNA methylating agent clinically approved against glioblastoma multiforme (GBM), a type of human brain cancer, and TRA-8, an anti-DR5 monoclonal antibody produced synergistic cytotoxicity in GBM cells in culture and in mouse xenograft models through an yet unknown mechanism. I hypothesized that understanding of the synergy phenomenon can offer an insight into the nature of GBM vulnerability. The goals of this investigation were two-fold - to elucidate the mechanism of this enhancement in cytotoxicity, and to improve understanding of DR5 mediated apoptosis toward improvement of current anti-GBM therapies. In GBM cells in culture, enhancement of apoptosis induced by TRA-8 was contingent upon pre-treatment with Tmz. Tmz induced cell cycle arrest and senescence, but not apoptosis, at therapeutically relevant doses. Thus, the requirement for pre-treatment implicated genotoxic stress response and cell cycle regulation in the mechanism of sensitization. These were investigated along with the key events of apoptosis. As a result, a segment of the apoptotic cascade downstream of mitochondrial depolarization was eliminated as extraneous to the mechanism of enhancement. Additionally, enhancement of apoptosis induced by genotoxic stress appears to be a general phenomenon independent of the mode/source of genotoxicity. Since Tmz pre-treatment is required for enhancement, I investigated global changes in the cellular proteome induced by Tmz using 2-dimensional difference gel electrophoresis (2D DIGE). This approach detected a change in abundance of Apoptosis Inducing Mitochondria Associated Factor (AIF), among other proteins. The necessity of AIF for enhancement was examined with a knockdown experiment. Finally, I developed a mechanistic model of enhancement based on these results. The pro-apoptotic signals generated by DNA damage are transmitted through the Bcl-2 family of proteins. However, they cannot induce apoptosis because genetic alterations in GBM make it highly resistant. This creates a cumulative conditioning effect that "primes" cells for apoptosis. The subsequent treatment with TRA-8 induces apoptosis to a greater extent due to this "priming". Therefore, I propose that the control of mitochondrial permeability by the Bcl-2 family of proteins is the principal mechanistic event causing the enhancement.

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