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Date of Award
Doctor of Philosophy (PhD)
Among the three morphologically distinct forms of cell death (apoptosis; autophagic cell death; necrosis), necrosis is the least understood and generally perceived as a passive cellular demise resulted from unmanageable physical damages. Our study concluded an active de novo genetic program underlying DNA damage-induced necrosis. We demonstrated that cells deficient of the essential mitochondrial apoptotic effectors, BAX and BAK, ultimately succumbed to DNA damage-induced necrosis. Importantly, this genotoxic stress-triggered necrotic death was completely abrogated when either transcription or translation was inhibited. Mechanistically, we pinpointed the p53-cathepsin axis cooperating with reactive oxygen species (ROS) to execute necrosis. Furthermore, p53 activates cancer cell necrosis in vivo. In a tumor allograft model, chemotherapy resulted in the shrinkage of Bax and Bak double knockout (DKO) cell derived tumors, but not the tumors derived from Bax and Bak DKO cells overexpressing p53 dominant negative mutants. Lastly, we demonstrate that p53, Bax and Bak triple knockout (TKO) cells were more resistant to DNA damage-induced necrosis than Bax and Bak DKO cells. Inhibition of ROS production further protected p53, Bax and Bak TKO cells from genotoxic stress.
We further extend our investigation in vivo using mouse models, and found that irradiation induces necrotic cell death of the cerebellar neurons at the external granule layer of BaxF/-Bak-/-Nestin-Cre+ animals. Importantly, deficiency of p53 in combination with antioxidant protected DKO neurons form necrotic death, providing an in vivo proof of the p53 and ROS mediated programmed necrotic death triggered by DNA damage in vivo. Necrotic death can also be induced in BaxF/-Bak-/-CD19-Cre+ B lymphocytes upon DNA damage. Interestingly, DNA damage induced necrosis of Bax/Bak DKO B lymphocyte is p53-independent, but still ROS dependent. In summary, our study offers experimental and therapeutic basis for future discoveries aimed at interfering or triggering "programmed necrotic death" in diseases with excessive or defective cellular demise.
Lastly, our laboratory showed that the activator BH3-only proteins activate a BAX/BAK-dependent but caspase-independent cell death by triggering mitochondrial dysfunction. Using a BH3-inducible system in Apaf-1 knockout cells, we have demonstrated that this caspase-independent cell death is blocked by a number of chemicals such as serine protease inhibitors, proteasome inhibitors, antioxidants, iron chelators, and autophagy inhibitors. We have performed a siRNA library screen against all the serine proteases in the mouse genome, and identified five potential regulators of the caspase-independent cell death pathway. Further characterization of these candidate serine proteases and their roles in cell death will provide new insights into the mechanisms of mitochondrial dysfunction regulated by the BCL-2 family proteins.
Chair and Committee
David A Harris
Eugene Johnson, Heather True-Krob
Tu, Ho-Chou, "Investigation of Caspase-Independent Cell Death Pathways" (2010). Arts & Sciences Electronic Theses and Dissertations. 520.
Available for download on Friday, August 15, 2110