| Summary: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013. === Cataloged from PDF version of thesis. Vita. === Includes bibliographical references. === The retinoblastoma protein (pRB) tumor suppressor is deregulated in the vast majority of human tumors. pRB is a well-established transcriptional co-regulator that influences many fundamental cellular processes. It has been most well characterized in its ability to block cell proliferation by inhibiting the E2F family of transcription factors. Importantly, pRB also plays a pivotal role in apoptosis. This function has been extensively characterized in the context of genotoxic stress. Specifically, these studies have revealed that pRB can act in both an anti-apoptotic manner by inducing cell cycle arrest, and a pro-apoptotic manner by transcriptionally co-activating proapoptotic genes. Here, we show that pRB can also promote TNF[alpha]-induced apoptosis. Moreover, this investigation led us to uncover a novel, non-transcriptional and non-nuclear role of pRB in the induction of apoptosis. Specifically, we found that pRB can enhance TNFainduced apoptosis even in the presence of an inhibitor of translation, and that a fraction of endogenous pRB is localized at the mitochondria both in the absence and presence of treatment with apoptotic stimuli. Further characterization revealed that pRB can directly bind to and activate BAX, resulting in mitochondrial outer membrane permeabilization and apoptosis. Importantly, targeting ectopically expressed pRB specifically to the mitochondria generated a separation-of-function mutant deficient for pRB's classic, nuclear roles. Remarkably, we found that this mito-tagged pRB mutant can promote apoptosis in response to many apoptotic stimuli, arguing that mitochondrial pRB is a general mediator of apoptosis. Moreover, expression of this mito-pRB mutant in vivo was sufficient to suppress tumorigenesis. Taken together, our data uncover a role for pRB in the direct activation of mitochondrial apoptosis. To our knowledge, this is the first characterization of a non-nuclear and transcription-independent function for pRB. Moreover, most human tumors are wild-type for pRB, but contain alterations that result in constitutive phosphorylation of pRB. While this functionally inactivates pRB's cell cycle function, we show that pRB's mitochondrial role is unaffected. This raises the possibility that this novel pro-apoptotic pRB mechanism can be exploited for chemotherapeutic treatment. === by Keren Ita Hilgendorf. === Ph.D.
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