Apoptosis is an evolutionarily conserved mechanism in organisms that is essential for homeostasis. TNFalpha in combination with cyclohexamide initiates a canonical extrinsic pathway, but a more physiologically relevant system is TNFalpha treatment in combination with IFNgamma, which together synergistically induce apoptosis in cells. Evidence is accumulating about the pathway induced in this system, however, the mechanism by which this occurs is still emerging.;One candidate mediator of this pathway is Siva-1, a pro-apoptotic protein known to sensitize cells to a variety of apoptotic signals. Because Siva-1 is regulated by p53 and is known to downmodulate NFkappaB, we hypothesized that Siva-1 is a mediator of TNFalpha/IFNgamma induced apoptosis. In this thesis, we wanted to address three central questions. What is the mechanism by which TNFalpha/IFNgamma induces apoptosis? Does Siva-1 play a role in this system? How does Siva-1 mediate this effect?;Results show several interesting points. First, TNFalpha/IFNgamma induced apoptosis is distinct from that induced by TNFalpha/CX. TNFalpha/IFNgamma induced apoptosis requires endogenous p53, whereas TNFalpha/CX does not. In addition, endogenous Bax was required in both models, but PUMA was required in neither. Inhibition of JNK or ROS partially rescued cells from apoptosis induced by TNFalpha/CX. Lastly, while NFkappaB reporter activity was high upon treatment with TNFalpha, cotreatment with IFNgamma reduced this activity at 24 hours. Because p53 is specifically induced in this model and its target protein Bax is essential for apoptosis, we looked at the requirement for Siva-1 in this system. Siva-1 was specifically upregulated in p53 dependent manner upon treatment with TNFalpha/IFNgamma. Further, knockdown of endogenous Siva-1 protected cells from apoptosis and overexpression of Siva-1 rescued the p53 null phenotype in a Bax dependent manner. Knockdown of endogenous Siva-1 was also shown to bring TNFalpha/IFNgamma induced NFkappaB activity back on par with TNFalpha stimulation alone. Overexpression of Siva-1 induces apoptosis and does so by an ROS dependent mechanism, indicating that Siva-1 can potentially modulate NFkappaB activity by its upregulation of ROS.;These results begin to describe a pathway that does not seem to fit into either extrinsic or intrinsic mediated apoptosis, rather a model in which external signals initiate an intrinsic like apoptosis. Because current strategies aim to regulate important mediators of apoptosis and/or cell survival, revealing novel mechanisms could potentially serve to offer new approaches to therapeutic design.
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