Mechanisms of mitochondria-mediated apoptosis induced by cytotoxic stress

Defects within the apoptotic pathway are thought to contribute to tumorigenesis and therapeutic resistance. Most cytotoxic anti-cancer drugs are thought to activate the mitochondria-mediated apoptotic pathway; however, the precise mechanistic details remain unclear and, in some instances, controversial. The prevailing view of mitochondria-mediated apoptosis is that the process occurs through a series of events that are orchestrated in a linear step-wise fashion. From this perspective, initiating events include the activation of a BH3-only family member which then activates Bax and/or Bak resulting in their homo-oligomerization and pore formation spanning the outer mitochondrial membrane to induce mitochondrial outer membrane permeabilization (MOMP). Subsequently, cytochrome c and other pro-apoptotic factors are released from the intermembrane space of the mitochondria into the cytosol through this pore. Cytosolic cytochrome c interacts with Apoptotic protease activating factor-1 (Apaf-1) and initiator caspase-9 where this complex serves as the activating platform for initiator caspase-9. Active caspase-9 then activates downstream effector caspase-3 and -7, which produce the biochemical and morphological features associated with apoptosis.;For my dissertation research, I have investigated the underlying molecular requirements necessary for mitochondria-mediated apoptosis induced by a DNA topoisomerase II inhibitor, a novel heat shock protein 90 (Hsp90) inhibitor, and elevated temperature in Jurkat T-lymphocytes. Combined, the data suggest that different cytotoxic stressors use similar as well as distinct mechanisms to execute mitochondria-mediated apoptosis. In addition, the data provide new insights into the mechanistic details utilized by these three different stressors, which may prove useful for future drug design and therapeutic approaches.;In the first Specific Aim, I investigated the molecular requirements necessary for mitochondria-mediated apoptosis in response to the DNAdamaging drug etoposide. The data suggest that the BH3-only family member Bid is important during DNA-damage induced apoptosis where it functions to facilitate MOMP. These studies also found that the active form of the protein, tBid, is generated downstream of initiator caspase-9 activation by executioner caspase-3 and/or -7. Taken together, these data suggest that effector caspases, Bid, and mitochondria forge an amplification loop to irreversibly commit a cell to apoptosis.;The antiproliferative effects of a novel C-terminal inhibitor of Hsp90, designated KU135, were characterized in the second Specific Aim. This study compared the effects of an N-terminal Hsp90 inhibitor, 17-AAG, to the effects of the C-terminal Hsp90 inhibitor, KU135. KU135 was found to have more potent antiproliferative effects than 17-AAG, and KU135 activated the mitochondria-mediated apoptotic pathway, whereas 17-AAG was primarily cytostatic. Finally, 17-AAG caused robust induction of the cytoprotective proteins Hsp90 and Hsp70 which could account for its lack of an apoptotic response.;The molecular requirements for heat-induced apoptosis were examined in the third Specific Aim. The data showed that heat-induced apoptosis is a mitochondria-mediated event and that caspase-9, and not caspase-2, is the most apically activated caspase. The BH3-only protein, Bid, was found to be important during heat-induced apoptosis where it was needed to facilitate MOMP. However, we found that tBid was generated downstream of caspase-9 suggesting that it may function to promote cytochrome c release during this process as part of a feed forward amplification loop.