Apoptosis is a type of programmed cell death that is essential for the development and maintenance of tissue homeostasis in multicellular organisms. Because apoptosis is important in many biological situations, laboratories are working to delineate the signaling pathways responsible for this process. This thesis work attempts to identify new components of the signaling pathways by examining a role for mitochondria and proteasomes in T cell apoptosis.
Interest in mitochondria was initiated when a library screen performed in the laboratory revealed that portions of the mitochondrial genome, called apt-1 and apt-3, were downregulated in thymocytes during negative selection. In this thesis work, two aspects of mitochondrial function were examined in the T cell hybridoma, DO11.10: the integrity of the mitochondrial genome as assessed by levels of apt-l and apt-3 and the integrity of the mitochondrial membrane as assessed by membrane potential. Both the steady-state levels of apt-l and apt-3 RNA and mitochondrial transmembrane potential declined rapidly in apoptotic cells. Recent studies from other laboratories indicate that these changes are the result of the active participation of mitochondria in apoptosis.
Apoptosis relies heavily on proteolysis, and this dependence has encouraged investigators to analyze the significance of many proteases and proteolytic pathways. The second part of this thesis work focused on describing a role for the ubiquitin-proteasome pathway in apoptosis. Proteasome inhibitors were used to determine whether the proteasome is required in thymocyte apoptosis. Treatment of thymocytes with acetyl-leu-leu-methioninal (LLM), acetyl-leu-leu-norleucinal (LLnL), carbobenzoxyl-leu-leu-leucinal (MG132), or lactacystin inhibited death induced by dexamethasone, ionizing radiation, or phorbol 12-myristate 13-acetate (PMA). These results suggest that proteasome activity is necessary for the progression of many apoptotic pathways in thymocytes. Inhibitor studies also indicate that the proteasome functions upstream of many apoptotic events, including: PARP cleavage, caspase-3 activation, and mitochondrial transmembrane potential depolarization. Although a precise role for the proteasome in apoptosis cannot be assigned without an identification of its proteolytic targets, the work described in this thesis provides the first definitive link between proteasomes and apoptosis in mammalian systems.
