The role of caspases in lytic mechanisms of tumor necrosis factor-mediated cytolysis

Chang, Wei-Chuan
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University of Hawaii at Manoa
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TNF activates several signaling pathways, some lead to cell death by apoptosis or necrosis. For our cell lines (B/C-N, 10ME, and L88.3), which represent a tumor progression model, it has not been determined whether TNF induces apoptosis or necrosis. B/C-N is not tumorigenic and represents normal cells; it is TNF-resistant. 10ME forms tumors only in immunodeficient animals and thus is intermediate on the pathway to cancer; it is TNF-sensitive. L88.3 forms tumors in normal mice and thus is cancerous; it is TNF resistant. TNF resistant cells can be rendered TNF-sensitive by addition of transcription or translation inhibitors. Recent findings indicate that TNF-induced cytolysis involves several initiator and effector caspases. Caspase comprise a family of proteases known to be involved apoptotic cell death. Thus, we investigated the correlation between TNF-mediated cytolysis and caspases activation in our cell lines. Our findings indicated that TNF induces activation of two initiator caspases (caspase-8 and caspase-9) and an effector caspase (caspase-3). Caspase-8 was activated first and may be involved in activating caspase-9. This suggests there is a link between the death receptor pathway (associated with caspase-8) and the mitochondrial pathway (associated with caspase-9). Activity levels of caspases correlated with TNF sensitivity of each cell line. TNF-induced resistance mechanisms also affect the level of caspase activities. In TNF-resistant cell lines B/C-N and L88.3, addition of a translation inhibitor caused an increase in TNF-mediated cytolysis and caspase activity. However in order to see an increase in TNF-mediated cytolysis, activity of caspase-3, -8 and -9 had to reach a threshold-level. When caspase activities were inhibited, TNF treatment activated a caspase-independent lytic pathway, perhaps involving generation of oxygen radicals or activation of phospholipase A2. In TNF-sensitive 10ME, inhibition of caspases caused a switch from apoptotic death to necrotic death in some cells. These experiments also revealed the presence of caspase-dependent and caspase-independent resistance mechanisms. Thus, the data indicate that TNF induces apoptosis through a caspase-dependent lytic mechanism. When caspase activities are inhibited, TNF induces a caspase-independent lytic mechanism. TNF also induces caspase-dependent and caspase-independent resistance mechanisms.
Thesis (Ph. D.)--University of Hawaii at Manoa, 2002.
Includes bibliographical references (leaves 134-151).
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xvii, 151 leaves, bound ill. 29 cm
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Theses for the degree of Doctor of Philosophy (University of Hawaii at Manoa). Microbiology; no. 4236
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