Apoptosis is a natural form of cell death by which damaged, defective or otherwise incompetent cells are efficiently removed without inflammation in multicellular organisms. The understanding of the mechanism and regulation of this active physiological process offers new possibilities for the manipulation of apoptosis for cancer therapy and prevention. Based on the hypothesis that a defective regulation of apoptosis may play a significant part in the etiology of cancer, this research project will use nuclear magnetic resonance (NMR) and other biophysical and biochemical techniques to determine the contributions of polyamines to the mechanism of apoptosis, and assess any contributions of polyamines to the mechanism of glucocorticoid resistance in human leukemic cells. The hypothesis that polyamines, due to their ability to promote cellular proliferation, may provide a mechanism for therapeutic resistance by inhibiting apoptotic cell death will be tested. The effects of intracellular level of polyamine on the activities of caspases, phospholipid metabolism and DNA fragmentation, which are key factors in apoptosis, will be determined. Using NMR and 15N-labeled polyamines, the metabolism of polyamines will be measured, and the connection between polyamine and the activity of transglutaminase (an enzyme involved in the crosslinking of proteins during apoptosis) will be evaluated as well as the time-scale of the actions of polyamines. Since intracellular pro-oxidant states are known contributing factors to cell proliferation and carcinogenesis, the assumption that antioxidants may be required during apoptosis to enhance apoptotic efficiency will also be tested by this research. The contributions of common vitamin antioxidants (e.g., vitamin A, vitamin C, and vitamin E) to the mechanism of apoptosis and glucocorticoid resistance, and the extent of the involvement of oxidative stress in the mechanism of apoptosis and glucocorticoid resistance will be evaluated by specifically determining the effects of these antioxidants on phospholipid metabolism, cellular activity of caspases, the level of DNA degradation, the level of intracellular free Ca(II), cell membrane potential, and cellular energy state. The ability of antioxidants to work synergistically with glucocorticoid to induce apoptosis or reduce resistance will also be evaluated. One glucocorticoid-sensitive and three different glucocorticoid-resistant variants will be used. The results obtained for all the four cell lines will be analyzed for the roles of polyamines and antioxidants in apoptosis, and for new insight into the mechanisms of apoptosis and glucocorticoid resistance in leukemic cell lines.
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