Despite strong evidence that omega-3 fatty acids play a significant role in alleviating various human afflictions including cancer, little is known about their molecular mode of action. Because docosahexaenoic acid, with 22 carbons and 6 double bonds, is the extreme example of an omega-3 fatty acid, deducing its mode of action should be easier than for other shorter and less unsaturated fatty acids. Recently DHA has been linked to alterations in a variety of membrane properties. The project proposed herein extends prior work by monitoring DHA-induced lipid domain formation in phospholipid monolayers and bilayers composed of DHA-containing phosphatidyicholines (PCs) and phosphatidylethanolamines (PEs) mixed with other types of PCs and PEs, as well as cholesterol and sphingomyelin. This work will be done using differential scanning calorimetry, fluorescence resonance energy transfer, and fluorescence digital imaging microscopy. Once the nature of the various lipid domains is established, a membrane protein known to play an essential role in cancer surveillance, the major histocompatibility complex class I (MHC I) protein, will be reconstituted into the model membranes. Partitioning of this protein into specific lipid domains and the effect of domain composition (particularly DHA level) on MHC I conformation and activity will be measured with a variety of fluorescence and radiometric assays. Of particular importance is how DHA-induced lipid domains affect MHC I's ability to generate effector and memory cytotoxic T lymphocytes (CTL). Effector CTL, which are active killers, and memory CTL, which are long lived and may be activated to kill, are key players in the immune attack on cancer. Thus, this project traces DHA's effects from the molecular level, i.e., membrane structure, through to the activation and maintenance of CTL. These results will set the stage for creating novel methods of activating tumor-reactive CTL in vitro for the purpose of generating memory CTL to patrol for cancer cells in vivo.
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