| This thesis describes the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in low density lipoprotein (LDL)-derived cholesterol trafficking. The cholesterol trafficking or CT mutants, selected by their ability to survive cholesterol starvation conditions, possess distinct phase-dense particles that stain intensely with acridine orange, suggesting that they are acidic. Co-localization with filipin, a cholesterol-specific fluorescent dye, and an antibody directed against a lysosomal/endosomal preparation, suggest that the phase-dense particles are lysosomes/endosomes filled with cholesterol. The mutants bind, internalize, and process LDL normally, but the cholesterol resulting from hydrolysis of cholesterol linoleate accumulates in the acidic compartments. The inability to transport the free cholesterol to its normal intracellular destination results in a decrease in activation of cholesterol ester synthesis in response to LDL compared to the parental cell line, 25-RA, despite the fact that the reconstituted activity of the enzyme catalyzing the esterification reaction is similar in the mutant and parental cells.;Fusion of the CT mutants with the 25-RA cell line indicated that the defect in the CT cell lines resulted from a recessive mutation. Hybridizations between one mutant, CT60, and the other CT cell lines revealed that all the CT mutants available in our laboratory belong to one complementation group. Fusions between normal human fibroblasts and one of the CT cell lines, CT60, demonstrated that human DNA could provide a factor (s) necessary for correcting the CT defect.;Transfection of either DNA from hamster/human hybrids or mutant CT60 into the CT60 cell line resulted in the isolation a cell line possessing a limited ability to mobilize LDL-derived cholesterol. Selection of the transfectants utilized the fluorescence activated cell sorter and nile red, a fluorescent dye which preferentially stains neutral lipids such as cholesterol esters. The transfectants which were able to utilized LDL-derived cholesterol at 30-40% the rate seen in 25-RA cells, were isolated by their high nile red fluorescence relative to CT60 cells. The fact that CT60 DNA transfected into the CT60 cell line results in a cell line with increased cholesterol mobilization indicates that a CHO gene different than the one encoding the CT factor has been altered during transfection. |
